Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications Boon M

207

10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications Boon M. Teo, Martin E. Lynge, Leticia Hosta-Rigau, and Brigitte Städler

10.1 Introduction

Integrating (bio)materials into living organisms heavily relies on the properties of the biointerface. Polymer coatings are successful tools to engineer this interface from protein repelling over selective cell adhesion to antimicrobial. Therefore, polymer thin films are essential building blocks for devices with applications ranging from tissue engineering to drug delivery [1]. There is a wide range of opportunities to modify different materials with polymer thin films including spray coating, dip coating, self-assembled monolayers, surface grafted or tethered polymer layers, as well as polymer multilayer assembly. The latter concept, the layer-by- layer (LbL) assembly technique, is among those approaches which have the inherent power to be successful due its simplicity, flexibility, and reproducibility [2]. Further, the LbL assembly method offers precise control over the composition and properties of the films depending on the chosen building blocks during assembly. Although LbL coatings can be deposited on virtually any substrate, the focus of this chapter is on planar substrates, and the film deposition/characterization and their applications are discussed in this context. LbL films consisting only of polymers can be employed to control cell adhesion and differentiation depending on the type of polymer, cross-linking density, terminating layer, and so on [3]. While the success of polymer coatings is an incon- trovertible fact, plain polymer films are insufficient to address many challenges. Embedding of active compounds, for example, proteins [4] or genes [5], toward substrate-mediated drug delivery (SMDD) is an approach to enhance the functionality of LbL coatings, but it is limited in the type and amount of compounds that can be trapped this way. It can be in particular challenging for small molecular payload or cargo relying on its 3D structure such as enzymes. To this end, trapping larger carriers as drug deposits into the polymer films is a more recent concept that combines two different building components into the same hybrid film. The power of these composite coatings comes from the preservation of the advantages of the individual building blocks, while there is an option to overcome their shortcom- ings. Particularly promising are assemblies that employ fundamentally different

Layer-by-Layer Films for Biomedical Applications, First Edition. Edited by Catherine Picart, Frank Caruso, and Jean-Claude Voegel. © 2015 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2015 by Wiley-VCH Verlag GmbH & Co. KGaA. 208 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

Micelle

Cyclodextrin Liposome

Scheme 10.1 Cyclodextrins, block copolymer micelles, and liposomes as embedded drug deposits within a LbL ﬁlm toward the use in surface-mediated drug delivery.

building blocks to polymers yielding subcompartmentalized multilayered films. Although alternative films, for example, hydrogel-based ones, have been equipped with drug deposits [6], LbL coatings are inherently suitable to be extended in this direction using the deposits as a building block during the sequential assembly. The aim of this book chapter is to review the progress in subcompartmentalized surface-adhering LbL films for SMDD (Scheme 10.1). We aim to provide an overview over the predominant subcompartments used, namely cyclodextrins (CDs), block copolymer micelles (BCMs), and liposomes. Further, we will discuss miscellaneous subunits with potential in SMDD. Each of the approaches will be reviewed in terms of their assembly followed by their characterization relevant in SMDD. We will highlight those composite coatings successfully used to deliver (active) compounds to adhering cells, and emphasize the few films, which have been tested in vivo. In doing so, we hope to present the progress and impact these subcompartmentalized coatings have made over the past years as well as their potential and remaining challenges for their application in SMDD.

10.2 Cyclodextrin (CD)-Containing LbL Films

CDs are cyclic oligosaccharides with a unique cup-like morphology with a polar exterior and a hydrophobic interior [7]. The nature of CDs allows for the 10.2 Cyclodextrin (CD)-Containing LbL Films 209 encapsulation of small hydrophobic molecules in an inclusion complex while the exterior of the CD cups can be easily modified with various chemical groups. CDs have been used in pharmaceutical formulations due to their versatility, low toxicity, and potential to enhance therapeutic effects while reducing toxic side effects of drugs [8].

10.2.1 Assembly

For incorporation of drugs into LbL films, the ability of CDs to convert a small hydrophobic therapeutic into a charged or hydrogen-bonding species is a very promising feature. In fact, modified CDs have been used for multilayer incorporation of several drugs and model compounds toward a range of applications including drug delivery and sensing. One of the first examples of multilayered thin films composed of CDs was reported by Xia and colleagues [9] who constructed films of polystyrene sulfonate

(PSS) and a stable host–guest complex CBr2which was generated from sono- β chemical treatment of a bolaamphiphile, stilbenoid compound SBr2,and -CDs. In another early work, Park et al. [10] reported on the chemospecific immobilization of polyplexes on surfaces that take advantage of the ability of CD molecules to form inclusion complexes with hydrophobic organic compounds. They used polyethylene imine (PEI) functionalized with β-CD to form DNA/PEI polyplexes. Subsequent experiments demonstrated that these CD-functionalized polyplexes bind specifically to gold surfaces modified to present bulky hydrophobic adamantine groups. In another study utilizing β-CD, Van der Heyden et al. [11] constructed polyelectrolyte multilayers (PEMs) using host–guest interactions between β-CDs and adamantyl-grafted chitosans (Chi). The stability of the films was due to multivalent complexation occurring at each step of the construction. They also showed that these films were reversibly responsive to solvent change. Inamorerecentwork,Damet al. [12] reported on the preparation of degradable polyrotaxanes (PRXs) multilayer films based on positively and negatively charged disulfide-containing α-CD-poly(ethylene glycol) (PEG) PRXs (Figure 10.1a (i)). Film construction and degradation were monitored and film degradation was demonstrated upon exposure to reducing conditions, that is, glutathione (GSH) (Figure 10.1a (ii)). Although the work published by Xia, Park, Van der Heyden, and Dam was not evaluated in a biological context, they provided a framework for applications of this approach with potential in the context of drug delivery.

10.2.2 Drug Delivery Applications

This section highlights recent reports describing PEMs with CDs for drug delivery applications including at least preliminary biological characterization. One of the first examples was reported by Jessel et al. [14] who demonstrated that it was possible to fabricate anti-inflammatory films by incorporating the 210 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

(i) (ii) 1800 PEG 3 1600 1400

) 1200

Capping Positive Negative Glutathione –2 1000 group Disulfide αCD αCD 800 600 2 / (ng cm 400 1 m 200 0 SiO surface SiO surface (a) 2 2 50 150 250 350 450 550 650 750 t /(min) (i) (ii) (iii) 100 Day 1 Day 3 Day 7 A A (poly1/PAA /GS/PAA) n 80

(poly1/PAA) 5 60 B

(DMLPEI/PAA) 40 10 C 20

B COX enzyme inhibition (%) 0 (poly2/polyCD-DIC) M) D n μ

No drug (DMLPEI/PAA) 10 Day 1 releaseDay 2 releaseDay 3 releaseDay 5 releaseDay 7 releaseDay 9 release (b) Carrier (polyCD) only Diclofenac only (44

Figure 10.1 (a) (i) Schematic diagram of PEM-containing PRX, showing the degradation of the ﬁlms initiated by the reductive cleav- age of the PRX-capping groups. (ii) (PRX-DMEDA/PRX-Gly)4 multilayer formation and degradation initiated by the addition of GSH, as observed by optical waveguide lightmode spectroscopy. (b) (i) Schematic diagram of the combination ﬁlms. (A) Gentamicin-releasing

