US 2015 0320694A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0320694 A1 GU et al. (43) Pub. Date: Nov. 12, 2015

(54) MUCOADHESIVENANOPARTICLE Publication Classification DELIVERY SYSTEM (51) Int. Cl. A 6LX 9/5 (2006.01) (71) Applicants: Frank GU, Kitchener (CA); Lyndon A638/3 (2006.01) James William JONES. Waterloo (CA): A613 L/704 (2006.01) Shengyan (Sandy) LIU, Waterloo (CA) (52) U.S. Cl. CPC ...... A61K9/5 161 (2013.01); A61 K3I/704 (72) Inventors: Frank GU, Kitchener (CA); Lyndon (2013.01); A61 K9/5192 (2013.01); A61 K James WilliamO O JONES. Waterloo (CA): 9/5153 (2013.01); A6K9/523A61K 38/13 (2013.01);(2013.O1 Shengyan (Sandy) LIU, Waterloo (CA) ( .01) (57) ABSTRACT (21) Appl. No.: 14/410,521 The present disclosure relates generally to a mucoadhesive nanoparticle delivery system. The nanoparticles are formed 1-1. from amphiphilic macromolecules conjugated to a mucosal (22) PCT Filed: Jun. 20, 2013 targeting moiety in Such a manner that the Surface of the nanoparticle is coated with the targeting moiety. The Surface (86). PCT No.: PCT/CA2O13/OSO475 density of the targeting moiety can be tuned for adjustable S371 (c)(1) targeting of the nanoparticles to a mucosal site without Sub A ri stantially compromising the stability of the particles. The (2) Date: Dec. 22, 2014 particles were found to have high loading efficiency and Sustained release properties at the mucosal site. The present O O disclosure also relates to polymers and macromolecules use Related U.S. Application Data ful in the preparation of the mucoadhesive nanoparticles, as (60) Provisional application No. 61/690,127, filed on Jun. well as compositions, methods, commercial packages, kits 20, 2012. and uses related thereto.

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MUCOADHESIVENANOPARTICLE for example, in US. Pat. No. 2011/0300219. Amphiphilic DELIVERY SYSTEM compound assisted nanoparticles for targeted delivery have been disclosed, for example, in US. Pat. No. 2010/0203142. CROSS REFERENCE TO RELATED 0007 Targeting controlled release polymer systems (e.g., APPLICATIONS targeted to a particular tissue or cell type or targeted to a 0001. This application claims the benefit of priority of specific diseased tissue but not normal tissue) is desirable. It U.S. Provisional Patent Application No. 61/690,127, filed can enhance the drug effect at the target site and reduce the Jun. 20, 2012, which is incorporated herein by reference in its amount of a drug present in tissues of the body that are not entirety. targeted. Therefore, with effective drug targeting, it may be possible to reduce the amount of drug administered to treat a TECHNICAL FIELD particular disease or condition and undesirableside side effects may also be reduced. 0002 The present disclosure relates generally to a 0008 Various benefits can be obtained through delivery of mucoadhesive nanoparticle delivery system. The nanopar therapeutic agents through a mucosal tissue. For example, ticles can be tuned for controlled targeting and adhesion of the mucosal delivery is generally non-invasive, thereby avoiding nanoparticles at a mucosal site without Substantially compro uncomfortable aspects of intravenous, intramuscular, or Sub mising the stability of the particles. The present disclosure cutaneoud delivery means. Application of a therapeutic agent also relates to components useful in the preparation of the to a mucosal tissue can also reduce the effect of first-pass nanoparticles, as well as to compositions, methods, pro metabolism and clearance by circulating immune cells. How cesses, commercial packages, kits and uses related thereto. ever, given the tendency of natural bodily fluids to clear applied therapeutic agents from the site of administration, the BACKGROUND administration of therapeutic agents to mucosal sites, such as 0003. The delivery of a drug to a patient with controlled the eye, nose, mouth, stomach, intestine, rectum, vagina, or release of the active ingredient has been an active area of lungs, among others, can be problematic. research for decades and has been fueled by the many recent 0009 Topical administration is the most common delivery developments in polymer Science. Controlled release poly method employed for treating diseases and conditions affect mer systems can be designed to provide a drug level in the ing the eye, such as corneal diseases. Common topical for optimum range over a longer period of time than other drug mulations, such as eye drops or ointments, Suffer from low delivery methods, thus increasing the efficacy of the drug and ocular bioavailability due to rapid drainage through the naso minimizing problems with patient compliance. lacrimal duct, near constant dilution by tear turnover, and low 0004 Nanomedicine the fusion of nanotechnology and drug permeability across the corneal epithelium. As a result, medicine—is among the most promising approaches to topical formulations are normally administered multiple address challenges associated with conventional drug deliv times daily in order to achieve therapeutic efficacy, resulting ery methods. In the past decade, drug delivery systems con in a higher potential for side effects and lower patient com structed from polymeric nanoparticles (NPs) have been the pliance. cornerstone of progress in the field of nanomedicine. Various 0010 Recently, formulations using NPs as drug carriers types of polymeric materials have been studied for NP drug have been proposed to overcome the limitation associated delivery applications. with topical administration methods. NP carriers have been 0005 PLGA PEG is the most widely used polymer for shown to improve drug stability in water and also prolong making biodegradable drug delivery systems. The self-as drug activity by releasing encapsulated compounds in a con sembly of PLGA-PEG block copolymers generally yields trolled manner (Ludwig, 2005; Nagarwal 2009; Liu, 2012). NPs of sizes greater than 150 nm (Karnik, 2008). Although NPs formulated using biodegradable polymers, such as poly Smaller particles can be synthesized, they generally Suffer (lactic-co-glycolic acid) (PLGA), have been tested for ocular from low drug encapsulation and rapid drug release (Karnik, topical drug delivery applications (Diebold, 1990; Zimmer, 2008). The present inventors reported that typical maximum 1995). Poly(ethylene glycol)-based NPs have attracted sig drug loading in PLGA-PEG was found to be 7.1 wit/wt % nificant attention due to their ability to improve the stability of (Verma, 2012, incorporated herein by reference in its drug carrier systems in physiological environments (Bazile, entirely). Other PEG based polymers showed drug loading 1995; Dhar, 2008; Dong, 2007; Esmaeili, 2008). ranging from 4.3 to 11.2 wit/wt % (Shuai, 2004; He, 2010; 0011. The synthesis of surface-functionalized NP drug Missirlis, 2006). delivery systems has been explored. In order to achieve 0006 Nanoparticles have been developed as sustained mucoadhesion, the synthesis typically requires two-stage release vehicles used in the administration of small molecule synthesis whereby the first stage involves the formation of drugs as well as protein and peptide drugs and nucleic acids. NPs, while the second stage involves the conjugation of The drugs are typically encapsulated in a polymer matrix ligands on the surface of these NPs. Recently, a new technol which is biodegradable and biocompatible. As the polymer is ogy demonstrated the formation of targeting NPS using one degraded and/or as the drug diffuses out of the polymer, the step synthesis whereby the formation of the NP and the sur drug is released into the body. Typically, polymers used in face functionalization can be accomplished in one step (U.S. preparing these particles are polyesters such as poly(lactide Pat. No. 8,323,698, incorporated herein by reference). This co-glycolide) (PLGA), polyglycolic acid, poly-beta-hy technology is particularly useful for applications where mini droxybutyrate, polyacrylic acid ester, etc. These particles can mal targeting ligand is required, e.g. for systemic bolus injec also protect the drug from degradation by the body. Further tions where the number of targeting ligands on the Surface more, these particles can be administered using a wide variety must be controlled to minimize systemic immunogenicity. of administration routes. Various types of materials used for When nanoparticles are formed using the one-step method, synthesizing nanoparticle drug carriers have been disclosed, targeting ligands may be detected within the core of the US 2015/0320694 A1 Nov. 12, 2015 nanoparticles. Thus, this methodology may not be ideal 2006). A recent study also suggested that polymers with where maximum targeting is desired. acrylate end groups are also capable of binding to the thiol 0012. The surfaces of polymeric NPs have been function moieties of mucous membrane through Michael addition alized with molecular ligands that can selectively bind to the (Davidovich-Pinhas and Bianco-Peled 2010). The study ocular mucosa to increase precorneal drug retention (duToit, demonstrated that the poly(ethylene glycol) diacrylate 2011; Khutoryanskiy, 2011; Shaikh, 2011). To date, the most formed stable covalent linkage with thiol groups of freshly widely used method to achieve mucoadhesion exploits elec extracted porcine Small intestinal mucin under physiological trostatic interactions between the negatively charged sialic conditions, which was confirmed using NMR characteriza acid moieties of the corneal mucin and cationic polymers tion. such as chitosan (Sogias, 2008). However, the electrostatic 0015. It is desirable to provide targeted nanoparticle deliv interactions may be hindered by various counter ions in the ery systems for controlled delivery of a payload to a mucosal tear fluid, resulting in the clearance of these NPs by tear site. In particular, it is desirable to provide improved mucoad turnOVer. hesive delivery systems that can be retained at a mucosal site 0013. A number of molecular targeting groups have been for a sufficient period of time to provide sustained release of Suggested in the past for targeting the human mucosal lining: the payload. It is particularly desirable to be able to tune such U.S. Pat. No. 7,803,392 B2 filed Dec. 8, 2011, entitled “pH delivery systems such that the extent of targeting and adhe sensitive mucoadhesive film-forming gels and wax-film com sion can be controlled without Substantially compromising posites suitable for topical and mucosal delivery of mol the stability of the delivery system. ecules”; US Pat. 2005/0196440, filed Sep. 8, 2005, entitled “Mucoadhesive drug delivery devices and methods of making SUMMARY and using thereof: US Pat. 2005/028.1775, filed Dec. 22, 0016. The present disclosure relates generally to a 2005, entitled “Mucoadhesive and bioadhesive polymers'; mucoadhesive nanoparticle delivery system. EP2167044 A1, filed Dec. 11, 2008, entitled “Mucoadhesive 0017. In a first aspect, the present disclosure provides a vesicles for drug delivery': WO 2005/117844, filed Sep. 17, nanoparticle composition useful for delivery of a payload to a 2009, entitled “Mucoadhesive nanocomposite delivery sys mucosal site, the nanoparticle comprising a plurality of tem: WO 2010/096558, filed Feb. 18, 2010, entitled “Bi amphiphilic macromolecules, the macromolecules compris functional co-polymer use for ophthalmic and other topical ing: a hydrophobic portion; a hydrophilic portion comprising and local applications'; US Pat. 2013/0034602, filed Jul.30, multiple functional moieties; and a mucosal targeting moiety, 2012, entitled “Enteric-coated capsule containing cationic wherein at least a portion of said functional moieties on the nanoparticles for oral insulin delivery, EP Pat. 25.10930A1, hydrophilic portion are conjugated to the mucosal targeting filed Apr. 15, 2011, entitled “Nanoparticles comprising half moiety. esters of poly (methyl vinyl ether-co-maleic anhydride) and 0018. In further aspect, the present disclosure provides a uses thereof; U.S. Pat. No. 8,242,165 B2, filed Oct. 26, 2007, nanoparticle composition useful for delivery of a payload to a entitled “Mucoadhesive nanoparticles for cancer treatment': mucosal site, the nanoparticle comprising a plurality of EP Pat. 0516141 B1 filed May 29, 1992, entitled “Pharma amphiphilic macromolecules, the macromolecules compris ceutical controlled-release composition with bioadhesive ing: a hydrophobic portion comprising a biocompatible poly properties”: WO 1998/030207 A1, filed Jan. 14, 1998, mer selected from a from polylactide, a polyglycolide, poly entitled “Chitosan-gelatin a microparticles'; EP Pat. (lactide-co-glycolide), poly(e-caprolactone), O a 1652517 B1, filed Jun. 17, 2004, entitled “Hyaluronic acid combination thereof a hydrophilic portion comprising a bio nanoparticles”; U.S. Pat. No. 8,361,439 B1, filed Aug. 20, compatible polymer selected from polysaccharide, poly 2012, entitled “Pharmaceutical composition of nanopar nucleotide, polypeptide, or a combination thereof, the hydro ticles'. However, these documents only describe mucoadhe philic portion comprising multiple functional moieties; and a sive materials that undergo physical interaction with the mucosal targeting moiety selected from a phenylboronic acid mucous lining (e.g. electrostatic interaction between cationic (PBA) derivative, a thiol derivative or an acrylate derivative, chitosan materials with the negatively charged mucin layer). wherein at least a portion of said functional moieties of the The main disadvantage of physical interaction is that it is hydrophilic portion are conjugated to the mucosal targeting unspecific and much weaker compared to covalent interac moiety. tions. 0019. In a further embodiment, there is provided a nano 0014. A few studies have reported molecular targeting particle composition useful for delivery of a payload to a groups with potential to covalently bind to mucosal tissue. mucosal site, the nanoparticle comprising a plurality of Phenylboronic acid (PBA), which contains a phenyl substitu amphiphilic macromolecules, the macromolecules each com ent and two hydroxyl groups attached to boron, has been prising: a hydrophobic biocompatible polymer selected from reported to form a complex with the diol groups of sialic acid a from polylactide, a polyglycolide, poly(lactide-co-gly at physiological pH (Matsumoto, 2010; Matsumoto, 2010; collide), poly(e-caprolactone), or a combination thereof, the Matsumoto, 2009). Another class of molecules that can hydrophobic polymer forming the core of the nanoparticle; a covalently bind to the mucous membrane is polymeric thi hydrophilic biocompatible polymer selected from polysac omers (Ludwig, 2005). These thiomers are capable of form charide, polynucleotide, polypeptide, or a combination ing covalent disulfide linkage with cysteine-rich Subdomains thereof, having multiple functional moieties, the hydrophilic of the mucous membrane (Khutoryanskiy, 2010). Typical portion forming the shell of the nanoparticle; at least a portion examples of polymeric thiomers include the following con of the functional moieties being conjugated to a mucosal jugates: poly(acrylic acid)/cysteine (Gugg, 2004), chitosan/ targeting moiety selected from a phenylboronic acid (PBA) N-acetylcysteine (Schmitz, 2008), alginate/cysteine derivative, a thiol derivative or an acrylate derivative. (Bernkop-Schnurch, 2008) chitosan/thio-glycolic acid (Sak 0020. In a further embodiment, there is provided a nano loetsakun, 2009) and chitosan/thioethylamidine (Kafedjiiski, particle composition useful for delivery of a payload to a US 2015/0320694 A1 Nov. 12, 2015 mucosal site, the nanoparticle comprising a plurality of ceutical composition as described herein, together with amphiphilic macromolecules, the macromolecules compris instructions for use in treating a disease. ing: a hydrophobic portion comprising a polylactide; a hydro 0033. In another aspect, there is provided a method of philic portion having multiple functional moieties, said preparing a nanoparticle composition useful for delivery of a hydrophilic portion comprising dextran; and a mucosal tar payload to a mucosal site, the method comprising: a) prepar geting moiety being a phenylboronic acid (PBA) derivative, ing an amphiphilic macromolecule comprising a hydrophilic wherein at least a portion of said functional moieties of the portion and a hydrophobic portion, the hydrophilic portion hydrophilic portion are conjugated to the mucosal targeting comprising multiple functional moieties; b) assembling a moiety. plurality of said macromolecules under Suitable conditions to 0021. In a further embodiment, there is provided a nano form a nanoparticle having a hydrophobic core and a hydro particle composition useful for delivery of a payload to a philic shell; and c) conjugating at least a portion of said mucosal site, the nanoparticle comprising a plurality of functional moieties on the hydrophobic portion to a mucosal amphiphilic macromolecules, the macromolecules each com targeting moiety to provide a Surface-functionalized nanopar prising a hydrophobic polylactide polymer conjugated to a ticle. hydrophilic dextran polymer having multiple functional moi 0034. Other aspects and features of the present disclosure eties, at least a portion of said functional moieties being will become apparent to those ordinarily skilled in the art conjugated to a phenylboronic acid (PBA) derivative. upon review of the following description of specific embodi 0022. In a further embodiment, there is provided a Dext ments in conjunction with the accompanying figures. ran-p-PLA block copolymer, wherein at least a portion of the functional groups on the Dextran are conjugated to a targeting BRIEF DESCRIPTION OF THE DRAWINGS moiety capable of forming a high affinity bond with a target at a mucosal site. 0035 Embodiments of the present disclosure will now be 0023. In some embodiments, the nanoparticle is formed described, by way of example only, with reference to the by conjugating the polylactide to the dextran to form a nano attached Figures. particle and Subsequently surface-functionalizing the nano 0036 FIG. 1 is NMR spectra at various steps of block particle by conjugating at least a portion of the functional copolymer synthesis: a) Proton NMR of I. Dextran 6 kDa moieties of the dextran to the PBA derivative to achieve a (D2O), II. Dextran-NH-Et-NH-Boc (D2O), III. Dextran-NH desired surface density of the PBA derivative. Et-NH2 (D2O), IV. PLA 20 kDa (DMSO-d6), V. Dextran-Et 0024. In some embodiments, the nanoparticle is formed PLA, or PLA20-Dex6 (DMSO-d6); b) Carbon NMR of block by conjugating the polylactide to the dextran to form a nano copolymer I. PLA20kDa, II. Dextran 6 kDa, III Dex-Et-PLA particle and Subsequently reacting the functional moieties of (PLA20-Dex6) confirming conjugation of Dextran and PLA. the dextran with PBA such that substantially all of the PBA is 0037 FIG. 2 shows particle size and morphology of dex tran-b-PLANPs: a) Effect of MWs of PLA and Dextran on located in the shell?on the surface of the nanoparticle. the sizes of the NPs formed from nine different polymers 0025. In some embodiments, the core of the nanoparticle using PLA with MW 10 kDa (red), 20kDa (green) and 50kDa is Substantially free of targeting moiety. (blue), and Dextran with MW 1.5kDa, 6 kDa and 10kDa. The 0026. In another aspect, there is provided a pharmaceuti black bars represent the standard deviation of the particle cal composition comprising a nanoparticle composition as sizes of each block copolymer; b) TEM image of PLA20 defined in herein, and a pharmaceutically acceptable carrier. Dex6 NPs (Scale bar is 100 nm) to demonstrate spherical 0027. In another aspect, there is provided a mucoadhesive shape of the nanoparticles. delivery system for delivering a payload to a mucosal Surface, 0038 FIG. 3 is a graph of drug encapsulation in NPs: a) the delivery system comprising a nanoparticle composition as Doxorubicin encapsulation efficiency in Dex-b-PLA and defined herein; a pharmaceutically acceptable carrier, and a PLGA-PEG NPs using nanoprecipitation and b) the corre payload. sponding drug loading wt %. Solid gray columns are for 0028. In another aspect, there is provided a method of PLA20-Dex6 NPs, solid white columns are for PLA20 treating or preventing a disease or condition comprising Dex 10 NPs and columns with diagonal lines pattern are for administering to a Subject an effective amount of a nanopar PLGA-PEG NPs (n=3; meani.S.D). ticle composition or pharmaceutical composition as 0039 FIG. 4 is a graph of in vitro Doxorubicincumulative described herein. release profiles from Dex-b-PLA and PLGA-PEG NPs con 0029. In another aspect, there is provided a use of the ducted in PBS at 37° C. Solid square () are for PLA20 nanoparticle composition or pharmaceutical composition as Dex10, solid circles (O) are for PLA20-Dex6 and solid tri described herein for treating a disease capable of being treat angles (A) are for PLGA-PEG NPs (n=3; meantS.D). ing by administering a therapeutic agent to a mucosal site. 0040 FIG. 5 is a graph of hemolytic activity of Dex-b- 0030. In another aspect, there is provided a use of the PLA and PLGA-PEG NPs for concentrations relevant to nanoparticle composition as described herein in the manufac theoretical administered dose in blood. VBS was used as a ture of a medicament for treating a disease capable of being negative control and deionized water was used as positive treating by administering a therapeutic agent to a mucosal control in sheep erythrocytes. Solid gray columns are for site. PLA20-Dex6 NPs, solid white columns are for PLA20 0031. In another aspect, there is provided a nanoparticle Dex 10 NPs and columns with diagonal lines pattern are for composition or pharmaceutical composition as described PLGA-PEG NPs (n=3; meani.S.D). hereinforuse intreating a disease capable of being treating by 0041 FIG. 6 is a graph illustrating pharmacokinetic pro administering a therapeutic agent to a mucosal site. files of Dextran-b-PLA and PLGA-PEG NPS administered at 0032. In another aspect, there is provided a commercial 30 mg/kg i.v. to rats. The NP concentration in blood was package comprising the nanoparticle composition orpharma tracked using 3H-PLA-radiolabeled nanocrystals. Solid US 2015/0320694 A1 Nov. 12, 2015 square () are for PLA20-Dex10, solid circles (O) are for 0055 FIG. 21 is a schematic illustration of the partition of PLA20-Dex6 and solid triangles (A) are for PLGA-PEG NPs nanoparticle carriers across tear fluid lipid layer. (n=5, meantS.D). 0056 FIG. 22 demonstrates that these various types of 0042 FIG.7. Is a graph illustrating biodistribution of Dex nanoparticle carriers are capable of achieving high percent tran-b-PLA and PLGA-PEG NPs in various organs in rats 24 age partition across the tear fluid lipid layer. hour post-injection. Solid gray columns are for PLA20-Dex6 0057 FIG. 23 demonstrates the compatibility of the exem NPs, solid white columns are for PLA20-Dex10 NPs and plary NP formulation showing no short-term toxicity effect columns with diagonal lines pattern are for PLGA-PEG NPs on the ocular surface in rabbits. NP treated and the control (n=5, meani.S.D)*: p<0.01. eyes (contralateral eyes) after one-time administration of the 0.043 FIG. 8 is a schematic illustration of mucoadhesion NP formulation were graded using 7 different categories (dis using particulates onto ocular mucosa to circumvent the comfort, conjunctival redness and Swelling, lid Swelling, dis clearance mechanisms such as tear dilution and tear turnover. charge, corneal opacification, and infiltrate) by daily slit Mucoadhesive agents are present throughout the Surface of lamp examination. The grades demonstrate that there is no the nanoparticle carriers. significant increase interms of severity of each category in the 0044 FIG. 9 is a schematic illustration of mucoadhesion NP treated eye compared to the control eye. of PBA modified Dextran-b-PLA NPs onto sialic acid resi 0.058 FIG. 24 shows the histopathology analysis of the dues present on ocular mucosa to circumvent the clearance cornea, bulbar and tarsal conjunctiva, one week after the mechanisms such as tear dilution and tear turnover. administration of the exemplary NP formulation on rabbits. 004.5 FIG.10 is a schematic illustration of the structure of The results demonstrate that the structure and morphology of the mucoadhesive nanoparticles with variations of targeting the ocular tissues are well-preserved after NP formulation moieties on the Surface of the nanoparticles. The presence of and no sign of inflammation was observed. multiple sites for conjugation of targeting moiety to the Sur 0059 FIG. 25 demonstrates the compatibility of the exem face of the nanoparticle provides a high degree of tunability plary NP formulation showing no long-term toxicity effect on for targeting. the ocular surface in rabbits after weekly administration for up to 12 weeks. Chronic response of the ocular Surfaces 0046 FIG. 11 is a schematic illustration of one embodi between NP treated and the control eyes (contralateral eyes) ment showing the surface modified the NPs with PBA using were evaluated similarly using 7 different categories (dis two-step approach: periodate oxidation of the Dextran, and comfort, conjunctival redness and Swelling, lid Swelling, dis conjugation of the aldehyde groups on the oxidated Dextran charge, corneal opacification, and infiltrate) by slit-lamp with amine groups of PBA. examination. The grades demonstrate that there is no signifi 0047 FIG.12a demonstrates H NMR verification of the cant difference interms of severity of each category in the NP presence of PBA on the Dex-b-PLA polymer chains. FIG. treated eye compared to the control eye throughout the dura 12b demonstrates the spherical morphology of the Dex-b- tion of the study. PLA PBA NPs. 0060 FIG. 26 compares the chronic response of the ocular 0048 FIG. 13 demonstrates H NMR verification of the surfaces between NP-drug treated and the control eyes (con presence of cysteamine on the Dex-b-PLA polymer chains tralateral eyes) after weekly administration of formulation which would expose thiol groups on the surface of the NPs. containing Cyclosporine A encapsulated NPs on rabbits. The 0049 FIG. 14 demonstrates the enhanced mucoadhesion grades of 7 different categories (discomfort, conjunctival red property, measured using PAS staining method, of the Dex ness and Swelling, lid Swelling, discharge, corneal opacifica b-PLANPs after Surface modified with PBA. tion, and infiltrate) were obtained by daily slit-lamp exami 0050 FIGS. 15 and 16 demonstrate the ability of the Dex nation for up to 4 weeks. The grades demonstrate that there is b-PLA PBA NPs to load up to 13.7 wt/wt % of the drug no significant difference in terms of severity of each category Cyclosporine A, and their ability to release them in a sus in the NP treated eye compared to the control eye throughout tained manner for up to 5 days in in vitro experiment. the duration of the study. 0051 FIG. 17 demonstrates the ability of Dex-b-PLANPs to encapsulate various bioactive agents. Olopatadine and DETAILED DESCRIPTION Doxorubicin were encapsulated in Dex-b-PLA NPs. Dor 0061 Generally, the present disclosure relates to a Zolamide, Brinzolamide, and Natamycin were encapsulated mucoadhesive nanoparticle delivery system. The nanopar in the Dex-b-PLA PBA NPs. ticles are formed from amphiphilic macromolecules, such as 0052 FIG. 18 demonstrates the ability of the Dex-b-PLA block copolymers, comprising a hydrophilic portion and a PBA NPs to release Dorzolamide (used in treatment of glau hydrophobic portion. The hydrophobic portion comprises coma) in a Sustained manner for up to 18 hours in in vitro multiple functional groups capable of being conjugated to a experiment. The NPs were able to load up to 2.8 wit/wt % targeting moiety, such as a mucosal targeting moiety. In an Dorzolamide. aqueous environment, the hydrophilic portion forms the shell 0053 FIG. 19 demonstrates the ability of the Dex-b-PLA of the nanoparticle providing a Surface that can be fuinction PBANPs to release Brinzolamide (used in treatment of glau alized by coating the nanoparticle with a desired Surface coma) in a Sustained manner for up to 11 days in in vitro density of the targeting moiety. The size of the nanoparticles experiment. The NPs were able to load up to 6.54 wit/wt % and the Surface density of the targeting moieties can be tuned Brinzolamide. without Substantially compromising the stability of the par 0054 FIG. 20 demonstrates the ability of the Dex-b-PLA ticles. The nanoparticles are useful for delivering a wide PBA NPs to release Natamycin (used in treatment of ocular variety of payloads to a mucosal site in a Subject and are fungal infection) in a Sustained manner for up to 24 hours in capable of providing Sustained release of the payload. The in vitro experiment. The NPs were able to load up to 3.88 nanoparticles demonstrate good loading capacity and loading wit/wt % Natamycin. efficiency. US 2015/0320694 A1 Nov. 12, 2015

