(12) Patent Application Publication (10) Pub. No.: US 2014/0005379 A1 GU (43) Pub

Home , Zolamine

US 2014.0005379A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0005379 A1 GU (43) Pub. Date: Jan. 2, 2014

(54) NANOPARTICLE DELIVERY SYSTEMAND (52) U.S. Cl. COMPONENTS THEREOF CPC ...... A61K 47/26 (2013.01); A61 K3I/704 (2013.01) (71) Applicant: Frank GU, Waterloo (CA) USPC ...... 536/112 (72) Inventor: Frank GU, Waterloo (CA) (57) ABSTRACT (21) Appl. No.: 13/923,274 (22) Filed: Jun. 20, 2013 The present disclosure relates generally to a nanoparticle delivery system. The nanoparticles are formed from Related U.S. Application Data amphiphilic block copolymers comprising polylactide and dextran. The dextran may be conjugated to a targeting moiety (60) Provisional application No. 61/690,127, filed on Jun. in Such a manner that the Surface of the nanoparticle is coated 20, 2012. with the targeting moiety. The size and targeting of the nano Publication Classification particles can be tuned by controlling Surface density of the targeting moiety. The present disclosure also relates to poly (51) Int. Cl. mers and macromolecules useful in the preparation of the A6 IK 47/26 (2006.01) mucoadhesive nanoparticles, as well as compositions, meth A6 IK3I/704 (2006.01) ods, commercial packages, kits and uses related thereto. Patent Application Publication Jan. 2, 2014 Sheet 1 of 19 US 2014/0005379 A1

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NANOPARTICLE DELIVERY SYSTEMAND 0007 Targeting controlled release polymer systems (e.g., COMPONENTS THEREOF targeted to a particular tissue or cell type or targeted to a specific diseased tissue but not normal tissue) is desirable. It CROSS REFERENCE TO RELATED can enhance the drug effect at the target site and reduce the APPLICATIONS amount of a drug present in tissues of the body that are not 0001. This application claims the benefit of priority of targeted. Therefore, with effective drug targeting, it may be U.S. Provisional Patent Application No. 61/690,127, filed possible to reduce the amount of drug administered to treat a Jun. 20, 2012, which is incorporated herein by reference in its particular disease or condition and undesirable side effects entirety. may also be reduced. 0008 Various benefits can be obtained through delivery of TECHNICAL FIELD therapeutic agents through a mucosal tissue. For example, mucosal delivery is generally non-invasive, thereby avoiding 0002 The present disclosure relates generally to a tunable uncomfortable aspects of intravenous, intramuscular, or Sub nanoparticle delivery system. The present disclosure also cutaneous delivery means. Application of a therapeutic agent relates to components useful in the preparation of the nano to a mucosal tissue can also reduce the effect of first-pass particles, as well as to compositions, methods, processes, metabolism and clearance by circulating immune cells. How 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 types of polymeric materials have been studied for NP drug 0010 Recently, formulations using NPs as drug carriers delivery applications. have been proposed to overcome the limitation associated 0005 PLGA-PEG is the most widely used polymer for with topical administration methods. NP carriers have been making biodegradable drug delivery systems. The self-as shown to improve drug stability in water and also prolong sembly of PLGA-PEG block copolymers generally yields drug activity by releasing encapsulated compounds in a con NPs of sizes greater than 150 nm (Karnik, 2008). Although trolled manner (Ludwig, 2005; Nagarwal 2009; Liu, 2012). Smaller particles can be synthesized, they generally Suffer NPs formulated using biodegradable polymers, such as poly from low drug encapsulation and rapid drug release (Karnik, (lactic-co-glycolic acid) (PLGA), have been tested for ocular 2008). The present inventors reported that typical maximum topical drug delivery applications (Diebold, 1990; Zimmer, drug loading in PLGA-PEG was found to be 7.1 wit/wt % 1995). Poly(ethylene glycol)-based NPs have attracted sig (Verma, 2012, incorporated herein by reference in its nificant attention due to their ability to improve the stability of entirely). Other PEG based polymers showed drug loading drug carrier systems in physiological environments (Bazile, ranging from 4.3 to 11.2 wit/wt % (Shuai, 2004; He, 2010; 1995; Dhar, 2008; Dong, 2007; Esmaeili, 2008). Missirlis, 2006). 0011. The synthesis of surface-functionalized NP drug 0006 Nanoparticles have been developed as sustained delivery systems has been explored. In order to achieve release vehicles used in the administration of small molecule mucoadhesion, the synthesis typically requires two-stage drugs as well as protein and peptide drugs and nucleic acids. synthesis whereby the first stage involves the formation of The drugs are typically encapsulated in a polymer matrix NPs, while the second stage involves the conjugation of which is biodegradable and biocompatible. As the polymer is ligands on the surface of these NPs. Recently, a new technol degraded and/or as the drug diffuses out of the polymer, the ogy demonstrated the formation of targeting NPS using one drug is released into the body. Typically, polymers used in step synthesis whereby the formation of the NP and the sur preparing these particles are polyesters such as poly(lactide face functionalization can be accomplished in one step (U.S. co-glycolide) (PLGA), polyglycolic acid, poly-beta-hy Pat. No. 8,323,698, incorporated herein by reference). This droxybutyrate, polyacrylic acid ester, etc. These particles can technology is particularly useful for applications where mini also protect the drug from degradation by the body. Further mal targeting ligand is required, e.g. for systemic bolus injec more, these particles can be administered using a wide variety tions where the number of targeting ligands on the Surface of administration routes. Various types of materials used for must be controlled to minimize systemic immunogenicity. synthesizing nanoparticle drug carriers have been disclosed, When nanoparticles are formed using the one-step method, for example, in US. Pat. No. 2011/0300219. Amphiphilic targeting ligands may be detected within the core of the compound assisted nanoparticles for targeted delivery have nanoparticles. Thus, this methodology may not be ideal been disclosed, for example, in US. Pat. No. 2010/0203142. where maximum targeting is desired. US 2014/0005379 A1 Jan. 2, 2014

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 nano 2005, entitled “Mucoadhesive and bioadhesive polymers'; particle delivery system and components thereof. EP2167044 A1, filed Dec. 11, 2008, entitled “Mucoadhesive 0017. In a first aspect, there is provided a block copolymer vesicles for drug delivery': WO 2005/117844, filed Sep. 17, useful in the formation of a nanoparticle delivery system for 2009, entitled “Mucoadhesive nanocomposite delivery sys encapsulating a hydrophobic therapeutic agent, the block tem: WO 2010/096558, filed Feb. 18, 2010, entitled “Bi copolymer having a hydrophobic portion consisting of poly functional co-polymer use for ophthalmic and other topical lactide and a hydrophilic portion consisting of dextran, the and local applications'; US Pat. 2013/0034602, filed Jul.30, dextran having a plurality of functional groups capable of 2012, entitled “Enteric-coated capsule containing cationic conjugation to a targeting moiety. nanoparticles for oral insulin delivery”; EP Pat. 25.10930A1, 0018. In another aspect, there is nanoparticle useful for filed Apr. 15, 2011, entitled “Nanoparticles comprising half encapsulating a hydrophobic therapeutic agent, the nanopar esters of poly (methyl vinyl ether-co-maleic anhydride) and ticle comprising a plurality of amphiphilic block copolymers, uses thereof; U.S. Pat. No. 8,242,165 B2, filed Oct. 26, 2007, each copolymer having a hydrophobic portion consisting of entitled “Mucoadhesive nanoparticles for cancer treatment': polylactide and a hydrophilic portion consisting of dextran, EP Pat. 0516141 B1 filed May 29, 1992, entitled “Pharma the dextran having a plurality of functional groups capable of ceutical controlled-release composition with bioadhesive conjugation to a targeting moiety. properties”: WO 1998/030207 A1, filed Jan. 14, 1998, 0019. In a further aspect, the present disclosure provides a entitled “Chitosan-gelatin a microparticles'; EP Pat. nanoparticle composition useful for delivery of a payload to a 1652517 B1, filed Jun. 17, 2004, entitled “Hyaluronic acid mucosal site, the nanoparticle comprising a plurality of nanoparticles”; U.S. Pat. No. 8,361,439 B1, filed Aug. 20, amphiphilic macromolecules, the macromolecules compris 2012, entitled “Pharmaceutical composition of nanopar ing: a hydrophobic portion; a hydrophilic portion comprising ticles'. However, these documents only describe mucoadhe comprising multiple functional moieties; and a mucosal tar sive materials that undergo physical interaction with the geting moiety, wherein at least a portion of said functional mucous lining (e.g. electrostatic interaction between cationic moieties on the hydrophilic portion are conjugated to the chitosan materials with the negatively charged mucin layer). mucosal targeting moiety. The main disadvantage of physical interaction is that it is 0020. In further aspect, the present disclosure provides a a unspecific and much weaker compared to covalent interac nanoparticle composition useful for delivery of a payload to a tions. mucosal site, the nanoparticle comprising a plurality of 0014. A few studies have reported molecular targeting amphiphilic macromolecules, the macromolecules compris groups with potential to covalently bind to mucosal tissue. ing: a hydrophobic portion comprising a biocompatible poly Phenylboronic acid (PBA), which contains a phenyl substitu mer selected from a from polylactide, a polyglycolide, poly ent and two hydroxyl groups attached to boron, has been (lactide-co-glycolide), poly(e-caprolactone), O a reported to form a complex with the diol groups of sialic acid combination thereof a hydrophilic portion comprising a bio at physiological pH (Matsumoto, 2010; Matsumoto, 2010; compatible polymer selected from polysaccharide, poly Matsumoto, 2009). Another class of molecules that can nucleotide, polypeptide, or a combination thereof, the hydro covalently bind to the mucous membrane is polymeric thi philic portion comprising multiple functional moieties; and a omers (Ludwig, 2005). These thiomers are capable of form mucosal targeting moiety selected from a phenylboronic acid ing covalent disulfide linkage with cysteine-rich Subdomains (PBA) derivative, a thiol derivative or an acrylate derivative, of the mucous membrane (Khutoryanskiy, 2010). Typical wherein at least a portion of said functional moieties of the examples of polymeric thiomers include the following con hydrophilic portion are conjugated to the mucosal targeting jugates: poly(acrylic acid)/cysteine (Gugg, 2004), chitosan/ moiety. N-acetylcysteine (Schmitz, 2008), alginate/cysteine 0021. In a further embodiment, there is provided a nano (Bernkop-Schnurch, 2008) chitosan/thio-glycolic acid (Sak particle composition useful for delivery of a payload to a loetsakun, 2009) and chitosan/thioethylamidine (Kafedjiiski, mucosal site, the nanoparticle comprising a plurality of 2006). A recent study also suggested that polymers with amphiphilic macromolecules, the macromolecules each com acrylate end groups are also capable of binding to the thiol prising: a hydrophobic biocompatible polymer selected from US 2014/0005379 A1 Jan. 2, 2014 a from polylactide, a polyglycolide, poly(lactide-co-gly 0032. In another aspect, there is provided a use of the collide), poly(e-caprolactone), or a combination thereof, the nanoparticle composition as described herein in the manufac hydrophobic polymer forming the core of the nanoparticle; a ture of a medicament for treating a disease capable of being hydrophilic biocompatible polymer selected from polysac treating by administering a therapeutic agent to a mucosal charide, polynucleotide, polypeptide, or a combination site. thereof, having multiple functional moieties, the hydrophilic 0033. In another aspect, there is provided a nanoparticle portion forming the shell of the nanoparticle; at least a portion composition or pharmaceutical composition as described of the functional moieties being conjugated to a mucosal hereinforuse intreating a disease capable of being treating by targeting moiety selected from a phenylboronic acid (PBA) administering a therapeutic agent to a mucosal site. derivative, a thiol derivative or an acrylate derivative. 0034. In another aspect, there is provided a commercial 0022. In a further embodiment, there is provided a nano package comprising the nanoparticle composition orpharma particle composition useful for delivery of a payload to a ceutical composition as described herein, together with mucosal site, the nanoparticle comprising a plurality of instructions for use in treating a disease. amphiphilic macromolecules, the macromolecules compris 0035. In another aspect, there is provided a method of ing: a hydrophobic portion comprising a polylactide; a hydro preparing a nanoparticle composition useful for delivery of a philic portion having multiple functional moieties, said payload to a mucosal site, the method comprising: a) prepar hydrophilic portion comprising dextran; and a mucosal tar ing an ampliphilic macromolecule comprising a hydrophilic geting moiety being a phenylboronic acid (PBA) derivative, portion and a hydrophobic portion, the hydrophilic portion wherein at least a portion of said functional moieties of the comprising multiple functional moieties; b) assembling a hydrophilic portion are conjugated to the mucosal targeting plurality of said macromolecules under Suitable conditions to moiety. form a nanoparticle having a hydrophobic core and a hydro 0023. In a further embodiment, there is provided a nano philic shell; and c) conjugating at least a portion of said particle composition useful for delivery of a payload to a functional moieties on the hydrophobic portion to a mucosal mucosal site, the nanoparticle comprising a plurality of targeting moiety to provide a Surface-functionalized nanopar amphiphilic macromolecules, the macromolecules each com ticle. prising a hydrophobic polylactide polymer conjugated to a 0036. Other aspects and features of the present disclosure hydrophilic dextran polymer having multiple functional moi will become apparent to those ordinarily skilled in the art eties, at least a portion of said functional moieties being upon review of the following description of specific embodi conjugated to a phenylboronic acid (PBA) derivative. ments in conjunction with the accompanying figures. 0024. In a further embodiment, there is provided a Dext ran-p-PLA block copolymer, wherein at least a portion of the BRIEF DESCRIPTION OF THE DRAWINGS functional groups on the Dextran are conjugated to a targeting moiety capable of forming a high affinity bond with a target at 0037 Embodiments of the present disclosure will now be a mucosal site. described, by way of example only, with reference to the 0025. In some embodiments, the nanoparticle is formed attached Figures. by conjugating the polylactide to the dextran to form a nano 0038 FIGS. 1A and B present NMR spectra at various particle and Subsequently surface-functionalizing the nano steps of block copolymer synthesis. FIG. 1A: Proton NMR of particle by conjugating at least a portion of the functional I. Dextran 6 kDa (D2O), II. Dextran-NH-Et-NH-Boc (D2O), moieties of the dextran to the PBA derivative to achieve a III. Dextran-NH-Et-NH2 (D2O), IV. PLA 20 kDa (DMSO desired surface density of the PBA derivative. d6), V. Dextran-Et-PLA, or PLA20-Dex6 (DMSO-d6). FIG. 0026. In some embodiments, the nanoparticle is formed 1B: Carbon NMR of block copolymer I. PLA 20 kDa, II. by conjugating the polylactide to the dextran to form a nano Dextran 6 kDa, III Dex-Et-PLA (PLA20-Dex6) confirming particle and Subsequently reacting the functional moieties of conjugation of Dextran and PLA. the dextran with PBA such that substantially all of the PBA is 0039 FIGS. 2A and B show particle size and morphology located in the shell?on the surface of the nanoparticle. of dextran-b-PLANPs. FIG. 2A: Effect of MW’s of PLA and 0027. In some embodiments, the core of the nanoparticle Dextran on the sizes of the NPs formed from nine different is Substantially free of targeting moiety. polymers using PLA with MW 10 kDa (red), 20 kDa (green) 0028. In another aspect, there is provided a pharmaceuti and 50kDa (blue), and Dextran with MW 1.5 kDa, 6 kDa and cal composition comprising a nanoparticle composition as 10 kDa. The black bars represent the standard deviation of the defined in herein, and a pharmaceutically acceptable carrier. particle sizes of each block copolymer. FIG. 2B: TEM image 0029. In another aspect, there is provided a mucoadhesive of PLA20-Dex6 NPs (Scale bar is 100 nm) to demonstrate delivery system for delivering a payload to a mucosal Surface, spherical shape of the nanoparticles. the delivery system comprising a nanoparticle composition as 0040 FIGS. 3A and B are graphs of drug encapsulation in defined herein; a pharmaceutically acceptable carrier, and a NPs. FIG.3A: Doxorubicin encapsulation efficiency in Dex payload. b-PLA and PLGA-PEG NPs using nanoprecipitation. FIG. 0030. In another aspect, there is provided a method of 3B: the corresponding drug loading wt %. Solid gray columns treating or preventing a disease or condition comprising are for PLA20-Dex6 NPs, solid white columns are for administering to a Subject an effective amount of a nanopar PLA20-Dex10 NPs and columns with diagonal lines pattern ticle composition or pharmaceutical composition as are for PLGA-PEG NPs (n=3; meani.S.D). described herein. 0041 FIG. 4 is a graph of in vitro Doxorubicincumulative 0031. In another aspect, there is provided a use of the release profiles from Dex-b-PLA and PLGA-PEG NPs con nanoparticle composition or pharmaceutical composition as ducted in PBS at 37° C. Solid square () are for PLA20 described herein for treating a disease capable of being treat Dex10, solid circles (O) are for PLA20-Dex6 and solid tri ing by administering a therapeutic agent to a mucosal site. angles (A) are for PLGA-PEG NPs (n=3; meantS.D). US 2014/0005379 A1 Jan. 2, 2014