(poly1/PAA/GS/PAA)n and (B) diclofenac-releasing (poly2/polyCD-DIC)n combination films, built on top of the microbicidal (DMLPEI/PAA)10 films. (ii) Percentage of cyclooxygenase (COX) enzyme inhibition showing that diclofenac released from (DMLPEI/PAA)10(poly2/polyCD-DIC)10 is still active. (iii) Proliferation (days 1, 3, and 7) of MC3T3-E1 cells on (A) bare glass substrates and (B) (DMLPEI/PAA)10 films; proliferation of A549 cells on (C) bare glass substrates and (D) (DMLPEI/PAA)10. (Reproduced with permission from Ref. [13].) 10.2 Cyclodextrin (CD)-Containing LbL Films 211 drug piroxicam into PEMs through inclusion complexes using monomeric CDs. These films comprised of poly(L-lysine)(PLL) and poly(L-glutamic acid) (PGA) and up to three layers of CD-drug complexes. The drug release was monitored by suppression of the tumor necrosis factor alpha, an inflammatory cytokine, from stimulated monocytes. However, monomeric CDs were unable to properly trap small molecules, resulting in rapid drug release. The Hammond group [15] expanded on the initial work by using charged CD-polymers to stably trap drug molecules, ciprofloxacin, flurbiprofen, and diclofenac, showing that release kinetics were found to be independent of the therapeutic agent incorporated, and that the release kinetics could be regulated through the choice of degradable polycations. In a follow-up work, they proposed the use of a microbicidal polyelectrolyte film, comprised of linear N,N-dodecyl,methyl poly(ethyleneimine) (DMLPEI) and poly(acrylic acid) (PAA), as a thin permanent nondegradable microbicidal base film and a hydrolytically degradable top multilayer film incorporating cationic poly(β-amino ester) (PBAE) for medical implants to prevent bacterial attachment. The subsequent deposition of polyCDs complexed with the anti-inflammatory and antibiotic drugs demonstrated the bolus delivery of gentamicin (>90% release in 2.5 h) to eradicate infection and a retarded release of diclofenac (over a period of 10 days) to minimize foreign body response at implant sites (Figure 10.1b (i)) [13]. They compared the antibicibial activity of the combination film to a control coating, gentamicin sulfate (GS)-releasing multilayer without an underlying antimicrobial base. At 0 and 15 min, the zones of inhibition (ZOIs) of these two films were nearly identical; however, after 2 days, a smaller ZOI appears around the combination films, while the control film no longer exhibits any ZOI, confirming the extended activity in the former case. Diclofenac within the multilayered films displayed similar activity against the inhibition of cyclooxygenase to the standard uncomplexed diclofenac solution, indicating that the activity of diclofenac was not altered upon complexation with polyCDs (Figure 10.1b (ii)). They further conducted experiments to investigate the cytotoxicity and interaction of cells with their films with A549 epithelial cancer cells and MC3T3-E1 osteoprogenitor cells and demonstrated that there was no significant difference in cell adherence and cell viability between the coated substrates and the control, indicating that the films were biocompatible for the tested cells (Figure 10.1b (iii)). Their films were also preventing Staphylococcus aureus and S. aureus attachment. This dual functional platform film technology appears versatile enough to satisfy a variety of thin film medical device coating specifications. In another successful preclinical study, PLL was covalently grafted with CDs as a polycationic vector for risedronate, a common drug against cancer cells for bone metastasis prevention [16]. In vitro studies of this system were conducted with CHO-β3 tumor cells, demonstrating the efficacy of the vector:drug supramolecular assemblies at inhibiting cancer cell invasion. Further, to the best of our knowledge, this report appears to be the first and only in vivo study of antitumor activ- ities of PLL–CD and risedronate in PEMs. This technique of incorporating complexes into PEMs for bone implants to locally deliver antitumoral compounds is 212 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

promising and might pave the way for broader clinical applications. The same authors constructed multilayered films based on PGA, PLL, and PLL succiny- lated with monocarboxylic-β-CDs incorporated within the films for delivery of two growth factors bone morphogenetic protein 2 and transforming growth factor -β with the aim to control differentiation of embryonic cells into cartilage and bone tissue [17]. The synergistic effect was achieved only when both growth factors were incorporated, and an insignificant effect was detected when only one protein was embedded. Films containing DNA are of great interest for a range of applications such as diagnostic [18], sensing [19], and gene delivery. The potential of gene delivery from thin films realized by the work published by Lynn and coworkers [20] highlights the significant impact of localized gene therapies. Jessel et al. [21] fabricated PEMs PLL–PGA films as anchors for which cationic CDs and DNA were sequentially deposited. They reported 100% transfection efficiency from films containing CDs and no transfection efficiency from films without CDs, indicating the importance of the usage of CDs in gene delivery from surfaces. In another report by the same group [22], they further assessed the DNA delivery from multilayered films composed of PLL/hyaluronic acid (HA) as a reservoir for active DNA complexes with gene delivery vectors PLL, β-CDs, and PLL–CDs. Consistent with their earlier publication, they showed that surface-mediated transfection yielded much higher efficiencies for HeLa cells. They concluded from their results that the internaliza- tion pathway proceeds via a nonendocytic mechanism resulting in high level of transfection. In another study by Hu et al. [23], films consisting of PEI–CDs conjugates and plasmid DNA were sequentially deposited on poly(D, L -lactic acid) substrate via the LbL technique. Although PEI is generally known to promote gene delivery, the high molecular weight of PEI can result in (high) cytotoxicity, while low molecular weight PEI is less cytotoxic but displays lower gene delivery efficiency [24]. To circumvent this problem, the authors improved the gene delivery efficiency of low molecular weight PEI by forming PEI–CDs conjugates by 1.65 and 1.95 times compared to PEI for human embryonic kidney 293 and human hepatoma G2 cells, respectively.

10.3 Block Copolymer Micelle (BCM)-Containing LbL Films

The advancement in controlled radical polymerization techniques in recent years has allowed for the synthesis of a wide variety of amphiphilic block copolymers that are able to self-assemble into micellar structures in solution [25]. The attractive potential of BCMs as drug delivery vehicles has been extensively investigated in different formulations [26]. One of such formulations is the fabrication of BCMs within multilayered films assembled via the LbL technique. The assembly of BCMs within multilayered films has several attractive features: they have the capability to (i) carry large amounts of cargo within the micellar core, (ii) control the assembly of micellar carriers within LbL films 10.3 Block Copolymer Micelle (BCM)-Containing LbL Films 213 without affecting the functionality of the cargo within the micellar cores, and (iii) manipulate the chemistry of the micellar coronas to provide an additional useful triggered delivery function by switching the polymer between collapsed and soluble states in response to stimuli, such as pH, temperature, and so on.

10.3.1 Assembly

BCMs incorporated within LbL films can be glassy (i.e., hard and relatively brittle) or responsive to environmental stimuli. In recent years, there has been a flurry of studies reporting on the LbL assembly of thin films containing BCMs for potential biomedical applications. In the following section, we highlight a selection of recent important reports on the fabrication of glassy and responsive BCMs within PEMs and provide relevant illustrations of such systems in biomedical applications.

10.3.1.1 Glassy BCMs within LbL Films One of the first few studies on micelles incorporated within LbL films was performed by Ma et al. [27], where they demonstrated the electrostatic assembly of layers of BCMs made of poly(styrene)-block-poly(acrylic acid) (PS-b-PAA) with poly(diallyl-dimethylammonium chloride) (PDDA). Cho and coworkers [28] self-assembled protonated poly(styrene)-block-poly(4-vinylpyridine) (PS-b- P4VP) and anionic PS-b-PAA BCMs into multilayer films by electrostatic and hydrogen-bonding interactions between the oppositely charged BCMs. They further showed that the film thickness and porosity were controlled by the ionization of the coronal weak polyelectrolytes and the lengths of the hydrophilic and hydrophobic blocks. Nguyen et al. [29] fabricated LbL films consisting of positively charged poly(propylene oxide)-poly(amidoamine) BCMs and PAA to demonstrate the encapsulation and prolonged release of an antibacterial agent triclosan. In another work, BCMs made of sulfonated hyperbranched poly(3-ethyl-3-oxetanemethanol) (HBPO-SO3), were assembled with Chi into LbL films which were loaded with a hydrophobic antioxidant drug, probucal, by either the pre-encapsulation or post diffusion [30]. The drug release from heparin/Chi multilayered films and HBPO-SO3/Chi films was compared, and the former case exhibited a burst release of approximately 66% drug in the first 10 h, whiletherewascontinuousreleasefromtheHBPO-SO3 film over a period of 25 days likely through a passive diffusion mechanism.

10.3.1.2 Temperature and pH Responsive BCMs within LbL Films BCMs that can undergo collapse/swelling transitions in aqueous solutions in response to changes in temperature, pH, and so on have been assembled within LbL ﬁlms for the purpose of controlling the (sustained) drug release kinetics. Sukhishvili et al. assembledmultilayeredﬁlmsfromBCMsof poly(2-(dimethylamino)ethyl methacrylate)-block-poly(N-isopropylacrylamide) (PDAM-b-PNIPAM) with PSS [31] and poly(vinylpyrrolidone)-block-PNIPAM 214 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

(PVP-b-PNIPAM) with poly(methacrylic acid) (PMA) [32]. The obtained films showed reversible swelling and shrinking upon temperature changes as well as a temperature-dependent release kinetic of an encapsulated hydrophobic dye, with a fast release rate below the lower critical solution temperature, due to the reduced retention of the dye by the hydrated PNI- PAM chains. In another study by Cavalieri et al. [33], they synthesized novel DNA–graft–PNIPAM micelles and assembled them into PEMs by sequential deposition of these micelles with complementary DNA sequences. Such design of oligonucleotide polymer-functionalized building blocks might provide structural and functional advantages that can be exploited for future SMDD applications. Another approach to designing smart LbL films is the incorporation of pH-responsive BCMs into the multilayered films. Biggs and colleagues [34] reported on the assembly of multilayers via electrostatic interactions of pH-responsive cationic BCMs of poly((dimethylamino)ethyl methacrylate)- block-poly(2-(diethylamino)ethyl methacrylate) (PDMA-b-PDEA), which were able to selectively release encapsulated hydrophobic dyes to the bulk solution in acidic environments. In another study by Erel et al. [35], the preparation of PDEA-b-PNIPAM BCMs with a PDEA core and PNIPAM-corona that form monolayers by hydrogen-bonding interactions on oxidized silicon surfaces was reported. Protonation and dissolution of the core were assisted by the acidic conditions resulting in the formation of an irreversible monolayer film. However, in hydrogen-bonded multilayers of PDEA-b-PNIPAM BCMs with tannic acid (TA), they retained their structure at slightly acidic pH, despite charging of the PDEA core. Hammond and coworkers [36] demonstrated that polymeric BCMs can be incorporated within hydrogen-bonded films to encapsulate the hydrophobic antibacterial agent triclosan. Polymer BCMs assembled from a block copolymer of poly(caprolactone) (PCL) and PEG were loaded with triclosan and assembled with PAA at pH 3.5, where the protonated PAA hydrogen binds to the PEG corona of the BCMs. The films formed contained 90 wt% polymer BCMs, suggesting the potential for high drug loadings. To determine the efficacy of the release of triclosan from the micelle LbL films, the films with and without triclosan on a silicon substrate were assayed against Gram-positive S. aureus. The ZOI of the films containing triclosan was approximately 15.0 ± 0.7 mm, whereas the control film without triclosan did not show any bacterial growth inhibition. More recently, the Fery group [37] reported on the immobilization of BCMs of a linear ABC tri-block terpolymer consisting of hydrophobic polybutadiene (B), a pH-sensitive PMA shell, and a cationic corona of quaternized PDAM and linear PSS. It was possible to reversibly switch the micellar morphology and the charge density of the corona by simply controlling the solution pH at the solid–liquid interface. Furthermore, the swelling behavior and the water content in the hydrogel-like films could be tuned by adjusting the number of micelles/PSS deposition steps to >1200% swelling and 90% water content in the swollen films. 10.4 Liposome-Containing LbL Films 215