0062. The present disclosure also relates to components 0068 An amphiphilic block copolymer generally has a useful in the preparation of the mucoadhesive nanoparticles, hydrophobic portion and a hydrophilic portion, or at least a as well as compositions, methods, processes, commercial relatively hydrophilic portion and a relatively hydrophobic packages, kits and uses related thereto. portion when two portions are considered relative to each 0063 Macromolecules other. A hydrophilic polymer is one that generally attracts 0064. The nanoparticles of the present disclosure are gen water and a hydrophobic polymer is one that generally repels erally formed by the association or assembly of amphiphilic water. A hydrophilic or a hydrophobic polymer can be iden macromolecules. The macromolecules are composed of at tified, for example, by preparing a sample of the polymer and least a hydrophobic portion and at least one hydrophilic por measuring its contact angle with water (typically, the hydro tion. The macromolecule may comprise a hydrophobic poly philic polymer will have a contact angle of less than 60°. mer conjugated to a hydrophilic polymer. Such macromol while a hydrophobic polymer will have a contact angle of ecules are capable of self-assembly to form nanoparticles greater than about 60°). In some cases, the hydrophilicity of according to methods well known to those skilled in the art, two or more polymers may be measured relative to each other, including nanoprecipitation methods. i.e., a first polymer may be more hydrophilic than a second 0065. A "polymer,” as used herein, refers to a molecular polymer. structure comprising one or more repeat units (e.g. mono 0069. In some embodiments, the macromolecule is a mers), connected by covalent bonds. The repeat units may be copolymer comprising a hydrophobic portion and a hydro identical, or in some cases, there may be more than one type philic portion. In some embodiments, the macromolecule is a of repeat unit present within the polymer. Polymers may be diblock copolymer comprising a first hydrophilic polymer obtained from natural sources or they may be chemically and a second hydrophobic polymer. Such configurations are synthesized. In some cases, the polymer is a biopolymer, Such generally useful for forming nanoparticles for encapsulating as a polysaccharide, polypeptide or polynucleotide. Biopoly hydrophobic agents of interest in an aqueous environment, mers may comprise naturally-occurring monomers orderiva Such as under physiologic conditions, since the hydrophobic tives or analogs thereof, for example, derivatives or analogs portions will shelter the hydrophobic agent in the core region comprising modified Sugars, nucleotides oramino acids. Sev of the nanoparticle and the hydrophilic portion will form the eral such modifications are known to those skilled in the art. shell of the nanoparticle by orienting toward the aqueous In some cases, the polymer is a synthetic polymer, Such as environment. polylactide (PLA), polyglycolide (PGA), or poly(lactide-co 0070. In one embodiment, the macromolecule is a Dext glycolide) (PLGA) or poly(e-caprolactone) (PCL). ran-b-PLA (Dex-b-PLA) diblock copolymer, which may 0066. If more than one type of repeat unit is present within optionally be functionalized on the dextran portion with one the polymer, then the polymer is said to be a “copolymer.” The or more targeting moieties, such as a mucosal targeting moi repeat units forming a copolymer may be arranged in any ety. fashion. For example, the repeat units may be arranged in a (0071. In some embodiments, the macromolecule is a tri random order, in an alternating order, or in “blocks'. As used block copolymer comprising a first hydrophilic polymer, a herein, a “block copolymer comprises two or more distinct second hydrophobic polymer, and third hydrophilic polymer. blocks or regions, e.g. at least a first block comprising a first Such configurations are generally useful for forming nano polymer and a second block comprising a second polymer. It particles for encapsulating hydrophilic agents of interest in an should be understood that, in this context, the terms “first aqueous environment, Such as under physiologic conditions. and “second do not describe a particular order or number of 0072 Since the macromolecule will be exposed to bodily elements but are merely descriptive. A block copolymer may tissues, it is preferable that the macromolecule comprises a have two (a "diblock copolymer), three (a “triblock copoly biocompatible polymer, for example, the polymer does not mer'), or more distinct blocks. induce a significant adverse response when administered to a 0067 Block copolymers may be chemically synthesized living Subject, for example, it can be administered without or may be polymeric conjugates. As used herein, a “poly causing significant inflammation, irritation and/or acute meric conjugate' describes two or more polymers that have rejection by the immune system. been associated with each other, usually by covalent bonding 0073. In some embodiments, the biocompatible polymer of two or more polymers together. Thus, a polymeric conju is biodegradable, for example, the polymer is able to degrade, gate may comprise a first polymer and a second polymer, chemically and/or biologically, within a physiological envi which have been conjugated together to form a block copoly ronment, such as when exposed to a body tissue. For instance, mer where the first polymer is a first block of the block the polymer may be one that hydrolyzes spontaneously upon copolymer and the second polymer is a second block of the exposure to water (e.g., within a Subject), the polymer may block copolymer. Of course, those of ordinary skill in the art degrade upon exposure to heat (e.g., attemperatures of about will understand that a block copolymer may, in Some cases, 37°C.). Degradation of a polymer may occurat varying rates, contain multiple blocks of polymer. For instance, a block depending on the polymer or copolymer used. For example, copolymer may comprise a first block comprising a first poly the half-life of the polymer (the time at which 50% of the mer, a second block comprising a second polymer, and a third polymer is degraded into monomers and/or other nonpoly block comprising a third polymer or the first polymer, etc. In meric moieties) may be on the order of hours, days, weeks, addition, it should be noted that block copolymers can also be months, or years, depending on the polymer. The polymers formed, in some instances, from other block copolymers. For may be biologically degraded, e.g., by enzymatic activity or example, a first block copolymer may be conjugated to cellular machinery, in some cases, for example, through another polymer to form a new block copolymer containing exposure to a lysozyme (e.g., having relatively low pH). In multiple types of blocks. The polymers may be conjugated by Some cases, the polymers may be broken down into mono any means known in the art and may optionally be connected mers and/or other nonpolymeric moieties that cells can either by an appropriate linker moiety. reuse or dispose of without significant toxic effect on the cells US 2015/0320694 A1 Nov. 12, 2015

(for example, polylactide may be hydrolyzed to form lactic in this case refers to the weight of the hydrophobic portion per acid, polyglycolide may be hydrolyzed to form glycolic acid, mol of the macromolecule prior to conjugation with a target etc.). ing moiety. 0074 Non-limiting examples of biodegradable polymers 0080. In some embodiments, the molecular weight of the include, but are not limited to, polysaccharides, polynucle hydrophobic portion is about 0.1 kDa to about 2000 kDa. In otides, polypeptides, poly(lactide) (or poly(lactic acid)), poly some embodiments, the molecular weight of the hydrophobic (glycolide) (or poly(glycolic acid)), poly(orthoesters), poly portion is about 0.5 kDa to about 200 kDa. In some embodi (caprolactones), polylysine, poly(ethylene imine), poly ments, the molecular weight of the hydrophobic portion is (acrylic acid), poly(urethanes), poly(anhydrides), poly about 1 kDa to 100 kDa. These values represent ranges prior (esters), poly(trimethylene carbonate), poly(ethyleneimine), to conjugation with a targeting moiety. poly(acrylic acid), poly(urethane), poly(beta amino esters) or I0081. The hydrophilic portion of the macromolecule gen the like, and copolymers or derivatives of these and/or other erally comprises a polymer having multiple reactive func polymers, for example, poly(lactide-co-glycolide) (PLGA). tional groups capable of being coupled to a targeting moiety. 0075. In certain embodiments, copolymers may contain For example, the polymer may comprise a backbone made up poly(ester-ether)s, e.g., polymers having repeat units joined of multiple monomer units, each monomer unit having mul by ester bonds (e.g., R C(O) O—R' bonds) and ether tiple functional groups available for conjugation to a targeting bonds (e.g., R-O-R' bonds). In some embodiments, the moiety. Each monomer unit may, for example, have 2, 3, 4 or nanoparticle may further include a polymer able to reduce 5 functional groups. In some embodiments, each monomer immunogenicity, for example, a poly(alkylene glycol) Such unit has 4 functional groups. The functional groups may, for as poly(ethylene glycol) (“PEG'). The amount of PEG in the example, be independently selected from OH groups, thiol nanoparticle should be limited however, so as not to Substan groups, ketone groups, amine groups, and carboxylic acid tiality compromise the tunability of the nanoparticles, which groups, among others. For example, a Sugar moiety in a is enhanced by selection of a polymer with a backbone having dextran polymer may have 4 OH groups available for conju multiple functional groups per monomer unit, Such as a gation to a targeting moiety (see Sheme 1). polysaccharide, as compared to PEG which has only reactive I0082. The proportion of functional moieties conjugated to functional group per polymer chain. In some embodiments, a targeting moiety can be controlled to effect targeting. In the nanoparticle composition is free of PEG. some embodiments, at least 10%, 20%, 30%, 40%, 50%, 0076. The hydrophobic portion of the macromolecule 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% of the generally comprises a hydrophobic polymer, for example, a functional moieties on the surface of the nanoparticle are hydrophobic polymer selected from polyesters, polyorthoe conjugated to a targeting moiety. ster, polycarbonates, polyimides, polybenzimidazoles, poly I0083. The selection of a hydrophilic polymer having mul urethanes, polyureas, polysulfides, polyethers, polysulfones, tiple functional moieties per monomer unit allows for phenolic and amino plastics, chitin and lipopolysaccharides, enhanced tunability of the nanoparticles as compared to, for cholesterol, proteoglycans, and combinations thereof. In an example, conventional PEG-based nanoparticles having only aqueous environment, e.g. under physiological conditions, one reactive functional group at the terminal end of each PEG the hydrophobic portion will substantially form the core of chain. Furthermore, in some embodiments, the hydrophilic the nanoparticle. polymers of the present disclosure are more hydrophilic than a PEG polymer such that the hydrophilic portion of the mac 0077. In some embodiments, the hydrophobic portion of romolecule is less likely to orient toward the core of the the macromolecule comprises a biocompatible polymer, for nanoparticle during nanoparticle formation. Since the target example, selected from polylactide (PLA), polyglycolide ing moieties will typically be conjugated to the hydrophilic (PGA), poly(lactide-co-glycolide) (PLGA), poly(e-caprolac portion, this results in a nanoparticle wherein Substantially all tone) (PCL), and combinations thereof. Such polymers are of the targeting moiety is on the Surface of the nanoparticle) also biodegradable. In a PLGA polymer, the ratios of lactide for targeting. In such embodiments, the core of the nanopar to glycolide may be varied. In some embodiments, the hydro ticle is Substantially free of targeting moiety (i.e. Substantially phobic portion of the macromolecule comprises polylactide no targeting moiety in the core of the nanoparticle. (PLA). In some embodiments, the hydrophobic portion of the I0084. In some embodiments, the hydrophilic portion of macromolecule comprises polyglycolide (PGA). In some the macromolecule having multiple functional groups com embodiments, the hydrophobic portion of the macromolecule prises a polymer selected from a polysaccharide, a polynucle comprises poly(lactide-co-glycolide) (PLGA). In some otide, a polypeptide, or a combination thereof. The polysac embodiments, the hydrophobic portion of the macromolecule charide, polynucleotide, or polypeptide may be based on comprises poly(e-caprolactone) (PCL). naturally-occurring monomers, or derivatives or analogues 0078. In some embodiments, the hydrophobic portion is a thereof. Such derivatives and analogues are known to those polymer comprising 2 or more repeat units. The hydrophilic skilled in the art and can be readily obtained or synthesized. In portion may comprise, for example, from 2 to 200,000 repeat Some embodiments, polysaccharides, for example, dextran, units depending on the size of the hydrophobic portion are preferred since there are multiple functional groups per desired. each monomer unit. 0079. In some embodiments, the molecular weight of the I0085. In some embodiments, the hydrophilic portion of hydrophobic portion is in the range of about 100 g/mol to the macromolecule comprises a “polysaccharide', e.g. a about 2,000,000 g/mol. In some embodiments, the molecular polymer of monosaccharide units joined together by glyco weight of the hydrophobic portion is in the range of about 500 sidic linkages. Any suitable polysaccharide may be used g/mol to about 200,000 g/mol. In some embodiments, the accordance with the present disclosure. In some embodi molecular weight of the hydrophobic portion is in the range of ments, the polysaccharide is composed of 4- to 8-carbon ring about 1,000 g/mol to about 100,000 g/mol. The unit “g/mol. monomers, such as 5-carbon ring monomers. The monomer US 2015/0320694 A1 Nov. 12, 2015 rings may be heterocyclic, form example, comprising one or refers to the weight of the hydrophilic portion per mol of the more N, O or Satoms in the monomer ring. The polysaccha macromolecule prior to conjugation with a targeting moiety. ride may be a “homopolysaccharide', where all of the 0093. In some embodiments, the molecular weight of the monosaccharides in the polysaccharide are the same type, or hydrophilic portion ranges from about 0.1 kDa to about 1,000 a "heteropolysaccharide', where more than one type of kDa. In some embodiments, the molecular weight of the monosaccharide is present. In some embodiments, the hydrophilic portion ranges from about 0.5 kDa to 100 kDa. In polysaccharide is a “homopolysaccharide'. In some embodi some embodiments, the molecular weight of the hydrophilic ments, the polysaccharide is a "heteropolysaccharide'. In portion ranges from about 1 kDa to 50 kDa. These values Some embodiments, the polysaccharide is a linear polysac represent ranges prior to conjugation with a targeting moiety. charide. In some embodiments, the polysaccharide is a 0094. The relative amount of hydrophobic polymer to branched polysaccharide. In some embodiments, the polysac hydrophilic polymer in the macromolecule may be any Suit charide has a reducing end that can be modified for conjuga able ratio that provides the desired characteristics of the tion purposes. In some embodiments, the polysaccharide is a resulting nanoparticle. In some embodiments, the molecular homopolysaccharide with a reducing end. weight of the hydrophobic portion is larger than the molecular I0086. In some embodiments, the polysaccharide is com weight of the hydrophilic portion. In some embodiments, the posed of monomers of glucose, fructose, lactose or a combi molecular weight of the hydrophilic portion is larger than the nation thereof. molecular weight of the hydrophobic portion. 0087. In some embodiments, the polysaccharide is 0.095. In some embodiments, the ratio of the molecular selected from dextran, chitosan, alginate, hyaluronic acid, weight of the hydrophobic portion to the hydrophilic portion heparin, chondroitin Sulphate, pectin, pullulan, amylose, (hydrophobic portion:hydrophilic portion) is a about 0.1:1 to cyclodextrin, carboxymethylcellulose or a polysaccharide 100:1. In some embodiments, the molecular weight ratio is with thiol functional groups conjugated to the polymer back about 0.5:1 to about 50:1. In some embodiments, the molecu bone. lar weight ratio is about 1:1 to about 10:1. In some embodi 0088. In some embodiments, the polysaccharide is dext ments, the molecular weight ratio is about 1:1 to about 5:1, ran, alginate, hyaluronic acid, chitosan, cyclodextrin, or car about 1:1 to about 4:1, about 1:1 to about 3:1, or about 1:1 to boxymethylcellulose. In some embodiments, the polysaccha about 2:1. In some embodiments, the molecular weight ratio ride is dextran. is about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5. These 0089. In some embodiments, the hydrophilic portion com values prepresent ratios before conjugation of the targeting prises a polynucleotide, e.g. a polymer of nucleotides. As moiety. A skilled person will be able to determine a suitable used herein, a “nucleotide' refers to a molecule comprising a ratio based on the particular polymers selected and the agent Sugar moiety, a phosphate group, and a base (usually nitrog of interest to be encapsulated. enous). Typically, the nucleotide comprises one or more bases (0096 Targeting connected to a Sugar-phosphate backbone (a base connected 0097. The macromolecules described herein are conju only to a Sugar moiety, without the phosphate group, is a gated to a targeting moiety, such that the targeting moiety “nucleoside'). The sugars within the nucleotide may be, for located on the Surface of the nanoparticle when the nanopar example, ribose sugars (a “ribonucleic acid, or “RNA), or ticle is formed to thereby surface-functionalize nanoparticle. deoxyribose sugars (a "deoxyribonucleic acid.” or “DNA). The interaction between the targeting moiety and a target at In some cases, the polymer may comprise both ribose and the mucosal site directs the nanoparticle to a particular site deoxyribose Sugars. Examples of bases include, but not lim and/or increases the retention time of the nanoparticle at a ited to, the naturally-occurring bases (e.g., adenosine or “A.” particular site compared to a nanoparticle with no targeting thymidine or “T” guanosine or “G” cytidine or “C.” or uri moiety. Any Suitable targeting moiety may be selected. dine or “U”). The nucleotide may be a naturally-occurring Examples of targeting moieties include, but are not limited to, nucleotide or a derivative or analog thereof. Several deriva Small molecules, polynucleotides, polypeptides, polysaccha tives and analogs are known to those skilled in the art. rides, fatty acids, lipids, and antibodies. 0090. In some embodiments, the hydrophilic portion com 0098. The targeting moiety may be a mucosal targeting prises a polypeptide, e.g. a polymer of amino acids. The moiety. As used herein, a “mucosal targeting moiety' is a amino acid may be a naturally-occurring amino acid or a targeting moiety capable of binding to a target expressed at derivative or analog thereof. Several derivatives and analogs the mucosal site. In some embodiments, the nanoparticles are known to those skilled in the art. In some embodiments, at may comprise more than one type of mucosal targeting moi least a portion (e.g. greater than 50%, 60%, 70%, 80%, 90%, ety. For example, an individual macromolecule may be func 95%, 98%, 99%, 100%) of the nucleotides in the polynucle tionalized with two or more different targeting moieties, or otide have side chains with reactive functional groups capable the nanoparticle may be formed from two or more macromol of being conjugated to the targeting moiety. ecules, each being functionalized with a different targeting 0091. In some embodiments, the hydrophilic portion is a moiety. polymer comprising 2 or more repeat units. The hydrophilic (0099. The term “binding,” as used herein, refers to the portion may comprise, for example, 2 to 100,000 repeat units interaction between a corresponding pair of molecules or depending on desired size of the nanoparticle. portions thereofthat exhibit mutual affinity or binding capac 0092. In some embodiments, the molecular weight of the ity, typically due to specific or non-specific binding or inter hydrophilic portion ranges from about 100 g/mol to about action, including, but not limited to, biochemical, physiologi 1,000,000 g/mol. In some embodiments, the molecular cal, electrostatic and/or chemical interactions. In some cases, weight of the hydrophilic portion ranges from about 500 the targeting moiety is able to selectively bind to a target g/mol to 100,000 g/mol. In some embodiments, the molecular expressed at the mucosal site, for example, a molecule, recep weight of the hydrophilic portion ranges from about 1,000 tor or residue expressed at the mucosal site. “Selective bind g/mol to about 50,000 g/mol. The unit “g/mol in this case ing', as used herein, refers to a targeting moiety, which may US 2015/0320694 A1 Nov. 12, 2015

be a small molecule or a large molecule, that is able to pref targeting moiety may offer Smaller dissociation constants, erentially bind to or recognize a particular target or Subset of e.g., tighter binding. However, in other embodiments, the targets, to a Substantially higher degree than to others. The targeting moiety may be relatively small, for example, having target may, for example, be a biological Substrate that is a molecular weight of less than about 1,000 Da or less than preferentially expressed at the mucosal site. Such as mucin or about 500 Da. a receptor or a glycoprotein or a polysaccharide or residue 0105 Nanoparticles expressed on the Surface of an epithelial cell. In some cases, 010.6 Another aspect of the disclosure is directed to nano the binding is a high affinity binding the binding, Such as particles formed generally from the association of macromol covalent bonding, van der Waal force or hydrogen bonding. ecules, such as the macromolecules described above. The Preferably, the binding is covalent binding. For example, in nanoparticles demonstrated effective targeting and adhesion, Some cases, the target may possess functional groups reactive as well as Sustained release of payload at the mucosal site. with the targeting moiety and in aparticular configuration that 0107 Under appropriate conditions, the macromolecules permits covalent binding of the targeting moiety. are capable of assembling to form a nanoparticle of the core 0100. In some embodiments, the targeting moiety is shell type, where the core of the nanoparticle is relatively capable of binding to carbohydrate residues that contain cis hydrophobic in comparison to the shell. Alternatively, under diol groups, for example, galactose, N-acetylgalactosamine, different conditions, the core of the nanoparticle may be N-acetyl-glucosamine, fucose, and Sialic acids. Such carbo relatively hydrophilic in comparison to the shell. The shell hydrate residues may, for example, be present on mucin. In provides a Surface of the nanoparticles, which may comprise Some embodiments, the carbohydrate residue is a sialic acid a targeting moiety at a desired Surface density, Such that the residue. In some embodiments, the targeting moiety is a nanoparticles are coated with the targeting moiety. boronic acid derivative capable of binding a cis-diol group on 0108. The nanoparticles may have a substantially spheri a sialic acid residue. In some embodiments, the targeting cal shape (i.e., the particles generally appear to be spherical). moiety is a phenylboronic acid (PBA) derivative. Such nanoparticles may also be referred to as "nanospheres' 0101. In some embodiments, the targeting moiety is a thiol or "nanovesicles' due to their generally spherical shape and derivative oran acrylate derivative capable of binding to thiol the formation of a cavity within the nanoparticle. It will be groups of cysteine moieties. Cysteine moieties may, for understood that the particles, for example, upon Swelling or example, be present on mucin. In some embodiments, the shrinkage, may adopt a non-spherical configuration. targeting moiety is a thiol derivative. Such as cysteamine. In 0109 The nanoparticles formed have an average particle some embodiments, the targeting molecule may be cysteam size of less than about 1000 nm (1 micrometer). In some ine derivative capable of forming a disulfide linkage with a embodiments, the average particle size is less than about 500 cysteine moiety on the mucous membrane. In some embodi nm, less than about 300 nm, less than about 200 nm, less than ments, the targeting moiety is an acrylate derivative capable about 150 nm, less than about 100 nm, less than about 50 nm, of binding to hydroxyl groups of the glycoproteins on the less than about 30 nm, less than about 10 nm, less than about mucous membrane. Acrylate derivatives include, but are not 3 nm, or less than about 1 nm in Some cases. In some cases, limited to methacrylate, ethyl acrylate, and diacrylate. In particles less than 150 mn are preferred, for example, such Some embodiments, the targeting moiety is an acrylate particles are better able to penetrate the tear layer of the eye derivative selected from methacrylate, ethyl acrylate, and compared to larger particles. diacrylate. 0110. In some embodiments, the average particle size is 0102. In some embodiments, the targeting moiety is a between about 0.1 nm and about 1000 nm, about 1 nm and phenylboronic acid (PBA) derivative, a thol derivative or an about 500 nm, about 1 nm and about 300 nm, about 1 nm and acrylate derivative. about 200 nm, about 1 nm and about 150 nm, about 1 nm and 0103) In some embodiments, the targeting moiety is the about 100 nm, about 1 nm and about 50 nm, about 10 nm and hydrophobic portion of the macromolecule comprises PLA; about 150 nm, about 10 nm and about 100 nm, about 10 nm. the hydrophilic portion comprises dextran; and the targeting and about 75 nm, about 10 nm and about 60 nm, and about 10 moiety comprises PBA. In some embodiments, the targeting nm and about 50 nm, or about 20 and about 40 nm. moiety is the hydrophobic portion is PLA; the hydrophilic 0111. As used herein, “particle size' refers to the average portion is dextran, and the targeting moiety is PBA. characteristic dimension of a population of nanoparticles 0104. In some embodiments, the targeting moiety is a formed, where the characteristic dimension of a particle is the biological moiety. Non-limiting examples of biological moi diameter of a perfect sphere having the same Volume as the eties include a peptide, a protein, an enzyme, a nucleic acid, a particle. A population of nanoparticles may, for example, fatty acid, a hormone, an antibody, a carbohydrate, a pepti include at least 20 particles, at least 50 particles, at least 100 doglycan, a glycopeptide, or the like. In some cases, the particles, at least 300 particles, at least 1,000 particles, at least biological moiety may be relatively large, for example, for 3,000 particles, at least 5,000 particles, at least 10,000 par peptides, nucleic acids, or the like. For example, the biologi ticles, or at least 50,000 particles. Various embodiments of the cal moiety may have a molecular weight of at least about present invention are directed to Such populations of particles. 1,000 Da, at least about 2,500 Da, at least about 3000 Da, at 0112. In some embodiments, the particles may each be least about 4000 Da, or at least about 5,000 Da, etc. Relatively Substantially the same shape and/or size, in which case the large targeting moieties may be useful, in Some cases, for population is "monodisperse'. For example, the particles differentiating between cells. For instance, in some cases, may have a distribution of particle sizes Such that no more Smaller targeting moieties (e.g., less than about 1000 Da) may than about 5% or about 10% of the particles have a particle not have adequate specificity for certain targeting applica size greater than about 10% greater than the average particle tions, such as mucosal targeting applications. In contrast, size of the particles, and in Some cases, such that no more than larger molecular weight targeting moieties can offer a much about 8%, about 5%, about 3%, about 1%, about 0.3%, about higher targeting affinity and/or specificity. For example, a 0.1%, about 0.03%, or about 0.01% have a particle size US 2015/0320694 A1 Nov. 12, 2015 greater than about 10% greater than the average particle size polymer can also impact particle size. A skilled person will be of the particles. In some cases, no more than about 5% of the able to select Suitable polymers for a particular application. particles have aparticle size greater than about 5%, about 3%, 0.120. The hydrophilic potion of the macromolecules will about 1%, about 0.3%, about 0.1%, about 0.03%, or about form the shell of the nanoparticles in an aqueous environ 0.01% greater than the average particle size of the particles. ment. The hydrophilic portion is selected such that it has 0113. In some embodiments, the particles have an interior multiple functional moieties for conjugation to a targeting core and an exterior shell which forms the surface of the moiety. The proportion of functional moieties conjugated to a nanoparticle, where the shell has a composition different targeting moiety can be controlled, at least in part, by the from the core i.e., there may be at least one polymer or moiety amount of targeting moiety added to the conjugation reaction. present in shell but not in the core (or vice versa), and/or at In general, the more functional moieties present on the hydro least one polymer or moiety present in the core and/or the philic portion, the higher the degree of tunability of the nano shell at differing concentrations. particles. In general, the nanoparticles disclosed herein are 0114. In some cases, the core of the particle is more hydro thus more tunable than PEG-based nanoparticles having only phobic than the shell of the particle. In some cases, a drug or one functional moiety per PEG chain, or other similar poly other payload may be hydrophobic, and therefore readily CS. associates with the relatively hydrophobic interior of the par I0121 By adjusting the surface density of the targeting ticle. The drug or other payload may thus be contained within moiety, the extent of targeting can be controlled. A high the interior of the particle, which may thus shelter it from the Surface density of the targeting moiety can be achieved with external environment Surrounding the particle (or vice versa). the nanoparticles disclosed herein due to the presence of A targeting moiety present on the Surface of the particle may multiple functional moieties on the hydrophilic portion. allow the particle to become localized at a particular targeting Advantageously, a skilled person will be able to control the site, for instance, a mucosal site. The drug or other payload extent of targeting without Substantially compromising the may then, in Some cases, be released from the particle and stability of the nanoparticle delivery system. In some embodi allowed to interact with the particular targeting site. ments, an optimal density can be determined in which maxi 0115 Yet another aspect of the disclosure is directed to mum targeting is achieved without Substantially compromis nanoparticles having more than one polymer or macromol ing the stability of the nanoparticle. ecule present. For example, in Some embodiments, particles I0122. In some embodiments, a majority (e.g. at least 50%, may contain more than one distinguishable macromolecule, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%) of the and the ratios of the two (or more) macromolecules may be mucosal targeting moieties are located on the surface of the independently controlled, which allows for the control of nanoparticle. It is understood that, in Some cases, a portion of properties of the particle. For instance, a first macromolecule the mucosal targeting moieties may be located within the core may be a biocompatible polymeric conjugate. Such as a block of the nanoparticle when the nanoparticle forms, depending copolymer, comprising a targeting moiety, and a second mac on the components of the nanoparticle and the method uti romolecule may comprise a biocompatible polymer but no lized. For instance, where a one-step method is used, it is targeting moiety, or the second macromolecule may contain a possible that Some of the targeting moieties may orient distinguishable biocompatible polymer from the first macro toward the core of the particles. molecule. Control of the amounts of these macromolecules I0123. The selection of a hydrophilic polymer having mul within the polymeric particle may thus be used to control tiple functional moieties along the polymer backbone renders various physical, biological, or chemical properties of the the hydrophilic portion of the molecule more hydrophilic particle, for instance, the size of the particle (e.g., by varying than other polymers, such as PEG, thus the hydrophilic por the molecular weights of one or both polymers), the surface tion is more likely to orient toward an aqueous environment. charge (e.g., by controlling the ratios of the polymers if the Since the targeting moiety is conjugated to the hydrophilic polymers have different charges or terminal groups), the Sur face hydrophilicity (e.g., if the polymers have different portion, typically after formation of a nanoparticle, Substan molecular weights and/or hydrophilicities), the Surface den tially all of the targeting moieties are located on the Surface of sity of the targeting moiety (e.g., by controlling the ratios of the nanoparticle as opposed to the core. the two or more polymers), etc. 0.124. In some embodiments, substantially all (e.g. at least 95%, 96%, 97%, 98%, 99%, or 100%) of the mucosal target 0116 Tunable Nanoparticles ing moieties are located on the Surface of the nanoparticle. 0117 The nanoparticles described herein are highly tun Localizing Substantially all of the targeting moieties to the able. Surface of the nanoparticles enhances targeting efficiency. 0118 For example, the size of the nanoparticles can be The selection of a hydrophilic polymer having multiple func tuned by adjusting the molecular weight and/or composition tional moieties along the polymer backbone renders the of the hydrophobic portion and/or the hydrophilic portion. hydrophilic portion of the molecule more hydrophilic than The particular targeting moiety selected, as well as the Sur other polymers. Such as PEG having only one functional face density of the targeting moiety on the Surface of the moiety, thus the hydrophilic portion is more likely to orient nanoparticles, will also impact the particle size. toward an aqueous environment. Since the targeting moiety is 0119. It should be noted that increasing size of the hydro conjugated to the hydrophilic portion, typically after forma philic and/or hydrophobic polymer components does not tion of a nanoparticle, Substantially all of the targeting moi always result in a larger particle size. For example, in some eties are located on the Surface of the nanoparticle as opposed cases, longer polymer chains may be more flexible and to the core. capable of folding to produce a more compact particle 0.125. The nanoparticles can be tuned by controlling the whereas a shorter polymer chain may be confined to a more Surface density of the targeting moieties on the nanoparticle. linear configuration. Selection of a branched versus a linear A skilled person will be able to precisely tune the nanoparticle US 2015/0320694 A1 Nov. 12, 2015 for a particular application without Substantially compromis 0.138. In some embodiments, the density of a cysteamine ing the stability of the nanoparticles. derivative on the nanoparticle surface is tuneable by the 0126. In some embodiments, the surface density of the amount of cysteamine derivative added in the reaction to targeting moiety is about 1 per nm to 15 per nm, about 1 per control the extent of mucoadhesion properties of the nano nm to 10 per nm, about 1 per nm to 5 per nm, about 1 per particles. nm to about 15 pernm, about 3 pernm to about 12 pernm, 0.139. In some embodiments, the density of an acrylate or from about 5 per nm to about 10 per nm. derivative on the nanoparticle surface is tuneable by the 0127. In some embodiments, the surface density of the amount of acrylate derivative added in the reaction to control targeting moiety is about 1, 2, 3, 4, 5, 6, 7, 8, 8, 9, 11, 12, 13, the extent of mucoadhesion properties of the nanoparticles. 14 or 15 per nm. 0140. The optimal density of surface functional groups 0128. In some embodiments, the nanoparticle is approxi may be determined by those skilled in the art in order to mately 10 nm in size and the density of targeting moieties on achieve balance between the extent of mucoadhesion and the the Surface of the nanoparticle (i.e. Surface density) ranges colloidal stability of the nanoparticles. from about 50 to about 3,500, from about 500 to about 3500, 0.141. In some embodiments, the nanoparticles are dis or from about 1000 to about 3500 per nanoparticle. persed in aqueous medium. The aqueous medium may, for 0129. In some embodiments, the nanoparticle is approxi example, be a physiologically compatible aqueous medium. mately 30 nm in size and the density of targeting moieties on 0.142 Controlled Release the Surface of the nanoparticle (i.e. Surface density) ranges 0.143 A controlled release system, as used herein, refers to from about 50 to about 30000, from about 1000 to about a nanoparticle delivery system capable of delivering a pay 30000, or from about 10000 to about 30000 per nanoparticle. load. Such as a therapeutic agent, a diagnostic agent, a prog 0130. In some embodiments, the nanoparticle is approxi nostic, a prophylactic agent, to a body tissue. Such as a mately 50 nm in size and the density of targeting moieties on mucous membrane, where the payload is released in a prede the Surface of the nanoparticle (i.e. Surface density) ranges signed or controlled manner. For example, the active agent from about 50 to about 90000, from about 3000 to about may be released in a constant manner over a predetermined 90000, or from about 30000 to about 90000 per nanoparticle. period of time, the active agent may be released in a cyclic 0131. In some embodiments, the nanoparticle is approxi manner over a predetermined period of time, or an environ mately 100 nm in size and the density of targeting moieties on mental condition or external event may trigger the release of the Surface of the nanoparticle (i.e. Surface density) ranges the active agent. The controlled release polymer system may from about 50 to about 350000, from about 10000 to about include a polymer that is biocompatible, and in some cases, 350000, or from about 100000 to about 350000 per nanopar the polymer is biodegradable. In some cases, the nanopar ticle. ticles disclosed herein are part of a controlled release delivery 0.132. In some embodiments, the nanoparticle is approxi system. The nanoparticles disclosed herein demonstrated mately 150 nm in size and the density of targeting moieties on Sustained release of payload. the Surface of the nanoparticle (i.e. Surface density) ranges 0144. The mucosal targeting moiety assists in retaining from about 50 to about 800000, from about 30000 to about the nanoparticles at the mucosal site, i.e. for a longer time that 800000, or from about 300000 to about 800000 per nanopar the same nanoparticle without the targeting moiety, Such that ticle. controlled delivery of a payload at the mucosal site can be 0133. In some embodiments, the nanoparticle is approxi achieved. The controlled delivery may include sustained mately 200 nm in size and the density of targeting moieties on delivery. the Surface of the nanoparticle (i.e. Surface density) ranges 0145. In some embodiments, in the payload is released from about 50 to about 1,500,000, from about 60000 to about from the nanoparticle for a sustained period of at least 24, 36, 1,500,000, or from about 600000 to about 1,500,000 per 48, 60, 72,84, of 96 hours. In some embodiments, the payload nanoparticle. is released from the nanoparticle for a Sustained period of at 0134. In some embodiments, the nanoparticle is approxi least 1, 2, 3, 4, 5, 6, 7 or 8 days. In some embodiments, the mately 250 nm in size and the density of targeting moieties on payload is released is released from the nanoparticle for a the Surface of the nanoparticle (i.e. Surface density) ranges Sustained period of at least 1 week. In some embodiments, the from about 50 to about 2,500,000, from about 100,000 to payload is released is released from the nanoparticle for a about 2,500,000, or from about 1,000,000 to about 2,500,000 Sustained period of at least 1 month. per nanoparticle. 0146 In some embodiments at least 50% of the payload is 0135) In some embodiments, the nanoparticle is approxi released within the first 24 hours. In other embodiments, at mately 300 nm in size and the density of targeting moieties on least 10% the payload is released within the first 6 hours. the Surface of the nanoparticle (i.e. Surface density) ranges 0147 Payload from about 50 to about 3,500,000, from about 150,000 to 0.148. A wide variety of payloads can be loaded into the about 3,500,000, or from about 1,500,000 to about 3,500,000 nanoparticles described herein. As used herein, the “payload per nanoparticle. may be any agent of interest to be delivered to a mucosal site, 0136. In some embodiments, the surface density of the for example, a therapeutic agent (e.g. drug), a diagnostic targeting moiety tunable by the amount of targeting moiety agent, a prophylactic agent, an imaging agent, or a combina added in the reaction during the functionalization step(s). tion thereof. In some embodiments, the payload is a single 0.137 In some embodiments, the density of a phenylbo agent of interest. In other embodiments, the payload com ronic acid derivative on the nanoparticle Surface is tuneable prises more than one agent of interest, for example, a combi by the amount of phenylboronic acid added in the reaction to nation of two or more agents of interest. In some embodi control the extent of mucoadhesion properties of the nano ments, the payload comprises 2, 3 or 4 agents of interest. For particles. example, the payload may comprise two or more agents of US 2015/0320694 A1 Nov. 12, 2015