0042 FIG. 5 is a graph of hemolytic activity of Dex-b- coma) in a Sustained manner for up to 18 hours in in vitro PLA and PLGA-PEG NPs for concentrations relevant to experiment. The NPs were able to load up to 2.8 wit/wt % theoretical administered dose in blood. VBS was used as a Dorzolamide. negative control and deionized water was used as positive 0055 FIG. 19 demonstrates the ability of the Dex-b-PLA control in sheep erythrocytes. Solid gray columns are for PBA NPs to release Brinzolamide (used in treatment of glau PLA20-Dex6 NPs, solid white columns are for PLA20 coma) in a Sustained manner for up to 11 days in in vitro Dex 10 NPs and columns with diagonal lines pattern are for experiment. The NPs were able to load up to 6.54 wit/wt % PLGA-PEG NPs (n=3; meani.S.D). Brinzolamide. 0043 FIG. 6 is a graph illustrating pharmacokinetic pro 0056 FIG. 20 demonstrates the ability of the Dex-b-PLA files of Dextran-b-PLA and PLGA-PEG NPS administered at PBA NPs to release Natamycin (used in treatment of ocular 30 mg/kg i.v. to rats. The NP concentration in blood was fungal infection) in a Sustained manner for up to 24 hours in tracked using 3H-PLA-radiolabeled nanocrystals. Solid in vitro experiment. The NPs were able to load up to 3.88 square () are for PLA20-Dex10, solid circles (O) are for wit/wt % Natamycin. PLA20-Dex6 and solid triangles (A) are for PLGA-PEG NPs 0057 FIG. 21 is a schematic illustration of the partition of (n=5, meantS.D). nanoparticle carriers across tear fluid lipid layer. 0044 FIG.7. Is a graph illustrating biodistribution of Dex 0.058 FIG. 22 demonstrates that these various types of tran-b-PLA and PLGA-PEG NPs in various organs in rats 24 nanoparticle carriers are capable of achieving high percent hour post-injection. Solid gray columns are for PLA20-Dex6 age partition across the tear fluid lipid layer. NPs, solid white columns are for PLA20-Dex10 NPs and 0059 FIG. 23 demonstrates the compatibility of the exem columns with diagonal lines pattern are for PLGA-PEG NPs plary NP formulation showing no short-term toxicity effect (n=5, meani.S.D)*: p<0.01. on the ocular surface in rabbits. NP treated and the control eyes (contralateral eyes) after one-time administration of the 004.5 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 0046 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 0060 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. 0047 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 0061 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 0.048 FIG. 11 is a schematic illustration of one embodi up to 12 weeks. Chronic response of the ocular Surfaces ment showing the surface modified the NPs with PBA using between NP treated and the control eyes (contralateral eyes) two-step approach: periodate oxidation of the Dextran, and were evaluated similarly using 7 different categories (dis conjugation of the aldehyde groups on the oxidated Dextran comfort, conjunctival redness and Swelling, lid Swelling, dis with amine groups of PBA. charge, corneal opacification, and infiltrate) by slit-lamp 0049 FIG. 12A demonstrates "H NMR verification of the examination. The grades demonstrate that there is no signifi presence of PBA on the Dex-b-PLA polymer chains. FIG. cant difference interms of severity of each category in the NP 12B demonstrates the spherical morphology of the Dex-b- treated eye compared to the control eye throughout the dura PLA PBA NPs. tion of the study. 0062 FIG. 26 compares the chronic response of the ocular 0050 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 0051 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 0052 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. 0053 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 0063 Generally, the present disclosure relates to a nano Zolamide, Brinzolamide, and Natamycin were encapsulated particle delivery system. The nanoparticles are formed from in the Dex-b-PLA PBA NPs. amphiphilic macromolecules, such as block copolymers, 0054 FIG. 18 demonstrates the ability of the Dex-b-PLA comprising a hydrophilic portion and a hydrophobic portion. PBA NPs to release Dorzolamide (used in treatment of glau The hydrophobic portion comprises multiple functional US 2014/0005379 A1 Jan. 2, 2014

groups capable of being conjugated to a targeting moiety, will understand that a block copolymer may, in Some cases, Such as a mucosal targeting moiety. In an aqueous environ contain multiple blocks of polymer. For instance, a block ment, the hydrophilic portion forms the shell of the nanopar copolymer may comprise a first block comprising a first poly ticle providing a Surface that can be functionalized by coating mer, a second block comprising a second polymer, and a third the nanoparticle with a desired surface density of the targeting block comprising a third polymer or the first polymer, etc. In moiety. The size of the nanoparticles and the Surface density addition, it should be noted that block copolymers can also be of the targeting moieties can be tuned without Substantially formed, in some instances, from other block copolymers. For compromising the stability of the particles. The nanoparticles example, a first block copolymer may be conjugated to are useful for delivering a wide variety of payloads to a another polymer to form a new block copolymer containing mucosal site in a subject and are capable of providing Sus multiple types of blocks. The polymers may be conjugated by tained release of the payload. The nanoparticles demonstrate any means known in the art and may optionally be connected good loading capacity and loading efficiency. by an appropriate linker moiety. 0064. The present disclosure also relates to components 0070 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 0065. Macromolecules other. A hydrophilic polymer is one that generally attracts 0066. 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 0067. A "polymer,” as used herein, refers to a molecular polymer. structure comprising one or more repeat units (e.g. mono (0071. 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 0072. 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 0068. 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 0073. 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 0074 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 0069 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 0075. 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 US 2014/0005379 A1 Jan. 2, 2014

37°C.). Degradation of a polymer may occurat varying rates, 0080. In some embodiments, the hydrophobic portion is a depending on the polymer or copolymer used. For example, polymer comprising 2 or more repeat units. The hydrophilic the half-life of the polymer (the time at which 50% of the portion may comprise, for example, from 2 to 200,000 repeat polymer is degraded into monomers and/or other nonpoly units depending on the size of the hydrophobic portion meric moieties) may be on the order of hours, days, weeks, desired. months, or years, depending on the polymer. The polymers I0081. In some embodiments, the molecular weight of the may be biologically degraded, e.g., by enzymatic activity or hydrophobic portion is in the range of about 100 g/mol to cellular machinery, in Some cases, for example, through about 2,000,000 g/mol. In some embodiments, the molecular exposure to a lysozyme (e.g., having relatively low pH). In weight of the hydrophobic portion is in the range of about 500 Some cases, the polymers may be broken down into mono g/mol to about 200,000 g/mol. In some embodiments, the mers and/or other nonpolymeric moieties that cells can either molecular weight of the hydrophobic portion is in the range of reuse or dispose of without significant toxic effect on the cells about 1,000 g/mol to about 100,000 g/mol. The unit “g/mol” (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. 0076 Non-limiting examples of biodegradable polymers I0082 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 I0083. 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. 0077. 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 40H 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 I0084. 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%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% of the 0078. The hydrophobic portion of the macromolecule functional moieties on the Surface of the nanoparticle are generally comprises a hydrophobic polymer, for example, a conjugated to a targeting moiety. hydrophobic polymer selected from polyesters, polyorthoe I0085. The selection of a hydrophilic polymer having mul ster, polycarbonates, polyimides, polybenzimidazoles, poly tiple functional moieties per monomer unit allows for urethanes, polyureas, polysulfides, polyethers, polysulfones, enhanced tunability of the nanoparticles as compared to, for phenolic and amino plastics, chitin and lipopolysaccharides, example, conventional PEG-based nanoparticles having only cholesterol, proteoglycans, and combinations thereof. In an one reactive functional group at the terminal end of each PEG aqueous environment, e.g. under physiological conditions, chain. Furthermore, in some embodiments, the hydrophilic the hydrophobic portion will substantially form the core of polymers of the present disclosure are more hydrophilic than the nanoparticle. a PEG polymer such that the hydrophilic portion of the mac 0079. 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 I0086. 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 US 2014/0005379 A1 Jan. 2, 2014

thereof. Such derivatives and analogues are known to those otide have side chains with reactive functional groups capable skilled in the art and can be readily obtained or synthesized. In of being conjugated to the targeting moiety. Some embodiments, polysaccharides, for example, dextran, 0093. In some embodiments, the hydrophilic portion is a are preferred since there are multiple functional groups per polymer comprising 2 or more repeat units. The hydrophilic each monomer unit. portion may comprise, for example, 2 to 100,000 repeat units 0087. In some embodiments, the hydrophilic portion of depending on desired size of the nanoparticle. the macromolecule comprises a “polysaccharide', e.g. a 0094. In some embodiments, the molecular weight of the polymer of monosaccharide units joined together by glyco hydrophilic portion ranges from about 100 g/mol to about sidic linkages. Any suitable polysaccharide may be used 1,000,000 g/mol. In some embodiments, the molecular accordance with the present disclosure. In some embodi weight of the hydrophilic portion ranges from about 500 ments, the polysaccharide is composed of 4- to 8-carbon ring g/mol to 100,000 g/mol. In some embodiments, the molecular monomers, such as 5-carbon ring monomers. The monomer weight of the hydrophilic portion ranges from about 1,000 rings may be heterocyclic, form example, comprising one or g/mol to about 50,000 g/mol. The unit “g/mol in this case more N, O or Satoms in the monomer ring. The polysaccha refers to the weight of the hydrophilic portion per mol of the ride may be a “homopolysaccharide', where all of the macromolecule prior to conjugation with a targeting moiety. monosaccharides in the polysaccharide are the same type, or 0095. In some embodiments, the molecular weight of the a "heteropolysaccharide', where more than one type of hydrophilic portion ranges from about 0.1 kDa to about 1,000 monosaccharide is present. In some embodiments, the kDa. In some embodiments, the molecular weight of the polysaccharide is a “homopolysaccharide'. In some embodi hydrophilic portion ranges from about 0.5 kDa to 100 kDa. In ments, the polysaccharide is a "heteropolysaccharide'. In some embodiments, the molecular weight of the hydrophilic Some embodiments, the polysaccharide is a linear polysac portion ranges from about 1 kDa to 50 kDa. These values charide. In some embodiments, the polysaccharide is a represent ranges prior to conjugation with a targeting moiety. branched polysaccharide. In some embodiments, the polysac 0096. The relative amount of hydrophobic polymer to charide has a reducing end that can be modified for conjuga hydrophilic polymer in the macromolecule may be any Suit tion purposes. In some embodiments, the polysaccharide is a able ratio that provides the desired characteristics of the homopolysaccharide with a reducing end. resulting nanoparticle. In some embodiments, the molecular 0088. In some embodiments, the polysaccharide is com weight of the hydrophobic portion is larger than the molecular posed of monomers of glucose, fructose, lactose or a combi weight of the hydrophilic portion. In some embodiments, the nation thereof. molecular weight of the hydrophilic portion is larger than the 0089. In some embodiments, the polysaccharide is molecular weight of the hydrophobic portion. selected from dextran, chitosan, alginate, hyaluronic acid, 0097. In some embodiments, the ratio of the molecular heparin, chondroitin Sulphate, pectin, pullulan, amylose, weight of the hydrophobic portion to the hydrophilic portion cyclodextrin, carboxymethylcellulose or a polysaccharide (hydrophobic portion:hydrophilic portion) is a about 0.1:1 to with thiol functional groups conjugated to the polymer back 100:1. In some embodiments, the molecular weight ratio is bone. about 0.5:1 to about 50:1. In some embodiments, the molecu lar weight ratio is about 1:1 to about 10:1. In some embodi 0090. 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 0091. 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 albe 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 (0098. Targeting connected to a Sugar-phosphate backbone (a base connected 0099. 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. 0092. In some embodiments, the hydrophilic portion com 0100. 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 US 2014/0005379 A1 Jan. 2, 2014

the nanoparticle may beformed from two or more macromol doglycan, a glycopeptide, or the like. In some cases, the ecules, each being functionalized with a different targeting biological moiety may be relatively large, for example, for moiety. peptides, nucleic acids, or the like. For example, the biologi 0101 The term “binding,” as used herein, refers to the cal moiety may have a molecular weight of at least about interaction between a corresponding pair of molecules or 1,000 Da, at least about 2,500 Da, at least about 3000 Da, at portions thereofthat exhibit mutual affinity or binding capac least about 4000 Da, or at least about 5,000 Da, etc. Relatively ity, typically due to specific or non-specific binding or inter large targeting moieties may be useful, in Some cases, for action, including, but not limited to, biochemical, physiologi differentiating between cells. For instance, in some cases, cal, electrostatic and/or chemical interactions. In some cases, Smaller targeting moieties (e.g., less than about 1000 Da) may the targeting moiety is able to selectively bind to a target not have adequate specificity for certain targeting applica expressed at the mucosal site, for example, a molecule, recep tions, such as mucosal targeting applications. In contrast, tor or residue expressed at the mucosal site. “Selective bind larger molecular weight targeting moieties can offer a much ing', as used herein, refers to a targeting moiety, which may higher targeting affinity and/or specificity. For example, a 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 musosal site. Such as mucin or about 500 Da. a receptor or a glycoprotein or a polysaccharide or residue 0107 Nanoparticles expressed on the Surface of an epithelial cell. In some cases, 0108. 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 0109 Under appropriate conditions, the macromolecules permits covalent binding of the targeting moiety. are capable of assembling to form a nanoparticle of the core 0102. 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 0110. 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' 0103) 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 0111. 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 75 nm, of binding to hydroxyl groups of the glycoproteins on the less than about 60 nm, less than about 50 nm, less than about mucous membrane. Acrylate derivatives include, but are not 30 nm, less than about 10 nm, less than about 3 nm, or less limited to methacrylate, ethyl acrylate, and diacrylate. In than about 1 nm in some cases. In some cases, particles less Some embodiments, the targeting moiety is an acrylate than 150 nm are preferred, for example, such particles are derivative selected from methacrylate, ethyl acrylate, and better able to penetrate the tear layer of the eye compared to diacrylate. larger particles. 0104. In some embodiments, the targeting moiety is a 0112. In some embodiments, the average particle size is phenylboronic acid (PBA) derivative, a thol derivative or an between about 0.1 nm and about 1000 nm, about 1 nm and acrylate derivative. about 500 nm, about 1 nm and about 300 nm, about 1 nm and 0105. In some embodiments, the targeting moiety is the about 200 nm, about 1 nm and about 150 nm, about 1 nm and hydrophobic portion of the macromolecule comprises PLA; about 100 nm, about 1 nm and about 50 nm, about 10 nm and the hydrophilic portion comprises dextran; and the targeting about 150 nm, about 10 nm and about 100 nm, about 10 nm. moiety comprises PBA. In some embodiments, the targeting and about 75 nm, about 10 nm and about 60 nm, and about 10 moiety is the hydrophobic portion is PLA; the hydrophilic nm and about 50 nm, or about 20 and about 40 nm. portion is dextran, and the targeting moiety is PBA. 0113. As used herein, “particle size' refers to the average 0106. In some embodiments, the targeting moiety is a characteristic dimension of a population of nanoparticles biological moiety. Non-limiting examples of biological moi formed, where the characteristic dimension of a particle is the eties include a peptide, a protein, an enzyme, a nucleic acid, a diameter of a perfect sphere having the same Volume as the fatty acid, a hormone, an antibody, a carbohydrate, a pepti particle. A population of nanoparticles may, for example, US 2014/0005379 A1 Jan. 2, 2014 include at least 20 particles, at least 50 particles, at least 100 I0120 For example, the size of the nanoparticles can be particles, at least 300 particles, at least 1,000 particles, at least tuned by adjusting the molecular weight and/or composition 3,000 particles, at least 5,000 particles, at least 10,000 par of the hydrophobic portion and/or the hydrophilic portion. ticles, or at least 50,000 particles. Various embodiments of the The particular targeting moiety selected, as well as the Sur present invention are directed to such populations of particles. face density of the targeting moiety on the Surface of the 0114. In some embodiments, the particles may each be nanoparticles, will also impact the particle size. Substantially the same shape and/or size, in which case the I0121. It should be noted that increasing size of the hydro population is "monodisperse'. For example, the particles philic and/or hydrophobic polymer components does not may have a distribution of particle sizes such that no more always result in a larger particle size. For example, in some than about 5% or about 10% of the particles have a particle cases, longer polymer chains may be more flexible and size greater than about 10% greater than the average particle capable of folding to produce a more compact particle size of the particles, and in some cases, such that no more than whereas a shorter polymer chain may be confined to a more about 8%, about 5%, about 3%, about 1%, about 0.3%, about linear configuration. Selection of a branched verus a linear 0.1%, about 0.03%, or about 0.01% have a particle size polymer can also impact particle size. A skilled person will be greater than about 10% greater than the average particle size able to select Suitable polymers for a particular application. of the particles. In some cases, no more than about 5% of the 0.122 The hydrophilic portion of the macromolecules will particles have aparticle size greater than about 5%, about 3%, form the shell of the nanoparticles in an aqueous environ about 1%, about 0.3%, about 0.1%, about 0.03%, or about ment. The hydrophilic portion is selected such that it has 0.01% greater than the average particle size of the particles. multiple functional moieties for conjugation to a targeting 0115. In some embodiments, the particles have an interior moiety. The proportion of functional moieties conjugated to a core and an exterior shell which forms the surface of the targeting moiety can be controlled, at least in part, by the nanoparticle, where the shell has a composition different amount of targeting moiety added to the conjugation reaction. from the core i.e., there may be at least one polymer or moiety In general, the more functional moieties present on the hydro present in shell but not in the core (or vice versa), and/or at philic portion, the higher the degree of tunability of the nano least one polymer or moiety present in the core and/or the particles. In general, the nanoparticles disclosed herein are shell at differing concentrations. thus more tunable than PEG-based nanoparticles having only 0116. In some cases, the core of the particle is more hydro one functional moiety per PEG chain, or other similar poly phobic than the shell of the particle. In some cases, a drug or CS. other payload may be hydrophobic, and therefore readily 0123. By adjusting the surface density of the targeting associates with the relatively hydrophobic interior of the par moiety, the extent of targeting can be controlled. A high ticle. The drug or other payload may thus be contained within Surface density of the targeting moiety can be achieved with the interior of the particle, which may thus shelter it from the the nanoparticles disclosed herein due to the presence of external environment Surrounding the particle (or vice versa). multiple functional moieties on the hydrophilic portion. A targeting moiety present on the Surface of the particle may Advantageously, a skilled person will be able to control the allow the particle to become localized at a particular targeting extent of targeting without Substantially compromising the site, for instance, a mucosal site. The drug or other payload stability of the nanoparticle delivery system. In some embodi may then, in Some cases, be released from the particle and ments, an optimal density can be determined in which maxi allowed to interact with the particular targeting site. mum targeting is achieved without Substantially compromis 0117. Yet another aspect of the disclosure is directed to ing the stability of the nanoparticle. nanoparticles having more than one polymer or macromol 0.124. In some embodiments, a majority (e.g. at least 50%, ecule present. For example, in Some embodiments, particles 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100%) of the may contain more than one distinguishable macromolecule, mucosal targeting moieties are located on the Surface of the and the ratios of the two (or more) macromolecules may be nanoparticle. It is understood that, in Some cases, a portion of independently controlled, which allows for the control of the mucosal targeting moieties may be located within the core properties of the particle. For instance, a first macromolecule of the nanoparticle when the nanoparticle forms, depending may be a biocompatible polymeric conjugate. Such as a block on the components of the nanoparticle and the method uti copolymer, comprising a targeting moiety, and a second mac lized. For instance, where a one-step method is used, it is romolecule may comprise a biocompatible polymer but no possible that Some of the targeting moieties may orient targeting moiety, or the second macromolecule may contain a toward the core of the particles. distinguishable biocompatible polymer from the first macro 0.125. The selection of a hydrophilic polymer having mul molecule. Control of the amounts of these macromolecules tiple functional moieties along the polymer backbone renders within the polymeric particle may thus be used to control the hydrophilic portion of the molecule more hydrophilic various physical, biological, or chemical properties of the than other polymers, such as PEG, thus the hydrophilic por particle, for instance, the size of the particle (e.g., by varying tion is more likely to orient toward an aqueous environment. the molecular weights of one or both polymers), the surface Since the targeting moiety is conjugated to the hydrophilic charge (e.g., by controlling the ratios of the polymers if the portion, typically after formation of a nanoparticle, Substan polymers have different charges or terminal groups), the Sur tially all of the targeting moieties are located on the Surface of face hydrophilicity (e.g., if the polymers have different the nanoparticle as opposed to the core. molecular weights and/or hydrophilicities), the Surface den I0126. In some embodiments, substantially all (e.g. at least sity of the targeting moiety (e.g., by controlling the ratios of 95%,96%,97%.98%.99%, or 100%) of the mucosal target the two or more polymers), etc. ing moieties are located on the Surface of the nanoparticle. 0118 Tunable Nanoparticles Localizing Substantially all of the targeting moieties to the 0119 The nanoparticles described herein are highly tun Surface of the nanoparticles enhances targeting efficiency. able. The selection of a hydrophilic polymer having multiple func US 2014/0005379 A1 Jan. 2, 2014