10.3.2 Drug Delivery Applications

To the best of our knowledge, only single-digit studies on micelles-containing LbL films have been reported with drug delivery ability in vitro, with no studies demonstrating in vivo ability to date. Among the in vitro works is a study by Kim et al. [38], where they showed the delivery of a hydrophobic drug to a human cervical cancer cell line from a surface-assembled thin film. Their bioactive multilayer comprised of poly(ethylene oxide)-block-poly(2-hydroxylethylmethacrylate) (PEO-b-PHEMA) BCM conjugate of doxorubicin (Dox) which assembled into multilayers via hydrogen-bonding interactions between the corona of the BCMs and biologically active TA. Release of the drug from the Dox-loaded multilayers upon hydrolysis of the pH-sensitive carbamate linkage resulted in cancer cell death. Coating of metallic stents for patients with cardiovascular problems with drug-loaded BCMs to achieve sustained drug release is a potential application. Such stents would have dual functionalities; they would be capable of widening the coronary arteries and releasing the drug of choice in a controlled manner. Kim et al. [39] heparinized metallic stents to render them non-thrombogenic, and multilayers composed of anionic poly(lactic-co-glycolic acid)-grafted-hyaluronic acid (HA-g-PLGA) micelles encapsulating paclitaxel (PTX), heparin, and PLL were assembled (Figure 10.2a). The release of the drug from the coatings exhibited about 45% initial burst release within 1 day, followed by sustained releases up to 90% of the initially loaded PTX amount after 24 days (Figure 10.2b). These PTX- loaded stents also prevented human coronary artery muscle cell proliferation for 5 days (Figure 10.2c). In a recent elegant work by Hong et al. [40], PEMs composed of PMA, poly(acrylamide) (PAAm), and PEO-b-PCL micelles were assembled for dexam- ethasone (Dex) delivery to human mesenchymal stem cells (MSCs). Their films were composed of three regions: (i) a nondegradable layer comprising of PDDA and PSS, (ii) a payload region of Dex-loaded PEO-b-PCL micelles embedded within PMA/PAAm/PMA layers, and (iii) the top layer of cell adhesive prepared using collagen, polysaccharide, and disaccharide. They demonstrated that their PEMs can induce MSC differentiation into osteoblasts through the controlled release of Dex from the micelles.

10.4 Liposome-Containing LbL Films

Liposomes are attractive as drug deposits due to their biocompatibility, ease of preparation via self-assembly, and their inherent capability to entrap both hydrophilic cargo in their lumen and/or hydrophobic cargo in their lipid membrane [41]. These features allow utilization of liposomes in various applications ranging from drug delivery [42], cosmetics [43], food science [44], and biosensing [45] to agriculture [44, 46]. Due to their advantageous nature, liposomes have also been used as components of multilayered ﬁlms. 216 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

L605 alloy Thin heparin hydrogel layer Co Cr W Ni Biomimetic Hep–DA coating

PLL/Hep/PLL LbL assembly

Anionic heparin Cationic PLL PTX loaded HA-G-PLGA nanoparticles

Repeat assembly Therapeutic dual multilayer (a) process

3.5 5 layers 100 10 layers Control 15 layers 3.0 Blank Hep 80 2.5 PTX 20 Hep/PTX 60 16 2.0 ) 2 12 1.5

40 g cm 8 μ ( 1.0 4

20 Cumulative release 0 0 510152025 Normalized cell number 0.5 Time (day) 0 0.0

Cumulative pacitaxel release (%) 0 5101520 25 135 (b) Time (day) (c) Incubation time (day)

Figure 10.2 (a) Schematic diagram (5, 10, and 15 layers; inset: the cumulative of heparin (Hep) coating on L605 with amount release profiles of PTX). (c) Inhibition heparin−dopamine conjugate (Hep–DA) and test of human coronary artery smooth mus- subsequent LbL assembly of Hep and PTX- cle cell proliferation on different therapeutic loaded HA-g-PLGA micellar nanoparticles. (b) coatings. (Reproduced with permission from Cumulative percent release profiles of PTX Ref. [39].) from three different PTX deposition cycles

10.4.1 Assembly

Liposomes can change their aggregate morphology by collapsing and/or fusion when coming in contact with either solid surfaces [47] or polyelectrolytes with the opposite charge [48], creating supported lipid bilayers. Therefore, diﬀer- ent strategies have been proposed to overcome this aspect when assembling liposome-containing surface-adherent ﬁlms [49]. The deposition of liposomes on solid surfaces includes liposome surface-tethering using the avidin–biotin [50] or DNA–DNA interaction [51] or chemical attachment [52]. While these bare liposome coatings are valid in their own way, they are not suitable for SMDD. The liposomes are not stable enough to withstand the biological environment to allow for controlled drug delivery, making their embedding into polymer coatings necessary. 10.4 Liposome-Containing LbL Films 217

The first study on the incorporation of liposomes within LbL films was reported in 2002 by Katagiri et al. [53]. They deposited intact vesicles on surfaces of poly(vinyl sulfate) (PVS)/PDDA. The liposomes were stabilized by the formation of a polymerized silica layer prior to their embedding into themultilayeredfilm.Michelet al. [54] presented an alternative route to adsorb and embed liposomes made only from phospholipids and, instead of stabilizing them with an inorganic layer, the liposomes were rigidified by the adsorption of polyelectrolytes. In particular, the liposomes were coated with a polycationic layer of poly-D-Lysine (PDL) and incorporated without rupture into PGA/poly(allylamine) (PAH) or HA/PLL-multilayered films. The same group also showed the possibility of depositing the liposomes by spraying [55]. As opposed to the common dipping method, spraying is a faster, reliable, and easily controllable procedure. It also allows achieving regular multilayer growth even under conditions for which dipping fails to produce homogeneous films (e.g., extremely short contact times) [56]. By charging the liposomes with ferrocyanide ions, an electro-active compound, and making use of cyclic voltammetry, the authors could prove that the liposomes were not disrupted during spraying. The possibility of depositing more than one layer of liposomes employing the spraying method has also been demonstrated [55]. In a follow-up work, they reported the utilization of the liposome-containing multilayered films as microreactors [57]. Liposomes containing Ca2+, spermine, and alkaline phosphatase were embedded in PGA/PAH multilayers (Figure 10.3a (i)). When bringing the films in contact with the substrate paranitrophenyl phosphate (PNP), which is able to diffuse across the lipid membrane, PNP was hydrolyzed by the encapsulated enzyme into active phosphates. These phosphates interacted with the encapsulated Ca2+ and spermine to produce calcium phosphate crystals in the liposomes lumen (Figure 10.3a (ii)). This study showed that liposomes containing active payload can also be embedded within the films, opening the route toward the engineering of reactor multilayers to perform localized chemical reactions. Studies in which the liposomes did not require stabilization prior to their immobilization within the multilayered coatings include the electrostatic embedding of zwitterionic liposomes in a PAH/PSS film [58] or negatively charged vesicles in a PLL/PSS coating [59]. The latter assembly allows for the formation of liposome multilayers due to the diffusion of PLL [59]. We recently reported an alternative approach by making use of cholesterol- [60] or oleic acid- [60b] modified polymers to embed uncoated liposomes within LbL films. The incorporation of cholesterol [61], a natural constituent of lipid membranes, or oleic acid provides a noncovalent linkage to the liposomes which is independent of the lipid composition and insensitive to the solution properties (pH, presence of salts, and/or reactive groups) [60a]. Typically, liposomes were deposited onto a polymer precursor layer. A polymer separation layer was required prior to the second liposome deposition step, and the assembly was terminated by adding a polymer-capping layer. Cholesterol-modified PLL

(PLLc) or poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMAc)andoleic 218 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