interest selected from a therapeutic agent, a diagnostic agent, 0.155. In some embodiments the payload has a molecular a prophylactic agent, an imaging agent and combinations weight of about 0.001 kDa to 100 kDa, about 0.01 kDa to 50 thereof. kDa, about 0.1 kDa to 10 kDa. 0149 When combined with a payload, the nanoparticles 0156. In some embodiments, the payload has a diameter of described herein are useful as a mucoadhesive nanoparticle about 0.01 nm to about 300 nm, about 0.01 nm to about 100 delivery system for delivering the payload to a mucosal site. nm, about 0.01 nm to about 50 nm. In some embodiments, the payload is predominantly encap 0157. Non-limiting examples of potentially suitable thera sulated within the core of the nanoparticle. By “predomi peutic agents include antimicrobial agents, analgesics, anti nantly” it is meant that more than 60%, 70%, 80%, 90%.95% inflammatory agents, IOP lowering agents, counterirritants, or 99% of the payload is encapsulated within the core of the coagulation modifying agents, diuretics, sympathomimetics, nanoparticle. It will be understood that, depending on the anorexics, antacids and other gastrointestinal agents; anti composition of the nanoparticle and the payload, a portion of parasitics, antidepressants, antihypertensives, anticholin the payload could also be distributed within the shell of the ergics, stimulants, antihormones, central and respiratory nanoparticle and/or on the Surface of the nanoparticle. Stimulants, drug antagonists, lipid-regulating agents, urico 0150. In some embodiments, the payload comprises a Surics, cardiac glycosides, electrolytes, ergot and derivatives hydrophobic agent. For example, the payload may be a hydro thereof, expectorants, hypnotics and sedatives, antidiabetic phobic therapeutic agent, a hydrophobic diagnostic agent, a agents, dopaminergic agents, antiemetics, muscle relaxants, hydrophobic prophylactic agent or a hydrophobic imaging para-sympathomimetics, anticonvulsants, antihistamines, agent. In one embodiment, the payload is a hydrophobic beta-blockers, purgatives, antiarrhythmics, contrast materi therapeutic agent. In one embodiment, the payload is a hydro als, radiopharmaceuticals, antiallergic agents, tranquilizers, phobic diagnostic agent. In one embodiment, the payload is a vasodilators, antiviral agents, and antineoplastic or cytostatic hydrophobic prophylactic agent. In one embodiment, the agents or other agents with anticancer properties, or a com payload is a hydrophobic imaging agent. The encapsulation bination thereof. Other suitable therapeutic agents may be of hydrophobic compounds in the nanoparticles is due to the selected from contraceptives and vitamins as well as micro hydrophobic interaction between the hydrophobic agent and and macronutrients. Still other examples include antiinfec the hydrophobic portions of the copolymers that form the tives such as antibiotics and antiviral agents; analgesics and core of the nanoparticles. analgesic combinations; anorexics; antihelmintics; antiar 0151. In some embodiments, the payload comprises a thritics; antiasthmatic agents; anticonvulsants; antidepres hydrophilic agent. For example, the payload may be a hydro sants; antidiuretic agents; antidiarrleals; antihistamines; anti philic therapeutic agent, a hydrophilic diagnostic agent, a inflammatory agents: antimigraine preparations; hydrophilic prophylactic agent or a hydrophilic imaging antinauseants; antineoplastics; antiparkinsonism drugs; anti agent. In one embodiment, the payload is a hydrophilic thera pruritics; antipsychotics; antipyretics, antispasmodics; anti peutic agent. In one embodiment, the payload is a hydrophilic cholinergics; sympathomimetics: Xanthine derivatives; car diagnostic agent. In one embodiment, the payload is a hydro diovascular preparations including calcium channel blockers philic prophylactic agent. In one embodiment, the payload is and beta-blockers such as pindolol and antiarrhythmics; anti a hydrophilic imaging agent. It will be understood that the hypertensives: diuretics; vasodilators including general coro composition of the nanoparticle would be modified to encap nary, peripheral and cerebral; central nervous system stimu Sulate a hydrophilic payload, for example, a triblock copoly lants; cough and cold preparations, including decongestants; mer comprising a first hydrophilic block, and second hydro hormones Such as estradiol and other steroids, including cor phobic block and a third hydrophilic block could be used. ticosteroids; hypnotics; immunosuppressives; muscle relax Such modifications are well known to those skilled in the art. ants; parasympatholytics; psychoStimulants; sedatives; and 0152 The nanoparticles described herein were found to tranquilizers; and naturally derived or genetically engineered have good loading capacity and efficiency. The loading proteins, polysaccharides, glycoproteins, or lipoproteins. capacity of various drugs using exemplary Dex-b-PLA (op 0158. Further non-limiting examples of drugs include tionally surface functionalized with PBA) nanoparticles is timolol, betaxolol, metipranolol, dorzolamide, brinzolamide, demonstrated in the Examples. In some embodiments, the neptazane, acetazolamide, alphagan, Xalatan, bimatoprost, nanoparticles disclosed herein have higher loading capacity travaprost, olopatadine, ketotifen, acyclovir, gancyclovir, Val than reported for conventional PEG-based polymers. Natu cyclovir, doxorubicin, mitomycin, cisplatin, daunorubicin, rally, loading capacity will be affected by the composition of bleomycin, actinomycin D. neocarzinostatin, carboplatin, the nanoparticles and the choice of payload. stratoplatin, Ara-C. Other examples include Capoten, Mono 0153. In some embodiments, the loading capacity is in the pril, Pravachol, Avapro, Plavix, Cefail. Durice?/Ultracef. range of about 1 to about 40% wit/wt, about 1 to about 30% Azactam, Videx, Zerit, Maxipime, VePesid, Paraplatin, Plati wit/wt., about 1 to about 20%, 1 to about 10%, about 1% to nol, Taxol, UFT, Buspar, Serzone, Stadol NS, Estrace, Glu about 8%, about 1% to about 6%, about 1% to about 5%, cophage (Bristol-Myers Squibb); Ceclor, Lorabid, Dynabac, about 1% to about 3%, or about 1% to about 2%. The loading Prozac, Darvon, Permax, Zyprexa, Humalog, AXid, Gemzar, capacity (%) is calculated here as the molecular weight of Evista (Eli Lily); Vasotec/Vaseretic, Mevacor, Zocor, Prinivil/ encapsulated drug over the entire weight of the nanoparticle Prinizide, Plendil, Cozaar/Hyzaar, Pepcid, Prilosec, Pri multiplied by 100. The total weight of the nanoparticle refers maxin, Noroxin, Recombivax HB, Varivax, Timoptic/XE, to the weight of the nanoparticle including the targeting moi Trusopt, Proscar, Fosamax, Sinemet, Crixivan, Propecia, ety and the encapsulated drug. Vioxx. Singulair, Maxalt, Ivermectin (Merck & Co.); Diflu 0154 In some embodiments, the loading capacity is up to can, Unasyn, Sulperazon, Zithromax, Trovan, Procardia XL, about 40%, up to about 30% wit/wt., up to about 20%, up to Cardura, Norvasc, Dofetilide, Feldene, Zoloft, Zeldox, Glu about 10%, up to about 8%, up to about 6%, up tp about 5%, cotrol XL, Zyrtec, Eletriptan, Viagra, Droloxifene, Aricept, up to about 3%, up to about 2%, or up to about 1%. Lipitor (Pfizer); Vantin, Rescriptor, Vistide, Genotropin, Mic US 2015/0320694 A1 Nov. 12, 2015 ronase/Glyn./Glyb., Fragmin, Total Medrol, Xanax/alpra roterenol, metaxalone, methamphetamine, methaqualone, Zolam, Sermion, Halcion/triazolam, Freedox, Dostinex, metharbital, methenamine, methicillin, methocarbamol. Edronax, Mirapex, Pharmorubicin, Adriamycin, Camptosar, methotrexate, methSuximide, methyclothinzide, methylcel Remisar, Depo-Provera, Caverject, Detrusitol, Estring, Hea lulose, methyldopa, methylergonovine, methylphenidate, lon, Xalatan, Rogaine (Pharmacia & Upjohn); Lopid, methylprednisolone, methysergide, metoclopramide, mato Accrupil, Dilantin, Cognex, Neurontin, Loestrin, Dilzem, laZone, metoprolol, metronidazole, minoxidil, mitotane, Fempatch, Estrostep, Rezulin, Lipitor, Omnicef. FemHRT, monamine oxidase inhibitors, nadolol, nafcillin, nalidixic Suramin, or Clinafloxacin (Warner Lambert). acid, naproxen, narcotic analgesics, neomycin, neostigmine, 0159 Further non-limiting examples of therapeutic agents niacin, nicotine, nifedipine, nitrates, nitrofurantoin, nomifensine, norethindrone, norethindrone acetate, norg that can be included within a particle of the present invention estrel, nylidrin, nystafin, orphenadrine, oxacillin, oxazepam, include acebutolol, acetaminophen, acetohydroxamic acid, oXprenolol, oxymetazoline, oxyphenbutaZone, pancrelipase, acetophenazine, acyclovir, adrenocorticoids, allopurinol, pantothenic acid, papaverine, para-aminosalicylic acid, alprazolam, aluminum hydroxide, amantadine, ambenonium, paramethasone, paregoric, pemoline, penicillamine, penicil amiloride, aminobenzoate potassium, amobarbital, amoxicil lin, penicillin-V, pentobarbital, perphenazine, phenacetin, lin, amphetamine, amplicillin, androgens, anesthetics, antico phenaZopyridine, pheniramine, phenobarbital, phenolphtha agulants, anticonvulsants-dione type, antithyroid medicine, lein, phenprocoumon, phensuximide, phenylbutaZone, phe appetite Suppressants, aspirin, atenolol, atropine, azatadine, nylephrine, phenylpropanolamine, phenyl toloxamine, phe bacampicillin, baclofen, beclomethasone, belladonna, ben nyloin, pilocarpine, pindolol, piper acetazine, piroxicam, droflumethiazide, benzoyl peroxide, benzthiazide, benz poloxamer, polycarbophil calcium, polythiazide, potassium tropine, betamethasone, betha nechol, biperiden, bisacodyl, Supplements, pruzepam, prazosin, prednisolone, prednisone, bromocriptine, bromodiphenhydramine, brompheniramine, primidone, probenecid, probucol, procainamide, procarba buclizine, bumetanide, busulfan, butabarbital, butaperazine, Zine, prochlorperazine, procyclidine, promazine, promethaZ caffeine, calcium carbonate, captopril, carbamazepine, car ine, propantheline, propranolol, pseudoephedrine, psoralens, benicillin, carbidopa & levodopa, carbinoxamine inhibitors, carbonic anhydsase, carisoprodol, carphenazine, cascara, syllium, pyridostigmine, pyrodoxine, pyrilamine, pyrvinium, cefaclor, cefadroxil, cephalexin, cephradine, chlophedianol, quinestrol, quinethaZone, uinidine, quinine, ranitidine, rau chloral hydrate, chlorambucil, chloramphenicol, chlordiaz wolfia alkaloids, riboflavin, rifampin, ritodrine, alicylates, epoxide, chloroquine, chlorothiazide, chlorotrianisene, chlo Scopolamine, secobarbital, Senna, Sannosides a & b, simethi rpheniramine, 6x chlorpromazine, chlorpropamide, chlor cone, sodium bicarbonate, sodium phosphate, sodium fluo prothixene, chlorthalidone, chlorZoxaZone, cholestyramine, ride, Spironolactone, Sucrulfate, Sulfacytine, Sulfamethox cimetidine, cinoxacin, clemastine, clidinium, clindamycin, azole, SulfaSalazine, Sulfinpyrazone, Sulfisoxazole, Sulindac, clofibrate, clomiphere, clonidine, cloraZepate, cloxacillin, talbutal, tamazepam, terbutaline, terfenadine, terphinhydrate, colochicine, coloestlipol, conjugated estrogen, contracep teracyclines, thiabendazole, thiamine, thioridazine, thiothix tives, cortisone, cromolyn, cyclacillin, cyclandelate, cycliz ene, thyroblobulin, thyroid, thyroxine, ticarcillin, timolol, ine, cyclobenzaprine, cyclophosphamide, cyclothiazide, tocamide, tolaZamide, tolbutamide, tolmetin troZodone, tret cycrimine, cyproheptadine, danazol, danthron, dantrolene, inoin, triamcinolone, trianterene, triazolam, trichlormethiaz dapsone, dextroamphetamine, dexamethasone, dexchlorphe ide, tricyclic antidepressants, tridheXethyl, trifluoperazine, niramine, dextromethorphan, diazepan, dicloxacillin, dicy triflupromazine, trihexyphenidyl, trimeprazine, trimethoben clomine, diethylstilbestrol, diflunisal, digitalis, diltiazen, Zamine, trimethoprim, tripclennamine, triprolidine, Valproic dimenhydrinate, dimethindene, diphenhydramine, dipheni acid, Verapamil, Vitamin A, vitamin B12, Vitamin C, Vitamin dol, diphenoxylate & atrophive, diphenylopyraline, dipyrad D, vitamin E, vitamin K, Xanthine, and the like. amole, disopyramide, disulfuram, divalporex, docusate cal 0160 AS another example, if the targeting moiety targets a cium, docusate potassium, docusate Sodium, doxyloamine, cancer cell, then the payload may be an anti-cancer drug Such dronabinol ephedrine, epinephrine, ergoloidmesylates, as 20-epi-1.25 dihydroxyvitamin D3.4-ipomeanol, 5-ethyny ergonovine, ergotamine, erythromycins, esterified estrogens, luracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin, estradiol, estrogen, estrone, estropipute, etharynic acid, eth acodazole hydrochloride, acronine, acylfulvene, adecypenol, chlorVynol, ethinyl estradiol, ethopropazine, ethosaximide, adoZelesin, aldesleukin, all-tk antagonists, altretamine, ethotoin, fenoprofen, ferrous fumarate, ferrous gluconate, ambamustine, ambomycin, ametantrone acetate, amidox, ferrous sulfate, flavoxate, flecamide, fluphenazine, flupred amifostine, aminoglutethimide, aminolevulinic acid, amrubi nisolone, flurazepam, folic acid, furosemide, gemfibrozil, cin, amsacrine, anagrelide, anastrozole, andrographolide, glipizide, glyburide, glycopyrrolate, gold compounds, angiogenesis inhibitors, antagonist D, antagonist G, antar griseofiwin, guaifenesin, guanabenz, guanadrel, guanethi elix, anthramycin, anti-dorsalizing morphogenetic protein-1, dine, halazepam, haloperidol, hetacillin, hexobarbital, antiestrogen, antineoplaston, antisense oligonucleotides, hydralazine, hydrochlorothiazide, hydrocortisone (cortisol), aphidicolin glycinate, apoptosis gene modulators, apoptosis hydroflunethiazide, hydroxychloroquine, hydroxy Zine, regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine hyoscyamine, ibuprofen, indapamide, indomethacin, insulin, deaminase, asparaginase, asperlin, asulacrine, atamestane, iofoquinol, iron-polysaccharide, isoetharine, isoniazid, iso atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azac propamide isoproterenol, isotretinoin, isoxSuprine, kaolin & itidine, aZasetron, azatoxin, azatyrosine, azetepa, azotomy pectin, ketoconazole, lactulose, levodopa, lincomycin liothy cin, baccatin III derivatives, balanol, batimastat, benzochlo ronine, liotrix, lithium, loperamide, lorazepam, magnesium rins, benzodepa, benzoylstaurosporine, beta lactam hydroxide, magnesium sulfate, magnesium trisilicate, derivatives, beta-alethine, betaclamycin B, betulinic acid, maprotiline, meclizine, meclofenamate, medroxyproyester BFGF inhibitor, bicalutamide, bisantrene, bisantrene hydro one, melenamic acid, melphalan, mephenyloin, mephobar chloride, bisaziridinylspermine, bisnafide, bisnafide dimesy bital, meprobamate, mercaptopurine, mesoridazine, metap late, bistratene A, bizelesin, bleomycin, bleomycin Sulfate, US 2015/0320694 A1 Nov. 12, 2015