tional moieties along the polymer backbone renders the from about 50 to about 3,500,000, from about 150,000 to hydrophilic portion of the molecule more hydrophilic than about 3,500,000, or from about 1,500,000 to about 3,500,000 other polymers. Such as PEG having only one functional per nanoparticle. moiety, thus the hydrophilic portion is more likely to orient 0.138. In some embodiments, the surface density of the toward an aqueous environment. Since the targeting moiety is targeting moiety tunable by the amount of targeting moiety conjugated to the hydrophilic portion, typically after forma added in the reaction during the functionalization step(s). tion of a nanoparticle, Substantially all of the targeting moi 0.139. In some embodiments, the density of a phenylbo eties are located on the Surface of the nanoparticle as opposed ronic acid derivative on the nanoparticle Surface is tuneable to the core. by the amount of phenylboronic acid added in the reaction to 0127. The nanoparticles can be tuned by controlling the control the extent of mucoadhesion properties of the nano Surface density of the targeting moieties on the nanoparticle. particles. A skilled person will be able to precisely tune the nanoparticle for a particular application without Substantially compromis 0140. In some embodiments, the density of a cysteamine ing the stability of the nanoparticles. derivative on the nanoparticle surface is tuneable by the 0128. 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, 0.141. 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 0129. 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. 0142. The optimal density of surface functional groups 0130. 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, 0143. 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 0131. 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 Controlled Release the Surface of the nanoparticle (i.e. Surface density) ranges 0144) from about 50 to about 30000, from about 1000 to about 0145 A controlled release system, as used herein, refers to 30000, or from about 10000 to about 30000 per nanoparticle. a nanoparticle delivery system capable of delivering a pay 0.132. In some embodiments, the nanoparticle is approxi load. Such as a therapeutic agent, a diagnostic agent, a prog mately 50 nm in size and the density of targeting moieties on nostic, a prophylactic agent, to a body tissue. Such as a the Surface of the nanoparticle (i.e. Surface density) ranges mucous membrane, where the payload is released in a prede from about 50 to about 90000, from about 3000 to about signed or controlled manner. For example, the active agent 90000, or from about 30000 to about 90000 per nanoparticle. may be released in a constant manner over a predetermined 0133. In some embodiments, the nanoparticle is approxi period of time, the active agent may be released in a cyclic mately 100 nm in size and the density of targeting moieties on manner over a predetermined period of time, or an environ the Surface of the nanoparticle (i.e. Surface density) ranges mental condition or external event may trigger the release of from about 50 to about 350000, from about 10000 to about the active agent. The controlled release polymer system may 350000, or from about 100000 to about 350000 per nanopar include a polymer that is biocompatible, and in Some cases, ticle. the polymer is biodegradable. In some cases, the nanopar 0134. In some embodiments, the nanoparticle is approxi ticles disclosed herein are part of a controlled release delivery mately 150 nm in size and the density of targeting moieties on system. The nanoparticles disclosed herein demonstrated the Surface of the nanoparticle (i.e. Surface density) ranges Sustained release of payload. from about 50 to about 800000, from about 30000 to about 0146 The mucosal targeting moiety assists in retaining 800000, or from about 300000 to about 800000 per nanopar the nanoparticles at the mucosal site, i.e. for a longer time that ticle. the same nanoparticle without the targeting moiety, Such that 0135) In some embodiments, the nanoparticle is approxi controlled delivery of a payload at the mucosal site can be mately 200 nm in size and the density of targeting moieties on achieved. The controlled delivery may include sustained the Surface of the nanoparticle (i.e. Surface density) ranges delivery. from about 50 to about 1,500,000, from about 60000 to about 0.147. In some embodiments, in the payload is released 1,500,000, or from about 600000 to about 1,500,000 per from the nanoparticle for a sustained period of at least 24, 36, nanoparticle. 48, 60, 72,84, of 96 hours. In some embodiments, the payload 0136. In some embodiments, the nanoparticle is approxi is released from the nanoparticle for a Sustained period of at mately 250 nm in size and the density of targeting moieties on least 1, 2, 3, 4, 5, 6, 7 or 8 days. In some embodiments, the the Surface of the nanoparticle (i.e. Surface density) ranges payload is released is released from the nanoparticle for a from about 50 to about 2,500,000, from about 100,000 to Sustained period of at least 1 week. In some embodiments, the about 2,500,000, or from about 1,000,000 to about 2,500,000 payload is released is released from the nanoparticle for a per nanoparticle. Sustained period of at least 1 month. 0.137 In some embodiments, the nanoparticle is approxi 0.148. In some embodiments at least 50% of the payload is mately 300 nm in size and the density of targeting moieties on released within the first 24 hours. In other embodiments, at the Surface of the nanoparticle (i.e. Surface density) ranges least 10% the payload is released within the first 6 hours. US 2014/0005379 A1 Jan. 2, 2014

0149 Payload wit/wt., about 1 to about 20%, 1 to about 10%, about 1% to 0150. A wide variety of payloads can be loaded into the about 8%, about 1% to about 6%, about 1% to about 5%, nanoparticles described herein. As used herein, the “payload about 1% to about 3%, or about 1% to about 2%. The loading may be any agent of interest to be delivered to a mucosal site, capacity (%) is calculated here as the molecular weight of for example, a therapeutic agent (e.g. drug), a diagnostic encapsulated drug over the entire weight of the nanoparticle agent, a prophylactic agent, an imaging agent, or a combina multiplied by 100. The total weight of the nanoparticle refers tion thereof. In some embodiments, the payload is a single to the weight of the nanoparticle including the targeting moi agent of interest. In other embodiments, the payload com ety and the encapsulated drug. prises more than one agent of interest, for example, a combi 0156. In some embodiments, the loading capacity is up to nation of two or more agents of interest. In some embodi about 40%, up to about 30% wit/wt., up to about 20%, up to ments, the payload comprises 2, 3 or 4 agents of interest. For about 10%, up to about 8%, up to about 6%, up to about 5%, example, the payload may comprise two or more agents of up to about 3%, up to about 2%, or up to about 1%. interest selected from a therapeutic agent, a diagnostic agent, 0157. 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. 0151. When combined with a payload, the nanoparticles 0158. 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 0159. 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 0152. 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; antiheimintics; antiar 0153. 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 0154 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 0.160) 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 0155. 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 US 2014/0005379 A1 Jan. 2, 2014

nol, Taxol, UFT, Buspar, Serzone, Stadol NS, Estrace, Glu dine, halazepam, haloperidol, hetacillin, hexobarbital, cophage (Bristol-Myers Squibb); Ceclor, Lorabid, Dynabac, hydralazine, hydrochlorothiazide, hydrocortisone (cortisol), Prozac, Darvon, Permax, Zyprexa, Humalog, AXid, Gemzar, hydroflunethiazide, hydroxychloroquine, hydroxy Zine, Evista (Eli Lily); Vasotec/Vaseretic, Mevacor, Zocor, Prinivil/ hyoscyamine, ibuprofen, indapamide, indomethacin, insulin, Prinizide, Plendil, Cozaar/Hyzaar, Pepcid, Prilosec, Pri iofoquinol, iron-polysaccharide, isoetharine, isoniazid, iso maxin, Noroxin, Recombivax HB, Varivax, Timoptic/XE, propamide isoproterenol, isotretinoin, isoXSuprine, kaolin & Trusopt, Proscar, Fosamax, Sinemet, Crixivan, Propecia, pectin, ketoconazole, lactulose, levodopa, lincomycin liothy Vioxx. Singulair, Maxalt, Ivermectin (Merck & Co.); Diflu ronine, liotrix, lithium, loperamide, lorazepam, magnesium can, Unasyn, Sulperazon, Zithromax, Trovan, Procardia XL, hydroxide, magnesium Sulfate, magnesium trisilicate, Cardura, Norvasc, Dofetilide, Feldene, Zoloft, Zeldox, Glu maprotiline, meclizine, meclofenamate, medroxyproyester cotrol XL, Zyrtec, Eletriptan, Viagra, Droloxifene, Aricept, one, melenamic acid, melphalan, mephenyloin, mephobar Lipitor (Pfizer); Vantin, Rescriptor, Vistide, Genotropin, Mic bital, meprobamate, mercaptopurine, mesoridazine, metap 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, 0161 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 0162. 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, US 2014/0005379 A1 Jan. 2, 2014

antiestrogen, antineoplaston, antisense oligonucleotides, larin-N triacetate, lanreotide, lanreotide acetate, leinamycin, aphidicolin glycinate, apoptosis gene modulators, apoptosis lenograstim, lentinan Sulfate, leptolstatin, letrozole, leukemia regulators, apurinic acid, ARA-CDP-DL-PTBA, arginine inhibiting factor, leukocyte alpha interferon, leuprolide deaminase, asparaginase, asperlin, asulacrine, atameStane, acetate, leuprolide/estrogen/progesterone, leuprorelin, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azac levamisole, liarozole, liarozole hydrochloride, linear itidine, aZasetron, azatoxin, azatyrosine, azetepa, azotomy polyamine analog, lipophilic disaccharide peptide, lipophilic cin, baccatin III derivatives, balanol, batimastat, benzochlo platinum compounds, lissoclinamide 7, lobaplatin, lombri rins, benzodepa, benzoylstaurosporine, beta lactam cine, lometrexol, lometrexol Sodium, lomustine, lonidamine, derivatives, beta-alethine, betaclamycin B, betulinic acid, losoxantrone, losoxantrone hydrochloride, lovastatin, loxor BFGF inhibitor, bicalutamide, bisantrene, bisantrene hydro ibine, lurtotecan, lutetium texaphyrin, lysofylline, lytic pep chloride, bisaziridinylspermine, bisnafide, bisnafide dimesy tides, maitansine, mannostatin A, marimastat, masoprocol, late, bistratene A, bizelesin, bleomycin, bleomycin Sulfate, maspin, matrilysin inhibitors, matrix metalloproteinase 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, 06-benzylguanine, octreotide, docosanol, dolasetron, doxifluridine, doxorubicin, doxorubi okicenone, oligonucleotides, onapristone, ondansetron, ora cin hydrochloride, droloxifene, droloxifene citrate, dromo cin, oral cytokine inducer, ormaplatin, osaterone, Oxaliplatin, stanolone propionate, dronabinol, duaZomycin, duocarmycin oxaunomycin, OXisuran, paclitaxel, paclitaxel analogs, pacli SA, ebselen, ecomustine, edatrexate, edelfosine, edrecolo taxel derivatives, palauamine, palmitoylrhizoxin, pamidronic mab, eflornithine, eflornithine hydrochloride, elemene, acid, panaxytriol, panomifene, parabactin, paZelliptine, elsamitrucin, emitefur, enloplatin, enpromate, epipropidine, pegaspargase, peldesine, peliomycin, pentamustine, pen epirubicin, epirubicin hydrochloride, epristeride, erbulozole, tosan polysulfate Sodium, pentostatin, pentroZole, peplomy erythrocyte gene therapy vector system, esorubicin hydro cin Sulfate, perflubron, perfosfamide, perillyl alcohol, phena chloride, estramustine, estramustine analog, estramustine Zinomycin, phenylacetate, phosphatase inhibitors, picibanil, phosphate Sodium, estrogen agonists, estrogen antagonists, pilocarpine hydrochloride, pipobroman, piposulfan, piraru etanidazole, etoposide, etoposide phosphate, etoprine, bicin, piritrexim, piroXantrone hydrochloride, placetin A, exemestane, fadrozole, fadrozole hydrochloride, fazarabine, placetin B, plasminogen activator inhibitor, platinum com fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, plex, platinum compounds, platinum-triamine complex, pli floxuridine, fluasterone, fludarabine, fludarabine phosphate, camycin, plomestane, porfimer Sodium, porfiromycin, pred fluorodaunorunicin hydrochloride, fluorouracil, fluorocitab nimustine, procarbazine hydrochloride, propyl bis-acridone, ine, forfenimex, formestane, fosquidone, fostriecin, fostrie prostaglandin J2, prostatic carcinoma antiandrogen, protea cin Sodium, fotemustine, gadolinium texaphyrin, gallium Some inhibitors, protein A-based immune modulator, protein nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcit kinase C inhibitor, protein tyrosine phosphatase inhibitors, abine, gemcitabine hydrochloride, glutathione inhibitors, purine nucleoside phosphorylase inhibitors, puromycin, hepsulfam, heregulin, hexamethylene bisacetamide, hydrox puromycin hydrochloride, purpurins, pyrazofurin, pyrazolo yurea, hypericin, ibandronic acid, idarubicin, idarubicin acridine, pyridoxylated hemoglobin polyoxyethylene conju hydrochloride, idoxifene, idramantone, ifosfamide, ilmofos gate, RAFantagonists, raltitrexed, ramosetron, RASfarnesyl ine, illomastat, imidazoacridones, imiquimod, immunostimu protein transferase inhibitors, RAS inhibitors, RAS-GAP lant peptides, insulin-like growth factor-1 receptor inhibitor, inhibitor, retelliptine demethylated, rhenium RE 186 etidr interferonagonists, interferon alpha-2A, interferon alpha-2B, onate, rhizoxin, riboprine; ribozymes, RII retinamide, RNAi, interferon alpha-N1, interferon alpha-N3, interferon beta-IA, rogletimide, rohitukine, romurtide, roquinimex, rubiginone interferon gamma-IB, interferons, interleukins, iobenguane, B1, ruboxyl, Safingol, Safingol hydrochloride, Saintopin, iododoxorubicin, iproplatin, irinotecan, irinotecan hydro sarcinu, sarcophytol A, Sargramostim, SDI 1 mimetics, chloride, iroplact, irsogladine, isobengaZole, isohomohali Semustine, senescence derived inhibitor 1, sense oligonucle condrin B, itasetron, jasplakinolide, kahalalide F. lamel otides, signal transduction inhibitors, signal transduction US 2014/0005379 A1 Jan. 2, 2014 modulators, simtraZene, single chain antigenbinding protein, compounds, as well as new active pharmaceutical ingredients sizofuran, Sobuzoxane, Sodium borocaptate, sodium pheny can be used with the delivery system described herein. lacetate, Solverol. Somatomedin binding protein, Sonermin, 0164. In one embodiment, the therapeutic agent is an oph sparfosate sodium, Sparfosic acid, sparsomycin, spicamycin thalmic agent selected from cycloprorinA, timolol, betaxolol. D. spirogermanium hydrochloride, Spiromustine, spiroplatin, metipranolol, dorzolamide, brinzolamide, natamycin, nepta splenopentin, spongistatin 1, squalamine, stem cell inhibitor, Zane, acetazolamide, alphagan, Xalatan, bimatoprost, stem-cell division inhibitors, stipiamide, Streptonigrin, Strep travaprost, olopatadine, ketotifen, acyclovir, gancyclovir, Val toZocin, Stromelysin inhibitors, Sulfinosine, Sulofenur, Super cyclovir. In one embodiment, the therapeutic agent is active vasoactive intestinal peptide antagonist, Suradista, cyclosporine A, natamycin, olopatadine, brinzolamide or dor Suramin, Swainsonine, synthetic glycosaminoglycans, taliso Zolamine. mycin, tallimustine, tamoxifen methiodide, tauromustine, 0.165. In one embodiment, the therapeutic agent is an oph tazarotene, tecogalan Sodium, tegafur, tellurapyrylium, thalmic agent used to treat glaucoma, Such as an agent used to telomerase inhibitors, teloxantrone hydrochloride, temopor reduce a sign and/or symptom of glaucoma, for example, and fin, temozolomide, teniposide, teroxirone, testolactone, tet agent used to reduce intraocular pressure associated with rachlorodecaoxide, tetraZomine, thaliblastine, thalidomide, ocular hypertension. In some embodiments, the therapeutic thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoi agent is a glaucoma medication, Such as a prostaglandin, etin, thrombopoietin mimetic, thymalfasin, thymopoietin carbonic anhydrase inhibitor, epinephrine or alpha-agonist, receptor agonist, thymotrinan, thyroid stimulating hormone, or a beta-blocker. In some embodiments, the therapeutic tiazofurin, tin ethyl etiopurpurin, tirapazamine, titanocene agent is Dorzolamide, Brinzolamide, Brimonidine, timolol. dichloride, topotecan hydrochloride, topsentin, toremifene, or latanoprost. toremifene citrate, totipotent stem cell factor, translation 0166 In one embodiment, the therapeutic agent is an oph inhibitors, trestolone acetate, tretinoin, triacetyluridine, thalmic agent used to treat allergic conjunctivitis, Such as an triciribine, triciribine phosphate, trimetrexate, trimetrexate agent used to reduce a sign and/or symptom of allergic con glucuronate, triptorelin, tropisetron, tubuloZole hydrochlo junctivitis. In one embodiment, the therapeutic agent is olo ride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBC patadine. inhibitors, ubenimex, uracil mustard, uredepa, urogenital 0167. In one embodiment, the therapeutic agent is an oph sinus-derived growth inhibitory factor, urokinase receptor thalmic agent used to treat keratoconjunctivitis sicca (KCS) antagonists, vapreotide, variolin B. velaresol, Veramine, Ver or “dry eye'. Such as an agent used to reduce a sign and/or dins, verteporfin, vinblastine sulfate, Vincristine sulfate, Vin symptom of KCS. In one embodiment, the therapeutic agent desine, Vindesine Sulfate, vinepidine Sulfate, Vinglycinate is cyclosporine A. sulfate, Vinleurosine sulfate, vinorelbine, vinorelbinetartrate, 0.168. In one embodiment, the therapeutic agent is Vinrosidine Sulfate, Vinxaltine, Vinzolidine Sulfate, vitaxin, cyclosporine A. In one embodiment, the therapeutic agent is Vorozole, Zanoterone, Zeniplatin, Zilascorb, Zinostatin, dorzolamide. In one embodiment, the therapeutic agent is Zinostatin stimalamer, or Zorubicin hydrochloride. In one natamycin. In one embodiment, the therapeutic agent is olo embodiment, the therapeutic agent is doxorubicin. patadine. 0163. In some embodiments, the therapeutic agent is an 0169. 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 0170 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, 0171 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 0172. 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, 0173. 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. 0.174 Diagnostic Agent levocabastine, levofloxin, lotepprednol, medrysone, meth 0.175. 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 US 2014/0005379 A1 Jan. 2, 2014