(i) p-Nitrophenyl phosphate p-Nitrophenyl phosphate

Alkaline phosphatase 2 PAH Calcium PGA Spermine

PGA 0.04 t PAH –1 = 19 h 2 PGA 1035 cm PEI 0.03 p p -Nitrophenyl + Alkaline -Nitrophenol + phosphates 0.02 phosphate phosphatase –1 (ii) 1095 cm 964 cm–1 t = 18 h 0.01 Absorbance 0.00 t = 18 h –0.01 PAH 2 PGA 1200 1150 1100 1050 1000 950 900 (b) Wavenumber (cm–1) PGA PAH 2 PGA PET (a) Phosphates + Calcium + Spermine Inorganic particles

Figure 10.3 (a) The polyelectrolyte multilay- the inside of the liposomes (ii). (b) Mineral- ered film containing liposomes with encap- ization kinetics of inorganic particles within sulated species was put in contact with a the embedded vesicles followed by atten- solution of paranitrophenyl phosphate, which tuated total reflectance Fourier transform diffuses into the lumen of the liposome (i). infrared spectroscopy. (Reproduced with After 18 h, inorganic particles are formed in permission from Ref. [57].)

acid-modiﬁed poly(methacrylic acid)-co-(oleyl methacrylate) (PMAoa)were tailor-made, and their ability to successfully anchor liposomes was demonstrated [60, 62]. While PMA did not allow for the adsorption of liposomes, the adsorption

of liposomes onto a negatively charged PMAc was possible, demonstrating the superiority of cholesterol anchoring over electrostatic interactions only [60a].

The ability of PLL, PMAc, or their combination to eﬃciently attach a second liposome layer was conﬁrmed, depending on the assembly conditions [62].

Additionally, when comparing PMAc and PMAoa, while both types of polymers were equally suited as precursor layers, PMAc was superior as capping layer [60b]. It is worth noting that solution conditions such as buﬀer concentration or pH also have an inﬂuence on the amount of deposited liposomes. When

employing the combination of PLL and PMAc as precursor layers, a higher amount of liposomes was deposited in tris(hydroxymethyl)aminomethane (TRIS) 100 mM as compared to TRIS 10 mM buﬀer conditions. Additionally, when

employing PMAc as separation layer to enable a second liposome deposition step, similar results were observed. Higher liposome incorporation was obtained when employing TRIS 100 mM as compared to TRIS 10 mM buﬀer conditions [63]. 10.4 Liposome-Containing LbL Films 219

10.4.2 Cargo Release Capability from Liposomes within LbL Films

The assembly of thin films, which are able to retain/release a controlled amount of cargo, is an important aspect in SMDD. Several groups have considered mechanisms to trigger the release of cargo from liposomes within LbL films. Temperature was used as a trigger to release a dye from liposomes embedded in HA/PLL films by increasing the temperature above the liquid- to gel-phase transition temperature (Tm) of the liposomes [64]. Temperature was also used as a trigger to release silver ions from PLL-coated silver nitrate-loaded liposomes embedded in PLL/HA multilayered films that promoted a strong and rapid antibacterial effect against Escherichia coli seeded onto the multilayered assembly [65]. Alternatively, an electrochemical stimuli is also an attractive trigger, in particular for surface-based systems, due to the potential to allow for a low-cost, spatially controlled cargo release as demonstrated by Graf et al. [59]

10.4.3 Drug Delivery Applications

Only a few liposome-containing films have been characterized in vitro. To the best of our knowledge, Lynge et al. [66] were the first ones to consider the delivery of the fluorescent model cargo loaded into the liposomal compart- ments to mammalian cells seeded on top of the multilayered film assembled by adsorbing zwitterionic liposomes (Lzw) onto PLL pre-coated glass slide and capping with a poly(dopamine) (PDA) layer. PDA [67], deposited by oxidative self-polymerization of dopamine first reported by Lee et al. [68], has many unique properties including thickness control via the deposition conditions or the possibility to co-deposit PDA with another polymer/biomolecule [69]. The latter aspect has initially been demonstrated for amines containing PEI-g-PEG [70], but was recently expanded by blending nonionic polymers PEG and poly(vinyl alcohol) [71] or temperature-responsive linear [72] and highly branched PNIPAM [73]. Therefore, PDA-based films can be considered as a versatile building block in LbL films and/or as a possible alternative to multilayers. We demonstrated that liposomes can be coated with PDA in solution [74] and immobilized on colloidal [63] or planar substrates [66]. Further, we showed that C2C12 mouse myoblast cells adhering to PLL/Lzw/PDA films, that contained liposomes loaded with a fluorescent lipid as a model cargo, were able to associate with the fluorescent lipids. The cell mean fluorescence (CMF) could be controlled by varying the PDA thickness of the PDA capping layer, and it decreased with increasing cell adhesion time, indicating a depletion or degradation of fluorescent lipids on the surface. Along the same line, we varied the composition of the liposomes and type of capping layers to assess the cell response [73]. The effect of the charge of the carrier for the fluorescent lipids as well as the type of capping layer (PDA, highly branched PNIPAM, or their mixture) on the ability of adhering cells to associate with the fluorescent cargo was assessed using myoblasts, hepatocytes, 220 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

and RAW264.7 mouse leukemic monocyte macrophages. The results showed that the use of positively charged liposomes capped with PNIPAM exhibited a significantly higher myoblast CMF compared to all the other tested combinations of cells and films, demonstrating the ability to engineer the biointerface. We further aimed to facilitate the assembly, and instead of sequentially depositing the liposomes and the PDA, we mixed the dopamine and the liposomes prior to adsorption and successfully deposited liposome-containing films [75]. We showed that these films were able to sustain the delivery of the fluorescent cargo over 24 h. Further, it was possible to obtain a different cellular response depending on the film deposition conditions, for example, dopamine concentration. We also demonstrated for the first time the delivery of an active compound from liposome-containing thin films to adhering mammalian cells by embedding thiocoraline (TC) in the liposome membrane. A significant decrease in cell viability measured from myoblasts adhering to these active films was found, demonstrating the potential of these coatings in SMDD. Alternatively, the response of mammalian cells to liposome-containing PEMs has been considered. The first report by Graf et al. [76] employed pH- sensitive, negatively charged liposomes loaded with calcein deposited onto a

(PLL/PSS)9 –PLL film and capped with (PLL/PGA)2. By applying a galvanostatic current that promotes the hydrolysis of water, which, in turn, created a pH gradient, the liposomes were disrupted and their cargo released. The uptake of the released dye by the adhering myoblast cells was observed by optical microscopy. Importantly, dead staining after the experiment indicated that the application of the current had no significant effect on the cell viability. We recently

reported on the response of myoblasts and hepatocytes to PLL/Lzw/PEMs (PEMs: (PLL/PMAc)x or (PAH/PSS)x) films) (Figure 10.4a (i)) [77]. We found that the CMF of the hepatocytes was higher than for myoblast adhering to the same type of fluorescent lipid-containing film for the same time (Figure 10.4a (ii)). Interestingly, when visualizing the cells, the fluorescence was evenly distributed across the cell interior of the hepatocytes, while there was an accumulation of fluorescence in the proximity of the nuclei for the myoblasts (Figure 10.4a (iii)). When TC was entrapped in the liposomes, a significant decrease in viability for

both cell types adhering to PLL/LTC/PMAc/(PLL/PMAc)x films was found as compared to the control coatings. Surprisingly, the cell viability was found to be more reduced for the myoblasts than for the hepatocytes, although the CMF due to the fluorescent lipid model cargo would have indicated the opposite. We further showed that the delivery from the surface was more efficient compared to the solution-based administration for both cell types. Inafollow-upwork,wecombinedthePDA-basedandtheLbL-basedassembly

[75]. We assembled PLL/Lzw /PEMs/(PDA/Lzw) films with the aim to control the amount of deposited Lzw/PDA in the second adsorption step, and we found that terminating the separation layers with PMAc led to higher liposome deposition in the second step as compared to PLL (Figure 10.4b (i)), and subsequently to a higher CMF of the adhering myoblasts due to the association with the fluorescent lipids (Figure 10.4b (ii)). While there was no significant difference in amount of adsorbed 10.4 Liposome-Containing LbL Films 221

Hepatocytes 35 000 PLL/PMA PAH/PSS O 30 000 700 C OH OH HO O 25 000 OH O HO O O 600 HO O 20 000 HO n O OH m Myoblasts 15 000 500 NH2 N H ALG 10 000 O n Mean fluorescence (a.u.) 5000

= 1 = 2 = 4 400

O = 1 = 2 = 4 PGA X X X 0 X X X f (Hz)

PLL/L /PMA /(PEMs)X Δ 300 zw C – O O HO O NH O OH Hepatocytes x n n ( = 1) 200 C m PLL H H 3 H H 3 100 H CH CH2 n 3 CH PMA 0 C X ∗ PMA 3 = 0.5 1 1.5 2 4 4 C CH H 3 10 PLL/L10/PMAC/(mSL)X/ M2.5