BRC/ABL antagonists, breflate, brequinar sodium, bropir inhibitors, maytansine, mechlorethamine hydrochloride, imine, budotitane, buSulfan, buthionine Sulfoximine, cactino megestrol acetate, melengestrol acetate, melphalan, menog mycin, calcipotriol, calphostin C, calusterone, camptothecin aril, merbarone, mercaptopurine, meterelin, methioninase, derivatives, canarypox IL-2, capecitabine, caracemide, car methotrexate, methotrexate Sodium, metoclopramide, meto betimer, carboplatin, carboxamide-amino-triazole, car prine, meturedepa, microalgal protein kinase C inhibitors, boxyamidotriazole, carest M3, carmustine, carn 700, carti MIF inhibitor, mifepristone, miltefosine, mirimostim, mis lage derived inhibitor, carubicin hydrochloride, carzelesin, matched double stranded RNA, mitindomide, mitocarcin, casein kinase inhibitors, castanospermine, cecropin B, cedef mitocromin, mitogillin, mitoguaZone, mitolactol, mitomal ingol, cetrorelix, chlorambucil, chlorins, chloroquinoxaline cin, mitomycin, mitomycin analogs, mitonafide, mitosper, Sulfonamide, cicaprost, cirolemycin, cisplatin, cis-porphyrin, mitotane, mitotoxin fibroblast growth factor-Saporin, mitox cladribine, clomifene analogs, clotrimazole, collismycin A, antrone, mitoxantrone hydrochloride, mofarotene, molgra collismycin B, combretastatin A4, combretastatin analog, mostim, monoclonal antibody, human chorionic gonadotro conagenin, crambescidin 816, crisinatol, crisinatol mesylate, phin, monophosphoryl lipid a? myobacterium cell wall SK, cryptophycin 8, cryptophycin Aderivatives, curacinA, cyclo mopidamol, multiple drug resistance gene inhibitor, multiple pentanthraquinones, cyclophosphamide, cycloplatam, cype tumor Suppressor 1-based therapy, mustard anticancer agent, mycin, cytarabine, cytarabine ocfosfate, cytolytic factor, my caperoxide B, mycobacterial cell wall extract, mycophe cytostatin, dacarbazine, dacliximab, dactinomycin, daunoru nolic acid, myriaporone, n-acetyldinaline, nafarelin, bicin hydrochloride, decitabine, dehydrodidemnin B, nagrestip, naloxone/pentazocine, napavin, naphterpin, nar deslorelin, dexifosfamide, dexormaplatin, dexraZOxane, dex tograstim, medaplatin, memorubicin, neridronic acid, neutral Verapamil, deZaguanine, deZaguanine mesylate, diaziquone, endopeptidase, nilutamide, nisamycin, nitric oxide modula didemnin B, didox, diethylnorspermine, dihydro-5-azacyti tors, nitroxide antioxidant, nitrullyn, nocodazole, nogalamy dine, dioxamycin, diphenyl spiromustine, docetaxel, cin, n-substituted benzamides, O6-benzylguanine, oct docosanol, dolasetron, doxifluridine, doxorubicin, doxorubi reotide, okicenone, oligonucleotides, onapristone, cin hydrochloride, droloxifene, droloxifene citrate, dromo ondansetron, oracin, oral cytokine inducer, ormaplatin, stanolone propionate, dronabinol, duaZomycin, duocarmycin oSaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel, SA, ebselen, ecomustine, edatrexate, edelfosine, edrecolo paclitaxel analogs, paclitaxel derivatives, palauamine, palmi mab, eflornithine, eflornithine hydrochloride, elemene, toylrhizoxin, pamidronic acid, panaxytriol, panomifene, elsamitrucin, emitefur, enloplatin, enpromate, epipropidine, parabactin, paZelliptine, pegaspargase, peldesine, peliomy epirubicin, epirubicin hydrochloride, epristeride, erbulozole, cin, pentamustine, pentosan polysulfate sodium, pentostatin, erythrocyte gene therapy vector system, esorubicin hydro pentroZole, peplomycin Sulfate, perflubron, perfosfamide, chloride, estramustine, estramustine analog, estramustine perillyl alcohol, phenazinomycin, phenylacetate, phos phosphate Sodium, estrogen agonists, estrogen antagonists, phatase inhibitors, picibanil, pilocarpine hydrochloride, etanidazole, etoposide, etoposide phosphate, etoprine, pipobroman, piposulfan, pirarubicin, piritrexim, piroX exemestane, fadrozole, fadrozole hydrochloride, fazarabine, antrone hydrochloride, placetin A, placetin B, plasminogen fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, activator inhibitor, platinum complex, platinum compounds, floxuridine, fluasterone, fludarabine, fludarabine phosphate, platinum-triamine complex, plicamycin, plomestane, por fluorodaunorunicin hydrochloride, fluorouracil, fluorocitab fimer Sodium, porfiromycin, prednimustine, procarbazine ine, forfenimex, formestane, fosquidone, fostriecin, fostrie hydrochloride, propyl bis-acridone, prostaglandin J2, pros cin Sodium, fotemustine, gadolinium texaphyrin, gallium tatic carcinoma antiandrogen, proteasome inhibitors, protein nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcit A-based immune modulator, protein kinase C inhibitor, pro abine, gemcitabine hydrochloride, glutathione inhibitors, tein tyrosine phosphatase inhibitors, purine nucleoside phos hepsulfam, heregulin, hexamethylene bisacetamide, hydrox phorylase inhibitors, puromycin, puromycin hydrochloride, yurea, hypericin, ibandronic acid, idarubicin, idarubicin purpurins, pyrazofurin, pyrazoloacridine, pyridoxylated hydrochloride, idoxifene, idramantone, ifosfamide, ilmofos hemoglobin polyoxyethylene conjugate, RAF antagonists, ine, illomastat, imidazoacridones, imiquimod, immunostimu raltitrexed, ramosetron, RAS farnesyl protein transferase lant peptides, insulin-like growth factor-1 receptor inhibitor, inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptine interferonagonists, interferon alpha-2A, interferon alpha-2B, demethylated, rhenium RE 186 etidronate, rhizoxin, ribo interferon alpha-N1, interferon alpha-N3, interferon beta-IA, prine; ribozymes, RII retinamide, RNAi rogletimide, rohi interferon gamma-IB, interferons, interleukins, iobenguane, tukine, romurtide, roquinimex, rubiginone B1, ruboxyl, saf iododoxorubicin, iproplatin, irinotecan, irinotecan hydro ingol, Safingol hydrochloride, Saintopin, sarcinu, Sarcophytol chloride, iroplact, irsogladine, isobengaZole, isohomohali A, SargramoStim, SDI 1 mimetics, Semustine, senescence condrin B, itasetron, jasplakinolide, kahalalide F. lamel derived inhibitor 1, sense oligonucleotides, signal transduc larin-N triacetate, lanreotide, lanreotide acetate, leinamycin, tion inhibitors, signal transduction modulators, simtraZene, lenograstim, lentinan Sulfate, leptolstatin, letrozole, leukemia single chain antigen binding protein, sizofuran, Sobuzoxane, inhibiting factor, leukocyte alpha interferon, leuprolide Sodium borocaptate, sodium phenylacetate, Solverol, acetate, leuprolide/estrogen/progesterone, leuprorelin, Somatomedin binding protein, Sonermin, sparfosate Sodium, levamisole, liarozole, liarozole hydrochloride, linear sparfosic acid, sparsomycin, Spicamycin D. Spirogermanium polyamine analog, lipophilic disaccharide peptide, lipophilic hydrochloride, spiromustine, Spiroplatin, splenopentin, platinum compounds, lissoclinamide 7, lobaplatin, lombri spongistatin 1, squalamine, stem cell inhibitor, stem-cell divi cine, lometrexol, lometrexol Sodium, lomustine, lonidamine, sion inhibitors, stipiamide, Streptonigrin, Streptozocin, losoxantrone, losoxantrone hydrochloride, lovastatin, loxor stromelysin inhibitors, Sulfinosine, Sulofenur, Superactive ibine, lurtotecan, lutetium texaphyrin, lysofylline, lytic pep vasoactive intestinal peptide antagonist, Suradista, Suramin, tides, maitansine, mannostatin A, marimastat, masoprocol, Swainsonine, synthetic glycosaminoglycans, talisomycin, maspin, matrilysin inhibitors, matrix metalloproteinase tallimustine, tamoxifen methiodide, tauromustine, tazaro US 2015/0320694 A1 Nov. 12, 2015 tene, tecogalan Sodium, tegafur, tellurapyrylium, telomerase 0163. In one embodiment, the therapeutic agent is an oph inhibitors, teloxantrone hydrochloride, temoporfin, temoZo thalmic agent used to treat glaucoma, Such as an agent used to lomide, teniposide, teroxirone, testolactone, tetrachlorodeca reduce a sign and/or symptom of glaucoma, for example, and oxide, tetraZomine, thaliblastine, thalidomide, thiamiprine, agent used to reduce intraocular pressure associated with thiocoraline, thioguanine, thiotepa, thrombopoietin, throm ocular hypertension. In some embodiments, the therapeutic bopoietin mimetic, thymalfasin, thymopoietin receptor ago agent is a glaucoma medication, Such as a prostaglandin, nist, thymotrinan, thyroid stimulating hormone, tiazofurin, carbonic anhydrase inhibitor, epinephrine or alpha-agonist, tin ethyl etiopurpurin, tirapazamine, titanocene dichloride, or a beta-blocker. In some embodiments, the therapeutic agent is Dorzolamide, Brinzolamide, Brimonidine, timolol. topotecan hydrochloride, topsentin, toremifene, toremifene or latanoprost. citrate, totipotent stem cell factor, translation inhibitors, tre 0164. In one embodiment, the therapeutic agent is an oph stolone acetate, tretinoin, triacetyluridine, triciribine, tricir thalmic agent used to treat allergic conjunctivitis, Such as an ibine phosphate, trimetrexate, trimetrexate glucuronate, trip agent used to reduce a sign and/or symptom of allergic con torelin, tropisetron, tubulozole hydrochloride, turosteride, junctivitis. In one embodiment, the therapeutic agent is olo tyrosine kinase inhibitors, tyrphostins, UBC inhibitors, ube patadine. nimex, uracil mustard, uredepa, urogenital sinus-derived 0.165. In one embodiment, the therapeutic agent is an oph growth inhibitory factor, urokinase receptor antagonists, vap thalmic agent used to treat keratoconjunctivitis sicca (KCS) reotide, variolin B. velaresol, Veramine, verdins, verteporfin, or “dry eye'. Such as an agent used to reduce a sign and/or vinblastine sulfate, Vincristine sulfate, vindesine, vindesine symptom of KCS. In one embodiment, the therapeutic agent Sulfate, vinepidine Sulfate, Vinglycinate Sulfate, Vinleurosine is cyclosporine A. sulfate, vinorelbine, vinorelbine tartrate, Vinrosidine sulfate, 0166 In one embodiment, the therapeutic agent is Vinxaltine, Vinzolidine Sulfate, vitaxin, Vorozole, Zanoterone, cyclosporine A. In one embodiment, the therapeutic agent is Zeniplatin, Zilascorb, Zinostatin, Zinostatin stimalamer, or dorzolamide. In one embodiment, the therapeutic agent is Zorubicin hydrochloride. In one embodiment, the therapeutic natamycin. In one embodiment, the therapeutic agent is olo agent is doxorubicin. patadine. 0161 In some embodiments, the therapeutic agent is an 0167. In some embodiments, the therapeutic agent is an agent used for treating or preventing a disease or condition antibiotic, for example, a fluoroquinolone, Vancomycin, that affects the eye (e.g. an ophthalmic agent). Non-limiting cephalosporin, gentamycin, erythromycin, azithromycin, a examples of ophthalmic agents include lubricants, demul Sulfa drug, bacitracin, gatifloxacin, levofloxin, moxifloxacin, cents, antibiotics, antivirals (e.g. acyclovir, gancyclovir, Val or of oxacin. cyclovir), antiallergic agents (e.g. antihistamine, e.g. olopata 0.168. In some embodiments, the therapeutic agent is an dine), IOP lowering agents, counterirritants, acetazolamide, antiviral, for example, acyclovir, gancyclovir, Valcyclovir. alphagan, antazoline, aspirin, atropine, azelastine, bacitracin, 0169. In some embodiments, the therapeutic agent is an betaxolol, bimatoprost, botanical drugs including Zeaxan antiallergy agent, for example, an antihistamine. In one thine lutein, lycopenebrimonodine, brinzolamide, carbachol, embodiment, the therapeutic agent is olopatadine. carteolol, ciprofloxacin, ofloxacin, cromalyn, cyclosporine 0170 In some embodiments, the nanoparticle composi (including cyclosporine pro-drugs and cyclosporine deriva tion comprising the therapeutic agent is administered to the tives), other immunomodulators, dapiprazole, dexametha anterior surface of the eye. Sone, diclofenac, diplivifren, dorzolamide, epinephrine, 0171 In some embodiments, the ophthalmic agent is for erythromycin, fluoromethalone, flurbiprofen, gentamycin, mulated in a dosage form for administration to the eye Sur glaucoma medications (e.g. prostaglandins, carbonic anhy face, such as a drop, ointment or gel. In some embodiments, drase inhibitors, epinephrine or alpha-agonists, beta-block the ophthalmic agent is formulated in a dosage form for ers), gramicidin, homatropine, hydrocortisone, hyoscine, ket administration to the eye via a contact lens. erolac, ibuprofen, ketotifen, latanaprost, levobunolol. 0172 Diagnostic Agent levocabastine, levofloxin, lotepprednol, medrysone, meth 0173. In another embodiment, the payload is a diagnostic aZolamide, metipranolol, naphazoline, natamycin, nedocro agent. For example, the payload may be a fluorescent mol mil, neomycin, neptazane, neuroprotective agents, nonsteroi ecule; a gas; a metal; a commercially available imaging agent dal anti-inflammatory agents, nepafanec, norfloxacin, used in positron emissions tomography (PET), computer ofloxacin, olopatadine, oxymetazoline, pemirolast, phe assisted tomography (CAT), single photon emission comput niramine, phenylephrine, pilocarpine, povidone, predniso erized tomography, X-ray, fluoroscopy, and magnetic reso lone, proparacaine, Scopolamine, tetracaine, Steroids, Sulfac nance imaging (MRI); or a contrast agents. Non-limiting etamide, tetrahydrozoline, hypertonic tears, timolal, examples of Suitable materials for use as contrast agents in tobramycin, travaprost, trifluridine, trimethiprim, tropicam MRI include gadolinium chelates, as well as iron, magne ide, unoprostone, Xalatan, and zinc. Prodrugs and related sium, manganese, copper, and chromium. Examples of mate compounds, as well as new active pharmaceutical ingredients rials useful for CAT and X-ray imaging include, but are not can be used with the delivery system described herein. limited to, iodine-based materials. 0162. In one embodiment, the therapeutic agent is an oph 0.174 Radionucleotide thalmic agent selected from cycloprorin A, timolol, betaxolol. 0.175. As another example, the payload may include a metipranolol, dorzolamide, brinzolamide, natamycin, nepta radionuclide, e.g., for use as a therapeutic, diagnostic, or Zane, acetazolamide, alphagan, Xalatan, bimatoprost, prognostic agent. Among the radionuclides used, gamma travaprost, olopatadine, ketotifen, acyclovir, gancyclovir, Val emitters, positron-emitters, and X-ray emitters are Suitable cyclovir. In one embodiment, the therapeutic agent is for diagnostic and/or therapy, while beta emitters and alpha cyclosporine A, natamycin, olopatadine, brinzolamide or dor emitters may also be used for therapy. Suitable radionuclides Zolamine. for use with various embodiments of the present invention US 2015/0320694 A1 Nov. 12, 2015 include, but are not limited to, I, I, I, I, I, I, in the form of a cream, ointment or gel, which may optionally *7Sc, 7°As, 72Sc, 90Y. 88Y. 7Ru, 100Pd, mRh, 19Sb, 128Ba, be applied using an applicator. '7Hg, 21 At 212Bi, 212Pb, 10°Pd, In, 7Ga, Ga, 67Cu, 0183 In some embodiments, the composition is in a dos 7Br 77Br mTc, “C, N, O, P, 33P or *F. The radio age form Suitable for nasal or pulmonary administration. In nucleotide may be contained within the nanoparticle (e.g., as Some embodiments, the dosage form for nasal or pulmonary a separate species), and/or form part of a macromolecule or administration is a spray or inhalant. In one embodiment, the polymer that forms the nanoparticle. dosage form is a spray. In one embodiment, the dosage form (0176) Pharmaceutical Compositions in an inhalant, which may be administered with an inhaler. 0177. Another aspect of the disclosure is related to phar 0184. In some embodiments, the composition is in a dos maceutical compositions comprising a nanoparticle compo age form Suitable for ocular orotic administration, i.e. admin sition as defined herein, and a pharmaceutically acceptable istration to the eye or ear. In some embodiments, the dosage carrier. Pharmaceutical compositions can be prepared in a form for ocular or otic administration is a drop. In some manner well known in the pharmaceutical art, and can be embodiments, the composition is in a dosage form Suitable administered by a variety of routes of administration, depend for ocular administration, such as a drop, gel or ointment. ing upon whether local or systemic effect is desired and upon Such drop, gel or ointment may, for example, be applied to the the area to be treated. anterior surface of the eye. 0178. In some embodiments, the pharmaceutical compo 0185. Parenteral routes of administration includes intrave sition is administered to a desired mucosal site in a subject. nous, intraarterial, Subcutaneous, intraperitoneal intramuscu The pharmaceutical composition can be administered to a lar or injection or infusion; or intracranial, e.g., intrathecal or desired mucosal site by any suitable route of administration. intraventricular, administration. Parenteral administration In some embodiments, the route of administration is non can be in the form of a single bolus dose, or may be, for parenteral. Such as topical. As used herein, topical adminis example, by a continuous perfusion pump. tration may include, for example, administration to a mucous 0186. In some embodiments, parenteral routes are desir membrane via the mouth, eye, ear, nose, esophagus, stomach, able since they avoid contact with the digestive enzymes that Small intestine, large intestine, rectum, vagina, urethra, penis, are found in the alimentary canal. According to Such embodi uterus, etc. It is understood that administration of a therapeu ments, the nanoparticle compositions may be administered tic agent to a mucosal site may provide local and/or systemic by injection (e.g., intravenous, Subcutaneous or intramuscu effect, for example, depending on the ability of the agent to be lar, intraperitoneal injection), rectally, vaginally, topically (as by powders, creams, ointments, or drops), or by inhalation (as absorbed into the circulation via the mucous membrane. by sprays). 0179 Pharmaceutical compositions and formulations for topical administration generally include ointments, lotions, 0187. Injectable preparations, for example, sterile inject creams, gels, drops, Suppositories, sprays, liquids and pow able aqueous or oleaginous Suspensions may be formulated ders. For topical administration to a mucous membrane of the according to the known art using Suitable dispersing or wet gut, an oral dosage form Such as a liquid, emulsion, tablet, ting agents and Suspending agents. The sterile injectable caplet or capsule may be used. Conventional pharmaceutical preparation may also be a sterile injectable solution, Suspen carriers, excipients and dulients may be employed. Sion, or emulsion in a nontoxic parenterally acceptable dilu ent or solvent, for example, as a solution in 1,3-butanediol. 0180. In some embodiments, the compositions are admin Among the acceptable vehicles and solvents that may be istered in a dosage form Suitable for topical or transdermal employed are water, Ringer's Solution, U.S.P., and isotonic administration. Non-limiting examples of dosage forms Suit sodium chloride solution. In addition, sterile, fixed oils are able for topical or transdermal administration of a pharma conventionally employed as a solvent or Suspending medium. ceutical composition as disclosed herein include ointments, For this purpose any bland fixed oil can be employed includ pastes, creams, lotions, gels, powders, Solutions, Suspen ing synthetic mono- or diglycerides. In addition, fatty acids sions, emulsions, sprays, inhalants, or patches. The compo Such as oleic acid are used in the preparation of injectables. In sition is typically admixed under sterile conditions with a one embodiment, the inventive conjugate is Suspended in a pharmaceutically acceptable carrier and any needed preser carrier fluid comprising 1% (w/v) sodium carboxymethyl vatives or buffers as may be required. cellulose and 0.1% (v/v) TWEENTM 80. The injectable for 0181. In some embodiments, the composition is in a dos mulations can be sterilized, for example, by filtration through age form Suitable for oral administration. Such a dosage form a bacteria-retaining filter, or by incorporating sterilizing may, for example, be useful for administration to an oral, agents in the form of sterile Solid compositions which can be esophageal, gastric or intestinal mucosal site. The composi dissolved or dispersed in sterile water or other sterile inject tion may or may not be Swallowed depending on the target able medium prior to use. mucosal site. For example, the dosage form could be a mouth 0188 Ointments, pastes, creams, and gels may contain, in wash. In some embodiments, the oral dosage form is a liquid addition to the nanoparticle delivery system of the present dosage form, Such as a Suspension, Solution or emulsion. In disclosure, excipients such as animal and vegetable fats, oils, Some embodiments, the dosage form is a solid dosage from, waxes, paraffins, starch, tragacanth, cellulose derivatives, Such as a powder, tablet, capsule or caplet. polyethylene glycols, silicones, bentonites, silicic acid, talc, 0182. In some embodiments, the composition is in a dos and Zinc oxide, or mixtures thereof. age form Suitable for rectal or vaginal administration. In some 0189 Powders and sprays can contain, in addition to the embodiments, the composition for rectal or vaginal adminis inventive conjugates of this invention, excipients such as tration is in the form of a Suppository. In some embodiments, lactose, talc, silicic acid, aluminum hydroxide, calcium sili the composition for rectal or vaginal administration is in the cates, and polyamide powder, or mixtures thereof. Sprays can form of a liquid. Such as a douche or enema. In some embodi additionally contain customary propellants such as chlorof ments, the composition for rectal or vaginal administration is luorohydrocarbons. US 2015/0320694 A1 Nov. 12, 2015

0190. Pharmaceutical compositions for oral administra depending on Such factors as the desired biological endpoint, tion can be liquid or Solid. Liquid dosage forms suitable for the drug to be delivered, the target tissue, the route of admin oral administration of inventive compositions include phar istration, etc. Additional factors which may be taken into maceutically acceptable emulsions, microemulsions, Solu account include the severity of the disease state; age, weight tions, Suspensions, syrups, and elixirs. In addition to an and gender of the patient being treated; diet, time and fre encapsulated or unencapsulated conjugate, the liquid dosage quency of administration; drug combinations; reaction sensi forms may contain inert diluents commonly used in the art tivities; and tolerance/response to therapy. Such as, for example, water or other solvents, Solubilizing 0.195 The compositions described herein may be formu agents and emulsifiers such as ethyl alcohol, isopropyl alco lated in unit dosage form for ease of administration and uni hol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl formity of dosage. The expression “unit dosage form as used benzoate, propylene glycol, 1,3-butylene glycol, dimethyl herein refers to a physically discrete unit appropriate for the formamide, oils (in particular, cottonseed, groundnut, corn, patient to be treated. It will be understood, however, that the germ, olive, castor, and sesame oils), glycerol, tetrahydrofur total daily dosage of the composition of the present invention furyl alcohol, polyethylene glycols and fatty acid esters of may be decided by a physician. For any composition, the sorbitan, and mixtures thereof. Besides inert diluents, the oral therapeutically effective dose can be estimated initially either compositions can also include adjuvants, wetting agents, in cell culture assays or in animal models, usually mice, emulsifying and Suspending agents, Sweetening, flavoring, rabbits, dogs, or pigs. The animal model is also used to and perfuming agents. As used herein, the term “adjuvant” achieve a desirable concentration range and route of admin refers to any compound which is a nonspecific modulator of istration. Such information can then be used to determine the immune response. In certain embodiments, the adjuvant useful doses and routes for administration in humans. Thera stimulates the immune response. Any adjuvant may be used in peutic efficacy and toxicity of conjugates can be determined accordance with the present invention. A large number of by standard pharmaceutical procedures in cell cultures or adjuvant compounds is known in the art (Allison Dev. Biol. experimental animals, e.g., ED50 (the dose is therapeutically Stand. 92:3-11, 1998: Unkeless et al. Annu. Rev. Immunol. effective in 50% of the population) and LD50 (the dose is 6:251-281, 1998; and Phillips et al. Vaccine 10:151-158, lethal to 50% of the population). The dose ratio of toxic to 1992). therapeutic effects is the therapeutic index, and it can be 0191 Solid dosage forms for oral administration include expressed as the ratio, LD50/ED50. Pharmaceutical compo capsules, tablets, caplets, pills, powders, and granules. In sitions which exhibit large therapeutic indices may be useful such solid dosage forms, the encapsulated or unencapsulated in some embodiments. The data obtained from cell culture conjugate is mixed with at least one inert, pharmaceutically assays and animal studies can be used in formulating a range acceptable excipient or carrier Such as Sodium citrate ordical of dosage for human use. cium phosphate and/or (a) fillers or extenders such as 0196. Kits and Commercial Packages starches, lactose, Sucrose, glucose, mannitol, and silicic acid, 0197) The present disclosure also provides any of the (b) binders such as, for example, carboxymethylcellulose, above-mentioned compositions in kits or commercial pack alginates, gelatin, polyvinylpyrrolidinone. Sucrose, and aca ages, optionally with instructions for use or administration of cia, (c) humectants such as glycerol, (d) disintegrating agents any of the compositions described herein by any suitable Such as agar-agar, calcium carbonate, potato or tapioca starch, technique as previously described. “Instructions' can define a alginic acid, certain silicates, and sodium carbonate, (e) solu component of promotion, and typically involve written tion retarding agents such as paraffin, (f) absorption accelera instructions on or associated with packaging of compositions tors such as quaternary ammonium compounds, (g) wetting of the invention. Instructions also can include any oral or agents such as, for example, cetyl alcohol and glycerol electronic instructions provided in any manner. The “kit monostearate, (h) absorbents such as kaolin and bentonite typically defines a package including any one or a combina clay, and (i) lubricants such as talc, calcium Stearate, magne tion of the compositions of the invention and the instructions, sium Stearate, Solid polyethylene glycols, sodium lauryl Sul but can also include the composition of the invention and fate, and mixtures thereof. In the case of capsules, tablets, and instructions of any form that are provided in connection with pills, the dosage form may also comprise buffering agents. the composition in a manner Such that a clinical professional 0.192 Solid compositions of a similar type may also be will clearly recognize that the instructions are to be associated employed as fillers in Soft and hard-filled gelatin capsules with the specific composition. using such excipients as lactose or milk Sugar as well as high 0198 The kits described herein may also contain one or molecular weight polyethylene glycols and the like. The solid more containers, which may contain the inventive composi dosage forms of tablets, dragees, capsules, pills, and granules tion and other ingredients as previously described. The kits can be prepared with coatings and shells Such as enteric also may contain instructions for mixing, diluting, and/or coatings and other coatings well known in the pharmaceutical administrating the compositions of the invention in some formulating art. cases. The kits also can include other containers with one or (0193 Dosage more solvents, surfactants, preservative and/or diluents (e.g., 0194 It will be appreciated that the exact dosage of the normal saline (0.9% NaCl), or 5% dextrose) as well as con nanoparticle or components thereof. Such as a therapeutic tainers for mixing, diluting or administering the components agent, may be determined by a physician in view of the patient in a sample or to a Subject in need of Such treatment. to be treated. In general, dosage and administration are 0199 The compositions of the kit may be provided as any adjusted to provide an effective amount of the inventive con Suitable form, for example, as liquid solutions or as dried jugate to the patient being treated. As used herein, the “effec powders. When the composition provided is a dry powder, the tive amount” refers to the amount necessary to elicit the composition may be reconstituted by the addition of a Suit desired biological response. As will be appreciated by those able diluent, which may also be provided. In embodiments of ordinary skill in this art, the effective amount may vary where liquid forms of the composition are used, the liquid US 2015/0320694 A1 Nov. 12, 2015

form may be concentrated or ready to use. The diluent will actinic keratosis, acute allergic blepharoconjunctivitis, aller depend on the components of the composition and the mode gic conjunctivitis, adenoviral keratoconjunctivitis, aniridia, of use or administration. Suitable diluents for drug composi atopic keratoconjunctivitis, bacterial conjunctivitis, bacterial tions are well known, for example as previously described, keratitis, bandkeratopathy, basal cell carcinoma, Bell’s palsy, and are available in the literature. The diluent will depend on blepharitis, bullous keratopathy, canaliculitis, caruncular the conjugate and the mode of use or administration. cyst, cataract, chalazion, chlamydial conjunctivitis, climatic 0200. The present disclosure also encompasses, in another droplet keratopathy, concretions, conjunctival intraepithelial aspect, promotion of the administration of the nanoparticle neoplasia, conjunctival lymphoma, conjunctival papilloma, delivery system described herein. In some embodiments, one conjunctival pigmented lesions, conjunctival scarring, con or more compositions of the invention are promoted for the junctivitis, conjunctivochalasia and chemosis, corneal col prevention or treatment of various diseases such as those lagen cross-linking, corneal edema, corneal graft-lamellar described herein via administration of any one of the compo keratoplasty, corneal graft rejection, corneal infiltrates, sitions of the present invention. As used herein, “promoted crocodile shagreen, crystalline keratopathy, cysts of the eye includes all methods of doing business including methods of lids, dacryocystitis, dellen, dendritic ulcer, dermatochalasis education, hospital and other clinical instruction, pharmaceu and blepharochalasis, Descemet's membrane breaks, disci tical industry activity including pharmaceutical sales, and any form keratitis, disciform keratitis, keratoconjunctivitis sicca, advertising or other promotional activity including written, ectopia lentis, ectropion, endophthalmitis, entropion, epible oral and electronic communication of any form, associated pharon and epicanthic folds, epibulbar choristomas, epi with compositions of the invention. phora, episcleritis, epithelial and fibrous ingrowth, epithelial 0201 Methods of Treatment and Use basement membrane dystrophy, exposure keratopathy, eyelid 0202 The nanoparticle compositions disclosed herein trauma, filamentary keratopathy, filtering bleb, flash burns, may be useful in the treatment or prevention of any disease or floppy eyelid syndrome, follicular conjunctivitis, Fuchs condition capable of being treated via controlled delivery of a endothelial dystrophy, Fuchs heterochromic iridocyclitis, therapeutic agent to a mucosal site. As used herein “treating fungal keratitis, giant papillary conjunctivitis, glaucoma includes preventing, reducing oralleviating one or more signs acute angle closure, gonococcal keratoconjunctivitis, granu and/or symptoms of the disease or condition. The nanopar lar dystrophy, hemangioma, herpes simplex keratitis, herpes ticle compositions provide controlled release of the therapeu simplex primary blepharokeratoconjunctivitis, herpes Zoster tic agent and are Surface-functionalized for targeting and ophthalmicus, hordeolum-internal and external, hyphema retention of the nanoparticles at the mucosal site such that blunt trauma, hypopyon, infectious crystalline keratopathy, Sustained release at the mucosal site can be achieved. interstitial keratitis, iridocorneal dysgenesis, iridocorneal 0203. In some embodiments, there are provided methods endotheliopathy, iris cysts, iritis, iron lines, keratoconus, of treating a disease or conditionina Subject by administering keratoconus forme frusta, keratoglobus, lattice stromal dys an effective amount of a composition or component thereofas trophy, leukocoria, lice, limbal stem cell deficiency, lipid defined herein. In other embodiments, there are provided uses keratopathy, macular stromal dystrophy, marginal keratitis, of the compositions or components thereofas defined herein meesmann's dystrophy, melanoma-conjunctival and eyelid, for treating and/or preventing a disease or condition. In other melanoma and nevus of the iris, membranous and embodiments, there are provided uses of the compositions or pseudomembranous conjunctivitis, molluscum contagiosum, components thereofas defined herein for the manufacture of mooren’s ulcer, nasolacrimal duct obstruction-congenital, a medicament to treating and/or preventing a disease or con neurotrophic keratopathy, nevus-eyelid, ocular cicatricial dition. In other embodiments, there are provided composi pemphigold, ophthalmia neonatorum, pannus and pseudop tions or components thereof disclosed herein for the manu terygia, pellucid marginal degeneration, perforation-corneal, facture of a medicament to treating and/or preventing a peripheral ulcerative keratitis, persistent epithelial defect, disease or condition. In other embodiments, there are pro phlyctenulosis, pingueculum, posterior capsular opacifica vided compositions or components thereofas defined herein tion, posterior polymorphous dystrophy, preseptal cellulitis, for the treatment of a disease or condition. pseudoexfoliation of the lens capsule, pterygium, ptosis and 0204. In some embodiments, the disease or condition to be pseudoptosis, punctual Stenosis, pyogenic granuloma, recur treated is a disease or condition capable of being treated via rent corneal erosion syndrome, Reis-Buckler's dystrophy, delivery of a therapeutic agent to a mucosal site. Such as a retention cyst and lymphangiectasia, rheumatoid arthritis, mucosal site of the mouth, eye, ear, nose, esophagus, stom rosacea keratitis, Salzmann nodular degeneration, Scleritis, ach, Small intestine, large intestine, rectum, vagina, urethra, sebaceous cell carcinoma, seborrheic keratosis, squamous penis or uterus. In some embodiments, the disease or condi cell carcinoma-lid, Stevens-Johnson syndrome, Sub-conjunc tion to be treated is a disease or condition affecting the mouth, tival hemorrhage, Superficial punctate keratopathy, Superior eye, ear, nose, esophagus, stomach, Small intestine, large limbic keratoconjunctivitis, Synechia, Terrien’s marginal intestine, rectum, vagina, urethra, penis, or uterus. In some degeneration, Thygeson's Superficial punctate keratopathy, embodiments, the disease or condition to be treated is a dis toxic keratopathy, trachoma, trichiasis, pseudotrichiasis, dis ease or condition affecting the mouth, eye, ear or nose. In tachiasis, metaplastic lashes, trichotillomania, uveitis, Vernal Some embodiments, the disease or condition to be treated is a keratoconjunctivitis, vitamin A deficiency, Vortex keratopa disease or condition affecting the rectum, vagina, urethra, thy, Xanthelasma. penis, or uterus. In some embodiments, the disease or condi 0206. In some embodiments, the disease or condition is a tion to be treated is a disease or condition affecting the disease or condition of the eye is glaucoma, keratoconjunc esophagus, stomach, Small intestine or large intestine. tivitis sicca or allergic conjunctivitis, fungal infection, viral 0205. In some embodiments, the disease or condition to be infection or bacterial infection. In some embodiments, the treated is a disease or condition affecting the eye. Non-limit disease or condition of the eye is glaucoma, keratoconjunc ing examples include abrasion, acanthamoeba keratitis, tivitis sicca or allergic conjunctivitis. In some embodiments, US 2015/0320694 A1 Nov. 12, 2015