rials useful for CAT and X-ray imaging include, but are not Some embodiments, the dosage form is a solid dosage from, limited to, iodine-based materials. Such as a powder, tablet, capsule or caplet. (0176 Radionucleotide 0184. In some embodiments, the composition is in a dos 0177. As another example, the payload may include a age form Suitable for rectal or vaginal administration. In some radionuclide, e.g., for use as a therapeutic, diagnostic, or embodiments, the composition for rectal or vaginal adminis prognostic agent. Among the radionuclides used, gamma tration is in the form of a Suppository. In some embodiments, emitters, positron-emitters, and X-ray emitters are suitable the composition for rectal or vaginal administration is in the for diagnostic and/or therapy, while beta emitters and alpha form of a liquid. Such as a douche or enema. In some embodi emitters may also be used for therapy. Suitable radionuclides ments, the composition for rectal or vaginal administration is for use with various embodiments of the present invention in the form of a cream, ointment or gel, which may optionally include, but are not limited to, I, II, I, II, I, I, be applied using an applicator. 7Sc, 7As, 72Sc, 90Y. 88y. 7Ru, 100Pd, 'mRh, Sb, 12 Ba, 0185. In some embodiments, the composition is in a dos '7Hg, 21 At 212Bi, 212Pb, 10°Pd, In, 7Ga, Ga, 67Cu, age form Suitable for nasal or pulmonary administration. In 7Br 77Br mTc, “C, N, O, P, 33P or *F. The radio Some embodiments, the dosage form for nasal or pulmonary nucleotide may be contained within the nanoparticle (e.g., as administration is a spray or inhalant. In one embodiment, the a separate species), and/or form part of a macromolecule or dosage form is a spray. In one embodiment, the dosage form polymer that forms the nanoparticle. in an inhalant, which may be administered with an inhaler. 0.178 Pharmaceutical Compositions 0186. In some embodiments, the composition is in a dos 0179 Another aspect of the disclosure is related to phar age form Suitable for ocular orotic administration, i.e. admin maceutical compositions comprising a nanoparticle compo istration to the eye or ear. In some embodiments, the dosage sition as defined herein, and a pharmaceutically acceptable form for ocular or otic administration is a drop. In some carrier. Pharmaceutical compositions can be prepared in a embodiments, the composition is in a dosage form Suitable manner well known in the pharmaceutical art, and can be for ocular administration, such as a drop, gel or ointment. administered by a variety of routes of administration, depend Such drop, gel or ointment may, for example, be applied to the ing upon whether local or systemic effect is desired and upon anterior surface of the eye. the area to be treated. 0187 Parenteral routes of administration includes intrave 0180. In some embodiments, the pharmaceutical compo nous, intraarterial, Subcutaneous, intraperitoneal intramuscu sition is administered to a desired mucosal site in a subject. lar or injection or infusion; or intracranial, e.g., intrathecal or The pharmaceutical composition can be administered to a intraventricular, administration. Parenteral administration desired mucosal site by any suitable route of administration. can be in the form of a single bolus dose, or may be, for In some embodiments, the route of administration is non example, by a continuous perfusion pump. parenteral. Such as topical. As used herein, topical adminis 0188 In some embodiments, parenteral routes are desir tration may include, for example, administration to a mucous able since they avoid contact with the digestive enzymes that membrane via the mouth, eye, ear, nose, esophagus, stomach, are found in the alimentary canal. According to Such embodi Small intestine, large intestine, rectum, vagina, urethra, penis, ments, the nanoparticle compositions may be administered uterus, etc. It is understood that administration of a therapeu by injection (e.g., intravenous, Subcutaneous or intramuscu tic agent to a mucosal site may provide local and/or systemic lar, intraperitoneal injection), rectally, vaginally, topically (as effect, for example, depending on the ability of the agent to be by powders, creams, ointments, or drops), or by inhalation (as absorbed into the circulation via the mucous membrane. by sprays). 0181 Pharmaceutical compositions and formulations for 0189 Injectable preparations, for example, sterile inject topical administration generally include ointments, lotions, able aqueous or oleaginous Suspensions may be formulated creams, gels, drops, Suppositories, sprays, liquids and pow according to the known art using Suitable dispersing or wet ders. For topical administration to a mucous membrane of the ting agents and Suspending agents. The sterile injectable gut, an oral dosage form such as a liquid, emulsion, tablet, preparation may also be a sterile injectable solution, Suspen caplet or capsule may be used. Conventional pharmaceutical Sion, or emulsion in a nontoxic parenterally acceptable dilu carriers, excipients and dulients may be employed. ent or solvent, for example, as a solution in 1,3-butanediol. 0182. 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 0183 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 0.190 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, US 2014/0005379 A1 Jan. 2, 2014

waxes, paraffins, starch, tragacanth, cellulose derivatives, (0195 Dosage polyethylene glycols, silicones, bentonites, silicic acid, talc, 0196. It will be appreciated that the exact dosage of the and Zinc oxide, or mixtures thereof. nanoparticle or components thereof. Such as a therapeutic agent, may be determined by a physician in view of the patient 0191 Powders and sprays can contain, in addition to the to be treated. In general, dosage and administration are inventive conjugates of this invention, excipients such as adjusted to provide an effective amount of the inventive con lactose, talc, silicic acid, aluminum hydroxide, calcium sili jugate to the patient being treated. As used herein, the “effec cates, and polyamide powder, or mixtures thereof. Sprays can tive amount” refers to the amount necessary to elicit the additionally contain customary propellants such as chlorof desired biological response. As will be appreciated by those luorohydrocarbons. of ordinary skill in this art, the effective amount may vary 0.192 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 0197) 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 expressed as the ratio, LD50/ED50. Pharmaceutical compo 0193 Solid dosage forms for oral administration include sitions which exhibit large therapeutic indices may be useful capsules, tablets, caplets, pills, powders, and granules. In in some embodiments. The data obtained from cell culture Such solid dosage forms, the encapsulated or unencapsulated assays and animal studies can be used in formulating a range conjugate is mixed with at least one inert, pharmaceutically of dosage for human use. acceptable excipient or carrier Such as Sodium citrate ordical 0198 Kits and Commercial Packages cium phosphate and/or (a) fillers or extenders such as 0199 The present disclosure also provides any of the starches, lactose, Sucrose, glucose, mannitol, and silicic acid, above-mentioned compositions in kits or commercial pack (b) binders such as, for example, carboxymethylcellulose, ages, optionally with instructions for use or administration of alginates, gelatin, polyvinylpyrrolidinone. Sucrose, and aca any of the compositions described herein by any suitable cia, (c) humectants such as glycerol, (d) disintegrating agents technique as previously described. “Instructions' can define a Such as agar-agar, calcium carbonate, potato or tapioca starch, component of promotion, and typically involve written alginic acid, certain silicates, and sodium carbonate, (e) solu instructions on or associated with packaging of compositions tion retarding agents such as paraffin, (f) absorption accelera of the invention. Instructions also can include any oral or tors such as quaternary ammonium compounds, (g) wetting electronic instructions provided in any manner. The “kit agents such as, for example, cetyl alcohol and glycerol typically defines a package including any one or a combina monostearate, (h) absorbents such as kaolin and bentonite tion of the compositions of the invention and the instructions, clay, and (i) lubricants such as talc, calcium Stearate, magne but can also include the composition of the invention and sium Stearate, Solid polyethylene glycols, sodium lauryl Sul instructions of any form that are provided in connection with fate, and mixtures thereof. In the case of capsules, tablets, and the composition in a manner Such that a clinical professional pills, the dosage form may also comprise buffering agents. will clearly recognize that the instructions are to be associated 0194 Solid compositions of a similar type may also be with the specific composition. employed as fillers in Soft and hard-filled gelatin capsules 0200. The kits described herein may also contain one or using such excipients as lactose or milk Sugar as well as high more containers, which may contain the inventive composi molecular weight polyethylene glycols and the like. The solid tion and other ingredients as previously described. The kits dosage forms of tablets, dragees, capsules, pills, and granules also may contain instructions for mixing, diluting, and/or can be prepared with coatings and shells Such as enteric administrating the compositions of the invention in some coatings and other coatings well known in the pharmaceutical cases. The kits also can include other containers with one or formulating art. more solvents, surfactants, preservative and/or diluents (e.g., US 2014/0005379 A1 Jan. 2, 2014

normal saline (0.9% NaCl), or 5% dextrose) as well as con ease or condition affecting the mouth, eye, ear or nose. In tainers for mixing, diluting or administering the components Some embodiments, the disease or condition to be treated is a in a sample or to a Subject in need of Such treatment. disease or condition affecting the rectum, vagina, urethra, 0201 The compositions of the kit may be provided as any penis, or uterus. In some embodiments, the disease or condi Suitable form, for example, as liquid solutions or as dried tion to be treated is a disease or condition affecting the powders. When the composition provided is a dry powder, the esophagus, stomach, Small intestine or large intestine. composition may be reconstituted by the addition of a Suit 0207. In some embodiments, the disease or condition to be able diluent, which may also be provided. In embodiments treated is a disease or condition affecting the eye. Non-limit where liquid forms of the composition are used, the liquid ing examples include abrasion, acanthamoeba keratitis, 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 0202 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, Descernet'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 0203 Methods of Treatment and Use basement membrane dystrophy, exposure keratopathy, eyelid 0204 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, retention of the nanoparticles at the mucosal site Such that hyphema blunt trauma, hypopyon, infectious crystalline Sustained release at the mucosal site can be achieved. keratopathy, interstitial keratitis, iridocorneal dysgenesis, iri 0205. In some embodiments, there are provided methods docorneal endotheliopathy, iris cysts, iritis, iron lines, kera of treating a disease or conditionina Subject by administering toconus, keratoconus forme frusta, keratoglobus, lattice stro an effective amount of a composition or component thereofas mal dystrophy, leukocoria, lice, limbal stem cell deficiency, defined herein. In other embodiments, there are provided uses lipid keratopathy, macular stromal dystrophy, marginal of the compositions or components thereofas defined herein keratitis, meesmann's dystrophy, melanoma—conjunctival for treating and/or preventing a disease or condition. In other and eyelid, 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—cor facture of a medicament to treating and/or preventing a neal, peripheral ulcerative keratitis, persistent epithelial disease or condition. In other embodiments, there are pro defect, phlyctenulosis, pingueculum, posterior capsular vided compositions or components thereofas defined herein opacification, posterior polymorphous dystrophy, preseptal for the treatment of a disease or condition. cellulitis, pseudoexfoliation of the lens capsule, pterygium, 0206. In some embodiments, the disease or condition to be ptosis and pseudoptosis, punctual Stenosis, pyogenic granu treated is a disease or condition capable of being treated via loma, recurrent corneal erosion syndrome, Reis-Buckler's delivery of a therapeutic agent to a mucosal site. Such as a dystrophy, retention cyst and lymphangiectasia, rheumatoid mucosal site of the mouth, eye, ear, nose, esophagus, stom arthritis, rosacea keratitis, Salzmann nodular degeneration, ach, Small intestine, large intestine, rectum, vagina, urethra, Scleritis, sebaceous cell carcinoma, Seborrheic keratosis, penis or uterus. In some embodiments, the disease or condi squamous cell carcinoma—lid, Stevens-Johnson syndrome, tion to be treated is a disease or condition affecting the mouth, Sub-conjunctival hemorrhage, Superficial punctate keratopa eye, ear, nose, esophagus, stomach, Small intestine, large thy, Superior limbic keratoconjunctivitis, Synechia, Terrien’s intestine, rectum, vagina, urethra, penis, or uterus. In some marginal degeneration, Thygeson's Superficial punctate embodiments, the disease or condition to be treated is a dis keratopathy, toxic keratopathy, trachoma, trichiasis, pseudot US 2014/0005379 A1 Jan. 2, 2014

richiasis, distachiasis, metaplastic lashes, trichotillomania, polyposis, Fenwick ulcer, fissured tongue, flu, folicular con uveitis, Vernal keratoconjunctivitis, vitamin A deficiency, junctivitis, follicular hamartoma, food allergy related asthma, Vortex keratopathy, Xanthelasma. Fordyce's disease, Gardner syndrome, gastresophageal 0208. In some embodiments, the disease or condition is a reflux-related chronic cough, gastric erosion, gastric reflux, disease or condition of the eye is glaucoma, keratoconjunc gastric ulcer, gastritis, gastritis, gastroesophageal reflux dis tivitis sicca or allergic conjunctivitis, fungal infection, viral ease, giant papillary conjunctivitis, gonorrhea, growth-hor infection or bacterial infection. In some embodiments, the mone secreting pituitary adenoma, hairy leukoplakia, hemo disease or condition of the eye is glaucoma, keratoconjunc philus influenzae B, hemorrhagic conjunctivitis, hemorrhagic tivitis sicca or allergic conjunctivitis. In some embodiments, proctocolitis, herpes, human papillomavirus, immotile cilia 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, 0209. 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, 0210. 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, 0211. 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 0212. 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, 0213 Subject cronkhite-Canada syndrome, cystic Fibrosis, cystitis, derma 0214. 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, 0215 Methods of Manufacture eruptive lingual papillitis, erythroplakia, esophageal ulcer, 0216. In another aspect, the present disclosure provides a esophagitis, extrinsic asthma, familial adenomatous polypo process for the preparation of macromolecules useful in the sis, familial intestinal polyposis, familial nasal acilia, familial formation of a mucoadhesive nanoparticle delivery system. US 2014/0005379 A1 Jan. 2, 2014