6h 24 h PMAC PAH/PSS Lipids 160000 PLL/PMAC 140000 Myoblasts x O O ( = 1) 120000

H 100000 H H3C H H 80000 CH3

CH3 60000 H3C CH 3 40000

Mean fluorescence (a.u.) 20000 0 X = 0.5 1 1.5 2 4 4∗ 10 PLL/L10/PMAC/(PEM)X/ M2.5

Figure 10.4 (a) (i) Schematic showing the formation of the polymer thin film containing fluorescently labeled liposomes. (ii) CMF and (iii) confocal laser scanning microscopy images of hepatocytes (top) and myoblasts (bottom) grown on liposome-containing polymer thin films are shown. (b)

(i) Change in frequency for adsorption of a mixed liposome/PDA layer onto PLL/L/PMAc/(mSL)x depending on the number of mSLs (mSLs: multiple 10 separation layers). (ii) CMF of myoblasts grown on a PLL/L/PMAc/(PEM)x/ M2.5 film with various numbers of separation PEMs of different composi- 10 tion, PLL/PMAc, or PAH/PSS. ( M2.5 refers to a specific mixture of dopamine and Lzw.) (Adapted with permission from Ref. [77].) 222 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

liposomes measured when terminating the separation layers with PMAc or PSS, the CMF of the adhering myoblasts was signiﬁcantly lower when PSS/PAH layers

were used instead of PMAc/PLL due to the fact that PAH/PSS formed a dense nondegradable film, hindering the access of the cells to the underlying liposomes. This sophisticated composite film showed the potential of these coatings with diverse building blocks to act as a controllable interface in SMDD. Another interesting example of multilayered assemblies containing liposomes for drug delivery applications was recently reported by DeMuth et al. [78], employing polymer/liposome-coated microneedles as a vaccination tool. Microneedles have shown promise for vaccination purposes to improve safety and eliminate pain upon treatment [79]. In this particular study, poly(lactide-co- glycolide) (PLGA) microneedle arrays were pre-coated first by protamine sulfate (PS) and sulfonated poly(styrene) (SPS) to form a base layer of uniform surface charge. Then, multilayers of the biodegradable cationic PBAE and negatively charged interbilayer-cross-linked liposomes (ICLs) were assembled. In ICLs, maleimide covalent cross-links had been introduced between adjacent phospholipid bilayers in the walls of the liposomes to create robust liposomes which can be embedded within polymer films without being ruptured (Figure 10.5a (i)) [78, 80]. The ICLs were loaded with an antigen, DiI as a fluorescent tracer for the lipid walls, and an adjuvant. The microneedle surface was then injected into the cutaneous tissue of mice, a site known for high frequency of antigen-presenting cells (APCs). The PBAE/ICLs films were then transferred from the microneedle surface into the mice cutaneous tissue and remained in the mice skin following the removal of the microneedle arrays, (Figure 10.5a (ii)) allowing for ICLs’ release due to hydrolytic degradation of PBAE (Figure 10.5a (iii)). The ICLs were taken up by the APCs (Figure 10.5a (iv)) in the local tissue, which were activated in situ by the adjuvants delivered by the ICLs. This setup was tested in vivo using fluorescent ovoalbumin (AF647-OVA) as the antigen and the Toll-like receptor -4 agonist monophosphoryl lipid A. The coated microneedles were applied to the skin of MHC II-GFP mice, which express all major histocompatibility class II (MHC II) molecules as a fusion with green fluorescent protein (GFP), allowing MHC II+ APCs in the epidermis to be observed through microscopy. After 6 or 24 h, AF647-OVA and Dil fluorescence was observed around the microneedle insertion sites (Figure 10.5b) which was co-localized in the same z-plane as APCs expressing MHC II-GFP. These results showed that transcutaneous vaccination with ICLs embedded in microneedle-based multilayers significantly enhanced humoral immune responses to a protein antigen.

10.5 LbL Films Containing Miscellaneous Drug Deposits

In this section, other types of drug deposits that have been immobilized in LbL ﬁlms for drug delivery purposes will be highlighted. Although for most of the 10.5 LbL Films Containing Miscellaneous Drug Deposits 223

R1 (i) HN O ICL /

MMM PBAE / O N PLGA Microneedle array M M M O SPS

/ Antigen S

PS R Adjuvant S Microneedle surface O N O (ii) Application + Film delivery (iii)Film degradation (iv) Delivery to APCs in situ

O NH Δt R1

Skin

6 h 24 h (a) MHC II-GFP, Dil-ICL, MHC II-GFP, Dil-ICL, AF647-OVA AF647-OVA

(b)

Figure 10.5 (a) Schematic representation delivery to skin-resident APCs provides coin- of PBAE/ICLs multilayers deposited onto cident antigen exposure and immunostim- PLGA microneedles. ICLs are prepared with ulation for initiation of adaptive immunity. interbilayer covalent cross-links between (b) ICL delivery at microneedle insertion sites maleimide head groups (M) of adjacent (outlined) after 6 and 24 h. Shown is fluores- phospholipid. (PBAE/ICL) PEMs were con- cent signal from (top to bottom) MHC II-GFP structed on microneedles after (PS/SPS) (green), DiI-ICLs (red), AF647-OVA (pink), and base layer deposition (i). Microneedles trans- overlay (yellow) at low (left) and high (right) fer (PBAE/ICL) coatings into the skin and magnification (scale bars 100 μm). (Repro- hydrolytic degradation of PBAE leads to ICL duced with permission from Ref. [78].) release into the surrounding tissue (iii), and examples only their assembly and no biological evaluation have been reported, it nonetheless illustrates the versatility of engineering composite PEMs. Cubic lipid mesophase nanoparticles, typically known as cubosomes [81], have been deposited in LbL films with the aim to improve their cargo retention 224 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

properties, but experimental functional characterization has yet to be provided [82]. Polymersomes made from amphiphilic synthetic block copolymers have recently attracted massive research attention as effective drug carriers due to their attractive benefits over their lipid counterparts, for example, a thicker and mechanically more stable membrane [83, 84] or better loading opportunities compared to micelles. Lomas et al. [85] deposited 100 nm sized stimuli-responsive polymersomes in LbL polymer-based planar films comprising of PVP and TA, demonstrating that polymersomes could potentially be used in SMDD. In a work published by the Picart group [86], PTX loaded in hydrophobic nanodomains were successfully trapped within PEM films made of an alkylated derivative of HA as polyanion and either PLL or quaternized Chi. The efficiency of PTX loading in their system was dependent on the number of deposition layers and type of poly- cation. The retention of the drug was good without initial burst release, but the use of hyaluronidase for enzymatic hydrolysis resulted in an accelerated release of PTX upon film degradation. In another piece of work performed by the Hammond group [88], the spray-assisted LbL process was combined together with the par- ticle replication in non-wetting templates (PRINT) technology [87] to fabricate thin films with the aim to provide a platform for precise control and large-scale production of multifunctional nanoparticle systems. In vitro tests demonstrated that there was enhanced cell uptake when HA was used as compared to other tested PEMs. The spray-LbL on PRINT technology is a highly advanced platform. In another piece of research conducted by Kim et al. [89], they reported on a simple approach for spatially patterned gene delivery inspired by adhesion of marine mussels. The adhesive polymer used in this study was PEI–catechol, which was complexed with adeno-associated viruses (AAV) to form sticky viruses that can stably adhere onto surfaces. The addition of AAV/PEI-catechol vectors to HEK293T cells resulted in a significant twofold increase in cellular transduction compared with the unmodified vectors AAV and AAV-PEI. The authors attributed the enhancement to the ability of the AAV/PEI-catechol vectors to interact between the receptor proteins. They further showed that unlike high cationic charge densities of PEI chains that can cause cellular toxicity, their AAV/PEI-catechol hybrid was able to reduce cellular toxicity. Further, due to the adhesive nature of the hybrid vectors, they can be simply drawn on to surfaces and remained stable, thus providing the opportunity for spatial patterning of viruses. Moreover, when cells were seeded onto the spatially patterned hybrid vectors, the resulting gene expression corresponded to the drawn patterns, highlighting the potential of the PEI-catechol hybrid vectors to guide directed or patterned gene expressions for tissue growth.

10.6 Conclusion/Outlook

In this chapter, we reviewed the assembly of multilayered thin films with entrapped drug deposits assembled on planar substrates toward SMDD. The References 225 predominantly employed drug deposits to date are CDs, micelles, and liposomes. The majority of the background literature cited in this chapter showed that these aforementioned nanocontainers embedded within thin films have already been critically evaluated and extensively characterized toward their potential in SMDD. Much of the literature on this topic has shown that these nanocontainers can be assembled together with PEMs. However, their functional characterization is lagging behind. While improved drug retention/release is claimed, the experimental proof has yet to be delivered for most systems. In vitro studies remain surprisingly scarce, with even fewer in vivo studies reported. However, the few successful preliminary biological characterization studies highlighted in this chapter also demonstrate the potential of these composite films for biomedical applications. The field is still in its infancy, but the interest in this research area is rapidly growing, as supported by the increase in the number of scientific reports. The studies reported here are mainly providing a framework, and there is still need for major improvements of these hybrid coatings prior to their consideration of potential translation to clinics. The true potential of many drug deposits has barely been touched. For instance, polymersomes are a very powerful alternative to liposomes, and their polymeric nature should make them amendable for many purposes. Future studies will have to focus not only on the assembly but also on the evaluation of biological response in the relevant settings. Long-term effects and sustained delivery remain to be achieved. Understanding the difference between administration from the substrate in contrast to solution-based delivery will have to be a focus aspect to address. However, despite all the missing pieces, even these first reports revealed the potential of hybrid coatings for SMDD. We envision that the next years will bring considerable progress and breakthroughs, and we will witness a substantial contribution of this field to novel (sustained) drug delivery approaches for medical therapies.