the disease or condition of the eye is a fungal infection, viral syndrome, inclusion conjunctivitis, influenza A, influenza B, infection or bacterial infection. interstitial cystitis, intraoral dental sinus, intrinsic asthma, 0207. In some embodiments, Cyclosporine A is adminis invasive candidiasis, irritative conjunctivitis, Jadassohn-Le tered for the treatment of keratoconjunctivitis sicca. In some wandowsky Syndrome, kaposiform hemangio-endothelioma, embodiment, Olopatadine is administered for the treatment keratoconjunctivitis, keratosis pharynges, laryngopharyn of allergic conjunctivitis. In some embodiment, Brinzola geal reflux, leprosy, leukoencephalopathy, leukoplakia, leu mide, Brimonidine, or Dorzolamide, are administered for the koplakia with tylosis and esophageal carcinoma, lipogranu treatment of glaucoma. lomatosis, logic syndrome, lower esophageal ulcer, 0208. In some embodiments, the composition is adminis lymphocytic colitis, lymphoma, mucosa-associated lym tered topically on the surface of the eye for treatment of phoid tissue, major ulcerative stomatitis, malignant peptic diseases associated with the anterior segments of the eye. ulcer, Melkersson-Rosenthal syndrome, membranous con junctivitis, mouth ulcers, mucinous carcinoma, mucocele, 0209. In some embodiments, the composition is adminis mucoepidermoid, mucoepidermoid carcinoma, mucoepithe tered topically on the surface of the eye for treatment of lial dysplasia, Witkop type, mucosal leishmaniasis, mucosal diseases associated with the posterior segments of the eye. In lichen planus, mucosal squamous cell carcinoma, mucositis, Some embodiments, the composition is administered intrana mucous cyst of oral mucosa, Nagayama's spots, nasal polyp. Sally to target the nasal mucosa. In some embodiments, the necrotizing entercolitis, necrotizing periodontal diseases, composition is administered orally to target the oral mucosa. nicotine stomatitis, ophthalmia neonatorum, oral Crohn's In some embodiments, the composition is administered intra disease, oral florid papillomatosis, oral fordyce granules, oral venously to target the gastrointestinal mucin for treatment of thrush, oral ulcer, orthomyxovirus-related cold, Osler diseases associated with the intestine. In some embodiments, Rendu-Weber syndrome, pancolitis, papillary conjunctivitis, the composition is administered vaginally to target the vagi parainfluenza, paramyxovirus-related cold, paucigranulo nal mucosa. In some embodiments, the composition admin cytic asthma, pemphigus, pemphigus foliaceus, pemphigus istered rectally to target the rectal mucosa. Volgaris, Penign peptic ulcer, penphigus Vulgaris, peptic 0210. In some embodiments, the disease or condition to be ulcer, periadenitis mucosa necrotica, periodic fever, pharyn treated is selected from one or more of acquired angioedema, goconjunctival fever, Pinguecula, plasma cell cheilitis, plas acrodermatitis enteropathica, acute serous conjunctivitis, moacanthoma/plasma cell gingivitis, primary ciliary dyski adenomatous polyposis of the colon, adenoviridae infections, nesia, proctitis pseudomembranous colitis, pseudomycoma adenovirus-related cold, allergic asthma, allergic contact peritonei, psoriasis on mucous membranes, psychiatric dis cheilitis, allergic rhinitis, allergies, amyloidosis of gingiva orders associated celiac disease, pterygium, pterygium of the and conjunctiva mental retardation, analgesic asthma Syn conjunctiva, purulent conjunctivitis, recurring scarring aph drome, Anderson's triad, angina bullosa haemorrhagica, thae, reflux laryngitis, refractory celiac disease, Rhinitis, rhi angular conjunctivitis, asthma, asthmatic Bronchitis, atro nosporidiosis, ritter syndrome, rostan asthma, Salicylate-sen phic glossitis, atrophic rhinitis, attenuated familial polyposis, sitive asthma, Schafer syndrome, sinusitis, Sjogren Behcet’s disease, benign migratory glossitis, benign mucosal syndrome, spring catarrh, sprue, Stevens-Johnson syndrome, penphigoid, black hairy tongue, Brodie pile, bronchitis, stomal ulcer, Stomatitis, Superior limbic keratoconjunctivitis, bullous penphigoid, candidiasis, canker Sores, carbon baby Sutton disease, Swime flu, systemic candidiasis, Takahara's syndrome, cariomegaly, catarrh, catarrhal or mucopurulent disease, the clap, thrush, trumpeter's wart, tuberculous dis conjunctivitis, central papillary atrophy, cervical polyps, ease of the mucous, ulcerative colitis, ulcerative conjunctivi cheilitis, cheilitis exfoliativa, cheilitis glandularis, cheilitis tis, ulcerative proctosigmoiditis, urban Schosser Spohn Syn granulomatosa, cholecystitis, cicatrizing conjunctivitis, cili frome, vaginal candidiasis, vasomotor rhinitis, Vestibular ary discoordination due to random ciliary orientation, ciliary papillomatosis, Vincents angina, Vulvovaginal gingival Syn dyskinesia, colitis, colorectal adenomatous polyposis, col orectal polyps, conjunctivitis ligneous, conjunctivitis with drome, white sponge nevus, Xanthogranulomatous cholecys pseudomembrane, coronavirus-related cold, costello syn titis, Xerostomia drome, coxsackievirus-related cold, Crohn's disease, 0211 Subject cronkhite-Canada syndrome, cystic Fibrosis, cystitis, derma 0212. The Subject may be a human or non-human animal. tostomatitis, desquamative gingivitis, dextrocardia-bron In some embodiments, the Subject is a mammal. Non-limiting chiectasis-sinusitis, drug-induced ulcer of the lip, duodenal examples of mammals include human, dog, cat, horse, don ulcer, dyskeratosis congenital, dyskeratosis congenita of Zin key, rabbit, cow, pig, sheep, goat, rat, mouse, guinea pig, sser-Cole-Engman, echovirus-related cold, Ectodermal dys hamster, and primate. In some embodiments, the Subject is a plasia, enterocolitis, eosinophilic cystitis, epidemic kaposi's human. sarcoma, epulis, epulis fissuratum, eruptive hemangioma, eruptive lingual papillitis, erythroplakia, esophageal ulcer, 0213 Methods of Manufacture esophagitis, extrinsic asthma, familial adenomatous polypo 0214. In another aspect, the present disclosure provides a sis, familial intestinal polyposis, familial nasal acilia, familial process for the preparation of macromolecules useful in the polyposis, Fenwick ulcer, fissured tongue, flu, folicular con formation of a mucoadhesive nanoparticle delivery system. junctivitis, follicular hamartoma, food allergy related asthma, The macromolecule is typically an amphiphilic copolymer, in Fordyce's disease, Gardner syndrome, gastresophageal particular, a block copolymer, which is conjugated to a plu reflux-related chronic cough, gastric erosion, gastric reflux, rality of mucosal targeting moieties. The macromolecules are gastric ulcer, gastritis, gastritis, gastroesophageal reflux dis capable of assembly under Suitable conditions to form a nano ease, giant papillary conjunctivitis, gonorrhea, growth-hor particle, i.e. of the core-shell type. In an aqueous environ mone secreting pituitary adenoma, hairy leukoplakia, hemo ment, the nanoparticle has a hydrophobic core and a hydro philus influenzae B, hemorrhagic conjunctivitis, hemorrhagic philic shell, the shell providing a Surface of the nanoparticle, proctocolitis, herpes, human papillomavirus, immotile cilia the Surface of the nanoparticle being coated in a desired US 2015/0320694 A1 Nov. 12, 2015

amount (i.e. Surface density) of the mucosal targeting moiety Some embodiments, the reaction takes place between the for controlled targeting and adhesion of the nanoparticle. amine end of N-protected-ethylenediamine and the reducing 0215. The macromolecules disclosed herein may be made end of a multimer having multiple functional groups per by any suitable process known to those skilled in the art, for monomer unit, Such as a polysaccharide, a polynucleotide or example, using Suitable conjugation techniques. Starting a polypeptide. Any suitable N-protecting group can be used. materials, including hydrophobic polymer and hydrophilic In some embodiments, the N-protecting group is tert-butoxy polymer, may be purchased from various commercial Suppli carbonyl (BOC). ers. Where desired, the starting materials can be prepared by 0225. The choice of a hydrophilic polymer having mul those of skill in the art. For example, where polymers com tiple functional groups per monomer unit enables tuning of prising modified backbone residues are desired. Exemplary the resulting nanoparticle to control particle size, targeting methods for making macromolecules useful in the formation and/or adhesion at a mucosal site, as described further below. of a mucoadhesive delivery system are described below. In some embodiments, the hydrophilic polymer is a polysac 0216. In some embodiments, there is provided a method of charide. In some embodiments, the hydrophilic polymer is preparing a nanoparticle composition. dextran. Therefore, in Some embodiments, the reaction takes 0217. In some embodiments, the method is carried out in a place between N-BOC-ethylenediamine and an aldehyde of series of steps, such as, preparation of an amphiphilic mac the reducing end of a dextran polymer. The reaction may be romolecule, nanoparticle formation, and conjugation to a tar carried out in a suitable solvent, such as a borate buffer geting moiety (i.e. coating of the Surface of the nanoparticle Solution, in the presence of a reducing agent, Such as NaC with a desired Surface density of the targeting moiety). Alter NBH. The mixture is stirred for a sufficient amount of time natively, the hydrophilic portion comprising multiple func to complete the reaction, for example, about 24 to 120 hours. tional groups may first be coupled to a desired amount of the In one embodiment the mixture is stirred for about 24, 48,72, targeting moiety, followed by conjugation of the functional 96, or 120 hours. In some embodiments, this step is carried ized hydrophilic portion to a hydrophobic polymer, which out at room temperature. In some embodiments, this step is may be in the form of a hydrophobic nanoparticle (i.e. coating carried out in the dark. the surface of a hydrophobic nanoparticle with a functional 0226. The mixture may then be washed to remove any ized hydrophilic polymer). When the hydrophobic polymer is unreacted molecules or catalysts. In one embodiment metha modified for conjugation, one end of the polymer will typi nol is used in the washing step. The end-modified dextran can cally become more hydrophilic (e.g. presence of a carboxyl optionally be dried before continuing the process. group). Such polymers can assemble to form hydrophobic 0227. The protecting group is then removed followed by nanoparticles in aqueous medium. Preparation of the nano conjugation of the amine-terminated multimer to a hydropho particles in a controlled sequence results in Surface-function bic polymer in a suitable solvent to provide an amphiphilic alized nanoparticles wherein substantially all (e.g. greater macromolecule. In one embodiment, hydrochloric acid and than 90%, 95%, 96%, 97%, 98%, 99%) of the targeting moi triethyl amien are used for the removal of the protecting eties are located on the surface of the nanoparticle formed by group. The macromolecule may be washed, for example, the hydrophilic portion of the macromolecules. using methanol, to remove unreacted polymer. 0218. In one embodiment, the method of preparing a nano 0228. The conjugation of the amine-terminated multimer particle composition useful for delivery of a payload to a with a hydrophobic polymer takes place in a suitable solvent. mucosal site comprises preparing an amphiphilic macromol In one embodiment, the solvent is DMSO, acetone, or aceto ecule comprising a hydrophilic portion and a hydrophobic nitrile. Catalysts may be employed to drive the reaction. In portion, the hydrophilic portion comprising multiple func one embodiment, the catalysts are EDC and Sulfo-NHS. tional moieties; b) assembling a plurality of said macromol 0229. The mixture may then be washed to remove any ecules under Suitable conditions to form a nanoparticle hav unreacted molecules or catalysts. In one embodiment metha ing a hydrophobic core and a hydrophilic shell; and c) nol is used in the washing step. Additional washing step may conjugating at least a portion of said functional moieties on be used to remove unreacted polymer. In one embodiment, the hydrophobic portion to a mucosal targeting moiety, to the unreacted polymer is dextran. The final mixture is dis thereby provide a Surface-functionalized nanoparticle. Solved in a suitable solvent, centrifuged and the resulting 0219. In some embodiments, a) comprises conjugation of Supernatant is collected. In one embodiment, the Suitable a hydrophilic polymer to a hydrophobic polymer to form a solvent is acetone oracetonitrile. The final product is dried. In diblock copolymer. one embodiment, vacuum dessicator is used to dry the prod 0220. In some embodiments, the hydrophilic polymer is uct dextran and the hydrophobic polymer is PLA. 0230. In another aspect, the present disclosure provides a 0221. In some embodiments, the targeting moiety is a process for the preparation of nanoparticles useful in the phenylboronic acid derivative, a thiol derivative or an acrylate formation of a mucoadhesive nanoparticle delivery system. derivative. In some embodiments, the targeting moiety is a The polymers or macromolecules described herein may be phenylboronic acid (PBA) derivative. formed into a nanoparticle using techniques known to those 0222. In some embodiments, step b) is performed before skilled in the art. The geometry formed by the particle from step c). However, in other embodiments, step c) is performed the macromolecule may depend on factors such as the size before step b). and composition of the polymers that form the macromol 0223) In some embodiments, the surface density of the ecule. In addition, also as discussed below, in Some cases, the mucosal targeting moiety on the nanoparticle is controlled by particle may include an agent of interest, such as a therapeu the amount of mucosal targeting moiety introduced into the tic, diagnostic or imaging agent. For example, in some reaction. embodiments, the nanoparticle may contain a therapeutic 0224. In some embodiments, the process comprises reduc agent, such as a drug. The agent of interest may be incorpo tive animation between the multimer and a suitable linker. In rated into the particle during formation of the particle, e.g., by US 2015/0320694 A1 Nov. 12, 2015 20 including the agent in a solution containing the polymers that methanol and dried in vacuum desiccator. The sample is then are used to form the particle, and/or the agent may be incor dissolved in DI-HO and treated with hydrochloric acid and porated in the particle after its formation. triethyl amine for the deprotection of the Boc group. The 0231. In addition, the method may employ additional conjugation of amine-terminated Dextran and PLA was car polymers or macromolecules distinguishable from the poly ried out in DMSO with EDC and Sulfo-NHS as catalysts for mers or macromolecules discussed above. As previously dis about 4 hrs. The final product was washed several times with cussed, first and second (or more) macromolecules may be methanol. The wash sample was further dissolved in acetone combined together at different ratios to produce particles and centrifuged. The Supernatant was extracted carefully in comprising the first and second (or more) macromolecules, order to separate from free unreacted Dextran that have been keeping in mind that, in Some embodiments, it is desirable to precipitated. Finally, the Supernatant containing Dex-b-PLA have hydrophilic portions with multiple functional groups was dried in vacuum desiccator. present in the shell of the nanoparticle fortunable targeting of 0238. Thus, in some embodiments, there is provided a the nanoparticles via coupling of the functional groups to a method of preparing a Dex-b-PLA macromolecule, the mucosal targeting moiety, Such as a targeting moiety capable method comprising: 1) reductive amination between Dextran of forming high affinity binding to a target at the mucosal site. and N-Boc-ethylenediamine, 2) deprotection of the Boc 0232. In some embodiments, the targeting moieties are group, and 3) conjugation of the end modified Dextran with conjugated to the macromolecules following nanoparticle PLA (Scheme 1). The first step of the synthesis involves formation. reductive amination between the aldehyde on the reducing 0233. The present disclosure also provides a process for end of Dextran and the amine group of N-Boc-ethylenedi conjugating targeting moieties on the Surface of the nanopar amine cross-linker. The reducing agent, NaCNBH was ticles formed using the amphiphilic macromolecules added to the borate buffer solution and the mixture was stirred described herein. In some embodiments, the conjugation is for 72 hours in dark conditions at room temperature. The between the functional groups of the hydrophilic portion (e.g. mixture was then washed in methanol to remove any unre the backbone of a hydrophilic polymer) and the functional acted molecules or catalysts. The end-modified Dextran was groups of the targeting moieties. In some embodiment, cata dried overnight in vacuo. The dried Dextran was re-dissolved lysts, for example, EDC, are used for the conjugation reac in de-ionized water (D1-HO). The deprotection of Boc tion. In some embodiments, the functional groups of the group was performed first by adding HCl for 1 hour to cleave polymer backbone are modified into other types of functional the amide bond between the Boc group and the protected groups prior to the conjugation reaction. In one embodiment, amine moiety. Subsequently, TEA was added to increase the NaIO is used to oxidize hydroxyl groups into aldehyde pH of the solution up to 9 to deprotonate the NH' end groups groups. The mixture may be washed, for example, using which were deprotected. The mixture was then washed twice methanol, to remove nonconjugated targeting moieties. In using methanol and dried in vacuo. An NMR sample of the Some embodiment, dialysis is used to remove the unreacted dried product was prepared in DO (). The amine terminated molecules. Dextran and carboxyl terminated PLA20 (Mw-20 kDa, 6 g. 0234. In some cases, the method may include conjugation 0.3 mmol) were dissolved in DMSO. The conjugation with more than one type of targeting moiety. The Surface between the two polymers was facilitated by adding catalysts density of targeting moiety on the resulting nanoparticles EDC (120 mg. 0.773 mmol) and Sulfo-NHS (300 mg, 1.38 may be controlled by adjusting the amount of material in the mmol) and allowing reaction to proceed for 4 hours at room reaction mixture. temperature. The resulting Dex-b-PLA was twice precipi 0235. In some embodiments, conjugation and nanopar tated and purified using excess methanol. In order to remove ticle formation may occur as a single-step reaction, for free Dextran, the mixture was dissolved in acetone (30 mL) to example, according to a single-step reaction as described in form a cloudy suspension. This was centrifuged at 4000 rpm U.S. Pat. No. 8.323,698. However, a single-step reaction such for 10 minutes and the Supernatant was extracted carefully. as this will resultina nanoparticle having a detectable amount The Supernatant was purged with air to remove the solvent of targeting moieties located within the core of the nanopar and then dried overnight in vacuo to obtain the final copoly ticle, thereby decreasing the targeting efficiency of the par CS. ticles compared to a more controlled sequence as described 0239. To functionalize the polymers, Dex-b-PLA may be above. dissolved in DMSO (30 mg/ml), and added slowly into water 0236 Specific reaction conditions can be determined by under mild stirring. Periodate oxidation of the Dextran sur those of ordinary skill in the art using no more than routine face was carried out by adding 60mg of NaIO and stirring for experimentation. an hour. Subsequently, glycerol was added to quench the unreacted NaIO. Various amounts of PBA (i.e. 40 mg for Embodiment of the Method Dex-b-PLA 40 PBA) were added to the mixture, along with 0237. An exemplary method of preparing a Dextran-b- NaCNBH for 24 hours. All reactions were carried out in the PLA (Dex-b-PLA) block copolymer is described below in dark. The mixture was then dialyzed in water for 24 hrs to Example 1 (Verma, 2012). Briefly, an exemplary procedure remove any unreacted Solutes, through changing the wash for the synthesis of Dex-b-PLA may be divided into three medium 4 times. stages: reductive amination between Dextran and N-Boc 0240. The polymers or macromolecules described herein ethylenediamine, deprotection of the Boc group, and conju may be formed into a nanoparticle using techniques know to gation of the amine-modified Dextran end group with car those skilled in the art, including those discussed in detail boxyl-terminated PLA. Reductive amination may be carried below. The geometry formed by the particle from the polymer out by dissolving Dex in aborate buffer and mixing it with or macromolecule may depend on factors such as the poly N-Boc-ethylenediamine and NaCNBH in dark condition for mers that form the particle. In addition, also as discussed about 72 hrs. After the reaction, the mixture is washed with below, in Some cases, the particle may include a hydrophilic US 2015/0320694 A1 Nov. 12, 2015 agent or a hydrophobic agent of interest, depending on the collide), poly(e-caprolactone), or a combination thereof, the structure of the particle. For example, the particle may con hydrophobic polymer forming the core of the nanoparticle; a tain a drug or other therapeutic agent. The hydrophilic or hydrophilic biocompatible polymer selected from polysac hydrophobic agent may be incorporated in the particle during charide, polynucleotide, polypeptide, or a combination formation of the particle, e.g., by including the agent in a thereof, having multiple functional moieties, the hydrophilic Solution containing the polymers that are used to form the portion forming the shell of the nanoparticle; at least a portion particle, and/or the agent may be incorporated in the particle of the functional moieties being conjugated to a mucosal after its formation. targeting moiety selected from a phenylboronic acid (PBA) 0241 The Dex-b-PLANPs were prepared using nanopre cipitation method: 1 mL of Dex-b-PLA in DMSO (10 derivative, a thiol derivative or an acrylate derivative. mg/mL) was added in a drop-wise manner to 10 mL of 0248. In some embodiments, there is provided a nanopar DI-HO under constant stirring in order to form NPs. This ticle composition useful for delivery of a payload to a was stirred for 30 minutes and then dynamic light scattering mucosal site, the nanoparticle comprising a plurality of (DLS) samples were prepared by extracting 3 mL samples amphiphilic macromolecules, the macromolecules compris into polystyrene cuvettes. The sizes of the NPs were analyzed ing: a hydrophobic portion comprising a polylactide; a hydro using 90Plus Particle Size Analyzer (Brookhaven, A=659 nm philic portion having multiple functional moieties, said at 90°). The volume averaged multimode size distribution hydrophilic portion comprising dextran; and a mucosal tar (MSD) mean diameters were used from the results. geting moiety being a phenylboronic acid (PBA) derivative, 0242. In addition, the method may employ additional wherein at least a portion of said functional moieties of the polymers or macromolecules, which may be distinguishable hydrophilic portion are conjugated to the mucosal targeting from the polymers or macromolecules discussed above. As moiety. previously discussed, the first and second macromolecules may be combined together at different ratios to produce par 0249. In some embodiments, there is provided a nanopar ticles comprising the first and second macromolecules. ticle composition useful for delivery of a payload to a 0243 In some cases, the method may include conjugation mucosal site, the nanoparticle comprising a plurality of with more than one type of targeting moiety. The Surface amphiphilic macromolecules, the macromolecules each com density of targeting moiety on the resulting nanoparticles prising a hydrophobic polylactide polymer conjugated to a may be controlled by adjusting the amount of material in the hydrophilic dextran polymer having multiple functional moi reaction mixture. eties, at least a portion of said functional moieties being 0244 Alternatively, the reaction may occur as a single conjugated to a phenylboronic acid (PBA) derivative. step reaction, i.e., the conjugation is performed without using intermediates Such as N-hydroxySuccinimide or a maleimide, (0250. In some embodiments, the macromolecule is Dext such as that described in U.S. Pat. No. 8,323,698. However, ran-p-PLA. In some embodiment, the functionalized macro Such method results in a nanoparticle having a portion of the molecule is Dextran-p-PLA PBA. targeting moiety located in the core of the particle. Thus, typically, a multi-step approach will be used to achieve higher 0251. In some embodiments, the nanoparticle is formed targeting efficiently. by conjugating the polylactide to the dextran to form macro 0245 Specific reaction conditions can be determined by molecule, then forming a nanoparticle, and Subsequently Sur those of ordinary skill in the art using no more than routine face-functionalizing the nanoparticle by conjugating at least a experimentation. portion of the functional moieties of the dextran to the PBA derivative to achieve a desired surface density of the PBA Particular Embodiments derivative. 0246. In some embodiments, there is provided a nanopar 0252 All definitions, as defined and used herein, should ticle composition useful for delivery of a payload to a be understood to control over dictionary definitions, defini mucosal site, the nanoparticle comprising a plurality of tions in documents incorporated by reference, and/or ordi amphiphilic macromolecules, the macromolecules compris nary meanings of the defined terms. ing: a hydrophobic portion comprising a biocompatible poly mer selected from a from polylactide, a polyglycolide, poly 0253) The indefinite articles“a” and “an as used herein in (lactide-co-glycolide), poly(e-caprolactone), or a the specification and in the claims, unless clearly indicated to combination thereof; a hydrophilic portion comprising a bio the contrary, should be understood to mean “at least one.” compatible polymer selected from polysaccharide, poly 0254. In the claims, as well as in the specification above, nucleotide, polypeptide, or a combination thereof, the hydro all transitional phrases such as “comprising.” “including.” philic portion comprising multiple functional moieties; and a “carrying.” “having.” “containing.” “involving,” “holding.” mucosal targeting moiety selected from a phenylboronic acid “formed from, “composed of and the like are to be under (PBA) derivative, a thiol derivative or an acrylate derivative, stood to be open-ended, i.e., to mean including but not limited wherein at least a portion of said functional moieties of the tO. hydrophilic portion are conjugated to the mucosal targeting moiety. 0255 Only the transitional phrases “consisting of and 0247. In some embodiments, there is provided a nanopar “consisting essentially of shall be closed or semi-closed ticle composition useful for delivery of a payload to a transitional phrases, respectively. mucosal site, the nanoparticle comprising a plurality of 0256 The following examples are intended to illustrate amphiphilic macromolecules, the macromolecules each com certain exemplary embodiments of the present disclosure. prising: a hydrophobic biocompatible polymer selected from However, the scope of the present disclosure is not limited to a from polylactide, a polyglycolide, poly(lactide-co-gly the following examples. US 2015/0320694 A1 Nov. 12, 2015 22

EXAMPLES with PLA (Scheme 1). The first step of the synthesis involves reductive amination between the aldehyde on the reducing Example 1 end of Dextran and the amine group of N-Boc-ethylenedi Synthesis and Characterisation of Dex-b-PLA amine cross-linker. In a typical reaction, Dex6 (M-6 kDa, 6 g, 1 mmol) was dissolved in 15 mL of borate buffer (0.05 M. 1.1 Materials pH 8.2) with 4 g (2.5 mmol) of N-Boc-ethylenediamine. The 0257 Acid-terminated poly(D.L-lactide) (PLA, M-10, reducing agent, NaCNBH (1 g, 15 mmol), was added to the 20 and 50 kDa) and PLGA-PEG (PLGA M-40 kDa, PEG borate buffersolution and the mixture was stirred for 72 hours M-6 kDa) were purchased from Lakeshore Biomaterials in dark conditions at room temperature. The mixture was then (Birmingham, Ala., USA). PLA was purified by dissolving in washed in methanol to remove any unreacted molecules or dimethylsulfoxide (DMSO) and precipitating in methanol to catalysts. The end-modified Dextran was dried overnight in remove residual monomers. Dextran (Dex, M. 1.5, 6, and 10 vacuo. H NMR samples were prepared by dissolving the kDa), hydrochloric acid (HCl), triethylamine (TEA), N-(3- end-modified Dextran in DO (30 mg/mL). The dried Dext dimethylaminopropyl)-N-ethylcarbodiimide (EDC), and ran was re-dissolved in de-ionized water (DI-HO). The sodium cyanoborohydride (NaCNBH) were purchased from deprotection of Boc group was performed first by adding HCl Sigma Aldrich (Oakville, ON, Canada), and used without (~4M) for 1 hour to cleave the amide bond between the Boc further purification. N-Hydroxysulfosuccinimide (Sulfo group and the protected amine moiety. Subsequently, TEA NHS) and N-Boc-ethylenediamine were purchased from was added to increase the pH of the solution up to 9 to CNH Technologies (Massachusetts, USA). Doxorubicin deprotonate the NH' end groups which were deprotected. HCl (MW=580 Da, Intatrade GmBH, Bitterfield, Germany) The mixture was then washed twice using methanol and dried was deprotonated by adding TEA (2M equivalent) in the in vacuo. An NMR sample of the dried product was prepared aqueous solution of Doxorubicin-HCl, and the hydrophobic in DO (30 mg/mL). The amine terminated Dextran and car form of Doxorubicin was extracted using Dichloromethane boxyl terminated PLA20 (Mw-20 kDa, 6 g., 0.3 mmol) were (DCM) (Chittasupho, 2009). Borate buffer was prepared at a dissolved in DMSO. The conjugation between the two poly concentration of 0.05M with pH of 8.2 by mixing boric acid mers was facilitated by adding catalysts EDC (120 mg. 0.773 and sodium hydroxide. Whole sheep blood (in Alsevers) was mmol) and Sulfo-NHS (300 mg, 1.38 mmol) and allowing purchased from Cedarlane (Burlington, ON, Canada). reaction to proceed for 4 hours at room temperature. The Veronal Buffer solution (VBS, 5x) was purchased from resulting Dex-b-PLA was twice precipitated and purified Lonza Walkersville Inc (Walkersville, Md., USA). Tritium using excess methanol. In order to remove free Dextran, the HI-PLA-radiolabeled nanocrystals were purchased from mixture was dissolved in acetone (30 mL) to form a cloudy PerkinElmer (Boston, Mass., USA). suspension. This was centrifuged at 4000 rpm for 10 minutes and the Supernatant was extracted carefully. The Supernatant 1.2 Synthesis of Dex-b-PLA was purged with air to remove the solvent and then dried 0258. The synthesis of the linear block copolymer is overnight in vacuo to obtain the final copolymers. NMR divided into three stages: 1) reductive amination between samples were prepared at a concentration of 30 mg/mL in Dextran and N-Boc-ethylenediamine, 2) deprotection of the DMSO-d6 for proton NMR and 150 mg/mL in DMSO-d6 for Boc group, and 3) conjugation of the end modified Dextran carbon NMR.