The macromolecule is typically an amphiphilic copolymer, in 0224. In some embodiments, step b) is performed before particular, a block copolymer, which is conjugated to a plu step c). However, in other embodiments, step c) is performed rality of mucosal targeting moieties. The macromolecules are before step b). capable of assembly under Suitable conditions to form a nano 0225. In some embodiments, the surface density of the particle, i.e. of the core-shell type. In an aqueous environ mucosal targeting moiety on the nanoparticle is controlled by ment, the nanoparticle has a hydrophobic core and a hydro the amount of mucosal targeting moiety introduced into the philic shell, the shell providing a surface of the nanoparticle, reaction. the Surface of the nanoparticle being coated in a desired 0226. In some embodiments, the process comprises reduc amount (i.e. Surface density) of the mucosal targeting moiety tive animation between the multimer and a suitable linker. In for controlled targeting and adhesion of the nanoparticle. Some embodiments, the reaction takes place between the 0217. The macromolecules disclosed herein may be made amine end of N-protected-ethylenediamine and the reducing by any suitable process known to those skilled in the art, for end of a multimer having multiple functional groups per example, using Suitable conjugation techniques. Starting monomer unit, Such as a polysaccharide, a polynucleotide or materials, including hydrophobic polymer and hydrophilic a polypeptide. Any suitable N-protecting group can be used. polymer, may be purchased from various commercial Suppli In some embodiments, the N-protecting group is tert-butoxy ers. Where desired, the starting materials can be prepared by carbonyl (BOC). those of skill in the art. For example, where polymers com 0227. The choice of a hydrophilic polymer having mul prising modified backbone residues are desired. Exemplary tiple functional groups per monomer unit enables tuning of methods for making macromolecules useful in the formation the resulting nanoparticle to control particle size, targeting of a mucoadhesive delivery system are described below. and/or adhesion at a mucosal site, as described further below. 0218. In some embodiments, there is provided a method of In some embodiments, the hydrophilic polymer is a polysac preparing a nanoparticle composition. charide. In some embodiments, the hydrophilic polymer is 0219. In some embodiments, the method is carried out in a dextran. Therefore, in Some embodiments, the reaction takes series of steps, such as, preparation of an amphiphilic mac place between N BOC-ethylenediamine and an aldehyde of romolecule, nanoparticle formation, and conjugation to a tar the reducing end of a dextran polymer. The reaction may be geting moiety (i.e. coating of the Surface of the nanoparticle carried out in a suitable solvent, such as a borate buffer with a desired Surface density of the targeting moiety). Alter Solution, in the presence of a reducing agent, Such as NaC natively, the hydrophilic portion comprising multiple func NBH. The mixture is stirred for a sufficient amount of time tional groups may first be coupled to a desired amount of the to complete the reaction, for example, about 24 to 120 hours targeting moiety, followed by conjugation of the functional hours. In one embodiment the mixture is stirred for about 24, ized hydrophilic portion to a hydrophobic polymer, which 48, 72, 96, or 120 hours. In some embodiments, this step is may be in the form of a hydrophobic nanoparticle (i.e. coating carried out at room temperature. In some embodiments, this the surface of a hydrophobic nanoparticle with a functional step is carried out in the dark. ized hydrophilic polymer). When the hydrophobic polymer is 0228. The mixture may then be washed to remove any modified for conjugation, one end of the polymer will typi unreacted molecules or catalysts. In one embodiment metha cally become more hydrophilic (e.g. presence of a carboxyl nol is used in the washing step. The end-modified dextran can group). Such polymers can assemble to form hydrophobic optionally be dried before continuing the process. nanoparticles in aqueous medium. Preparation of the nano 0229. The protecting group is then removed followed by particles in a controlled sequence results in Surface-function conjugation of the amine-terminated multimer to a hydropho alized nanoparticles wherein substantially all (e.g. greater bic polymer in a suitable solvent to provide an amphiphilic than 90%, 95%, 96%, 97%, 98%, 99%) of the targeting moi macromolecule. In one embodiment, hydrochloric acid and eties are located on the surface of the nanoparticle formed by triethyl amien are used for the removal of the protecting the hydrophilic portion of the macromolecules. group. The macromolecule may be washed, for example, 0220. In one embodiment, the method of preparing a nano using methanol, to remove unreacted polymer. particle composition useful for delivery of a payload to a 0230. The conjugation of the amine-terminated multimer mucosal site comprises preparing an ampliphilic macromol with a hydrophobic polymer takes place in a suitable solvent. ecule comprising a hydrophilic portion and a hydrophobic In one embodiment, the solvent is DMSO, acetone, or aceto portion, the hydrophilic portion comprising multiple func nitrile. Catalysts may be employed to drive the reaction. In tional moieties; b) assembling a plurality of said macromol one embodiment, the catalysts are EDC and Sulfo-NHS. ecules under Suitable conditions to form a nanoparticle hav 0231. The mixture may then be washed to remove any ing a hydrophobic core and a hydrophilic shell; and c) unreacted molecules or catalysts. In one embodiment metha conjugating at least a portion of said functional moieties on nol is used in the washing step. Additional washing step may the hydrophobic portion to a mucosal targeting moiety, to be used to remove unreacted polymer. In one embodiment, thereby provide a Surface-functionalized nanoparticle. the unreacted polymer is dextran. The final mixture is dis Solved in a suitable solvent, centrifuged and the resulting 0221. 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 0222. In some embodiments, the hydrophilic polymer is uct. dextran and the hydrophobic polymer is PLA. 0232. In another aspect, the present disclosure provides a 0223. 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 US 2014/0005379 A1 Jan. 2, 2014 20 skilled in the art. The geometry formed by the particle from for the synthesis of Dex-b-PLA may be divided into three the macromolecule may depend on factors such as the size stages: reductive amination between Dextran and N-Boc and composition of the polymers that form the macromol ethylenediamine, deprotection of the Boc group, and conju ecule. In addition, also as discussed below, in Some cases, the gation of the amine-modified Dextran end group with car particle may include an agent of interest. Such as a therapeu boxyl-terminated PLA. Reductive amination may be carried tic, diagnostic or imaging agent. For example, in some out by dissolving Dex in aborate buffer and mixing it with embodiments, the nanoparticle may contain a therapeutic N-Boc-ethylenediamine and NaCNBH in dark condition for agent, such as a drug. The agent of interest may be incorpo rated into the particle during formation of the particle, e.g., by about 72 hrs. After the reaction, the mixture is washed with 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 0233. 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 the nanoparticles via coupling of the functional groups to a 0241 Thus, in some embodiments, there is provided a mucosal targeting moiety, Such as a targeting moiety capable method of preparing a Dex-b-PLA macromolecule, the of forming high affinity binding to a target at the mucosal site. method comprising: 1) reductive amination between Dextran 0234. In some embodiments, the targeting moieties are and N-Boc-ethylenediamine, 2) deprotection of the Boc conjugated to the macromolecules following nanoparticle group, and 3) conjugation of the end modified Dextran with formation. PLA (Scheme 1). The first step of the synthesis involves 0235. The present disclosure also provides a process for reductive amination between the aldehyde on the reducing conjugating targeting moieties on the surface of the nanopar end of Dextran and the amine group of N-Boc-ethylenedi ticles formed using the amphiphilic macromolecules amine cross-linker. The reducing agent, NaCNBH was described herein. In some embodiments, the conjugation is added to the borate buffer solution and the mixture was stirred between the functional groups of the hydrophilic portion (e.g. for 72 hours in dark conditions at room temperature. The the backbone of a hydrophilic polymer) and the functional mixture was then washed in methanol to remove any unre groups of the targeting moieties. In some embodiment, cata acted molecules or catalysts. The end-modified Dextran was lysts, for example, EDC, are used for the conjugation reac dried overnight in vacuo. The dried Dextran was re-dissolved tion. In some embodiments, the functional groups of the in de-ionized water (DI-HO). The deprotection of Boc group polymer backbone are modified into other types of functional was performed first by adding HCl for 1 hour to cleave the groups prior to the conjugation reaction. In one embodiment, amide bond between the Boc group and the protected amine NaIO is used to oxidize hydroxyl groups into aldehyde moiety. Subsequently, TEA was added to increase the pH of groups. The mixture may be washed, for example, using the solution up to 9 to deprotonate the NH' end groups which methanol, to remove nonconjugated targeting moieties. In were deprotected. The mixture was then washed twice using Some embodiment, dialysis is used to remove the unreacted methanol and dried in vacuo. An NMR sample of the dried molecules. product was prepared in DO (). The amine terminated Dex 0236. In some cases, the method may include conjugation tran and carboxyl terminated PLA20 (Mw-20 kDa, 6 g., 0.3 with more than one type of targeting moiety. The Surface mmol) were dissolved in DMSO. The conjugation between density of targeting moiety on the resulting nanoparticles the two polymers was facilitated by adding catalysts EDC may be controlled by adjusting the amount of material in the (120 mg, 0.773 mmol) and Sulfo-NHS (300 mg, 1.38 mmol) reaction mixture. and allowing reaction to proceed for 4 hours at room tem 0237. In some embodiments, conjugation and nanopar perature. The resulting Dex-b-PLA was twice precipitated ticle formation may occur as a single-step reaction, for and purified using excess methanol. In order to remove free example, according to a single-step reaction as described in Dextran, the mixture was dissolved in acetone (30 mL) to U.S. Pat. No. 8.323,698. However, a single-step reaction such form a cloudy suspension. This was centrifuged at 4000 rpm as this will resultina nanoparticle having a detectable amount for 10 minutes and the Supernatant was extracted carefully. of targeting moieties located within the core of the nanopar The Supernatant was purged with air to remove the solvent ticle, thereby decreasing the targeting efficiency of the par and then dried overnight in vacuo to obtain the final copoly ticles compared to a more controlled sequence as described CS. above. 0242 To functionalize the polymers, Dex-b-PLA may be 0238 Specific reaction conditions can be determined by dissolved in DMSO (30 mg/ml), and added slowly into water those of ordinary skill in the art using no more than routine under mild stirring. Periodate oxidation of the Dextran sur experimentation. face was carried out by adding 60mg of NaIO and stirring for 0239 Embodiment of the Method an hour. Subsequently, glycerol was added to quench the 0240 An exemplary method of preparing a Dextran-b- unreacted NaIO. Various amounts of PBA (i.e. 40 mg for PLA (Dex-b-PLA) block copolymer is described below in Dex-b-PLA 40PBA) were added to the mixture, along with Example 1 (Verma, 2012). Briefly, an exemplary procedure NaCNBH for 24 hours. All reactions were carried out in the US 2014/0005379 A1 Jan. 2, 2014

dark. The mixture was then dialyzed in water for 24 hrs to (PBA) derivative, a thiol derivative or an acrylate derivative, remove any unreacted Solutes, through changing the wash wherein at least a portion of said functional moieties of the medium 4 times. hydrophilic portion are conjugated to the mucosal targeting 0243 The polymers or macromolecules described herein moiety. may be formed into a nanoparticle using techniques know to 0250 In some embodiments, there is provided a nanopar those skilled in the art, including those discussed in detail ticle composition useful for delivery of a payload to a below. The geometry formed by the particle from the polymer mucosal site, the nanoparticle comprising a plurality of or macromolecule may depend on factors such as the poly amphiphilic macromolecules, the macromolecules each com mers that form the particle. In addition, also as discussed prising: a hydrophobic biocompatible polymer selected from below, in some cases, the particle may include a hydrophilic a from polylactide, a polyglycolide, poly(lactide-co-gly 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) 0244. The Dex-b-PLANPs were prepared using nanopre derivative, a thiol derivative or an acrylate derivative. cipitation method: 1 mL of Dex-b-PLA in DMSO (10 0251. In some embodiments, there is provided a nanopar mg/mL) was added in a drop-wise manner to 10 mL of ticle composition useful for delivery of a payload to a DI-HO under constant stirring in order to form NPs. This mucosal site, the nanoparticle comprising a plurality of was stirred for 30 minutes and then dynamic light scattering amphiphilic macromolecules, the macromolecules compris (DLS) samples were prepared by extracting 3 mL samples ing: a hydrophobic portion comprising a polylactide; a hydro into polystyrene cuvettes. The sizes of the NPs were analyzed philic portion having multiple functional moieties, said using 90Plus Particle Size Analyzer (Brookhaven, v=659 nm hydrophilic portion comprising dextran; and a mucosal tar at 90°). The volume averaged multimode size distribution geting moiety being a phenylboronic acid (PBA) derivative, (MSD) mean diameters were used from the results. wherein at least a portion of said functional moieties of the 0245. In addition, the method may employ additional hydrophilic portion are conjugated to the mucosal targeting polymers or macromolecules, which may be distinguishable moiety. from the polymers or macromolecules discussed above. As 0252. In some embodiments, there is provided a nanopar previously discussed, the first and second macromolecules ticle composition useful for delivery of a payload to a may be combined together at different ratios to produce par mucosal site, the nanoparticle comprising a plurality of ticles comprising the first and second macromolecules. amphiphilic macromolecules, the macromolecules each com 0246. In some cases, the method may include conjugation prising a hydrophobic polylactide polymer conjugated to a with more than one type of targeting moiety. The Surface hydrophilic dextran polymer having multiple functional moi density of targeting moiety on the resulting nanoparticles eties, at least a portion of said functional moieties being may be controlled by adjusting the amount of material in the conjugated to a phenylboronic acid (PBA) derivative. reaction mixture. 0253. In some embodiments, the macromolecule is Dex 0247 Alternatively, the reaction may occur as a single tan-p-PLA. In some embodiment, the functionalized macro step reaction, i.e., the conjugation is performed without using molecule is Dextran-p-PLA PBA. intermediates Such as N-hydroxySuccinimide or a maleimide, 0254. In some embodiments, the nanoparticle is formed such as that described in U.S. Pat. No. 8,323,698. However, by conjugating the polylactide to the dextran to form macro Such method results in a nanoparticle having a portion of the molecule, then forming a nanoparticle, and Subsequently Sur targeting moiety located in the core of the particle. Thus, face-functionalizing the nanoparticle by conjugating at least a typically, a multi-step approach will be used to achieve higher portion of the functional moieties of the dextran to the PBA targeting efficiently. derivative to achieve a desired surface density of the PBA 0248 Specific reaction conditions can be determined by derivative. those of ordinary skill in the art using no more than routine 0255 All definitions, as defined and used herein, should experimentation. be understood to control over dictionary definitions, defini tions in documents incorporated by reference, and/or ordi Particular Embodiments nary meanings of the defined terms. 0249. In some embodiments, there is provided a nanopar 0256 The indefinite articles“a” and “an as used herein in ticle composition useful for delivery of a payload to a the specification and in the claims, unless clearly indicated to mucosal site, the nanoparticle comprising a plurality of the contrary, should be understood to mean “at least one.” amphiphilic macromolecules, the macromolecules compris 0257. In the claims, as well as in the specification above, ing: a hydrophobic portion comprising a biocompatible poly all transitional phrases such as “comprising.” “including.” mer selected from a from polylactide, a polyglycolide, poly “carrying.” “having.” “containing.” “involving,” “holding.” (lactide-co-glycolide), poly(e-caprolactone), or a “formed from, “composed of and the like are to be under combination thereof; a hydrophilic portion comprising a bio stood to be open-ended, i.e., to mean including but not limited compatible polymer selected from polysaccharide, poly tO. nucleotide, polypeptide, or a combination thereof, the hydro 0258. Only the transitional phrases “consisting of and philic portion comprising multiple functional moieties; and a “consisting essentially of shall be closed or semi-closed mucosal targeting moiety selected from a phenylboronic acid transitional phrases, respectively. US 2014/0005379 A1 Jan. 2, 2014 22

0259. The following examples are intended to illustrate Dextran and N-Boc-ethylenediamine, 2) deprotection of the certain exemplary embodiments of the present disclosure. Boc group, and 3) conjugation of the end modified Dextran However, the scope of the present disclosure is not limited to with PLA (Scheme 1). The first step of the synthesis involves the following examples. reductive amination between the aldehyde on the reducing end of Dextran and the amine group of N-Boc-ethylenedi EXAMPLES amine cross-linker. In a typical reaction, Dex6 (M-6 kDa, 6 Example 1 g, 1 mmol) was dissolved in 15 mL of borate buffer (0.05 M. pH 8.2) with 4 g (2.5 mmol) of N-Boc-ethylenediamine. The Synthesis and Characterisation of Dex-b-PLA reducing agent, NaCNBH (1 g, 15 mmol), was added to the 1.1 Materials borate buffersolution and the mixture was stirred for 72 hours in dark conditions at room temperature. The mixture was then 0260 Acid-terminated poly(D, L-lactide) (PLA, M-10, washed in methanol to remove any unreacted molecules or 20 and 50 kDa) and PLGA-PEG (PLGA M-40 kDa, PEG catalysts. The end-modified Dextran was dried overnight in M-6 kDa) were purchased from Lakeshore Biomaterials (Birmingham, Ala., USA). PLA was purified by dissolving in vacuo. H NMR samples were prepared by dissolving the dimethylsulfoxide (DMSO) and precipitating in methanol to end-modified Dextran in DO (30 mg/mL). The dried Dext remove residual monomers. Dextran (Dex, M-1.5, 6, and 10 ran was re-dissolved in de-ionized water (DI-HO). The kDa), hydrochloric acid (HCl), triethylamine (TEA), N-(3- deprotection of Boc group was performed first by adding HCl dimethylaminopropyl)-N-ethylcarbodiimide (EDC), and (~4M) for 1 hour to cleave the amide bond between the Boc sodium cyanoborohydride (NaCNBH) were purchased from group and the protected amine moiety. Subsequently, TEA Sigma Aldrich (Oakville, ON, Canada), and used without was added to increase the pH of the solution up to 9 to further purification. N-Hydroxysulfosuccinimide (Sulfo deprotonate the NH' end groups which were deprotected. NHS) and N-Boc-ethylenediamine were purchased from The mixture was then washed twice using methanol and dried CNH Technologies (Massachusetts, USA). Doxorubicin in vacuo. An NMR sample of the dried product was prepared HCl (MW=580 Da, Intatrade GmBH, Bitterfield, Germany) in DO (30 mg/mL). The amine terminated Dextran and car was deprotonated by adding TEA (2M equivalent) in the boxyl terminated PLA20 (Mw-20 kDa, 6 g., 0.3 mmol) were aqueous solution of Doxorubicin-HCl, and the hydrophobic dissolved in DMSO. The conjugation between the two poly form of Doxorubicin was extracted using Dichloromethane mers was facilitated by adding catalysts EDC (120 mg. 0.773 (DCM) (Chittasupho. 2009). Borate buffer was prepared at a mmol) and Sulfo-NHS (300 mg, 1.38 mmol) and allowing concentration of 0.05M with pH of 8.2 by mixing boric acid reaction to proceed for 4 hours at room temperature. The and sodium hydroxide. Whole sheep blood (in Alsevers) was resulting Dex-b-PLA was twice precipitated and purified purchased from Cedarlane (Burlington, ON, Canada). using excess methanol. In order to remove free Dextran, the Veronal Buffer solution (VBS, 5x) was purchased from mixture was dissolved in acetone (30 mL) to form a cloudy Lonza Walkersville Inc (Walkersville, Md., USA). Tritium suspension. This was centrifuged at 4000 rpm for 10 minutes HI-PLA-radiolabeled nanocrystals were purchased from and the Supernatant was extracted carefully. The Supernatant PerkinElmer (Boston, Mass., USA). was purged with air to remove the solvent and then dried overnight in vacuo to obtain the final copolymers. NMR 1.2 Synthesis of Dex-b-PLA samples were prepared at a concentration of 30 mg/mL in 0261 The synthesis of the linear block copolymer is DMSO-d6 for proton NMR and 150 mg/mL in DMSO-d6 for divided into three stages: 1) reductive amination between carbon NMR.