References

1. Stuart, M.A.C., Huck, W.T.S., Genzer, tissue engineering. Chem. Mater., 24 (5), J., Muller, M., Ober, C., Stamm, M., 854–869. Sukhorukov, G.B., Szleifer, I., Tsukruk, 4. Ariga, K., Ji, Q.M., and Hill, J.P. (2010) V.V.,Urban,M.,Winnik,F.,Zauscher,S., Enzyme-encapsulated layer-by-layer Luzinov, I., and Minko, S. (2010) Emerg- assemblies: current status and chal- ing applications of stimuli-responsive lenges toward ultimate nanodevices, polymer materials. Nat. Mater., 9 (2), in Modern Techniques for Nano- and 101–113. Microreactors/-Reactions,vol.229 (ed 2. Hammond, P.T. (2012) Building biomedical materials layer-by-layer. Mater. F. Caruso), Springer-Verlag, Berlin, pp. Today, 15 (5), 196–206. 51–87. 3. (a) Zelikin, A.N. (2010) Drug releas- 5. (a) Li, P. and Zhang, N. (2011) Layer- ing polymer thin ﬁlms: new era of by-layer self-assembly vectors for surface-mediated drug delivery. ACS gene delivery. Curr. Gene Ther., 11 Nano, 4 (5), 2494–2509.(b) Gribova, V., (1), 58–73.(b) DeMuth, P.C., Min, Y.J., Auzely-Velty, R., and Picart, C. (2012) Huang, B., Kramer, J.A., Miller, A.D., Polyelectrolyte multilayer assemblies on Barouch, D.H., Hammond, P.T., and materials surfaces: from cell adhesion to Irvine, D.J. (2013) Polymer multilayer 226 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

tattooing for enhanced DNA vaccina- assembly of cyclodextrin-based nanopar- tion. Nat. Mater., 12 (4), 367–376.(c) ticles on solid surfaces for gene delivery. Chang, H., Ren, K.F., Wang, J.L., Zhang, Langmuir, 22 (20), 8478–8484. H., Wang, B.L., Zheng, S.M., Zhou, 11. Van der Heyden, A., Wilczewski, Y.Y., and Ji, J. (2013) Surface-mediated M., Labbe, P., and Auzely, R. (2006) functional gene delivery: an effective Multilayer films based on host-guest strategy for enhancing competitiveness interactions between biocompatible poly- of endothelial cells over smooth muscle mers. Chem. Commun., (30), 3220–3222. cells. Biomaterials, 34 (13), 3345–3354. 12. Dam, H.H. and Caruso, F. (2011) 6. (a) Schexnailder, P. and Schmidt, Construction and degradation of polyro- G. (2009) Nanocomposite polymer taxane multilayers. Adv. Mater., 23 (27), 287 hydrogels. Colloid Polym. Sci., 3026–3029. (1), 1–11.(b) Hosta-Rigau, L., Jensen, 13. Wong, S.Y., Moskowitz, J.S., Veselinovic, B.E.B., Fjeldso, K.S., Postma, A., Li, G.X., J., Rosario, R.A., Timachova, K., Blaisse, Goldie, K.N., Albericio, F., Zelikin, A.N., M.R., Fuller, R.C., Klibanov, A.M., and and Stadler, B. (2012) Surface-adhered Hammond, P.T. (2010) Dual functional composite poly(Vinyl Alcohol) physical polyelectrolyte multilayer coatings for hydrogels: polymersome-aided delivery implants: permanent microbicidal base of therapeutic small molecules. Adv. with controlled release of therapeutic 1 Healthcare Mater., (6), 791–795.(c) agents. J. Am. Chem. Soc., 132 (50), Jensen, B.E.B., Hosta-Rigau, L., Spycher, 17840–17848. P.R., Reimhult, E., Stadler, B., and 14. Benkirane-Jessel, N., Schwinté, P., Zelikin, A.N. (2013) Lipogels: surface- Falvey, P., Darcy, R., Haïkel, Y., Schaaf, adherent composite hydrogels assembled P., Voegel, J.C., and Ogier, J. (2004) from poly(vinyl alcohol) and liposomes. Build-up of polypeptide multilayer Nanoscale, 5 (15), 6758–6766. coatings with anti-inflammatory prop- 7. Zhang, J. and Ma, P.X. (2010) erties based on the embedding of Host–guest interactions mediated nano- piroxicam–cyclodextrin complexes. assemblies using cyclodextrin-containing Adv. Funct. Mater., 14 (2), 174–182. hydrophilic polymers and their biomed- 15. Smith, R.C., Riollano, M., Leung, A., and ical applications. Nano Today, 5 (4), Hammond, P.T. (2009) Layer-by-layer 337–350. platform technology for small-molecule 8. (a) Sun, T., Guo, Q., Zhang, C., Hao, 48 J.,Xing,P.,Su,J.,Li,S.,Hao,A.,and delivery. Angew. Chem. Int. Ed., (47), Liu, G. (2012) Self-assembled vesicles 8974–8977. prepared from amphiphilic cyclodex- 16. Daubiné, F., Cortial, D., Ladam, G., trinsasdrugcarriers.Langmuir, 28 Atmani, H., Haïkel, Y., Voegel, J.-C., (23), 8625–8636.(b) Li, J., Xiao, H., Li, Clézardin, P., and Benkirane-Jessel, N. J., and Zhong, Y. (2004) Drug carrier (2009) Nanostructured polyelectrolyte systems based on water-soluble cationic multilayer drug delivery systems for β-cyclodextrin polymers. Int. J. Pharm., bone metastasis prevention. Biomateri- 30 278 (2), 329–342.(c) Davis, M.E. and als, (31), 6367–6373. Brewster, M.E. (2004) Cyclodextrin- 17. Dierich, A., Le Guen, E., Messaddeq, N., based pharmaceutics: past, present and Stoltz, J.F., Netter, P., Schaaf, P., Voegel, future. Nat. Rev. Drug Discovery, 3 (12), J.C., and Benkirane-Jessel, N. (2007) 1023–1035. Bone formation mediated by synergy- 9. Dreja, M., Kim, I.T., Yin, Y., and Xia, acting growth factors embedded in a Y. (2000) Multilayered supermolecular polyelectrolyte multilayer film. Adv. structures self-assembled from poly- Mater., 19 (5), 693–697. electrolytes and cyclodextrin host-guest 18. Schouten, S., Stroeve, P., and Longo, complexes. J. Mater. Chem., 10 (3), M.L. (1999) DNA adsorption and 603–605. cationic bilayer deposition on self- 10. Park, I.-K., von Recum, H.A., Jiang, S., assembled monolayers. Langmuir, 15 and Pun, S.H. (2006) Supramolecular (23), 8133–8139. References 227