Scheme 1. Synthesis of Dex-b-PLA block copolymers. H OH 1 HO HO \-NOH O

HO O H 2 HO

(2) OH O OH OH

Ho \-N O OH (2)

(2) US 2015/0320694 A1 Nov. 12, 2015

-continued

Dextran NH Et (2)

b OH (2) HO O OH HO HO O HO OH O C OH O HO HO Y-N (3) HO OH O HO V-No HO \ O O OH HO O OH HO O M-NOH Dextran NH Et ()

OH (?) -- HN (2) d CH, O CH3 HC 4fne 3 O O OH -- CH, O 2 a) Synthesis of Dextran-NH-Et-NH-Boc. Conditions: NaCNBH in Borate buffer (pH8.2) for 72 hr at RT in dark. b): Synthesis of Dextran-NH-Et-NH2. Conditions: HCITEA in DI-HO for 1 hr each at RT, c): Synthesis of Dextran-NH-Et-NH-PLA. Conditions: EDC/Sulfo-NHS RT for 4 hrs. (2) indicates text missing or illegible when filed

1.3 Characterization of Dex-b-PLA Using Nuclear Magnetic DI-HO under constant stirring in order to form NPs. This Resonance (NMR) was stirred for 30 minutes and then dynamic light scattering (DLS) samples were prepared by extracting 3 mL samples 0259. The various stages of Dex-b-PLA synthesis were into polystyrene cuvettes. The sizes of the NPs were analyzed verified using H NMR spectroscopy (Bruker 300 MHz). The using 90Plus Particle Size Analyzer (Brookhaven, A=659 nm final polymer conjugation was also verified using C NMR at 90°). The volume averaged multimode size distribution spectroscopy (Bruker 300 MHz). Before any modification, (MSD) mean diameters were used from the results. Dextran was dissolved in DO (30 mg/mL) and acid termi nated PLA was dissolved in CDC1 (5 mg/mL) for preparing 1.5 Transmission Electron Microscopy NMR samples. As mentioned in the previous synthesis meth ods, the end products from the first two steps were dissolved 0261 The particle size and the morphology of the Dex-b- in DO, whereas the final product, Dex-b-PLA, was dissolved PLANPs were further verified using Transmission Electron in DMSO-d6 for the NMR analysis. Microscopy (TEM, Philips CM10) with the accelerating volt age of 60 kV and the Lanthanum Hexaboride filament 1.4 Dex-b-PLANP Formation by Nanoprecipitation (LaB6).300 Mesh Formvar coated copper grids (Canemco & Marivac) were used for this experiment. The NP suspension 0260 The Dex-b-PLANPs were prepared using nanopre in water was prepared using the nanoprecipitation method as cipitation method: 1 mL of Dex-b-PLA in DMSO (10 mentioned above. A drop of the NP suspension was placed mg/mL) was added in a drop-wise manner to 10 mL of onto the grid, and the grid was briefly stained with aqueous US 2015/0320694 A1 Nov. 12, 2015 24 phosphotungstic acid solution. The copper grid with the NP PLA NPs, on the other hand, can be controlled simply by Suspension was dried under ambient environment overnight changing the MW of the compositional polymers as exempli before imaging under TEM. fied in FIG. 2a.

1.6 Results and Discussion Example 2 0262 The synthesis of Dex-b-PLA block copolymers was Encapsulation and In Vitro of Doxorubicin in analyzed using H NMR spectrometer. As shown in FIG. 1a I, Dex-b-PLANPs Via Nanoprecipitation the 4.86 ppm multiplet was assigned to the proton on carbon 0265. The encapsulation of Doxorubicin in the Dex-b- 1 of Dextran repeating units. The 3.14 ppm multiplet was PLANPs was accomplished using nanoprecipitation method. assigned to the proton on carbon 5 of the non-reducing end the Dex-b-PLA and Doxorubicin were both dissolved in DMSO integral ratio between these two multiplets was used to con (Dex-b-PLA concentration of 7 mg/mL, with varying drug firm the MW of Dextran. The reductive amination reaction of concentrations). 1 mL of the DMSO solution is added drop Dextran and N-Boc-ethylenediamine was confirmed by the wise into 10 mL of water under stirring and continued to stir presence of 1.3 ppm peak (Boc group) after removing unre for additional 30 minutes. The NPs in water were filtered acted free N-Boc-ethylenediamine (FIG. 1a II). The subse through syringe filter (pore size=200 nm) to remove the drug quent deprotection of Boc group exposing the —NH2 end aggregates and Subsequently filtered through Amicon filtra group on Dextran was verified by the disappearance of the 1.3 tion tubes (MWCO=10kDa, Millipore) to further remove any ppm peak (FIG. 1a III). It was shown that the 1.3 ppm peaks remaining free drugs in the suspension. The filtered NPs were completely removed after the deprotection steps using containing encapsulated Doxorubicin were resuspended and HCl and TEA. After the conjugation of the NH terminated diluted in DMSO. Consequently, the drug loading (wt %) in Dextran with COOH-terminated PLA (FIG. 1a IV), the the polymer matrix was calculated by measuring concentra excess free Dextran molecules were removed by precipitating tion of the Doxorubicin in the mixture by obtaining the absor in acetone. The final product shows peaks corresponding to bance of the solution at 480 nm using Epoch Multi-Volume both the Dextran (multiplets at 4.86 ppm) and the PLA (mul Spectrophotometer System (Biotek). The measurements tiplets at 5.2 ppm) which confirm the conjugation of the two were obtained in triplicates (n=3, mean+S.D). The absor polymers (FIG. 1a V). The linear end-to-end conjugation of bance measured from same procedure using the polymers PLA and Dextran was also confirmed by Carbon NMR (FIG. without the drugs was used as the baseline. The absorbance 1b). The peak at 166.81 ppm is assigned to the carbon on PLA was correlated with the concentration of the Doxorubicin in that attaches to the amine terminal of the ethylenediamine DMSO by using standard calibration obtained. The same linker, while 169 ppm peak is the carbonyl carbon on PLA procedure was used for PLGA-PEG to encapsulate Doxoru backbone (FIG. 1b). bicin for comparative analysis. The encapsulation efficiency 0263. The size and morphology of NPs using nine formu (%) and drug loading (wt %) were calculated using the two lations of Dex-b-PLA block copolymers are shown in FIG. 2. equations (Eq. 1 and Eq. 2). Varying the MW of PLA and Dextran resulted increating NPs with different sizes ranging from 15 to 70 nm. As shown in mass of drug encapsulated (1) FIG. 2a, increasing the MW of PLA increased the particle Encapsulation efficiency (%) = x 100% size whereas increasing the MW of Dextran decreased the mass of initial drug feed particle size. The NP core, formed by PLA, was predicted to f d lated 2 increase in size with increasing MW’s of the PLA chains as Drug loadi t 9%) = mass of drug encapsulate x 100% (2) demonstrated previously (Riley 1999; Riley 2001) and it was rug loading (wt %) mass of the nanoparticle confirmed here by the NPs composed of PLAMW of 10 kDa, 20 kDa and 50 kDa. We postulate that the effect of Dextran 0266 Based on the size tuning as shown in FIG.2, PLA20 MW on NP size is likely due to Dextran configuration on the Dex10 (MWPLA-20 kDa, MWDextran-10 kDa) and NP surface. Zahr et al. found that hydrophilic chains, such as PLA20-Dex6 were selected for analyzing the encapsulation PEG, at MW of 5kDa or longer would be able to “fold-down” efficiencies and the drug loading using Doxorubicin as a onto the particle Surface creating a mushroom conformation model hydrophobic drug (FIG. 3). The particle sizes for (Zahr 2006). Similarly, this phenomenon may explain why PLA20-Dex10 and PLA20-Dex6 were 20.5 and 30.1 nm the NPs with longer Dextran chains lead to smaller hydrody respectively. Doxorubicincompounds were incorporated into namic diameters. The shorter Dextran chain length has a NPs through nanoprecipitation method. Both Dextran based Smaller degree of freedom and confined to linear structure NPs, PLA20-Dex10 and PLA20-Dex6 NPs, were found to compared to those with longer chain length. The TEM image encapsulate large amounts of Doxorubicin with maximum of NPs composed of PLA20-Dex6 (MW-20 kDa, loadings of 21.2 and 10.5 wt % respectively. The maximum MW extrapa ~6 kDa) confirmed the particles exhibit spherical loadings were achieved at 40 wt % initial loading, and further structure (FIG.2b). increase in the initial loading did not increase the drug loading 0264. Dex-b-PLANPs with sizes under 50 nm have been in the NPS due to aggregation of the particles. It is speculated synthesized using the simple process of bulk nanoprecipita that PLA20-Dex10, with longer Dextran chain than PLA20 tion. PLGA-PEG block copolymer was used as a commercial Dex6, is likely to have more Doxorubicin weakly associated benchmark, which formed NPs with size 133.9-6.1 nm fol on the NP surface or encapsulated near the surface of the NPs lowing the same procedure. The particle size for PLGA-PEG during nanoprecipitation. This effect was minimized by con is in agreement to previous literature values (Dhar 2009). ducting ultrafiltration (MWCO=10 kDa) after the nanopre PLGA-PEG NPs have been able to achieve smaller particle cipitation ensuring that the non-specifically bound drugs sizes but it required the assistance of microfluidic devices for were removed from the NP suspension. The maximum drug enhanced control (Karnik 2008). The particle size of Dex-b- loading in PLGA-PEGNPs, used as a control, was found to be US 2015/0320694 A1 Nov. 12, 2015

7.1 wt.%. It was found that excess initial loading caused more release phase. It is possible that the burst-release region cor drug precipitation and particle aggregation during nanopre responds to drugs non-specifically bound on the Surface of the cipitation for PLGA-PEG NPs, whereas Dex-b-PLA NPs NPs, or drugs encapsulated near the surface of the NPs during showed negligible size increases even at their maximum drug the nanoprecipitation procedure (Magenheim 1993). PLA20 loading. The maximum Doxorubicin loading achieved with Dex6 and PLA20-Dex10 NPs exhibited burst-release region PLA20-Dex10 NPs were considerably higher than the most within the initial 24 hours, releasing up to 48% and 74% reported values using PEG based NPs in the literatures, which respectively. The Subsequent Sustained-release phase of varied over 4.3-11.2% for poly(e-caprolactone)-PEG copoly Doxorubicin from PLA20-Dex6 and PLA20-Dex10 NPs mers (Shuai 2004; He 2010), 8.7% for poloxamer 407 and continued for 192 hours with similar rate of release from both PEG hydrogel system (Missirlis 2006), and 18% for PEG NPs. The sustained-release phase may correspond to the dif poly(B-benzyl-L-aspartate) based NPs (Kataoka 2001). The fusional release of the drugs from the core of the NPs. In the increased drug loading is most likely due to the greater hydro control study using PLGA-PEG NPs, the burst-release phase philicity of Dextran compared to PEG (Alpert 1990), which of Doxorubicin was within the first 6 hours while steady in turn reduces the probability of Dextran chains from the release phase continued up to 96 hours, similar to what has block copolymers associating in the hydrophobic core of the been reported previously (Esmaeili 2008). NPs. The encapsulation efficiency and the total drug payload using the Dex-b-PLA system is comparable to commercially Example 4 available liposomal systems such as the FDA approved Doxil R, which has a drug loading of 12.5% and DaunoX Hemolysis Assay ome(R), which has Daunorubicin loading of 7.9% (Drummond 0269. Dex-b-PLA NPs were purified by using Amicon 1999). The concentration of Doxorubicin in the Doxil R for filtration tubes (MWCO=10 kDa) and centrifugation at 4100 mulation translates into 6.25 mg/m when Doxil R is admin rpm for 30 minutes. A concentration range of NPs was istered at 50 mg/m (Drummond 1999; Safra 2000). The same obtained by this process. These NPs were then incubated at physiological concentration of Doxorubicin can theoretically 37° C. for one hour with 200 uL of sheep erythrocytes with be achieved using only 30 mg/m of PLA20-Dex10 red blood cells concentration of 1x10 cells/mL to obtain a NP-Doxorubicinformulation. final Volume of 1 mL per sample. The percent hemolysis was calculated by measuring the absorbance at 415 nm and using Example 3 the absorbance at 500 nm as the baseline. The measurements were conducted in triplicates (meantS.D). Here, VBS solu In Vitro Release of Doxorubicin from Dex-b-PLA tion was used as the negative control and deionized water was NPS used as the positive control. PLGA-PEG NPs were also pre 0267. Using the procedure described in the previous pared and tested in a similar manner for comparison. example, drug encapsulated NPs were prepared and filtered to 0270 Previous work has considered hemolysis of NPs less remove non-encapsulated drug aggregates. A purified sample than 5% to be biocompatible (Dobrovoiskaia 2008). It has of NPs-drug Suspension was collected to measure the maxi been demonstrated that PLGANPs stabilized by surfactants mumabsorbance and this was used as the 100% release point. are severely hemolytic to 80% and hemolysis is reduced Subsequently, the NP-drug Suspension was injected into a considerably by using a hydrophilic PEG surface in the case Slide-a-Lyzer Dialysis cassette (MWCO=20kDa, Fisher Sci of PLGA-PEG NPs (Kim 2005). The same results were entific) and dialyzed against 200 mL of phosphate buffered expected from the use of Dextran based NP formulation since saline (PBS, pH 7.4) at 37°C. under mild stirring. At prede Dextran derivatives such as diethylaminoethyl-dextran have termined time intervals, 1 mL of the release medium was low (-5%) hemolysis (Fischer 2003). The block copolymer extracted and the same volume of fresh new PBS was added NPs formulated previously were tested for hemolytic activity to the release medium. The extracted release medium was at various concentrations (1-10 mg/mL). It was shown that all used to perform UV-Vis absorption measurements at 480 nm formulated NPs were not significantly hemolytic (<5%) up to in triplicates (n=3, meantS.D). The release medium was a concentration of 10 mg/mL in the blood (FIG. 5). The replaced several times to maintain the concentration of Doxo hemolysis by both PLA20-Dex6 and PLA20-Dex 10 were rubicin in the medium below 3 g/mL and to stay below the similar since they have the same component polymers. For solubility limit of the Doxorubicin in PBS. Replacing the comparison, Doxil(R) (a liposomal formulation of doxorubi medium was also expected to prevent the adhesion of released cin) is usually administered at the dose of 50 mg/m (Safra Doxorubicinto the glass walls of the beaker or the magnetic 2000). This dose translates to a concentration of 0.018 mg/mL Stir bar. The release of Doxorubicin from PLGA-PEG was in blood for an average human being (body surface area 1.79 also obtained with identical procedure for comparative analy m (Sacco 2010), and blood volume 5 L) (Kusnierz-Glaz sis. Free Doxorubicin, without any polymers, release was also 1997). The tested hemocompatible concentration (10 observed using the same procedure and all three release pro mg/mL) for PLA-b-Dex NPs is considerably higher than the files from the NPs were normalized using the free Doxorubi administered dose of Doxil (R). This suggests that PLA-b-Dex cin release data along with encapsulation efficiency data. This NPs are a safe system for intravenous administration. normalization resulted in a release curve for only encapsu lated Doxorubicin. All experiments were performed in dark Example 5 environment, and the beakers were sealed with Parafilm to prevent evaporation of PBS. Pharmacokinetics and Biodistribution of Dex-b-PLA 0268. The in vitro release of Doxorubicin from the NPs NPS was carried out in pH 7.4 PBS buffer at 37° C. As shown in 0271 To ensure that all radioactivity administered to rats FIG. 4, the release profile of Doxorubicin from NPs was was associated with the particles, tritium H-PLA-radiola characterized with an initial burst followed by a sustained beled nanocrystals were washed and purified in methanol US 2015/0320694 A1 Nov. 12, 2015 26 prior to NP formation. Albino Wistar rats, body weight of PLA20-Dex10 compared PLA20-Dex6 since the former between 200 and 250 g, were fasted overnight but had free has smaller particle size. In addition to their size effect on access to water. 200LL of the NP formulations were prepared protein adsorption, it is also hypothesized that the abundant in NaCl 0.9% and injected intravenously into the tail vein at a hydroxyl groups on the Dextran Surface may induce Sufficient dose of approximately 30 mg/kg. Blood (approximately 200 hydration layer around the NPs to limit protein adsorption LL) was collected in heparinized microcentrifuge tubes by (Portet 2001). It has been reported that accumulation rate in controlled bleeding of hind leg saphenous veins at the indi tissues such as spleen increases with increase in the particle cated time intervals. To characterize the biodistribution of sizes (Li 2008) which is consistent with the current findings. NPs, rats were euthanized at 24 h after NP injections. It has also been observed that PEG coating in PEGylated Approximately 200 uL of blood was drawn by cardiac punc particles can increase accumulation in the spleen (Peraccia ture from each mouse. Organs including heart, lungs, liver, 1999) whereas the neutrality (Chouly 1996) and flexibility spleen and kidneys were harvested from each animal as (Passirani 1998) of dextran chains on the NP surface can described previously (Gu 2008). The H content in the tissue cause lower protein absorption leading to lower spleen accu and blood were assayed in a Wallac 1414 Liquid Scintillation mulation. Dex-b-PLANPs are expected to have low comple Counter. ment activation as observed for dextran-poly(methyl meth 0272. The NP circulation half-life in vivo was character acrylate) NPs, whose behaviour was similar to soluble ized by measuring the amount of tritium H-PLA-radiola dextran (Passirani 1998). The lower accumulation of the Dex beled nanocrystals that were incorporated in the NP formu b-PLANPs in spleen along with lower complement activa lations. FIG. 6 shows NP concentration in blood circulation at tion may have attributed to their longer blood circulation predetermined time intervals after intravenous administra (Meerasa 2011). The long circulation half-life of NP drug tion. It is noted that the time-dependent NP concentration in carriers is a crucial parameter in cancer therapy since it the blood were characterized by two regions of distinct increases the probability of accumulating at cancerous tissues slopes. The first region (first ~18 hrs) corresponds to the due to EPR effect: particle size below 100 nm directly pro initial clearance of the NPs from the blood circulation, motes accumulation of NPs in the tumor sites since the vas whereas the second region indicates the terminal clearance of cular pores around tumor are at least 100 nm in size (Cho the NPs. The former region profiles the NP volume of distri 2008). The size-tuneable Dex-b-PLA system developed here bution among vascular and extravascular tissues, while the presents a polymeric platform for systematically studying the terminal half-life relates to the systemic clearance phase of effect of NP size on various in vivo characteristics such as the NPs from the body (Yang 2009). The initial half-life (t), biocompatibility, blood clearance, tumor accumulation and terminal half-life (t), the blood retention time for 90% of biodistribution and screening candidates for further clinical the NPs (ts), and AUC (Gaucher 2009) of the three NPs are evaluation. Summarized in Table 1. At 24 hours postinjection, rats were euthanized, and the major organs were harvested from the TABLE 1 animals to evaluate the biodistribution of the NPs (FIG. 7). It Blood pharmacokinetic parameters for PLA20-Dex10, PLA20-Dex6. was observed that all three NPs had maximum accumulation and PLGA-PEGNPs in the liver and the percent distribution was similar for each NP. Higher accumulations in the spleen were observed with to (hr) t12 (hr) too (hr) AUC PLGA-PEG NPs compared to both of Dex-b-PLANPs (p<0. PLA20-Dex10 12.3 2.2 29.81.O 38.3 21.5 1040 01). Accumulation of NPs in all other organs was below 5% PLA20-Dex6 7.20.4 26.63.1 17.98.6 691 with similar amount of accumulation among the NPS in each PLGA-PEG 3.7 0.6 27.02.3 5.024 287 Organ. to: initial half-life; 0273 Although all three types of NPs showed similart to: terminal half-life; values, both PLA20-Dex10 and PLA20-Dex6 NPs showed too: blood retention time for 90% of the NPs; significantly higher values oft, too, and AUC compared to AUC: Area under curve (% dose hr) that of the model NPs composed of PLGA-PEG. Previous 0274 Statistical analysis was performed using the student studies mainly focused ont, values for NPs but the present t-test and statistical significance was assessed with p<0.01. inventors extracted too values for comparison purposes. It was observed that to values were only about 2 hrs for PEG Example 6 b-PLANPs (Gaucher 2009), 6 hrs for polyvinylpyrrolidone based NPs (Gaur 2000) and about 8 hrs for chitosan based Synthesis and Characterization of Dex-b-PLA-BLA NPs (He 2010). Not only do Dex-b-PLA NPs outperform NPS these NPs with a to of 38.3 hrs, they are also comparable to 60 nm PEG-b-PCL system (Lee 2010) and StealthR) lipo 6.1 Synthesis of Dex-b-PLA somes (Allen 1991), both of which have to values over 48 hours. In this study, the longer blood circulation observed in 0275. The synthesis of Dex-b-PLA was carried out as Dex-b-PLANPs, compared to PLGA-PEG NPs, is believed described in Example 1 (see also, Verma, 2012). Briefly the to be partially due to the size difference. A recent study by procedure for the synthesis of Dex-b-PLA divides into three Rehor et al. showed that NPs with diameter of 40 nm had stages: reductive amination between Dextran and N-Boc longer circulation half-life compared to larger NPs with ethylenediamine, deprotection of the Boc group, and conju diameter of 100 nm (Rehor 2008). It is hypothesized that gation of the amine-modified Dextran end group with car Dex-b-PLANPs, having smaller sizes than PLGA-PEG NPs, boxyl-terminated PLA. Reductive amination was carried out have increased curvature that reduce protein adsorption, by dissolving Dex in borate buffer and mixing it with N-Boc which may in turn result in slower clearance rate by the RES. ethylenediamine and NaCNBH in dark condition for 72 hrs. This is further supported by the longer blood circulation time After the reaction the mixture is washed with methanol and US 2015/0320694 A1 Nov. 12, 2015 27 dried in vacuum desiccator. The sample is then dissolved in 1 and 2 of the cysteamine are shown as multiplets peaks near DI-HO and treated with hydrochloric acid and triethylamine 2.7 ppm. However, higher resolution NMR characterization for the deprotection of the Boc group. The conjugation of is required in the future to further differentiate the peaks from amine-terminated Dextran and PLA was carried out in other noise peaks from the polymer. DMSO with EDC and Sulfo-NHS as catalysts for 4 hrs. The 0280. The sizes and morphology of the nanoparticles were final product was washed several times with methanol. The analyzed using the procedure illustrated in Example 1. The wash sample was further dissolved in acetone and centri sizes of the NPs were in the range of 25 to 28 nm, which are fuged. The Supernatant was extracted carefully in order to smaller than the unmodified NPs of 47.9 mm. Without being separate from free unreacted Dextran that have been precipi bound by theory, it is postulated that the particle size reduc tated. Finally the supernatant containing Dex-b-PLA was tion is attributed by the PBA molecules causing the Dextran dried in vacuum desiccator. chains to be less hydrophilic, leading them to form more 6.2 Surface Functionalization of Dex-b-PLANPs with PBA compact shells around the PLA particle core. TEM images (0276 Dex-b-PLA was dissolved in DMSO (30 mg/ml), confirmed a spherical morphology, due to the formation of a and added slowly into water under mild stirring. Periodate core-shell structure of the amphiphilic block copolymers oxidation of the Dextran surface was carried out by adding 60 (FIG.12b). The sizes of Dex-b-PLA PBA NPs obtained are mg of NaIO and stirring for an hour. Subsequently, glycerol smaller than that normally achieved with PEG-based block was added to quench the unreacted NaIO. Various amounts copolymers such as PLGA-PEG (Karnik, 2008). We postu of PBA (i.e. 40 mg for Dex-b-PLA 40 PBA) were added to late that smaller NPs may be more desirable for mucoadhe the mixture, along with NaCNBH for 24 hours. All reactions Sion, since they provide greater Surface area for interaction were carried out in the dark. The mixture was then dialyzed in with the mucous membrane. water for 24 hrs to remove any unreacted Solutes, through changing the wash medium 4 times. Example 7 6.3 Characterization of Dex-b-PLA PBA NPs Drug Encapsulation in Dex-b-PLA PBA NPs (0277. The surface modification with PBA was verified 0281. The Cyclosporine A (CycA) encapsulation in the using H NMR spectroscopy (Bruker 300 MHz). Dex-b- Dex-b-PLA and Dex-b-PLA PBA NPs were measured using PLA PBA polymers were dissolved in DMSO-d6 (25 the procedure described in O. Maximum encapsulation of mg/ml) for the "H NMR characterization. UV-Vis absorption CycA was achieved at an initial feed of 40 wit/wt %: Dex-b- measurement at 291 nm was performed with Epoch Multi PLANPs encapsulated up to 10.8 wit/wt %, whereas Dex-b- Volume Spectrophotometer System (Biotek, USA) on the PLA 40 PBA and Dex-b-PLA 320 PBA encapsulated up to Dex-b-PLA PBA in order to quantify the amount of PBA 11.2 and 13.7 wt/wt %, respectively (FIG. 15). The 13.7 wt/wt attached to the Dextran chains. Dex-b-PLA solution with % encapsulation is equivalent to 2.38 ug of CycA in 28 Jull same concentration was used as the baseline for UV-vis formulation (which is the same as the administration Volume absorption study. The NPs of Dex-b-PLA PBA prepared of commercially available RESTASISR), whereas the com using nanoprecipitation were also analyzed using 90Plus Par mercial product contains 14 Jug. Therefore, a therapeutically ticleSize Analyzer (Brookhaven, A=659 nm at 90°) obtaining relevant dosage can be achieved by simply adjusting the poly volume-averaged multimode size distribution (MSD) mean mer and drug concentration in the formulation. diameter. The particle size and morphology of Dex-b-PLA 0282) Encapsulation of other types of bioactive agents in PBANPs were further confirmed verified using TEM (Philips the Dex-b-PLA PBA NPs has also been explored (FIG. 17). CM10) with the accelerating voltage of 60 kV and the Lan In one embodiment the bioactive agent is Dorzolamide, thanum Hexaboride filament (LaB6). 300 Mesh Formvar which is commonly used to treat glaucoma. In another coated copper grids (Canemco & Marivac) were used for this embodiment the bioactive agent is Brinzolamide, which is experiment. The NP Suspension in water was prepared using also used to treat glaucoma. Natamycin, which is an antifun the nanoprecipitation method as mentioned above. A drop of gal agent, is also used as a bioactive agent in the encapsulation the NP Suspension was placed onto the grid, and the grid was in the Dex-b-PLANPs. In other embodiments, Doxorubicn, briefly stained with aqueous phosphotungstic acid solution (2 an anti-cancer agent, and Olopatadine, antihistamine, were w/v '% in water). The copper grid with the NP suspension was also explored in the encapsulation in the PLA-Dex NPs. dried under ambient environment overnight before imaging 0283. The encapsulation of Cyclosporine A (CycA) in the under TEM. Dex-b-PLANPs was accomplished using nanoprecipitation (0278. The NMR spectrum of Dex-b-PLA PBA shows method. Dex-b-PLA and CycA were both dissolved in peaks corresponding to both PLA (multiplets at 5.2 ppm) and DMSO (Dex-b-PLA concentration of 7 mg/mL, with varying Dextran (multiplets at 4.86 ppm), while also showing multip drug concentrations). 1 mL of the DMSO solution is added let peaks at 6.6 ppm and 6.9 ppm, which correspond to the drop-wise into 10 mL of DI-HO under mild stirring and protons from carbon 2 to 6 in the phenyl group of the PBA continued to stir for additional 30 minutes. The NPs in water (FIG.12a). UV absorption at 291 nm was measured to quan were filtered through syringe filter (pore size=200 nm) to tify the amount of PBA on the NPs with respect to the Dextran remove the drug aggregates and Subsequently centrifuged monomers. Increasing the amount of PBA in the initial reac using Amicon filtration tubes (MWCO=10kDa, Millipore) to tion mixture proportionally increased the final PBA conjuga further remove any remaining free drugs in the Suspension. tion on the Dextran surface (Table 2) with the highest density The filtered NPs containing encapsulated CycA were resus of 34.6 mol% (equivalent of about 3.5 PBA conjugated per 10 pended and diluted in Acetonitrile. Consequently, the drug Dextran monomers) achieved for Dex-b-PLA 320PBANPs. loading (wt/wt %) in the polymer matrix was calculated by 0279 Conjugation of Cysteamine onto the Dex-b-PLA measuring concentration of the CycA in the mixture using NP surface was also demonstrated using "H NMR spectrum High-performance liquid chromatography (HPLC. Thermo (FIG. 13). The peaks that correspond to the protons on carbon Scientific). The measurements were obtained in triplicates US 2015/0320694 A1 Nov. 12, 2015 28