Scheme 1. H OH

HO O HO OH O

HO O H 2 HO OH O

HO O HO OH

Dextran

US 2014/0005379 A1 Jan. 2, 2014 24

-continued 1stCH3

a' Synthesis of Dex-b-PLA block copolymers, a) Synthesis of Dextran-NH-Et-NH-Boc. Conditions: NaCNBH in Borate buffer (pH 8.2) for 72 hr at RT in dark. b): Synthesis of Dextran-NH-Et-NH2. Conditions: HCITEA in DI-H2O for 1 hr each at RT. c): Synthesis of Dextran-NH-Et-NH-PLA. Conditions: EDC/Sulfo-NHS RT for 4 hrs.

1.3 Characterization of Dex-b-PLA Using Nuclear Magnetic presence of 1.3 ppm peak (Boc group) after removing unre Resonance (NMR) acted free N-Boc-ethylenediamine (FIG. 1a II). The subse quent deprotection of Boc group exposing the —NH2 end 0262 The various stages of Dex-b-PLA synthesis were group on Dextran was verified by the disappearance of the 1.3 verified using H NMR spectroscopy (Bruker 300 MHz). The ppm peak (FIG. 1a III). It was shown that the 1.3 ppm peaks final polymer conjugation was also verified using C NMR were completely removed after the deprotection steps using spectroscopy (Bruker 300 MHz). Before any modification, HCl and TEA. After the conjugation of the NH terminated Dextran was dissolved in DO (30 mg/mL) and acid termi Dextran with COOH-terminated PLA (FIG. 1a IV), the nated PLA was dissolved in CDC1 (5 mg/mL) for preparing excess free Dextran molecules were removed by precipitating NMR samples. As mentioned in the previous synthesis meth in acetone. The final product shows peaks corresponding to ods, the end products from the first two steps were dissolved both the Dextran (multiplets at 4.86 ppm) and the PLA (mul in DO, whereas the final product, Dex-b-PLA, was dissolved tiplets at 5.2 ppm) which confirm the conjugation of the two in DMSO-d6 for the NMR analysis. polymers (FIG. 1a V). The linear end-to-end conjugation of PLA and Dextran was also confirmed by Carbon NMR (FIG. 1.4 Dex-b-PLANP Formation by Nanoprecipitation 1b). The peak at 166.81 ppm is assigned to the carbon on PLA 0263. The Dex-b-PLANPs were prepared using nanopre that attaches to the amine terminal of the ethylenediamine cipitation method: 1 mL of Dex-b-PLA in DMSO (10 linker, while 169 ppm peak is the carbonyl carbon on PLA mg/mL) was added in a drop-wise manner to 10 mL of backbone (FIG. 1b). DI-HO under constant stirring in order to form NPs. This 0266 The size and morphology of NPs using nine formu was stirred for 30 minutes and then dynamic light scattering lations of Dex-b-PLA block copolymers are shown in FIG. 2. (DLS) samples were prepared by extracting 3 mL samples Varying the MW of PLA and Dextran resulted increating NPs into polystyrene cuvettes. The sizes of the NPs were analyzed with different sizes ranging from 15 to 70 nm. As shown in using 90Plus Particle Size Analyzer (Brookhaven, v=659 nm FIG. 2a, increasing the MW of PLA increased the particle at 90°). The volume averaged multimode size distribution size whereas increasing the MW of Dextran decreased the (MSD) mean diameters were used from the results. particle size. The NP core, formed by PLA, was predicted to increase in size with increasing MW’s of the PLA chains as 1.5 Transmission Electron Microscopy demonstrated previously (Riley 1999; Riley 2001) and it was confirmed here by the NPs composed of PLAMW of 10 kDa, 0264. The particle size and the morphology of the Dex-b- 20 kDa and 50 kDa. We postulate that the effect of Dextran PLANPs were further verified using Transmission Electron MW on NP size is likely due to Dextran configuration on the Microscopy (TEM, Philips CM10) with the accelerating volt NP surface. Zahr et al. found that hydrophilic chains, such as age of 60 kV and the Lanthanum Hexaboride filament PEG, at MW of 5kDa or longer would be able to “fold-down” (LaB6). 300 Mesh Formvar coated copper grids (Canemco & onto the particle Surface creating a mushroom conformation Marivac) were used for this experiment. The NP suspension (Zahr 2006). Similarly, this phenomenon may explain why in water was prepared using the nanoprecipitation method as mentioned above. A drop of the NP suspension was placed the NPs with longer Dextran chains lead to smaller hydrody onto the grid, and the grid was briefly stained with aqueous namic diameters. The shorter Dextran chain length has a phosphotungstic acid solution. The copper grid with the NP Smaller degree of freedom and confined to linear structure Suspension was dried under ambient environment overnight compared to those with longer chain length. The TEM image before imaging under TEM. of NPs composed of PLA20-Dex6 (MW-20 kDa, MW extrapa ~6 kDa) confirmed the particles exhibit spherical 1.6 Results and Discussion structure (FIG.2b). 0267 Dex-b-PLANPs with sizes under 50 nm have been 0265. The synthesis of Dex-b-PLA block copolymers was synthesized using the simple process of bulk nanoprecipita analyzed using H NMR spectrometer. As shown in FIG. 1a I, tion. PLGA-PEG block copolymer was used as a commercial the 4.86 ppm multiplet was assigned to the proton on carbon benchmark, which formed NPs with size 133.9-6.1 nm fol 1 of Dextran repeating units. The 3.14 ppm multiplet was lowing the same procedure. The particle size for PLGA-PEG assigned to the proton on carbon 5 of the non-reducing end the is in agreement to previous literature values (Dhar 2009). integral ratio between these two multiplets was used to con PLGA-PEG NPs have been able to achieve smaller particle firm the MW of Dextran. The reductive amination reaction of sizes but it required the assistance of microfluidic devices for Dextran and N-Boc-ethylenediamine was confirmed by the enhanced control (Karnik 2008). The particle size of Dex-b- US 2014/0005379 A1 Jan. 2, 2014

PLA NPs, on the other hand, can be controlled simply by 7.1 wt.%. It was found that excess initial loading caused more changing the MW of the compositional polymers as exempli drug precipitation and particle aggregation during nanopre fied in FIG. 2a. cipitation for PLGA-PEG NPs, whereas Dex-b-PLA NPs showed negligible size increases even at their maximum drug Example 2 loading. The maximum Doxorubicin loading achieved with PLA20-Dex10 NPs were considerably higher than the most Encapsulation and In Vitro of Doxorubicin in reported values using PEG based NPs in the literatures, which Dex-b-PLANPs Via Nanoprecipitation varied over 4.3-11.2% for poly(e-caprolactone)-PEG copoly 0268. The encapsulation of Doxorubicin in the Dex-b- mers (Shuai 2004; He 2010), 8.7% for poloxamer 407 and PLANPs was accomplished using nanoprecipitation method. PEG hydrogel system (Missirlis 2006), and 18% for PEG Dex-b-PLA and Doxorubicin were both dissolved in DMSO poly(3-benzyl-L-aspartate) based NPs (Kataoka 2001). The (Dex-b-PLA concentration of 7 mg/mL, with varying drug increased drug loading is most likely due to the greater hydro concentrations). 1 mL of the DMSO solution is added drop philicity of Dextran compared to PEG (Alpert 1990), which wise into 10 mL of water under stirring and continued to stir in turn reduces the probability of Dextran chains from the for additional 30 minutes. The NPs in water were filtered block copolymers associating in the hydrophobic core of the through syringe filter (pore size=200 nm) to remove the drug NPs. The encapsulation efficiency and the total drug payload aggregates and Subsequently filtered through Amicon filtra using the Dex-b-PLA system is comparable to commercially tion tubes (MWCO=10kDa, Millipore) to further remove any available liposomal systems such as the FDA approved remaining free drugs in the suspension. The filtered NPs Doxil R, which has a drug loading of 12.5% and DaunoX containing encapsulated Doxorubicin were resuspended and omeR), which has Daunorubicin loading of 7.9% (Drummond diluted in DMSO. Consequently, the drug loading (wt %) in 1999). The concentration of Doxorubicin in the Doxil(R) for the polymer matrix was calculated by measuring concentra mulation translates into 6.25 mg/m when Doxil R is admin tion of the Doxorubicin in the mixture by obtaining the absor istered at 50 mg/m (Drummond 1999; Safra 2000). The same bance of the solution at 480 nm using Epoch Multi-Volume physiological concentration of Doxorubicin can theoretically Spectrophotometer System (Biotek). The measurements be achieved using only 30 mg/m of PLA20-Dex10 were obtained in triplicates (n=3, mean+S.D). The absor NP-Doxorubicinformulation. bance measured from same procedure using the polymers Example 3 without the drugs was used as the baseline. The absorbance was correlated with the concentration of the Doxorubicin in In Vitro Release of Doxorubicin from Dex-b-PLA DMSO by using standard calibration obtained. The same procedure was used for PLGA-PEG to encapsulate Doxoru NPS bicin for comparative analysis. The encapsulation efficiency 0270. Using the procedure described in the previous (%) and drug loading (wt %) were calculated using the two example, drug encapsulated NPs were prepared and filtered to equations (Eq. 1 and Eq. 2). remove non-encapsulated drug aggregates. A purified sample of NPs-drug Suspension was collected to measure the maxi mumabsorbance and this was used as the 100% release point. mass of drug encapsulated (1) Encapsulation efficiency(%) = x 100% Subsequently, the NP-drug Suspension was injected into a mass of initial drug feed Slide-a-Lyzer Dialysis cassette (MWCO=20kDa, Fisher Sci mass of drug encapsulated (2) entific) and dialyzed against 200 mL of phosphate buffered Drug loading (wt %) = x 100% mass of the nanoparticle saline (PBS, pH 7.4) at 37°C. under mild stirring. At prede termined time intervals, 1 mL of the release medium was extracted and the same volume of fresh new PBS was added 0269 Based on the size tuning as shown in FIG.2, PLA20 to the release medium. The extracted release medium was Dex10 (MWPLA-20 kDa, MWDextran-10 kDa) and used to perform UV-Vis absorption measurements at 480 nm PLA20-Dex6 were selected for analyzing the encapsulation in triplicates (n=3, meantS.D). The release medium was efficiencies and the drug loading using Doxorubicin as a replaced several times to maintain the concentration of Doxo model hydrophobic drug (FIG. 3). The particle sizes for rubicin in the medium below 3 lug/mL and to stay below the PLA20-Dex10 and PLA20-Dex6 were 20.5 and 30.1 nm solubility limit of the Doxorubicin in PBS. Replacing the respectively. Doxorubicin compounds were incorporated into medium was also expected to prevent the adhesion of released NPs through nanoprecipitation method. Both Dextran based Doxorubicin to the glass walls of the beaker or the magnetic NPs, PLA20-Dex10 and PLA20-Dex6 NPs, were found to Stir bar. The release of Doxorubicin from PLGA-PEG was encapsulate large amounts of Doxorubicin with maximum also obtained with identical procedure for comparative analy loadings of 21.2 and 10.5 wt % respectively. The maximum sis. Free Doxorubicin, without any polymers, release was also loadings were achieved at 40 wt % initial loading, and further observed using the same procedure and all three release pro increase in the initial loading did not increase the drug loading files from the NPs were normalized using the free Doxorubi in the NPS due to aggregation of the particles. It is speculated cin release data along with encapsulation efficiency data. This that PLA20-Dex10, with longer Dextran chain than PLA20 normalization resulted in a release curve for only encapsu Dex6, is likely to have more Doxorubicin weakly associated lated Doxorubicin. All experiments were performed in dark on the NP surface or encapsulated near the surface of the NPs environment, and the beakers were sealed with Parafilm to during nanoprecipitation. This effect was minimized by con prevent evaporation of PBS. ducting ultrafiltration (MWCO=10 kDa) after the nanopre 0271 The in vitro release of Doxorubicin from the NPs cipitation ensuring that the non-specifically bound drugs was carried out in pH 7.4 PBS buffer at 37° C. As shown in were removed from the NP suspension. The maximum drug FIG. 4, the release profile of Doxorubicin from NPs was loading in PLGA-PEGNPs, used as a control, was found to be characterized with an initial burst followed by a sustained US 2014/0005379 A1 Jan. 2, 2014 26 release phase. It is possible that the burst-release region cor prior to NP formation. Albino Wistar rats, body weight responds to drugs non-specifically bound on the Surface of the between 200 and 250 g, were fasted overnight but had free NPs, or drugs encapsulated near the surface of the NPs during access to water. 200LL of the NP formulations were prepared the nanoprecipitation procedure (Magenheim 1993). PLA20 in NaCl 0.9% and injected intravenously into the tail vein at a Dex6 and PLA20-Dex10 NPs exhibited burst-release region dose of approximately 30 mg/kg. Blood (approximately 200 within the initial 24 hours, releasing up to 48% and 74% LL) was collected in heparinized microcentrifuge tubes by respectively. The Subsequent Sustained-release phase of controlled bleeding of hind leg saphenous veins at the indi Doxorubicin from PLA20-Dex6 and PLA20-Dex10 NPs cated time intervals. To characterize the biodistribution of continued for 192 hours with similar rate of release from both NPs, rats were euthanized at 24 h after NP injections. NPs. The sustained-release phase may correspond to the dif Approximately 200 uL of blood was drawn by cardiac punc fusional release of the drugs from the core of the NPs. In the ture from each mouse. Organs including heart, lungs, liver, control study using PLGA-PEG NPs, the burst-release phase spleen and kidneys were harvested from each animal as of Doxorubicin was within the first 6 hours while steady described previously (Gu 2008). The H content in the tissue release phase continued up to 96 hours, similar to what has and blood were assayed in a Wallac 1414 Liquid Scintillation been reported previously (Esmaeili 2008). Counter. 0275. The NP circulation half-life in vivo was character Example 4 ized by measuring the amount of tritium H-PLA-radiola beled nanocrystals that were incorporated in the NP formu Hemolysis Assay lations. FIG. 6 shows NP concentration in blood circulation at (0272 Dex-b-PLA NPs were purified by using Amicon predetermined time intervals after intravenous administra filtration tubes (MWCO=10 kDa) and centrifugation at 4100 tion. It is noted that the time-dependent NP concentration in rpm for 30 minutes. A concentration range of NPs was the blood were characterized by two regions of distinct obtained by this process. These NPs were then incubated at slopes. The first region (first ~18 hrs) corresponds to the 37° C. for one hour with 200 uL of sheep erythrocytes with initial clearance of the NPs from the blood circulation, red blood cells concentration of 1x10 cells/mL to obtain a whereas the second region indicates the terminal clearance of final Volume of 1 mL per sample. The percent hemolysis was the NPs. The former region profiles the NP volume of distri calculated by measuring the absorbance at 415 nm and using bution among vascular and extravascular tissues, while the the absorbance at 500 nm as the baseline. The measurements terminal half-life relates to the systemic clearance of the NPs were conducted in triplicates (meantS.D). Here, VBS solu from the body (Yang 2009). The initial half-life (t), termi tion was used as the negative control and deionized water was nal half-life t2), the blood retention time for 90% of the NPs used as the positive control. PLGA-PEG NPs were also pre (too), and AUC (Gaucher 2009) of the three NPs are summa pared and tested in a similar manner for comparison. rized in Table 1. At 24 hours postinjection, rats were eutha 0273 Previous work has considered hemolysis of NPs less nized, and the major organs were harvested from the animals than 5% to be biocompatible (Dobrovoiskaia 2008). It has to evaluate the biodistribution of the NPs (FIG. 7). It was been demonstrated that PLGA NPs stabilized by surfactants observed that all three NPs had maximum accumulation in the are severely hemolytic to 80% and hemolysis is reduced liver and the percent distribution was similar for each NP, considerably by using a hydrophilic PEG surface in the case Higher accumulations in the spleen were observed with of PLGA-PEG NPs (Kim 2005). The same results were PLGA-PEG NPs compared to both of Dex-b-PLANPs (p<0. expected from the use of Dextran based NP formulation since 01). Accumulation of NPs in all other organs was below 5% Dextran derivatives such as diethylaminoethyl-dextran have with similar amount of accumulation among the NPS in each low (-5%) hemolysis (Fischer 2003). The block copolymer Organ. NPs formulated previously were tested for hemolytic activity 0276 Although all three types of NPs showed similart at various concentrations (1-10 mg/mL). It was shown that all values, both PLA20-Dex10 and PLA20-Dex6 NPs showed formulated NPs were not significantly hemolytic (<5%) up to significantly higher values oft, too, and AUC compared to a concentration of 10 mg/mL in the blood (FIG. 5). The that of the model NPs composed of PLGA-PEG. Previous hemolysis by both PLA20-Dex6 and PLA20-Dex 10 were studies mainly focused on t values for NPs but the present similar since they have the same component polymers. For inventors extracted too values for comparison purposes. It comparison, Doxil(R) (a liposomal formulation of doxorubi was observed that to values were only about 2 hrs for PEG cin) is usually administered at the dose of 50 mg/m (Safra b-PLANPs (Gaucher 2009), 6 hrs for polyvinylpyrrolidone 2000). This dose translates to a concentration of 0.018 mg/mL based NPs (Gaur 2000) and about 8 hrs for chitosan based in blood for an average human being (body surface area 1.79 NPs (He 2010). Not only do Dex-b-PLA NPs outperform m (Sacco 2010), and blood volume 5 L) (Kusnierz-Glaz these NPs with a to of 38.3 hrs, they are also comparable to 1997). The tested hemocompatible concentration (10 60 nm PEG-b-PCL system (Lee 2010) and StealthR) lipo mg/mL) for PLA-b-Dex NPs is considerably higher than the somes (Allen 1991), both of which have to values over 48 administered dose of Doxil R. This suggests that PLA-b-Dex hours. In this study, the longer blood circulation observed in NPs are a safe system for intravenous administration. Dex-b-PLANPs, compared to PLGA-PEG NPs, is believed to be partially due to the size difference. A recent study by Example 5 Rehor et al. showed that NPs with diameter of 40 nm had longer circulation half-life compared to larger NPs with Pharmacokinetics and Biodistribution of Dex-b-PLA diameter of 100 nm (Rehor 2008). It is hypothesized that NPS Dex-b-PLANPs, having smaller sizes than PLGA-PEGNPs, 0274. To ensure that all radioactivity administered to rats have increased curvature that reduce protein adsorption, was associated with the particles, tritium H-PLA-radiola which may in turn result in slower clearance rate by the RES. beled nanocrystals were washed and purified in methanol This is further supported by the longer blood circulation time US 2014/0005379 A1 Jan. 2, 2014 27 of PLA20-Dex10 compared PLA20-Dex6 since the former dried in vacuum desiccator. The sample is then dissolved in has smaller particle size. In addition to their size effect on DI-HO and treated with hydrochloric acid and triethylamine protein adsorption, it is also hypothesized that the abundant for the deprotection of the Boc group. The conjugation of hydroxyl groups on the Dextran Surface may induce Sufficient amine-terminated Dextran and PLA was carried out in hydration layer around the NPs to limit protein adsorption DMSO with EDC and Sulfo-NHS as catalysts for 4 hrs. The (Portet 2001). It has been reported that accumulation rate in final product was washed several times with methanol. The tissues such as spleen increases with increase in the particle wash sample was further dissolved in acetone and centri sizes (Li 2008) which is consistent with the current findings. fuged. The Supernatant was extracted carefully in order to It has also been observed that PEG coating in PEGylated separate from free unreacted Dextran that have been precipi particles can increase accumulation in the spleen (Peraccia tated. Finally the supernatant containing Dex-b-PLA was 1999) whereas the neutrality (Chouly 1996) and flexibility dried in vacuum desiccator. (Passirani 1998) of dextran chains on the NP surface can 6.2 Surface Functionalization of Dex-b-PLA NPS with PBA cause lower protein absorption leading to lower spleen accu (0279 Dex-b-PLA was dissolved in DMSO (30 mg/ml), mulation. Dex-b-PLANPs are expected to have low comple and added slowly into water under mild stirring. Periodate ment activation as observed for dextran-poly(methyl meth oxidation of the Dextran surface was carried out by adding 60 acrylate) NPs, whose behaviour was similar to soluble mg of NaIO and stirring for an hour. Subsequently, glycerol dextran (Passirani 1998). The lower accumulation of the Dex was added to quench the unreacted NaIO. Various amounts b-PLANPs in spleen along with lower complement activa of PBA (i.e. 40 mg for Dex-b-PLA 40PBA) were added to tion may have attributed to their longer blood circulation the mixture, along with NaCNBH for 24 hours. All reactions (Meerasa 2011). The long circulation half-life of NP drug were carried out in the dark. The mixture was then dialyzed in carriers is a crucial parameter in cancer therapy since it water for 24 hrs to remove any unreacted Solutes, through increases the probability of accumulating at cancerous tissues changing the wash medium 4 times. due to EPR effect: particle size below 100 nm directly pro motes accumulation of NPs in the tumor sites since the vas 6.3 Characterization of Dex-b-PLA PBA NPs cular pores around tumor are at least 100 nm in size (Cho 0280. The surface modification with PBA was verified 2008). The size-tuneable Dex-b-PLA system developed here using H NMR spectroscopy (Bruker 300 MHz). Dex-b- presents a polymeric platform for systematically studying the PLA PBA polymers were dissolved in DMSO-d6 (25 effect of NP size on various in vivo characteristics such as mg/ml) for the "H NMR characterization. UV-Vis absorption biocompatibility, blood clearance, tumor accumulation and measurement at 291 nm was performed with Epoch Multi biodistribution and screening candidates for further clinical Volume Spectrophotometer System (Biotek, USA) on the evaluation. Dex-b-PLA PBA in order to quantify the amount of PBA attached to the Dextran chains. Dex-b-PLA solution with TABLE 1. same concentration was used as the baseline for UV-vis Blood pharmacokinetic parameters for PLA20-Dex10, PLA20-Dex6, and absorption study. The NPs of Dex-b-PLA PBA prepared PLGA-PEGNPs using nanoprecipitation were also analyzed using 90Plus Par ticle Size Analyzer (Brookhaven, v=659 nm at 90°) obtaining t12 (hr) t12 (hr) to g (hr) AUC volume-averaged multimode size distribution (MSD) mean PLA2O-Dex10 12.3 - 22 29.81.O 38.3 21.5 1040 diameter. The particle size and morphology of Dex-b-PLA PLA2O-Dex6 7.204 26.63.1 17.98.6 691 PBANPs were further confirmed verified using TEM (Philips PLGA-PEG 3.7 O.6 27.02.3 5.024 287 CM10) with the accelerating voltage of 60 kV and the Lan t2: initial half-life; thanum Hexaboride filament (LaB6). 300 Mesh Formvar to: terminal half-life; coated copper grids (Canemco & Marivac) were used for this too: blood retention time for 90% of the NPs; experiment. The NP Suspension in water was prepared using AUC: Area under curve (% dose hr) the nanoprecipitation method as mentioned above. A drop of the NP Suspension was placed onto the grid, and the grid was 0277 Statistical analysis was performed using the student briefly stained with aqueous phosphotungstic acid solution (2 t-test and statistical significance was assessed with p<0.01. w/v '% in water). The copper grid with the NP suspension was Example 6 dried under ambient environment overnight before imaging under TEM. Synthesis and Characterization of Dex-b-PLA-BLA (0281. The NMR spectrum of Dex-b-PLA PBA shows NPS peaks corresponding to both PLA (multiplets at 5.2 ppm) and Dextran (multiplets at 4.86 ppm), while also showing multip let peaks at 6.6 ppm and 6.9 ppm, which correspond to the 6.1 Synthesis of Dex-b-PLA protons from carbon 2 to 6 in the phenyl group of the PBA 0278. The synthesis of Dex-b-PLA was carried out as (FIG.12a). UV absorption at 291 nm was measured to quan described in Example 1 (see also, Verma, 2012). Briefly the tify the amount of PBA on the NPs with respect to the Dextran procedure for the synthesis of Dex-b-PLA divides into three monomers. Increasing the amount of PBA in the initial reac stages: reductive amination between Dextran and N-Boc tion mixture proportionally increased the final PBA conjuga ethylenediamine, deprotection of the Boc group, and conju tion on the Dextran surface (Table 2) with the highest density gation of the amine-modified Dextran end group with car of 34.6 mol% (equivalent of about 3.5 PBA conjugated per 10 boxyl-terminated PLA. Reductive amination was carried out Dextran monomers) achieved for Dex-b-PLA 320PBANPs. by dissolving Dex in borate buffer and mixing it with N-Boc 0282 Conjugation of Cysteamine onto the Dex-b-PLA ethylenediamine and NaCNBH in dark condition for 72 hrs. NP surface was also demonstrated using "H NMR spectrum After the reaction the mixture is washed with methanol and (FIG. 13). The peaks that correspond to the protons on carbon US 2014/0005379 A1 Jan. 2, 2014 28