19. Boon, E.M., Ceres, D.M., Drummond, as building blocks for layer-by-layer T.G., Hill, M.G., and Barton, J.K. (2000) assembly: an approach for incorporation Mutation detection by electrocataly- and controlled release of water-insoluble sis at DNA-modified electrodes. Nat. dyes. Chem. Mater., 17 (20), 5065–5069. Biotechnol., 18 (10), 1096–1100. 28. Cho, J., Hong, J., Char, K., and Caruso, 20. (a) Jewell, C.M., Zhang, J., Fredin, F. (2006) Nanoporous block copolymer N.J., and Lynn, D.M. (2005) Multi- micelle/micelle multilayer films with dual layered polyelectrolyte films promote optical properties. J. Am. Chem. Soc., the direct and localized delivery of 128 (30), 9935–9942. DNA to cells. J. Controlled Release, 106 29. Nguyen, P.M., Zacharia, N.S., (1–2), 214–223.(b) Zhang, J., Chua, Verploegen, E., and Hammond, P.T. L.S., and Lynn, D.M. (2004) Multilay- (2007) Extended release antibacterial ered thin films that sustain the release layer-by-layer films incorporating linear- of functional DNA under physiolog- dendritic block copolymer micelles. ical conditions. Langmuir, 20 (19), Chem. Mater., 19 (23), 5524–5530. 8015–8021. 30. Hu, X. and Ji, J. (2009) Construc- 21. Jessel, N., Oulad-Abdelghani, M., Meyer, tion of multifunctional coatings via F., Lavalle, P., Haîkel, Y., Schaaf, P., layer-by-layer assembly of sulfonated and Voegel, J.-C. (2006) Multiple and hyperbranched polyether and chitosan. time-scheduled in situ DNA delivery Langmuir, 26 (4), 2624–2629. mediated by β-cyclodextrin embedded in 31. Xu, L., Zhu, Z., and Sukhishvili, S.A. a polyelectrolyte multilayer. Proc. Natl. (2010) Polyelectrolyte multilayers Acad. Sci. U.S.A., 103 (23), 8618–8621. of diblock copolymer micelles with 22. Zhang, X., Sharma, K.K., Boeglin, M., temperature-responsive cores. Langmuir, Ogier, J., Mainard, D., Voegel, J.-C., 27 (1), 409–415. Mély, Y., and Benkirane-Jessel, N. (2008) 32. Zhu, Z. and Sukhishvili, S.A. (2009) Transfection ability and intracellular Temperature-induced swelling and small DNA pathway of nanostructured gene- molecule release with hydrogen-bonded delivery systems. Nano Lett., 8 (8), multilayers of block copolymer micelles. 2432–2436. ACS Nano, 3 (11), 3595–3605. 23. Hu, Y., Cai, K., Luo, Z., and 33. Cavalieri, F., Postma, A., Lee, L., and Hu, R. (2010) Construction of Caruso, F. (2009) Assembly and func- polyethyleneimine-β-cyclodextrin/pDNA tionalization of DNA polymer microcap- multilayer structure for improved in situ sules. ACS Nano, 3 (1), 234–240. gene transfection. Adv. Eng. Mater., 12 34. Addison, T., Cayre, O.J., Biggs, S., (1-2), B18–B25. Armes, S.P., and York, D. (2008) Incor- 24. Wen, Y., Pan, S., Luo, X., Zhang, X., poration of block copolymer micelles Zhang, W., and Feng, M. (2009) A into multilayer films for use as nan- biodegradable low molecular weight odelivery systems. Langmuir, 24 (23), polyethylenimine derivative as low 13328–13333. toxicity and efficient gene vector. Biocon- 35. Erel,I.,Zhu,Z.,Zhuk,A.,and jugate Chem., 20 (2), 322–332. Sukhishvili, S.A. (2011) Hydrogen- 25. Müller, A.H.E. and Borisov, O. (2011) bonded layer-by-layer films of block Self Organized Nanostructures of copolymer micelles with pH-responsive Amphiphilic Block Copolymers I, cores. J. Colloid Interface Sci., 355 (1), Springer. 61–69. 26. Rijcken, C.J.F., Soga, O., Hennink, W.E., 36. Kim, B.-S., Park, S.W., and Hammond, and van Nostrum, C.F. (2007) Triggered P.T. (2008) Hydrogen-bonding layer-by- destabilisation of polymeric micelles and layer-assembled biodegradable polymeric vesicles by changing polymers polarity: micelles as drug delivery vehicles from an attractive tool for drug delivery. J. surfaces. ACS Nano, 2 (2), 386–392. Controlled Release, 120 (3), 131–148. 37. Gensel, J., Dewald, I., Erath, J., 27. Ma, N., Zhang, H., Song, B., Wang, Z., Betthausen, E., Muller, A.H.E., and Fery, and Zhang, X. (2005) Polymer micelles A. (2013) Reversible swelling transitions 228 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

in stimuli-responsive layer-by-layer films nanocapsules in food science and agri- containing block copolymer micelles. culture. Crit. Rev. Food Sci. Nutr., 45 Chem. Sci., 4 (1), 325–334. (7-8), 587–605. 38. Kim, B.-S., Lee, H.-i., Min, Y., Poon, Z., 45. Bally, M., Bailey, K., Sugihara, K., and Hammond, P.T. (2009) Hydrogen- Grieshaber, D., Voeroes, J., and Stadler, bonded multilayer of pH-responsive B. (2010) Liposome and lipid bilayer polymeric micelles with tannic acid for arrays towards biosensing applications. surface drug delivery. Chem. Commun., Small, 6 (22), 2481–2497. (28), 4194–4196. 46. Barenholz, Y. (2001) Liposome appli- 39. Kim, T.G., Lee, H., Jang, Y., and Park, cation: problems and prospects. Curr. T.G. (2009) Controlled release of pacli- Opin. Colloid Interface Sci., 6 (1), 66–77. taxel from heparinized metal stent 47. Richter, R.P., Berat, R., and Brisson, A.R. fabricated by layer-by-layer assembly (2006) Formation of solid-supported of polylysine and hyaluronic acid-g- lipid bilayers: an integrated view. Lang- poly(lactic-co-glycolic acid) micelles muir, 22 (8), 3497–3505. encapsulating paclitaxel. Biomacro- 48. Cha, T., Guo, A., and Zhu, X.Y. (2006) 10 molecules, (6), 1532–1539. Formation of supported phospholipid 40. Hong, J., Alvarez, L., Shah, N., Cho, bilayers on molecular surfaces: role Y., Kim, B.-S., Griffith, L., Char, K., of surface charge density and electro- and Hammond, P. (2012) Multilayer static interaction. Biophys. J., 90 (4), thin-film coatings capable of extended 1270–1274. programmable drug release: application 49. Ruysschaert, T., Germain, M., Gomes, to human mesenchymal stem cell differ- J., Fournier, D., Sukhorukov, G.B., entiation. Drug Delivery Transl. Res., 2 Meier, W., and Winterhalter, M. (2004) (5), 375–383. Liposome-based nanocapsules. IEEE 41. (a) Petersen, A.L., Hansen, A.E., Trans. Nanobioscience, 3 (1), 49–55. Gabizon, A., and Andresen, T.L. (2012) 50. (a) Vermette, P., Meagher, L., Gagnon, Liposome imaging agents in personal- E., Griesser, H.J., and Doillon, C.J. (2002) ized medicine. Adv. Drug Delivery Rev., Immobilized liposome layers for drug 64 (13), 1417–1435.(b) Torchilin, V.P. delivery applications: inhibition of angio- (2005) Recent advances with liposomes genesis. J. Controlled Release, 80 (1-3), as pharmaceutical carriers. Nat. Rev. 179–195.(b) Patolsky, F., Lichtenstein, Drug Discovery, 4 (2), 145–160. A., and Willner, I. (2001) Electronic 42. (a) Thongborisute, J., Tsuruta, A., Kawabata, Y., and Takeuchi, H. (2006) transduction of DNA sensing processes Theeffectofparticlestructureof on surfaces: amplification of DNA chitosan-coated liposomes and type detection and analysis of single-base of chitosan on oral delivery of calcitonin. mismatches by tagged liposomes. J. Am. 123 J. Drug Targeting, 14 (3), 147–154.(b) Chem. Soc., (22), 5194–5205. Gerelli, Y., Barbieri, S., Di Bari, M.T., 51. (a) Yoshina-Ishii, C., Miller, G.P., Kraft, Deriu, A., Cantu, L., Brocca, P., Sonvico, M.L., Kool, E.T., and Boxer, S.G. (2005) F., Colombo, P., May, R., and Motta, S. General method for modification of (2008) Structure of self-organized mul- liposomes for encoded assembly on tilayer nanoparticles for drug delivery. supported bilayers. J. Am. Chem. Soc., Langmuir, 24 (20), 11378–11384. 127 (5), 1356–1357.(b) Stadler, B., 43. Goncalves Maia Campos, P.M.B., de Falconnet, D., Pfeiffer, I., Hook, F., and Camargo Junior, F.B., de Andrade, J.P., Voros, J. (2004) Micropatterning of and Gaspar, L.R. (2012) Efficacy of cos- DNA-tagged vesicles. Langmuir, 20 (26), metic formulations containing dispersion 11348–11354. of liposome with magnesium ascorbyl 52. Zhou, J., Loftus, A.L., Mulley, G., and phosphate, alpha-lipoic acid and kinetin. Jenkins, A.T.A. (2010) A thin film detec- Photochem. Photobiol., 88 (3), 748–752. tion/response system for pathogenic 44. Taylor, T.M., Davidson, P.M., Bruce, bacteria. J. Am. Chem. Soc., 132 (18), B.D., and Weiss, J. (2005) Liposomal 6566–6570. References 229