(n=3, mean+S.D). The absorbance measured from same pro glass walls of the beaker or the magnetic stir bar. The beakers cedure using the polymers without the drugs was used as the were sealed with Parafilm to prevent evaporation of water. baseline. The measurements were converted to the concen 0287. The invitro release study of Dorzolamide, Brinzola tration of the CycA using standard calibration obtained. mide, and Natamycin in the Dex-b-PLA NPs were accom 0284. The encapsulation of Dorzolamide, Brinzolamide, plished using the same method. The characterization of these and Natamycin in the Dex-b-PLA NPs were accomplished drugs was performed using Multi-Volume Spectrophotom using the same method. The characterization of these drugs eter System (Biotek, USA) instead of HPLC. was performed using Multi-Volume Spectrophotometer Sys tem (Biotek, USA) instead of HPLC. Example 9 Example 8 Mucoadhesion Tests Drug Release from Dex-b-PLA PBA NPs In Vitro 9.1 In Vitro Mucoadhesion Test—Zeta Potential 0285. The in vitro release phenomenon of CycA from the 0288 Zetapotential measurements were used to analyze Dex-b-PLA NPs were analyzed using the procedures the interaction between mucin particles and the NPs using the described in O. Both Dex-b-PLA and Dex-b-PLA PBA NPs procedures described in O. Several reports in the past have (Dex-b-PLA 40 PBA and Dex-b-PLA 320 PBA) showed a used Zeta potential to assess the mucoadhesive properties of total release point at around 120 hrs (FIG. 16), which is drug carriers (Khutoryanskiy, 2011; Shaikh, 2011: Sogias, significantly longer than previous studies involving in vitro 2008; du Toit 2011: Takeuchi, 2005). Mucin particles at release of CycA from micro or nanoparticles which showed physiological pH exhibit overall negative surface charge due up to 48 hours of sustained release (Li, 2012; Shen, 2010; to the presence of carboxylate groups (sialic acid) and ester Shen, 2010; Yuan, 2006). Moreover, the release rate may Sulfates at the terminus of Sugar units (Khutoryanskiy, 2011). potentially be a significant improvement over the commercial By adhering to the Sialic acid moieties of the mucin particles, product, which requires administering twice a day. Whereas the Dex-b-PLA PBA NPs may shield the negative charges Dex-b-PLANPs demonstrated a sustained release rate for up from the Surface of the mucin particles and also cause aggre to 120 hours, Dex-b-PLA PBA NPs showed two regions of gation of the mucin particles, thus increasing the overall slightly different release rate. In the first 48 hrs, the Dex-b- surface charge. Only Dex-b-PLA 160 PBA (22.9 mol % PLA PBA NPs released CycA at a faster rate compared to PBA) and Dex-b-PLA 320 PBA NPs (34.6 mol%) showed Dex-b-PLANPs, which may be due to the release of CycA significant interaction with mucin particles compared to the that were encapsulated near the slightly more hydrophobic control study (Table 2). Low PBA surface functionalization surface Dex-b-PLA PBA NPs. The subsequent slower densities do not appear to show a difference compared to release rate, compared to Dex-b-PLANPs, may be due to the unmodified NPs interms of mucin-NP interaction. It is there release of drugs from the core of the Dex-b-PLA PBA NPs, fore desirable to use NPs with abundant surface functional which need to diffuse through the more compact Dextran groups, such as Dextran-based NPs, to tune the functional Surface. When the volume of PBA modified NP formulation ization density where maximum mucoadhesion is desired. If were scaled to the administration volume of RESTASISR) (28 one was to functionalize the surface of PLGA-PEG NPs, LL), the CycA release rates were in the range of ug/day, which using one functional group per each PEG chain, the maxi is similar to the daily administration dosage of CycA in mum PBA modification can be achieved is only 0.44 mol % RESTASISR). Therefore, it is possible to optimize the formu (assuming the same MW of PEG, i.e. 10 kDa). An increased lation by changing the concentration of the PBA modified amount of PBA functionalization also increased the extent of NPs and/or the amount of CycA to achieve a clinically effec NP-mucin interaction, which allows potential increase of tive release rate and amount. mucin-NP interaction by saturating PBA on the surface. 0286. In vitro CycA release phenomena from both PBA However, the functionalization of PBA causes the Dextran modified and unmodified Dex-b-PLANPs in the STF at 35° Surface to be more hydrophobic, increasing the potential for C. were analyzed by quantifying the CycA in the STF at aggregation of the NPs. It is therefore ideal to tune the amount predetermined time intervals using High Performance Liquid of PBA functionalization to achieve optimal mucin-NP inter Chromatography (HPLC). Using the procedure described in action without compromising the NP colloidal stability. the previous section, drug encapsulated NPs, both Dex-b- PLA and Dex-b-PLA PBA, were prepared and filtered to TABLE 2 remove non-encapsulated drug aggregates. A purified sample PBA conjugation efficiency and diameter of unmodified and modified of NPs-drug Suspension was collected to measure the maxi Dex-b-PLANPs mumabsorbance and this was used as the 100% release point. Subsequently, the NP-drug Suspension was injected into a PBA:Dex) Diameter Zeta potential Slide-a-Lyzer Dialysis cassette (Molecular weight cut Formulation (mol%) (nm) (mV) off 20 kDa; Fisher Scientific, Canada) and dialyzed against Mucin -11.1 + 0.1 Dex-b-PLA O 47.9 O.S -10.7 O.6 200 mL of simulated tear fluid (STF) at 35°C. under stirring. Dex-b-PLA 10PBA 2.85 O.O3 27.5 - 0.9 -11.4 + 0.2 At predetermined time intervals, 1 mL of the release medium Dex-b-PLA 4OPBA 12.2 O2 26.7 0.1 -10.8 + 0.4 was extracted and the same volume of fresh new STF was Dex-b-PLA 160PBA 22.90.3 25.2 1.O -9.67 + O.76 added to the release medium. The extracted release medium Dex-b-PLA 32OPBA 34.6 O2 28.1 - 0.3 -8.32 O.28 was characterized using HPLC method (n=3, meant S.D). Mol% of PBA with respect to Dextran monomers; The release medium was replaced several times to maintain b)NP suspensions are mixed with mucin suspension in PBS the concentration of CycA in the medium in order to stay below its solubility limit in water. Replacing the medium was 0289. To assess the mucoadhesive properties of PBA also expected to prevent the adhesion of released CycA to the modified Dex-b-PLA, Zeta potential was measured for quan US 2015/0320694 A1 Nov. 12, 2015 29 titative analysis of interaction between mucin particles and to 4 (severe), the control eyes showed overall higher values Dex-b-PLA PBA NP suspension. 1 w/v '% mucin solution compared to the corresponding NP treated eyes (FIG. 23). was prepared in pH 7.4 PBS by stirring overnight and the Throughout the duration of the study, conjunctival Swelling, Solution was Subsequently Sonicated for 10 minutes (Branson corneal opacification, and infiltrate were not observed in any Digital Sonifier 450, USA). To 700 uL of mucin particle of the rabbits. solution were added 200 uL of 0.7 mg/ml Dex-b-PLA PBA 0293. Three female rabbits (New Zealand Albino) were NP suspension in PBS. A control study was also performed by used for this study. The rabbits were acclimated for one week adding 200 uL of PBS to the mucin particle solution. The Zeta prior to the experiment. The nanoparticles are prepared using potential of mucin particles with the NP suspension and the the nanoprecipitation method described in Example 4 but control study were determined using a Malvern ZetaSizer without the drug. The nanoparticles were filtered using 200 Nano ZS90 (Malvern Instruments, Worcestershire, U.K.). nm syringe filter, and further sterilized using UV irradiation inside a BioSafety Cabinet (BSC) for 1 hour. One eye was 9.2 In Vitro Mucoadhesion Test PAS Staining Method administered with NPs (28 Jul 19 ug of Dex-b-PLA PBA 0290 Mucoadhesion of the NPs was measured using the NPs) while the contra-lateral eye is used as control. Slit lamp in vitro PAS staining method as described above. Compared examination at 0, 1, 8, 24, 48, 72, 96, 120, 144, and 168 hr to the Dex-b-PLA and the PLGA-PEGNPs, the Dex-b-PLA after administration was used to evaluate 7 different catego PBANPs showed increased mucin adsorption (Table 2). Dex ries (Note that Ohr means before administration). These 7 b-PLA 10 PBA, Dex-b-PLA 40 PBA, and Dex-b-PLA categories (discomfort, conjunctival redness and Swelling, lid 160 PBA NPs showed a linear increase in mucin adsorption Swelling, discharge, corneal opacification, and infiltrate) from 0.575 to 0.605 mg/mg of NPs as the degree of PBA were graded from 0 (no sign) to 4 (severe). After 168 hrs, the surface functionalization increased. However, further rabbits were euthanized, and the ocular tissues were collected increase in PBA surface functionalization (i.e. Dex-b-PLA in formalin for further histopathology analysis. 320 PBA) decreased the amount of mucin adsorbed. Without wishing to be bound by theory, it is possible that excess functionalization of the NP surfaces with PBA causes the 10.2 Histopathology Analysis of Ocular Tissues Dextran to become more hydrophobic. This would increase 0294. After the duration of slit-lamp examination, the rab the potential for self-aggregation of the NPs, reducing the bits were euthanized and the ocular tissues (the entire ocular total available surface area for mucin adsorption. It is there globe, and upper and lower eyelids) were collected for histo fore ideal to tune the amount of PBA functionalization to pathology analysis (0). From examining the cornea, bulbar achieve optimal mucin-NP interaction without compromis and tarsal conjunctivas of all the eyes, normal ocular tissues ing the NP colloidal stability. It is also possible that smaller surfaces were observed in both the NP treated and the control NPs render higher mucin adsorption due to their larger total eyes (FIG. 24). All eyes showed anterior segment with pre surface area, as shown by comparing PLGA-PEG, Dex-b- served architecture and morphology. No sign of inflamma PLA, and Dex-b-PLA PBA NPs. However, as each type of tion, altered layer integrity, or presence of residual particles NP exhibit different surface properties, the trend is inconclu were detected in any of the eyes. Adequate number of goblet sive. The Dex-b-PLA PBA NPs all exhibited significantly cells with preserved morphology was also shown. Presence of higher mucin adsorption compared to the previous studies occasional intraepithelial and subepithelial eosinophils in tar involving chitosan based NPs and thiolated NPs, which sal conjunctiva were found in both NP treated and control showed about 0.25 and 0.13 mg/mg of NPs respectively at 1 eyes, suggesting that the phenomenon is not directly caused hr incubation (Lee, 2006). by the administration of the NP formulation. 0291. Mucoadhesion was calculated as the amount of mucin adsorbed per mg of NPs. NP suspension (1 ml) was 0295 The eyes were enucleated and collected immedi mixed with 1 ml of mucin solution (1 mg/ml in STF) and ately after euthanasia for histpathological evaluation. The incubated at 37°C. for 1 hr. The mixture was then centrifuged entire upper and lower eyelids were also dissected and col at 15,000 rpm for 1 hr and free mucin in the supernatant was lected for evaluation of the tarsal conjunctiva and underlying quantified using the periodic acid/Schiff (PAS) staining Soft tissues. Consecutive sections of the entire ocular globe method (Lee, 2006). Mucin adsorption was calculated by and eyelids were processed for microscopic analysis: after Subtracting the free mucin concentration from the initial initial fixation in 10% neutral buffered formalin, the tissue mucin concentration. Mucin standards (0.1, 0.25 and 0.5 was embedded in paraffin, serially sectioned in 5 um thick mg/ml) were determined using the same procedure to obtain sections, and stained with hematoxylin and eosin (H&E). The a calibration curve. histological slides were evaluated using bright field micros copy (Leica DM1000, ICC50 HD, Leica Microsystems Inc, Example 10 Concord, ON). In Vivo Studies 10.3 Chronic Response Study Using Dex-b-PLA PBA NPs 10.1 Acute Response Study. Using Dex-b-PLA PBA NPs 0296 To analyze the long-term biocompatibility of the NP 0292 To analyze the short-term biocompatibility of the formulation, the formulations were administered to rabbit NP formulation, the formulations were administered to rabbit eyes once a week for up to 12 weeks, while having contralat eyes, while having contralateral eyes as control, and daily eral eyes as control, and daily slit-lamp examination was slit-lamp examination for up to 7 days was performed to performed similar to above. Similar to acute response study, analyze the ocular Surface. Upon grading of the 7 categories no sign of conjunctival Swelling, corneal opacification, or (discomfort, conjunctival Swelling and redness, lid Swelling, infiltrate were observed in any of the rabbits at any point of the discharge, corneal opacification, and infiltrate) from 0 (none) time during the study. Overall, the difference in values US 2015/0320694 A1 Nov. 12, 2015 30 between the NP treated and control eyes were insignificant 2009). Add LSS into 2000 fold volume of CSS and bath across all of the 7 categories throughout the duration of the sonicate at 37°C. for 30 minutes. The mixture is allowed to study (12 weeks) (FIG. 25). settle overnight to form the lipid layer. Nanoparticles with 0297 Five female rabbits (New Zealand Albino) were Natamycin were prepared using the nanoprecipitation used for this study. The rabbits were acclimated for one week method described above. 1 ml of the NP-Natamycin formu prior to the experiment. The nanoparticles are prepared using lation was added onto 2 ml of CSS/LSS mixture. The mixture the nanoprecipitation method described in Example 4 but was then incubated at 37° C. for 10 minutes. The bottom 1.5 without the drug. The nanoparticles were filtered using 200 ml of the mixture was extracted without disturbing the top nm syringe filter, and further sterilized using UV irradiation layer, and dried overnight in vacuum desiccator. The precipi inside a BioSafety Cabinet (BSC) for 1 hour. One eye was tates were dissolved in DMSO again and UV-vis absorption administered with NPs (28 ul; 19 ug of Dex-b-PLA PBA was performed to calculate the concentration of Natamycin. NPs) once a week for 12 weeks while the contra-lateral eye is used as control. Slit lamp examination at 0, 1, 24, 48 hr after DISCUSSION administration each week was used to evaluate 7 different categories (Note that 0 hr means before administration). 0302) The present inventors synthesized a model linear These 7 categories (discomfort, conjunctival redness and block copolymer using PLA and Dextran (Dex-b-PLA), and Swelling, lid Swelling, discharge, corneal opacification, and demonstrated that NPs composed of Dex-b-PLA can self infiltrate) were graded from 0 (no sign) to 4 (severe). After 12 assemble into core-shell structured NPs of small particle size, weeks, the rabbits were euthanized, and the ocular tissues e.g. sizes less than 40 nm, without using any flow-focusing were collected for further histopathology analysis. devices. They further showed that the size of Dex-b-PLANPs 10.4 Chronic Response Study Using Dex-b-PLA PBA NPs can be precisely fine-tuned, e.g. between 15-70 nm by alter Encapsulated with Cyclosporine A ing the molecular weight of the component blocks (Verma, 0298 Similarly, the long-term biocompatibility of the NP 2012). Dextran, a natural polysaccharide composed of 1->6 formulation with encapsulation of Cyclosporine A was also linked C-D-glucopyranosyl units, was selected as a model examined using slit-lamp. The formulation containing both hydrophilic block because of its high hydrophilicity and bio the Dex-b-PLA PBA NPs and Cyclosporine A were admin compatibility. Dextran has an abundance of functional istered to rabbit eyes, while having contralateral eyes as con hydroxyl groups on its back bone. The higher density of trol. The slit-lamp examination for up to 4 weeks has shown Surface functional groups (as opposed to PEG, which has one no significant difference between the NP treated and the functional group per chain) can improve the efficiency of control eyes in any of the 7 categories (FIG. 26). surface functionalization, and thus, desirable surface proper 0299 Four female rabbits (New Zealand Albino) were ties are more easily achieved with Dextran based NPs. Dex used for this study. The rabbits were acclimated for one week tran coated NPs showed excellent colloidal stability in physi prior to the experiment. The nanoparticles are prepared using oloigcal media in vitro and long retention in the systemic the nanoprecipitation method described in Example 4 with circulation in vivo (Verma, 2012: Albert, 1990). Cyclosporine A. The nanoparticles were filtered using 200 0303. There is a another fundamental difference in the nm syringe filter, and further sterilized using UV irradiation structure of NPs composed of Dextran-PLA particles and inside a BioSafety Cabinet (BSC) for 1 hour. One eye was PEG-PLA particles, which is due to the greater hydrophilicity administered with NPs (28ul; 19 ug of Dex-b-PLA PBANPs of the Dextrans compared to that of PEG. The more hydro and 8 ug of Cyclosporine A) once a week for 12 weeks, while philic Dextran is less likely randomly associated in the hydro the contra-lateral eye is used as control. Slit lamp examina phobic core of the NPs compared to PEG, which could also tion at 0, 1, 24, 48 hr after administration each week was used explain the increased drug encapsulated in the Dextran-PLA to evaluate 7 different categories (Note that Ohr means before NPs compared to PLGA-PEG NPs (Verma, 2012). administration). 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Advanced Drug 0349 46. Peracchia, M. T.; Fattal, E.: Desmaele, D.; Delivery Reviews 57 2005, 1583-1594. Besnard, M.; Noel, J. P.: Gomis, J. M.; Appel, M.: 0366 63. Unkeless et al. Annu. Rev. Immunol. 6:251-281, d’Angelo, J.; Couvreur, P. Stealth R. PEGylated polycy 1998. anoacrylate nanoparticles for intravenous administration 0367 64. M. S. Verma, S. Liu, Y.Y. Chen, A. Meerasa, F. and splenic targeting.J. Control. Release 1999. 1, 121-128. X. Gu. Nano Research 2012, 5, 49. 0368 65. M. S. Verma, S. Liu, Y.Y. Chen, A. Meerasa, F. 0350 47. Phillips et al. Vaccine 10:151-158, 1992. X. Gu. Nano Research 2012. 0351) 48. Portet, D.; Denizot, B. Rump, E.: Hindre, F.: Le 0369 66. X. Yuan, H. Li, Y. Yuan. Carbohydr. Polym. Jeune, J. J.; Jallet, P. Comparative biodistribution of thin 2006, 65,337. coated iron oxide nanoparticles TCION: Effect of different 0370 67. Yang, Z. Leon, J.; Martin, M.: Harder, J. W.; bisphosphonate coatings. Drug Dev. Res. 2001, 4, 173 Zhang, R., Liang, D.; Lu, W.; Tian, M., Gelovani, J. G.; 181. Qiao, A., et al. Pharmacokinetics and biodistribution of 0352 49. Rehor, A.; Schmoekel, H.; Tirelli, N.; Hubbell, J. near-infrared fluorescence polymeric nanoparticles. Nano A. Functionalization of polysulfide nanoparticles and their technology 2009, 16, 165101-165101. performance as circulating carriers. Biomaterials 2008, 12, 0371 68. Zahr, A. S.; Davis, C. A.; Pishko, M. V. Mac 1958-1966. rophage uptake of core-shell nanoparticles Surface modi 0353 50. Riley, T: Govender, T.; Stolnik, S.:Xiong, C. D.; fied with poly(ethylene glycol). Langmuir 2006, 19, 8178 Garnett, M. C.; Illum, L.; Davis, S. S. Colloidal stability 81.85. and drug incorporation aspects of micellar-like PLA-PEG 0372 69. A. Zimmer, J. Kreuter, Adv. Drug Deliv. Rev. nanoparticles. Colloids Surf B Biointerfaces 1999, 1-4, 1995, 16, 61. 147-159. 0373 All references cited in this document are incorpo 0354) 51. Riley, T.; Stolnik, S.; Heald, C. R.: Xiong, C. D.; rated herein by reference in their entirety. Garnett, M. C.; Illum, L.; Davis, S. S.; Purkiss, S. C.; 0374. The above-described embodiments are intended to Barlow, R. J.; Gellert, P. R. Physicochemical evaluation of be examples only. Alterations, modifications and variations nanoparticles assembled from poly(lactic acid)-poly(eth can be effected to the particular embodiments by those of skill ylene glycol) (PLA-PEG) block copolymers as drug deliv in the art without departing from the scope of the disclosure, ery vehicles. Langmuir 2001, 11, 31 68-3174. which is defined solely by the claims appended hereto. 0355 52. Sacco, J. J.; Botten, J.; Macbeth, F.; Bagust, A.; What is claimed is: Clark, P. The Average Body Surface Area of Adult Cancer 1. A nanoparticle composition useful for delivery of a Patients in the UK: A Multicentre Retrospective Study. payload to a mucosal site, the nanoparticle comprising a PloS One 2010, 1, e8933-e8933. plurality of amphiphilic macromolecules, the macromol 0356 53. Safra, T: Muggia, F.; Jeffers, S.; Tsao-Wei, D. ecules comprising: D.; Groshen, S.; Lyass, O.; Henderson, R.; Berry, G.; Gabi a hydrophobic portion; zon, A. Pegylated liposomal doxorubicin (doxil): Reduced a hydrophilic portion comprising multiple functional moi clinical cardiotoxicity in patients reaching or exceeding eties; and cumulative doses of 500 mg/m(2). Ann. Oncol. 2000, 8, a mucosal targeting moiety, 1029-1033. wherein at least a portion of said functional moieties on the 0357 54. R. Shaikh, T. R. Raj Singh, M.J. Garland, A. D. hydrophilic portion are conjugated to the mucosal tar Woolfson, R. F. Donnelly. Journal of pharmacy & bioallied geting moiety. sciences 2011, 3,89. 2. The composition of claim 1, wherein the hydrophilic 0358 55. Shuai, X. T.; Ai, H.; Nasongkla, N.; Kim, S.; portion is a biocompatible polymer comprising 2, 3, or 4 Gao, J. M. Micellar carriers based on block copolymers of functional moieties per monomer unit. poly(e-caprolactone) and poly(ethylene glycol) for doxo 3. The composition of claim 1 or 2, wherein the hydrophilic rubicin delivery.J. Control. Release 2004, 3, 415-426. portion comprises a biocompatible polymer selected from a 0359 56. Sakloetsakun, et al. In situ gelling properties of polysaccharide, a polynucleotide, a polypeptide, or a combi chitosan-thioglycolic acid conjugate in the presence of nation thereof. oxidizing agents. Biomaterials 30, 2009, 6151–6157. 4. The composition of any one of claims 1 to 3, wherein the 0360 57. Schmitz, et al., Synthesis and characterization of hydrophilic portion comprises a polysaccharide. chitosan-N-acetyl cysteine conjugate. International Jour 5. The composition of claim 4, wherein the polysaccharide nal of Pharmaceutics 347 2008, 79-85. is selected from dextran, chitosan, alginate, hyaluronic acid, 0361 58. Shuai, X. T.; Ai. H.; Nasongkla, N.; Kim, S.; heparin, chondroitin Sulphate, pectin, pullulan, amylose, Gao, J. M. Micellar carriers based on block copolymers of cyclodextrin, carboxymethylcellulose or a polysaccharide poly(e-caprolactone) and poly(ethylene glycol) for doxo having thiol functional groups conjugated to the polymer rubicin delivery.J. Control. Release 2004, 3, 415-426. backbone. US 2015/0320694 A1 Nov. 12, 2015