1 and 2 of the cysteamine are shown as multiplets peaks near (n=3, mean+S.D). The absorbance measured from same pro 2.7 ppm. However, higher resolution NMR characterization cedure using the polymers without the drugs was used as the is required in the future to further differentiate the peaks from baseline. The measurements were converted to the concen other noise peaks from the polymer. tration of the CycA using standard calibration obtained. 0283. The sizes and morphology of the nanoparticles were 0287. The encapsulation of Dorzolamide, Brinzolamide, analyzed using the procedure illustrated in Example 1. The and Natamycin in the Dex-b-PLA NPs were accomplished sizes of the NPs were in the range of 25 to 28 nm, which are using the same method. The characterization of these drugs smaller than the unmodified NPs of 47.9 mm. Without being was performed using Multi-Volume Spectrophotometer Sys bound by theory, it is postulated that the particle size reduc tem (Biotek, USA) instead of HPLC. tion is attributed by the PBA molecules causing the Dextran chains to be less hydrophilic, leading them to form more Example 8 compact shells around the PLA particle core. TEM images confirmed a spherical morphology, due to the formation of a Drug Release from Dex-b-PLA PBA NPs In Vitro core-shell structure of the amphiphilic block copolymers 0288 The in vitro release phenomenon of CycA from the (FIG.12b). The sizes of Dex-b-PLA PBA NPs obtained are Dex-b-PLA NPs were analyzed using the procedures smaller than that normally achieved with PEG-based block described in O. Both Dex-b-PLA and Dex-b-PLA PBA NPs copolymers such as PLGA-PEG (Karnik, 2008). We postu (Dex-b-PLA 40PBA and Dex-b-PLA 320PBA) showed a late that smaller NPs may be more desirable for mucoadhe total release point at around 120 hrs (FIG. 16), which is Sion, since they provide greater Surface area for interaction significantly longer than previous studies involving in vitro with the mucous membrane. release of CycA from micro or nanoparticles which showed Example 7 up to 48 hours of sustained release (Li, 2012; Shen, 2010; Shen, 2010; Yuan, 2006). Moreover, the release rate may potentially be a significant improvement over the commercial Drug encapsulation in Dex-b-PLA PBA NPs product, which requires administering twice a day. Whereas 0284. The Cyclosporine A (CycA) encapsulation in the Dex-b-PLANPs demonstrated a sustained release rate for up Dex-b-PLA and Dex-b-PLA PBANPs were measured using to 120 hours, Dex-b-PLA PBA NPs showed two regions of the procedure described in O. Maximum encapsulation of slightly different release rate. In the first 48 hrs, the Dex-b- CycA was achieved at an initial feed of 40 wit/wt %: Dex-b- PLA PBA NPs released CycA at a faster rate compared to PLANPs encapsulated up to 10.8 wit/wt %, whereas Dex-b- Dex-b-PLANPs, which may be due to the release of CycA PLA 40PBA and Dex-b-PLA 320PBA encapsulated up to that were encapsulated near the slightly more hydrophobic 11.2 and 13.7 wt/wt %, respectively (FIG. 15). The 13.7 wt/wt surface Dex-b-PLA PBA NPs. The subsequent slower % encapsulation is equivalent to 2.38 ug of CycA in 28 Jull release rate, compared to Dex-b-PLANPs, may be due to the formulation (which is the same as the administration Volume release of drugs from the core of the Dex-b-PLA PBA NPs, of commercially available RESTASISR), whereas the com which need to diffuse through the more compact Dextran mercial product contains 14 Jug. Therefore, a therapeutically Surface. When the volume of PBA modified NP formulation relevant dosage can be achieved by simply adjusting the poly were scaled to the administration volume of RESTASISR) (28 mer and drug concentration in the formulation. LL), the CycA release rates were in the range of ug/day, which 0285 Encapsulation of other types of bioactive agents in is similar to the daily administration dosage of CycA in the Dex-b-PLA PBA NPs has also been explored (FIG. 17). RESTASISR). Therefore, it is possible to optimize the formu In one embodiment the bioactive agent is Dorzolamide, lation by changing the concentration of the PBA modified which is commonly used to treat glaucoma. In another NPs and/or the amount of CycA to achieve a clinically effec embodiment the bioactive agent is Brinzolamide, which is tive release rate and amount. also used to treat glaucoma. Natamycin, which is an antifun (0289. In vitro CycA release phenomena from both PBA gal agent, is also used as a bioactive agent in the encapsulation modified and unmodified Dex-b-PLANPs in the STF at 35° in the Dex-b-PLANPs. In other embodiments, Doxorubicin, C. were analyzed by quantifying the CycA in the STF at an anti-cancer agent, and Olopatadine, antihistamine, were predetermined time intervals using High Performance Liquid also explored in the encapsulation in the PLA-Dex NPs. Chromatography (HPLC). Using the procedure described in 0286 The encapsulation of Cyclosporine A (CycA) in the the previous section, drug encapsulated NPs, both Dex-b- Dex-b-PLANPs was accomplished using nanoprecipitation PLA and Dex-b-PLA PBA, were prepared and filtered to method. Dex-b-PLA and CycA were both dissolved in remove non-encapsulated drug aggregates. A purified sample DMSO (Dex-b-PLA concentration of 7 mg/mL, with varying of NPs-drug Suspension was collected to measure the maxi drug concentrations). 1 mL of the DMSO solution is added mumabsorbance and this was used as the 100% release point. drop-wise into 10 mL of DI-HO under mild stirring and Subsequently, the NP-drug Suspension was injected into a continued to stir for additional 30 minutes. The NPs in water Slide-a-Lyzer Dialysis cassette (Molecular weight cut were filtered through syringe filter (pore size=200 nm) to off 20 kDa; Fisher Scientific, Canada) and dialyzed against remove the drug aggregates and Subsequently centrifuged 200 mL of simulated tear fluid (STF) at 35°C. under stirring. using Amicon filtration tubes (MWCO=10kDa, Millipore) to At predetermined time intervals, 1 mL of the release medium further remove any remaining free drugs in the Suspension. was extracted and the same volume of fresh new STF was The filtered NPs containing encapsulated CycA were resus added to the release medium. The extracted release medium pended and diluted in Acetonitrile. Consequently, the drug was characterized using HPLC method (n=3, meant S.D). loading (wt/wt %) in the polymer matrix was calculated by The release medium was replaced several times to maintain measuring concentration of the CycA in the mixture using the concentration of CycA in the medium in order to stay High-performance liquid chromatography (HPLC, Thermo below its solubility limit in water. Replacing the medium was Scientific). The measurements were obtained in triplicates also expected to prevent the adhesion of released CycA to the US 2014/0005379 A1 Jan. 2, 2014 29 glass walls of the beaker or the magnetic stir bar. The beakers titative analysis of interaction between mucin particles and were sealed with Parafilm to prevent evaporation of water. Dex-b-PLA PBA NP suspension. 1 w/v '% mucin solution 0290 The invitro release study of Dorzolamide, Brinzola was prepared in pH 7.4 PBS by stirring overnight and the mide, and Natamycin in the Dex-b-PLA NPs were accom Solution was Subsequently Sonicated for 10 minutes (Branson plished using the same method. The characterization of these Digital Sonifier 450, USA). To 700 uL of mucin particle drugs was performed using Multi-Volume Spectrophotom solution were added 200 uL of 0.7 mg/ml Dex-b-PLA PBA eter System (Biotek, USA) instead of HPLC. NP suspension in PBS. A control study was also performed by adding 200 uL of PBS to the mucin particle solution. The Zeta Example 9 potential of mucin particles with the NP suspension and the control study were determined using a Malvern ZetaSizer Mucoadhesion Tests Nano ZS90 (Malvern Instruments, Worcestershire, U.K.). 9.1 In Vitro Mucoadhesion Test—Zeta Potential 9.2 In Vitro Mucoadhesion Test PAS Staining Method 0291 Zetapotential measurements were used to analyze 0293 Mucoadhesion of the NPs was measured using the the interaction between mucin particles and the NPs using the in vitro PAS staining method as described above. Compared procedures described in O. Several reports in the past have to the Dex-b-PLA and the PLGA-PEGNPs, the Dex-b-PLA used Zeta potential to assess the mucoadhesive properties of PBANPs showed increased mucin adsorption (Table 2). Dex drug carriers (Khutoryanskiy, 2011; Shaikh, 2011: Sogias, b-PLA 1 OPBA, Dex-b-PLA 40PBA, and Dex-b-PLA 2008; du Toit 2011: Takeuchi, 2005). Mucin particles at 160PBA NPs showed a linear increase in mucin adsorption physiological pH exhibit overall negative Surface charge due from 0.575 to 0.605 mg/mg of NPs as the degree of PBA to the presence of carboxylate groups (sialic acid) and ester surface functionalization increased. However, further Sulfates at the terminus of Sugar units (Khutoryanskiy, 2011). increase in PBA surface functionalization (i.e. Dex-b-PLA By adhering to the Sialic acid moieties of the mucin particles, 320PBA) decreased the amount of mucin adsorbed. Without the Dex-b-PLA PBA NPs may shield the negative charges wishing to be bound by theory, it is possible that excess from the Surface of the mucin particles and also cause aggre functionalization of the NP surfaces with PBA causes the gation of the mucin particles, thus increasing the overall Dextran to become more hydrophobic. This would increase surface charge. Only Dex-b-PLA 160PBA (22.9 mol % the potential for self-aggregation of the NPs, reducing the PBA) and Dex-b-PLA 320PBA NPs (34.6 mol%) showed total available surface area for mucin adsorption. It is there significant interaction with mucin particles compared to the fore ideal to tune the amount of PBA functionalization to control study (Table 2). Low PBA surface functionalization achieve optimal mucin-NP interaction without compromis densities do not appear to show a difference compared to ing the NP colloidal stability. It is also possible that smaller unmodified NPs interms of mucin-NP interaction. It is there NPs render higher mucin adsorption due to their larger total fore desirable to use NPs with abundant surface functional surface area, as shown by comparing PLGA-PEG, Dex-b- groups, such as Dextran-based NPs, to tune the functional PLA, and Dex-b-PLA PBA NPs. However, as each type of ization density where maximum mucoadhesion is desired. If NP exhibit different surface properties, the trend is inconclu one was to functionalize the surface of PLGA-PEG NPs, sive. The Dex-b-PLA PBA NPs all exhibited significantly using one functional group per each PEG chain, the maxi higher mucin adsorption compared to the previous studies mum PBA modification can be achieved is only 0.44 mol % involving chitosan based NPs and thiolated NPs, which (assuming the same MW of PEG, i.e. 10 kDa). An increased showed about 0.25 and 0.13 mg/mg of NPs respectively at 1 amount of PBA functionalization also increased the extent of hr incubation (Lee, 2006). NP-mucin interaction, which allows potential increase of 0294 Mucoadhesion was calculated as the amount of mucin-NP interaction by saturating PBA on the surface. mucin adsorbed per mg of NPs. NP suspension (1 ml) was However, the functionalization of PBA causes the Dextran mixed with 1 ml of mucin solution (1 mg/ml in STF) and Surface to be more hydrophobic, increasing the potential for incubated at 37°C. for 1 hr. The mixture was then centrifuged aggregation of the NPs. It is therefore ideal to tune the amount at 15,000 rpm for 1 hr and free mucin in the supernatant was of PBA functionalization to achieve optimal mucin-NP inter quantified using the periodic acid/Schiff (PAS) staining action without compromising the NP colloidal stability. method (Lee, 2006). Mucin adsorption was calculated by Subtracting the free mucin concentration from the initial TABLE 2 mucin concentration. Mucin standards (0.1, 0.25 and 0.5 PBA conjugation efficiency and diameter of unmodified and modified mg/ml) were determined using the same procedure to obtain Dex-b-PLANPs a calibration curve. PBA:Dex) Diameter Zeta potential Example 10 Formulation (mol%) (nm) (mV) Mucin -11.1 + 0.1 In Vivo Studies Dex-b-PLA O 47.9 O.S -10.7 O.6 Dex-b-PLA 10PBA 2.85 O.O3 27.50.9 -11.4 + 0.2 10.1 Acute Response Study. Using Dex-b-PLA PBA NPs Dex-b-PLA 4OPBA 12.2 O.2 26.7 0.1 -10.8 + 0.4 Dex-b-PLA 160PBA 22.90.3 25.2 10 -9.67 + O.76 0295) To analyze the short-term biocompatibility of the Dex-b-PLA 320PBA 34.6 0.2 28.10.3 -8.32 O.28 NP formulation, the formulations were administered to rabbit eyes, while having contralateral eyes as control, and daily Mol% of PBA with respect to Dextran monomers; slit-lamp examination for up to 7 days was performed to b)NP suspensions are mixed with mucin suspension in PBS analyze the ocular Surface. Upon grading of the 7 categories 0292 To assess the mucoadhesive properties of PBA (discomfort, conjunctival Swelling and redness, lid Swelling, modified Dex-b-PLA, Zeta potential was measured for quan discharge, corneal opacification, and infiltrate) from 0 (none) US 2014/0005379 A1 Jan. 2, 2014 30 to 4 (severe), the control eyes showed overall higher values between the NP treated and control eyes were insignificant compared to the corresponding NP treated eyes (FIG. 23). across all of the 7 categories throughout the duration of the Throughout the duration of the study, conjunctival Swelling, study (12 weeks) (FIG. 25). corneal opacification, and infiltrate were not observed in any (0300 Five female rabbits (New Zealand Albino) were of the rabbits. used for this study. The rabbits were acclimated for one week 0296. Three female rabbits (New Zealand Albino) were prior to the experiment. The nanoparticles are prepared using used for this study. The rabbits were acclimated for one week the nanoprecipitation method described in Example 4 but prior to the experiment. The nanoparticles are prepared using without the drug. The nanoparticles were filtered using 200 the nanoprecipitation method described in Example 4 but nm syringe filter, and further sterilized using UV irradiation without the drug. The nanoparticles were filtered using 200 inside a BioSafety Cabinet (BSC) for 1 hour. One eye was nm syringe filter, and further sterilized using UV irradiation administered with NPs (28 ul; 19 ug of Dex-b-PLA PBA inside a BioSafety Cabinet (BSC) for 1 hour. One eye was NPs) once a week for 12 weeks while the contra-lateral eye is administered with NPs (28 ul; 19 ug of Dex-b-PLA PBA used as control. Slit lamp examination at 0, 1, 24, 48 hr after NPs) while the contra-lateral eye is used as control. Slit lamp administration each week was used to evaluate 7 different examination at 0, 1, 8, 24, 48, 72, 96, 120, 144, and 168 hr categories (Note that 0 hr means before administration). after administration was used to evaluate 7 different catego These 7 categories (discomfort, conjunctival redness and ries (Note that Ohr means before administration). These 7 Swelling, lid Swelling, discharge, corneal opacification, and categories (discomfort, conjunctival redness and Swelling, lid infiltrate) were graded from 0 (no sign) to 4 (severe). After 12 weeks, the rabbits were euthanized, and the ocular tissues Swelling, discharge, corneal opacification, and infiltrate) were collected for further histopathology analysis. were graded from 0 (no sign) to 4 (severe). After 168 hrs, the 10.4 Chronic Chronic Response Study Using Dex-b-PLA rabbits were euthanized, and the ocular tissues were collected PBA NPs Encapsulated with Cyclosporine A in formalin for further histopathology analysis. 0301 Similarly, the long-term biocompatibility of the NP formulation with encapsulation of Cyclosporine A was also 10.2 Histopathology Analysis of Ocular Tissues examined using slit-lamp. The formulation containing both the Dex-b-PLA PBA NPs and Cyclosporine A were admin 0297. After the duration of slit-lamp examination, the rab istered to rabbit eyes, while having contralateral eyes as con bits were euthanized and the ocular tissues (the entire ocular trol. The slit-lamp examination for up to 4 weeks has shown globe, and upper and lower eyelids) were collected for histo no significant difference between the NP treated and the pathology analysis (0). From examining the cornea, bulbar control eyes in any of the 7 categories (FIG. 26). and tarsal conjunctivas of all the eyes, normal ocular tissues (0302) Four female rabbits (New Zealand Albino) were surfaces were observed in both the NP treated and the control used for this study. The rabbits were acclimated for one week eyes (FIG. 24). All eyes showed anterior segment with pre prior to the experiment. The nanoparticles are prepared using served architecture and morphology. No sign of inflamma the nanoprecipitation method described in Example 4 with tion, altered layer integrity, or presence of residual particles Cyclosporine A. The nanoparticles were filtered using 200 were detected in any of the eyes. Adequate number of goblet nm syringe filter, and further sterilized using UV irradiation cells with preserved morphology was also shown. Presence of inside a BioSafety Cabinet (BSC) for 1 hour. One eye was occasional intraepithelial and subepithelial eosinophils in tar administered with NPs (28ul; 19 ug of Dex-b-PLA PBANPs sal conjunctiva were found in both NP treated and control and 8 ug of Cyclosporine A) once a week for 12 weeks, while eyes, suggesting that the phenomenon is not directly caused the contra-lateral eye is used as control. Slit lamp examina by the administration of the NP formulation. tion at 0, 1, 24, 48 hr after administration each week was used 0298. The eyes were enucleated and collected immedi to evaluate 7 different categories (Note that Ohr means before ately after euthanasia for histpathological evaluation. The administration). These 7 categories (discomfort, conjunctival entire upper and lower eyelids were also dissected and col redness and Swelling, lid Swelling, discharge, corneal opaci lected for evaluation of the tarsal conjunctiva and underlying fication, and infiltrate) were graded from 0 (no sign) to 4 Soft tissues. Consecutive sections of the entire ocular globe (severe). After 12 weeks, the rabbits were euthanized, and the and eyelids were processed for microscopic analysis: after ocular tissues were collected for further histopathology initial fixation in 10% neutral buffered formalin, the tissue analysis. was embedded in paraffin, serially sectioned in 5 um thick sections, and stained with hematoxylin and eosin (H&E). The Example 11 histological slides were evaluated using bright field micros copy (Leica DM1000, ICC50 HD, Leica Microsystems Inc, In Vitro Nanoparticle Partition Across Tear Fluid Concord, ON). Lipid Layer (0303. The ability of different types of NPs to partition 10.3 Chronic Response Study Using Dex-b-PLA PBA NPs across tear fluid lipid membrane was studied using in vitro model. The types of surface properties seem to have very little 0299 To analyze the long-term biocompatibility of the NP effect on the '% of partition nanoparticle-drug complexes formulation, the formulations were administered to rabbit across the lipid membrane, with all of them achieve partition eyes once a week for up to 12 weeks, while having contralat of approximately 70%. However, further studies are required eral eyes as control, and daily slit-lamp examination was to better simulate the structure and the turnover phenomenon performed similar to above. Similar to acute response study, of the lipid layer in the tear fluid. no sign of conjunctival Swelling, corneal opacification, or 0304 Artificial tear fluid with lipid layer was prepared infiltrate were observed in any of the rabbits at any point of the using previously reported method by preparing complex Salt time during the study. Overall, the difference in values solution (CSS) and lipid stock solution (LSS) (Lorentz, US 2014/0005379 A1 Jan. 2, 2014

2009). Add LSS into 2000 fold volume of CSS and bath doxorubicin-loaded PLGA nanoparticles to lung epithelial sonicate at 37°C. for 30 minutes. The mixture is allowed to cells. Eur: J. Pharm. Sci. 2009, 2, 141-150. settle overnight to form the lipid layer. Nanoparticles with 0314 8. Cho, K. Wang, X.Nie, S.; Chen, Z.; Shin, D. M. Natamycin were prepared using the nanoprecipitation Therapeutic nanoparticles for drug delivery in cancer. Clin. method described above. 1 ml of the NP-Natamycin formu Cancer. Res. 2008, 5, 1310-1316. lation was added onto 2 ml of CSS/LSS mixture. The mixture 0315 9. Chouly, C.; Pouliquen, D.; Lucet, I.; Jeune, J. J.; was then incubated at 37° C. for 10 minutes. The bottom 1.5 Jallet, P. Development of Superparamagnetic nanoparticles ml of the mixture was extracted without disturbing the top for MRI: Effect of particle size, charge and surface nature layer, and dried overnight in vacuum desiccator. The precipi on biodistribution.J. Microencapsul. 1996, 3, 245-255. tates were dissolved in DMSO again and UV-vis absorption 0316 10. Dhar, S.: Gu, F. X.; Langer, R.; Farokhzad, O. was performed to calculate the concentration of Natamycin. C.; Lippard, S.J. Targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized Pt(IV) prodrug Discussion PLGA-PEG nanoparticles. Proc. Natl. Acad. Sci. U.S.A. 2008, 45, 17356-17361. 0305 The present inventors synthesized a model linear 0317 11. Diebold, Y. and Calonge. Applications of nano block copolymer using PLA and Dextran (Dex-b-PLA), and particles in ophthalmology. Progress in Retinal and Eye demonstrated that NPs composed of Dex-b-PLA can self Research 29 2010, 596-609. assemble into core-shell structured NPs of small particle size, 0318 12. Dobrovoiskaia, M.A.: Clogston, J. D.; Neun, B. e.g. sizes less than 40 nm, without using any flow-focusing W.; Hall, J. B. Patri, A. K. McNeil, S. E. Method for devices. They further showed that thesize of Dex-b-PLANPs analysis of nanoparticle hemolytic properties in vitro. can be precisely fine-tuned, e.g. between 15-70 nm by alter ing the molecular weight of the component blocks (Verma, Nano Lett. 2008, 8, 2180-2187. 2012). Dextran, a natural polysaccharide composed of 1->6 0319 13. Dong, Y. and Feng, S. In vitro and in vivo evalu linked C-D-glucopyranosyl units, was selected as a model ation of methoxy polyethylene glycol-polylactide (MPEG hydrophilic block because of its high hydrophilicity and bio PLA) nanoparticles for Small-molecule drug chemo compatibility. Dextran has an abundance of functional therapy. Biomaterials 2007, 28, 4154-4160. hydroxyl groups on its back bone. The higher density of 0320 14. Drummond, D.C.; Meyer, O.; Hong, K. L.; Surface functional groups (as opposed to PEG, which has one Kirpotin, D. B.: Papahadjopoulos, D. 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0365 59. R. Shaikh, T. R. Raj Singh, M.J. Garland, A. D. can be effected to the particular embodiments by those of skill Woolfson, R. F. Donnelly. Journal of pharmacy & bioallied in the art without departing from the scope of the disclosure, sciences 2011, 3,89. which is defined solely by the claims appended hereto. 0366 60. I. A. Sogias, A. C. Williams, V. V. Khutoryan What is claimed is: skiy. Biomacromolecules 2008, 9, 1837. 1. A block copolymer useful in the formation of a nanopar 0367 61.J. Shen, Y. Deng, X. Jin, Q. Ping, Z. Su, L. Li. Int. ticle delivery system for encapsulating a hydrophobic thera J. Pharm. 2010, 402, 248. peutic agent, the block copolymer having a hydrophobic por 0368 62. Takeuchi, et al., Novel mucoadhesion tests for tion consisting of polylactide and a hydrophilic portion polymers and polymer-coated particles to design optimal consisting of dextran, the dextran having a plurality of func mucoadhesive drug delivery systems. Advanced Drug tional groups capable of conjugation to a targeting moiety. Delivery Reviews 57 2005, 1583-1594. 2. The block copolymer of claim 1, which is poly(D.L- 0369. 63. Unkeless et al. Annu. Rev. Immunol. 6:251-281, lactide)-b-dextran. 1998. 3. The block copolymer of claim 2, wherein at least a 0370) 64. M. S. Verma, S. Liu, Y.Y. Chen, A. Meerasa, F. portion of the functional groups are conjugated to the target X. Gu. Nano Research 2012, 5, 49. ing moiety. 0371 65. M. S. Verma, S. Liu, Y.Y. Chen, A. Meerasa, F. 4. The block copolymer of claim 3, wherein the targeting X. Gu. Nano Research 2012. moiety is phenylboronic acid. 0372) 66. X. Yuan, H. Li, Y. Yuan. Carbohydr. Polym. 5. A nanoparticle useful for encapsulating a hydrophobic 2006, 65,337. therapeutic agent, the nanoparticle comprising a plurality of 0373 67. Yang, Z. Leon, J.; Martin, M.: Harder, J. W.; amphiphilic block copolymers, each copolymer having a Zhang, R., Liang, D.; Lu, W.; Tian, M., Gelovani, J. G.; hydrophobic portion consisting of polylactide and a hydro Qiao, A., et al. Pharmacokinetics and biodistribution of philic portion consisting of dextran, the dextran having a near-infrared fluorescence polymeric nanoparticles. Nano plurality of functional groups capable of conjugation to a technology 2009, 16, 165101-165101. targeting moiety. 0374 68. Zahr, A. S.; Davis, C. A.; Pishko, M. V. Mac 6. The nanoparticle of claim 5, wherein the block copoly rophage uptake of core-shell nanoparticles Surface modi mer is poly(D.L-lactide)-b-dextran. fied with poly(ethylene glycol). Langmuir 2006, 19, 8178 7. The nanoparticle of claim 6, wherein at least a portion of 81.85. the functional groups are conjugated to the targeting moiety, 0375 69. A. Zimmer, J. Kreuter, Adv. Drug Deliv. Rev. thereby providing a Surface-functionalized nanoparticle. 1995, 16, 61. 8. The nanoparticle of claim 7, wherein targeting moiety is 0376 All publications, patents, and patent applications phenylboronic acid (PBA). cited herein are hereby incorporated by reference in their 9. The nanoparticle of claim 8, wherein the size of the entirety for all purposes. nanoparticle is tunable by the surface density of phenylbo 0377 The above-described embodiments are intended to ronic acid. be examples only. Alterations, modifications and variations