6. The composition of claim 5, wherein the polysaccharide a hydrophilic portion comprising a biocompatible polymer is dextran, alginate, hyaluronic acid, chitosan, cyclodextrin, Selected from polysaccharide, polynucleotide, polypep or carboxymethylcellulose. tide, or a combination thereof, the hydrophilic portion 7. The composition of claim 6, wherein the polysaccharide comprising multiple functional moieties; and is dextran. a mucosal targeting moiety selected from a phenylboronic 8. The composition of any of the preceding claims, wherein acid (PBA) derivative, a thiol derivative or an acrylate the hydrophobic portion comprises a biocompatible polymer derivative, selected from a polyester, polyorthoester, polycarbonate, wherein at least a portion of said functional moieties of the polyimide, polybenzimidazole, polyurethane, polyurea, hydrophilic portion are conjugated to the mucosal tar polysulfide, polyether, polysulfone, phenolic and amino plas geting moiety. tic, chitin, lipopolysaccharide, cholesterol, proteoglycan, or a 25. A nanoparticle composition useful for delivery of a combination thereof. payload to a mucosal site, the nanoparticle comprising a 9. The composition of any of the preceding claims, wherein plurality of amphiphilic macromolecules, the macromol the hydrophobic portion comprises a biocompatible polymer ecules each comprising: selected from polylactide, a polyglycolide, poly(lactide-co a hydrophobic biocompatible polymer selected from a glycolide), poly(e-caprolactone), or a combination thereof. from polylactide, a polyglycolide, poly(lactide-co-gly 10. The composition of claim 9, wherein the hydrophobic collide), poly(e-caprolactone), or a combination thereof, portion comprises a polylactide polymer. the hydrophobic polymer forming the core of the nano 11. The composition of any of the preceding claims, particle; wherein the targeting moiety is capable of high affinity bind a hydrophilic biocompatible polymer selected from ing to a target present at the mucosal site. polysaccharide, polynucleotide, polypeptide, or a com 12. The composition of claim 11, wherein the high affinity bination thereof, having multiple functional moieties, binding is covalent binding. the hydrophilic portion forming the shell of the nano 13. The composition of any one of claims 11 or 12, wherein particle; at least a portion of the functional moieties the target is mucin or a receptor or a glycoprotein or a polysac being conjugated to a mucosal targeting moiety selected charide or residue expressed on the surface of an epithelial from a phenylboronic acid (PBA) derivative, a thiol cell. derivative or an acrylate derivative. 14. The composition of any one of claims 11 to 13, wherein 26. A nanoparticle composition useful for delivery of a the targeting moiety is a boronic acid derivative capable of payload to a mucosal site, the nanoparticle comprising a binding to carbohydrate residues. plurality of amphiphilic macromolecules, the macromol 15. The composition of claim 13, wherein the carbohydrate ecules comprising: residues are sialic acid residues on mucin. a hydrophobic portion comprising a polylactide; 16. The composition of claim 14 or 15, wherein the target a hydrophilic portion having multiple functional moieties, ing moiety is a phenylboronic acid (PBA) derivative. said hydrophilic portion comprising dextran; and 17. The composition of any one of claims 11 to 13, wherein a mucosal targeting moiety being a phenylboronic acid the targeting moiety is a thiol derivative oran acrylate deriva (PBA) derivative, tive capable of binding to thiol groups of cysteine moieties, wherein at least a portion of said functional moieties of the e.g. on the mucin. hydrophilic portion are conjugated to the mucosal tar 18. The composition of claim 17, wherein the targeting geting moiety. moiety is a thiol derivative, Such as cysteamine. 27. A nanoparticle composition useful for delivery of a 19. The composition of claim 17, wherein the targeting payload to a mucosal site, the nanoparticle comprising a moiety is an acrylate derivative, such as, methacrylate, ethyl plurality of amphiphilic macromolecules, the macromol acrylate, or diacrylate. ecules each comprising a hydrophobic polylactide polymer 20. The composition of any one of claims 11 to 13, wherein conjugated to a hydrophilic dextran polymer having multiple the targeting moiety is a phenylboronic acid (PBA) deriva functional moieties, at least a portion of said functional moi tive, a thol derivative or an acrylate derivative. eties being conjugated to a phenylboronic acid (PBA) deriva 21. The composition of claim 1, wherein the hydrophobic tive. portion comprises PLA; the hydrophilic portion comprises 28. The composition of claim 26 or 27, wherein the nano dextran; and the targeting moiety comprises PBA. particle is formed by conjugating the polylactide to the dex 22. The composition of claim 20, wherein the hydrophobic tran to form a nanoparticle and Subsequently surface-func portion is PLA; the hydrophilic portion is dextran, and the tionalizing the nanoparticle by conjugating at least a portion targeting moiety is PBA. of the functional moieties of the dextranto the PBA derivative 23. A Dextran-p-PLA block copolymer, wherein at least a to achieve a desired surface density of the PBA derivative. portion of the functional groups on the Dextran are conju 29. The composition of claim 26 or 27, wherein the nano gated to a targeting moiety capable of forming a high affinity particle is formed by conjugating the polylactide to the dex bond with a target at a mucosal site. tran to form a nanoparticle and Subsequently reacting the 24. A nanoparticle composition useful for delivery of a functional moieties of the dextran with PBA such that sub payload to a mucosal site, the nanoparticle comprising a stantially all of the PBA is located in the shell?on the surface plurality of amphiphilic macromolecules, the macromol of the nanoparticle. ecules comprising: 30. The composition of any preceding claim, wherein the a hydrophobic portion comprising a biocompatible poly molecular weight of the hydrophilic portion ranges from mer selected from a from polylactide, a polyglycolide, about 100 g/mol to about 1,000,000 g/mol, from about 500 poly(lactide-co-glycolide), poly(e-caprolactone), or a g/mol to 100,000 g/mol, or from about 1,000 g/mol to about combination thereof 50,000 g/mol. US 2015/0320694 A1 Nov. 12, 2015 34

31. The composition of any preceding claim, wherein the 44. The composition of claim 38, wherein the nanoparticle molecular weight of the hydrophilic portion ranges from is approximately 100 nm in size and the density of targeting about 0.1 kDa to about 1000kDa, from about 0.5kDato about moieties on the Surface of the nanoparticle ranges from about 100 kDa, or from about 1 kDa to about 50 kDa. 50 to about 350000, from about 10000 to about 350000, or 32. The composition of any preceding claim, wherein the from about 100000 to about 350000 per nanoparticle. molecular weight of the hydrophobic portion ranges from 100 45. The composition of any claim 38, wherein the nano g/mol to about 2,000,000 g/mol, from about 500 g/mol to particle is approximately 150 nm in size and the density of about 200,000 g/mol, or from about 1,000 g/mol to about targeting moieties on the Surface of the nanoparticle (i.e. 100,000 g/mol. surface density) ranges from about 50 to about 800000, from 33. The composition of any preceding claim, wherein the about 30000 to about 800000, or from about 300000 to about molecular weight of the hydrophobic portion ranges from 800000 per nanoparticle. about 0.1 kDa to about 2000 kDa, from about 0.5kDato about 46. The composition of any claim 38, wherein the nano 200 kDa, or from about 1 kDa to about 100 kDa. particle is approximately 200 nm in size and the density of 34. The composition of any preceding claim, wherein ratio targeting moieties on the Surface of the nanoparticle from of the molecular weight of the hydrophobic portion to the about 50 to about 1,500,000, from about 60000 to about hydrophilic portion is a about 0.1 to 100, about 0.5 to about 1,500,000, or from about 600000 to about 1,500,000 per 50, or about 1 to about 10. nanoparticle. 35. The composition of any preceding claim, wherein the 47. The composition of any claim 38, wherein the nano average particle size of the nanoparticles is less than about particle is approximately 250 nm in size and the density of 500 nm, less than about 300 nm, less than about 200 nm, less targeting moieties on the Surface of the nanoparticle ranges than about 150 nm, less than about 100 nm, less than about 50 from about 50 to about 2,500,000, from about 100,000 to nm, less than about 30 nm, less than about 10 nm, less than about 2,500,000, or from about 1,000,000 to about 2,500,000 about 3 nm, or less than about 1 nm. per nanoparticle. 36. The composition of any preceding claim, wherein the 48. The composition of any claim 38, wherein the nano average particle size of the nanoparticles is between about 0.1 particle is approximately 300 nm in size and the density of nm and about 1000 nm, about 1 nm and about 500 nm, about targeting moieties on the Surface of the nanoparticle (i.e. 1 nm and about 300 nm, about 1 nm and about 200 nm, about surface density) ranges from about 50 to about 3,500,000, 1 nm and about 150 nm, about 1 nm and about 100 nm, about from about 150,000 to about 3,500,000, or from about 1,500, 1 nm and about 50 nm, about 10 nm and about 150 nm, about 000 to about 3,500,000 per nanoparticle. 10 nm and about 100 nm, about 10 nm and about 75 nm, about 49. The composition of any preceding claim, wherein the 10 nm and about 60 nm, and about 10 nm and about 50 nm, or nanoparticle is formed by a process whereby substantially all about 20 and about 40 nm. of the targeting moiety is located on the Surface of the nano 37. The composition of any preceding claim, wherein sub particle. stantially all of the mucosal targeting moieties are on the 50. The composition of any preceding claim, further com Surface of the nanoparticle. prising a payload. 38. The composition of any preceding claim, wherein the 51. The composition of claim 50, wherein the payload is a nanoparticle has a Surface density of the mucosal targeting therapeutic agent, a diagnostic agent, a prophylactic agent or moiety, the Surface density being tunable for adjustable tar an imaging agent. geting of the nanoparticle to the mucosal site. 52. The composition of claim 50, wherein the payload is a 39. The composition of claim 38, wherein the surface den hydrophobic therapeutic agent, a diagnostic agent, a prophy sity of the targeting moieties on the Surface of the nanopar lactic agent or an imaging agent. ticle ranges from about 1 per nm2 to 15 per nm2, about 1 per 53. The composition of claim 50, wherein the payload is a nm2 to 10 pernm2, about 1 pernm2 to 5 pernm2, about 1 per therapeutic agent. nm2 to about 15 per nm2, about 3 per nm2 to about 12 per 54. The composition of claim 50, wherein the payload is nm2, or from about 5 per nm2 to about 10 per nm2. selected from antimicrobial agents, analgesics, antiinflam 40. The composition of claim 38, wherein the surface den matory agents, IOP lowering agents, counterirritants, coagul sity of the targeting moieties on the Surface of the nanopar lation modifying agents, diuretics, sympathomimetics, anor ticle is about 1, 2, 3, 4, 5, 6, 7, 8, 8, 9, 11, 12, 13, 14 or 15 per exics, antacids and other gastrointestinal agents; nm2. antiparasitics, antidepressants, antihypertensives, anticholin 41. The composition of claim 38, wherein the nanoparticle ergics, stimulants, antihormones, central and respiratory is approximately 10 nm in size and the density of targeting Stimulants, drug antagonists, lipid-regulating agents, urico moieties on the Surface of the nanoparticle ranges from about Surics, cardiac glycosides, electrolytes, ergot and derivatives 50 to about 3,500, from about 500 to about 3500, or from thereof, expectorants, hypnotics and sedatives, antidiabetic about 1000 to about 3500 per nanoparticle. agents, dopaminergic agents, antiemetics, muscle relaxants, 42. The composition of claim 38, where the nanoparticle is para-sympathomimetics, anticonvulsants, antihistamines, approximately 30 nm in size and the density of targeting beta-blockers, purgatives, antiarrhythmics, contrast materi moieties on the Surface of the nanoparticle ranges from about als, radiopharmaceuticals, antiallergic agents, tranquilizers, 50 to about 30000, from about 1000 to about 30000, or from vasodilators, antiviral agents, and antineoplastic or cytostatic about 10000 to about 30000 per nanoparticle. agents or other agents with anticancer properties, or a com 43. The composition of claim 38, wherein the nanoparticle bination thereof. Other suitable therapeutic agents may be is approximately 50 nm in size and the density of targeting selected from contraceptives and vitamins as well as micro moieties on the Surface of the nanoparticle ranges from about and macronutrients. Still other examples include antiinfec 50 to about 90000, from about 3000 to about 90000, or from tives such as antibiotics and antiviral agents; analgesics and about 30000 to about 90000 per nanoparticle. analgesic combinations; anorexics; antihelmintics; antiar US 2015/0320694 A1 Nov. 12, 2015 thritics; antiasthmatic agents; anticonvulsants; antidepres protective agents, nonsteroidal anti-inflammatory agents, sants; antidiuretic agents; antidiarrleals; antihistamines; anti nepafanec, norfloxacin, ofloxacin, olopatadine, oxymetazo inflammatory agents: antimigraine preparations; line, pemirolast, pheniramine, phenylephrine, pilocarpine, antinauseants; antineoplastics; antiparkinsonism drugs; anti poVidone, prednisolone, proparacaine, Scopolamine, tetra pruritics; antipsychotics; antipyretics, antispasmodics; anti caine, Steroids, Sulfacetamide, tetrahydrozoline, hypertonic cholinergics; sympathomimetics: Xanthine derivatives; car tears, timolal, tobramycin, travaprost, trifluridine, trime diovascular preparations including calcium channel blockers thiprim, tropicamide, unoprostone, Xalatan, and Zinc, as well and beta-blockers such as pindolol and antiarrhythmics; anti as pharmaceutically acceptable salts, esters and prodrugs hypertensives: diuretics; vasodilators including general coro thereof. nary, peripheral and cerebral; central nervous system stimu 58. The composition of claim 56, wherein the ophthalmic lants; cough and cold preparations, including decongestants; agent is timolol, betaxolol, metipranolol, dorzolamide, hormones Such as estradiol and other steroids, including cor cyclosporine, brinzolamide, neptazane, acetazolamide, ticosteroids; hypnotics; immunosuppressives; muscle relax alphagan, Xalatan, bimatoprost, travaprost, olopatadine, keto ants; parasympatholytics; psychoStimulants; sedatives; and tifen, acyclovir, gancyclovir, or Valcyclovir. tranquilizers; and naturally derived or genetically engineered 59. The composition of any preceding claim, wherein the proteins, polysaccharides, glycoproteins, or lipoproteins. payload is predominantly encapsulated within the core of the 55. The composition of claim 50, wherein the payload is nanoparticle. selected from timolol, betaxolol, metipranolol, dorzolamide, 60. The composition of any preceding claim, wherein the brinzolamide, neptazane, acetazolamide, alphagan, Xalatan, loading capacity of the nanoparticle is in the range of about 1 bimatoprost, travaprost, olopatadine, ketotifen, acyclovir, to about 30% wit/wt., about 1 to about 20%, 1 to about 10%, gancyclovir, Valcyclovir, doxorubicin, mitomycin, cisplatin, about 1% to about 8%, about 1% to about 6%, about 1% to daunorubicin, bleomycin, actinomycin D. neocarzinostatin, about 5%, about 1% to about 3%, or about 1% to about 2% carboplatin, stratoplatin, Ara-C, Capoten, Monopril, Prava 61. The composition of any preceding claim, wherein the chol, Avapro, Plavix, Cefail. Duricef/Ultracef, AZactam, loading capacity of the nanoparticle is up to about 40%, up to Videx, Zerit, Maxipime, VePesid, Paraplatin, Platinol, Taxol. about 30% wit/wt., up to about 20%, up to about 10%, up to UFT, Buspar, Serzone, Stadol NS, Estrace, Glucophage about 8%, up to about 6%, up tp about 5%, up to about 3%, up (Bristol-Myers Squibb); Ceclor, Lorabid, Dynabac, Prozac, to about 2%, or up to about 1%. Darvon, Permax, Zyprexa, Humalog, AXid, Gemzar, Evista; 62. The composition of any preceding claim, wherein the Vasotec/Vaseretic, Mevacor, Zocor, Prinivil/Prinizide, nanoparticles are dispersed in aqueous medium. Plendil, Cozaar/HyZaar, Pepcid, Prilosec, Primaxin, Noroxin, 63. The composition of any preceding claim, wherein the Recombivax HB, Varivax, Timoptic/XE. Trusopt, Proscar, payload is capable of being released from the nanoparticle for Fosamax. Sinemet, Crixivan, Propecia, Vioxx. Singulair, a sustained period of at least 24, 36, 48, 60, 72, 84, of 96 Maxalt, Ivermectin; Diflucan, Unasyn, Sulperazon, Zithro hours. max, Trovan, Procardia XL, Cardura, Norvasc, Dofetilide, 64. The composition of any one of claims 1 to 62, wherein Feldene, Zoloft, Zeldox, Glucotrol XL, Zyrtec, Eletriptan, the payload is capable of being released from the nanoparticle Viagra, Droloxifene, Aricept, Lipitor (Pfizer); Vantin, for a sustained period of at least 1, 2, 3, 4, 5, 6, 7 or 8 days. Rescriptor, Vistide, Genotropin, Micronase/Glyn./Glyb. 65. The composition of any one of claims 1 to 62, wherein Fragmin, Total Medrol, Xanax/alprazolam, Sermion, Hal the payload is released is capable of being released from the cion/triazolam, Freedox, Dostinex, Edronax, Mirapex, Phar nanoparticle for a Sustained period of at least 1 week. morubicin, Adriamycin, Camptosar, Remisar, Depo-Provera, 66. A pharmaceutical composition comprising a nanopar Caverject, Detrusitol, Estring, Healon, Xalatan, Rogaine; ticle composition as defined in any of the preceding claims, Lopid. Accrupil, Dilantin, Cognex, Neurontin, Loestrin, and a pharmaceutically acceptable carrier. Dilzem, Fempatch, Estrostep, Rezulin, Lipitor, Omnicef. 67. A mucoadhesive delivery system for delivering a pay FemHRT, Suramin, or Clinafloxacin. load to a mucosal Surface, the delivery system comprising a 56. The composition of claim 50, wherein the payload is an nanoparticle composition as defined in any of the preceding ophthalmic agent. claims; a pharmaceutically acceptable carrier, and a payload. 57. The composition of claim 56, wherein the ophthalmic 68. A method of treating or preventing a disease or condi agent is selected from selected from lubricants, demulcents, tion comprising administering to a subject an effective antibiotics, antivirals (e.g. acyclovir, gancyclovir, Valcyclo amount of a nanoparticle composition or pharmaceutical vir), antiallergic agents, acetazolamide, alphagan, antazoline, composition as defined in any of the preceding claims. aspirin, atropine, azelastine, bacitracin, betaxolol, bimato 69. The method of claim 68, wherein the disease or condi prost, botanical drugs including zeaxanthine lutein, lycopene tion is a disease or condition affecting the eye. brimonodine, brinzolamide, carbachol, carteolol, ciprofloxa 70. The method of claim 69, wherein the disease or condi cin, ofloxacin, cromalyn, cyclosporine, cyclosporine pro tion affecting the eye is selected from any one or more of drugs and cyclosporine derivatives, other immunomodula abrasion, acanthamoeba keratitis, actinic keratosis, acute tors, dapiprazole, dexamethasone, diclofenac, diplivifren, allergic blepharoconjunctivitis, allergic conjunctivitis, aden dorzolamide, epinephrine, erythromycin, fluoromethalone, Oviral keratoconjunctivitis, aniridia, atopic keratoconjunc flurbiprofen, gentamycin, glaucoma medications (e.g. pros tivitis, bacterial conjunctivitis, bacterial keratitis, band kerat taglandins, carbonic anhydrase inhibitors, epinephrine or opathy, basal cell carcinoma, Bell's palsy, blepharitis, bullous alpha-agonists, beta-blockers), gramicidin, homatropine, keratopathy, canaliculitis, caruncular cyst, cataract, cha hydrocortisone, hyoscine, keterolac, ibuprofen, ketotifen, lazion, chlamydial conjunctivitis, climatic droplet keratopa latanaprost, levobunolol, levocabastine, levofloxin, lotep thy, concretions, conjunctival intraepithelial neoplasia, con prednol, medrysone, methazolamide, metipranolol, naphazo junctival lymphoma, conjunctival papilloma, conjunctival line, natamycin, nedocromil, neomycin, neptazane, neuro pigmented lesions, conjunctival Scarring, conjunctivitis, con US 2015/0320694 A1 Nov. 12, 2015 36 junctivochalasia and chemosis, corneal collagen cross-link bullous penphigoid, candidiasis, canker Sores, carbon baby ing, corneal edema, corneal graft-lamellar keratoplasty, cor syndrome, cariomegaly, catarrh, catarrhal or mucopurulent neal graft rejection, corneal infiltrates, crocodile shagreen, conjunctivitis, central papillary atrophy, cervical polyps, crystalline keratopathy, cysts of the eyelids, dacryocystitis, cheilitis, cheilitis exfoliativa, cheilitis glandularis, cheilitis dellen, dendritic ulcer, dermatochalasis and blepharochala granulomatosa, cholecystitis, cicatrizing conjunctivitis, cili sis, Descemet's membrane breaks, disciform keratitis, disci ary discoordination due to random ciliary orientation, ciliary form keratitis, keratoconjunctivitis sicca, ectopia lentis, dyskinesia, colitis, colorectal adenomatous polyposis, col ectropion, endophthalmitis, entropion, epiblepharon and epi orectal polyps, conjunctivitis ligneous, conjunctivitis with canthic folds, epibulbar choristomas, epiphora, episcleritis, epithelial and fibrous ingrowth, epithelial basement mem pseudomembrane, coronavirus-related cold, costello syn brane dystrophy, exposure keratopathy, eyelid trauma, fila drome, coxsackievirus-related cold, Crohn's disease, mentary keratopathy, filtering bleb, flashburns, floppy eyelid cronkhite-Canada syndrome, cystic Fibrosis, cystitis, derma syndrome, follicular conjunctivitis, Fuchs' endothelial dys tostomatitis, desquamative gingivitis, dextrocardia-bron trophy, Fuchs heterochromic iridocyclitis, fungal keratitis, chiectasis-sinusitis, drug-induced ulcer of the lip, duodenal giant papillary conjunctivitis, glaucoma-acute angle closure, ulcer, dyskeratosis congenital, dyskeratosis congenita of Zin gonococcal keratoconjunctivitis, granular dystrophy, heman sser-Cole-Engman, echovirus-related cold, Ectodermal dys gioma, herpes simplex keratitis, herpes simplex primary ble plasia, enterocolitis, eosinophilic cystitis, epidemic kaposi's pharokeratoconjunctivitis, herpes Zoster ophthalmicus, sarcoma, epulis, epulis fissuratum, eruptive hemangioma, hordeolum-internal and external, hyphema-blunt trauma, eruptive lingual papillitis, erythroplakia, esophageal ulcer, hypopyon, infectious crystalline keratopathy, interstitial esophagitis, extrinsic asthma, familial adenomatous polypo keratitis, iridocorneal dysgenesis, iridocorneal endotheliopa sis, familial intestinal polyposis, familial nasal acilia, familial thy, iris cysts, iritis, iron lines, keratoconus, keratoconus polyposis, Fenwick ulcer, fissured tongue, flu, folicular con forme frusta, keratoglobus, lattice stromal dystrophy, leuko junctivitis, follicular hamartoma, food allergy related asthma, coria, lice, limbal stem cell deficiency, lipid keratopathy, Fordyce's disease, Gardner syndrome, gastresophageal macular stromal dystrophy, marginal keratitis, meesmann's reflux-related chronic cough, gastric erosion, gastric reflux, dystrophy, melanoma-conjunctival and eyelid, melanoma gastric ulcer, gastritis, gastritis, gastroesophageal reflux dis and nevus of the iris, membranous and pseudomembranous ease, giant papillary conjunctivitis, gonorrhea, growth-hor conjunctivitis, molluscum contagiosum, mooren’s ulcer, mone secreting pituitary adenoma, hairy leukoplakia, hemo nasolacrimal duct obstruction-congenital, neurotrophic philus influenzae B, hemorrhagic conjunctivitis, hemorrhagic keratopathy, nevus-eyelid, ocular cicatricial pemphigold, proctocolitis, herpes, human papillomavirus, immotile cilia ophthalmia neonatorum, pannus and pseudopterygia, pellu syndrome, inclusion conjunctivitis, influenza A, influenza B, cid marginal degeneration, perforation-corneal, peripheral interstitial cystitis, intraoral dental sinus, intrinsic asthma, ulcerative keratitis, persistent epithelial defect, phlyctenulo invasive candidiasis, irritative conjunctivitis, Jadassohn-Le sis, pingueculum, posterior capsular opacification, posterior wandowsky Syndrome, kaposiform hemangio-endothelioma, polymorphous dystrophy, preseptal cellulitis, pseudoexfolia keratoconjunctivitis, keratosis pharynges, laryngopharyn tion of the lens capsule, pterygium, ptosis and pseudoptosis, geal reflux, leprosy, leukoencephalopathy, leukoplakia, leu punctual Stenosis, pyogenic granuloma, recurrent corneal koplakia with tylosis and esophageal carcinoma, lipogranu erosion syndrome, Reis-Buckler's dystrophy, retention cyst lomatosis, logic syndrome, lower esophageal ulcer, and lymphangiectasia, rheumatoid arthritis, rosaceakeratitis, lymphocytic colitis, lymphoma, mucosa-associated lym Salzmann nodular degeneration, Scleritis, sebaceous cell car phoid tissue, major ulcerative stomatitis, malignant peptic cinoma, Seborrheic keratosis, squamous cell carcinoma-lid, ulcer, Melkersson-Rosenthal syndrome, membranous con Stevens-Johnson syndrome, Sub-conjunctival hemorrhage, junctivitis, mouth ulcers, mucinous carcinoma, mucocele, Superficial punctate keratopathy, Superior limbic keratocon mucoepidermoid, mucoepidermoid carcinoma, mucoepithe junctivitis, Synechia, Terrien’s marginal degeneration, lial dysplasia, Witkop type, mucosal leishmaniasis, mucosal Thygeson's Superficial punctate keratopathy, toxic keratopa lichen planus, mucosal squamous cell carcinoma, mucositis, thy, trachoma, trichiasis, pseudotrichiasis, distachiasis, meta mucous cyst of oral mucosa, Nagayama's spots, nasal polyp. plastic lashes, trichotillomania, uveitis, Vernal keratocon necrotizing entercolitis, necrotizing periodontal diseases, junctivitis, vitamin A deficiency, Vortex keratopathy, nicotine stomatitis, ophthalmia neonatorum, oral Crohn's disease, oral florid papillomatosis, oral fordyce granules, oral Xanthelasma. thrush, oral ulcer, orthomyxovirus-related cold, Osler 71. The method of claim 69, wherein the disease or condi Rendu-Weber syndrome, pancolitis, papillary conjunctivitis, tion affecting the eye is KCS, glaucoma or allergic conjunc parainfluenza, paramyxovirus-related cold, paucigranulo tivitis. cytic asthma, pemphigus, pemphigus foliaceus, pemphigus 72. The method of claim 68, wherein the disease or condi Volgaris, Penign peptic ulcer, penphigus Vulgaris, peptic tion is selected from any one or more of acquired angioedema, ulcer, periadenitis mucosa necrotica, periodic fever, pharyn acrodermatitis enteropathica, acute serous conjunctivitis, goconjunctival fever, Pinguecula, plasma cell cheilitis, plas adenomatous polyposis of the colon, adenoviridae infections, moacanthoma/plasma cell gingivitis, primary ciliary dyski adenovirus-related cold, allergic asthma, allergic contact nesia, proctitis pseudomembranous colitis, pseudomycoma cheilitis, allergic rhinitis, allergies, amyloidosis of gingiva peritonei, psoriasis on mucous membranes, psychiatric dis and conjunctiva mental retardation, analgesic asthma Syn orders associated celiac disease, pterygium, pterygium of the drome, Anderson's triad, angina bullosa haemorrhagica, conjunctiva, purulent conjunctivitis, recurring scarring aph angular conjunctivitis, asthma, asthmatic Bronchitis, atro thae, reflux laryngitis, refractory celiac disease, Rhinitis, rhi phic glossitis, atrophic rhinitis, attenuated familial polyposis, nosporidiosis, ritter syndrome, rostan asthma, Salicylate-sen Behcet’s disease, benign migratory glossitis, benign mucosal sitive asthma, Schafer syndrome, sinusitis, Sjogren penphigoid, black hairy tongue, Brodie pile, bronchitis, syndrome, spring catarrh, sprue, Stevens-Johnson syndrome, US 2015/0320694 A1 Nov. 12, 2015 37 stomal ulcer, Stomatitis, Superior limbic keratoconjunctivitis, a) preparing an amphiphilic macromolecule comprising a Sutton disease, Swime flu, systemic candidiasis, Takahara's hydrophilic portion and a hydrophobic portion, the disease, the clap, thrush, trumpeters wart, tuberculous dis hydrophilic portion comprising multiple functional ease of the mucous, ulcerative colitis, ulcerative conjunctivi moieties; tis, ulcerative proctosigmoiditis, urban Schosser Spohn Syn b) assembling a plurality of said macromolecules under frome, vaginal candidiasis, vasomotor rhinitis, Vestibular Suitable conditions to form a nanoparticle having a papillomatosis, Vincents angina, Vulvovaginal gingival Syn hydrophobic core and a hydrophilic shell; and drome, white sponge nevus, Xanthogranulomatous cholecys titis, Xerostomia. c) conjugating at least a portion of said functional moieties 73. A use of the nanoparticle composition or pharmaceu on the hydrophobic portion to a mucosal targeting moi tical composition according to any of the preceding claims for ety, treating a disease capable of being treating by administering to thereby provide a surface-functionalized nanoparticle. a therapeutic agent to a mucosal site. 79. The method of claim 78 wherein step a) comprises 74. A nanoparticle composition or pharmaceutical compo conjugation of a hydrophilic polymer to a hydrophobic poly sition as described herein for use in treating a disease capable mer to form a diblock copolymer. of being treating by administering a therapeutic agent to a mucosal site. 80. The method of claim 78 or 79 wherein the hydrophilic 75. A use of the nanoparticle composition according to any polymer is dextran and the hydrophobic polymer is PLA. of the preceding claims in the manufacture of a medicament 81. The method of any one of claims 78 to 80 wherein the for treating a disease capable of being treating by administer targeting moiety is a phenylboronic acid derivative, a thiol ing a therapeutic agent to a mucosal site. derivative or an acrylate derivative. 76. A commercial package comprising the nanoparticle 82. The method of claim 81 wherein the targeting moiety is composition orpharmaceutical composition according to any a phenylboronic acid derivative, of the preceding claims, together with instructions for use in 83. The method of any one of claims 78 to 82 wherein step treating a disease. b) is performed before step c). 77. The use, composition or commercial package of any one of claims 73 to 73, wherein the disease is as defined in 84. The method of any one of claims 78 to 83 wherein the claim 70 or 72. Surface density of the mucosal targeting moiety on the nano 78. A method of preparing a nanoparticle composition particle is controlled by the amount of mucosal targeting useful for delivery of a payload to a mucosal site, the method moiety introduced into the reaction. comprising: k k k k k