53. (a) Katagiri, K., Hamasaki, R., Ariga, K., L., Chandrawati, R., Saveriades, E., and Kikuchi, J. (2002) Layer-by-layer Odermatt, P.D., Postma, A., Ercole, self-assembling of liposomal nanohybrid F.,Breheney,K.,Wark,K.L.,Stadler, "cerasome" on substrates. Langmuir, B., and Caruso, F. (2010) Noncovalent 18 (17), 6709–6711.(b) Katagiri, K., liposome linkage and miniaturiza- Hamasaki, R., Ariga, K., and Kikuchi, tion of capsosomes for drug delivery. J. (2002) Layered paving of vesicular Biomacromolecules, 11 (12), 3548–3555. nanoparticles formed with cerasome as 61. Hosta-Rigau, L., Zhang, Y., Teo, B.M., a bioinspired organic-inorganic hybrid. J. Postma, A., and Staedler, B. (2013) 124 Am. Chem. Soc., (27), 7892–7893. Cholesterol - a biological compound as 54. Michel,M.,Vautier,D.,Voegel,J.C., a building block in bionanotechnology. Schaaf, P., and Ball, V. (2004) Layer by Nanoscale, 5 (1), 89–109. layer self-assembled polyelectrolyte mul- 62. Hosta-Rigau, L., Stadler, B., Yan, Y., tilayers with embedded phospholipid Nice,E.C.,Heath,J.K.,Albericio,F., vesicles. Langmuir, 20 (12), 4835–4839. and Caruso, F. (2010) Capsosomes with 55. Michel, A., Izquierdo, A., Decher, G., multilayered subcompartments: assembly Voegel, J.C., Schaaf, P., and Ball, V. (2005) Layer by layer self-assembled and loading with hydrophobic cargo. 20 polyelectrolyte multilayers with embed- Adv. Funct. Mater., (1), 59–66. ded phospholipid vesicles obtained 63. Hosta-Rigau, L., York-Duran, M.J., byspraying:integrityofthevesicles. Zhang, Y., Goldie, K.N., and Staedler, Langmuir, 21 (17), 7854–7859. B. (2013) Confined multiple enzy- 56. Izquierdo, A., Ono, S.S., Voegel, J.C., matic (cascade) reactions within Schaaf, P., and Decher, G. (2005) Dip- poly(dopamine)-based capsosomes. ping versus spraying: exploring the ACS Applied Mater.Interfaces,DOI: deposition conditions for speeding up 10.1021/am502743z. layer-by-layer assembly. Langmuir, 21 64. Volodkin, D., Arntz, Y., Schaaf, P., (16), 7558–7567. Moehwald,H.,Voegel,J.-C.,andBall, 57. Michel,M.,Arntz,Y.,Fleith,G., V. (2008) Composite multilayered bio- Toquant, J., Haikel, Y., Voegel, J.C., compatible polyelectrolyte films with Schaaf, P., and Ball, V. (2006) Layer- intact liposomes: stability and tempera- by-layer self-assembled polyelectrolyte ture triggered dye release. Soft Matter, 4 multilayers with embedded liposomes: (1), 122–130. immobilized submicronic reactors 65. Malcher, M., Volodkin, D., Heurtault, 22 for mineralization. Langmuir, (5), B., Andre, P., Schaaf, P., Moehwald, H., 2358–2364. Voegel, J.-C., Sokolowski, A., Ball, V., 58. Städler, B., Chandrawati, R., Goldie, Boulmedais, F., and Frisch, B. (2008) K., and Caruso, F. (2009) Capsosomes: Embedded silver ions-containing lipo- sub-compartmentalizing polyelectrolyte somes in polyelectrolyte multilayers: capsules using liposomes. Langmuir, 25 cargos films for antibacterial agents. (12), 6725–6732. Langmuir, 24 (18), 10209–10215. 59. Graf, N., Albertini, F., Petit, T., Reimhult, 66. Lynge, M.E., Ogaki, R., Laursen, E., Voeroes, J., and Zambelli, T. (2011) Electrochemically stimulated release A.O., Lovmand, J., Sutherland, from liposomes embedded in a polyelec- D.S., and Stadler, B. (2011) Poly- trolyte multilayer. Adv. Funct. Mater., 21 dopamine/liposome coatings and (9), 1666–1672. their interaction with myoblast cells. 3 60. (a) Chandrawati, R., Städler, B., Postma, ACS Appl. Mater. Interfaces, (6), A., Connal, L.A., Chong, S.-F., Zelikin, 2142–2147. A.N., and Caruso, F. (2009) Modeling 67. Lynge, M.E., van der Westen, R., capsosome assembly: from tailor-made Postma, A., and Stadler, B. (2011) building blocks to intact cargo-loaded Polydopamine–a nature-inspired poly- sub-compartmentalized capsules. Bioma- mer coating for biomedical science. terials, 30, 5988–5998.(b) Hosta-Rigau, Nanoscale, 3 (12), 4916–4928. 230 10 Subcompartmentalized Surface-Adhering Polymer Thin Films Toward Drug Delivery Applications

68. Lee, H., Dellatore, S.M., Miller, W.M., embedded in polyelectrolyte multilayers. and Messersmith, P.B. (2007) Mussel- Soft Matter, 8 (13), 3641–3648. inspired surface chemistry for multi- 77. Lynge, M.E., Laursen, M.B., Hosta-Rigau, functional coatings. Science, 318 (5849), L.,Jensen,B.E.B.,Ogaki,R.,Smith, 426–430. A.A.A., Zelikin, A.N., and Stadler, B. 69. Kang, S.M., Hwang, N.S., Yeom, J., (2013) Liposomes as drug deposits in Park, S.Y., Messersmith, P.B., Choi, I.S., multilayered polymer films. ACS Appl. Langer, R., Anderson, D.G., and Lee, H. Mater. Interfaces, 5 (8), 2967–2975. (2012) One-step multipurpose surface 78. DeMuth, P.C., Moon, J.J., Suh, H., functionalization by adhesive cate- Hammond, P.T., and Irvine, D.J. (2012) cholamine. Adv. Funct. Mater., 22 (14), Releasable layer-by-layer assembly 2949–2955. of stabilized lipid nanocapsules on 70. Tsai, W.-B., Chien, C.-Y., Thissen, microneedles for enhanced transcuta- 6 H., and Lai, J.-Y. (2011) Dopamine- neous vaccine delivery. ACS Nano, (9), assisted immobilization of poly(ethylene 8041–8051. imine) based polymers for control of 79. (a) DeMuth, P.C., Su, X., Samuel, cell–surface interactions. Acta Biomater., R.E., Hammond, P.T., and Irvine, D.J. 7 (6), 2518–2525. (2010) Nano-layered microneedles 71. Zhang, Y., Thingholm, B., Goldie, K.N., for transcutaneous delivery of poly- Ogaki, R., and Städler, B. (2012) Assem- mer nanoparticles and plasmid DNA. 22 bly of poly(dopamine) films mixed with Adv. Mater., (43), 4851–4856.(b) Ekwueme, D.U., Weniger, B.G., and a nonionic polymer. Langmuir, 28 (51), Chen, R.T. (2002) Model-based estimates 17585–17592. of risks of disease transmission and eco- 72. Zhang, Y., Panneerselvam, K., Ogaki, nomic costs of seven injection devices in R.,Hosta-Rigau,L.,vanderWesten, sub-saharan africa. Bull. World Health R., Jensen, B.E.B., Teo, B.M., Zhu, Organ., 80 (11), 859–870.(c) Chen, X., M., and Städler, B. (2013) Assem- Kask, A.S., Crichton, M.L., McNeilly, bly of poly(dopamine)/poly(N- C., Yukiko, S., Dong, L., Marshak, J.O., isopropylacrylamide) mixed films and Jarrahian, C., Fernando, G.J.P., Chen, their temperature-dependent interac- D., Koelle, D.M., and Kendall, M.A.F. tion with proteins, liposomes, and cells. (2010) Improved DNA vaccination by Langmuir, 29 (32), 10213–10222. skin-targeted delivery using dry-coated 73. Zhang, Y., Teo, B.M., Postma, A., Ercole, densely-packed microprojection arrays. J. F.,Ogaki,R.,Zhu,M.,andStädler, Controlled Release, 148 (3), 327–333. B. (2013) Highly-branched poly(N- 80. Moon, J.J., Suh, H., Bershteyn, A., isopropylacrylamide) as a component in Stephan, M.T., Liu, H., Huang, B., poly(dopamine) films. J. Phys. Chem. B, Sohail, M., Luo, S., Um, S.H., Khant, 117 (36), 10504–10512. H., Goodwin, J.T., Ramos, J., Chiu, W., 74. Westen, R., Hosta-Rigau, L., Sutherland, and Irvine, D.J. (2011) Interbilayer- D., Goldie, K., Albericio, F., Postma, crosslinked multilamellar vesicles as A., and Städler, B. (2012) Myoblast cell synthetic vaccines for potent humoral interaction with polydopamine coated and cellular immune responses. Nat. liposomes. Biointerphases, 7 (1–4), 1–9. Mater., 10 (3), 243–251. 75. Lynge, M.E., Teo, B.M., Laursen, M.B., 81. Spicer, P.T. (2005) Progress in liquid Zhang, Y., and Stadler, B. (2013) Cargo crystalline dispersions: cubosomes. Curr. delivery to adhering myoblast cells from Opin. Colloid Interface Sci., 10 (5–6), liposome-containing poly(dopamine) 274–279. composite coatings. Biomater. Sci., 1, 82. Driever, C.D., Mulet, X., Johnston, 1181–1192. A.P.R., Waddington, L.J., Thissen, H., 76. Graf, N., Tanno, A., Dochter, A., Caruso, F., and Drummond, C.J. (2011) Rothfuchs,N.,Voeroes,A.,and Converging layer-by-layer polyelectrolyte Zambelli, T. (2012) Electrochemically microcapsule and cubic lyotropic liquid driven delivery to cells from vesicles crystalline nanoparticle approaches for References 231

molecular encapsulation. Soft Matter, 7 87. Gratton, S.E.A., Pohlhaus, P.D., Lee, (9), 4257–4266. J., Guo, J., Cho, M.J., and DeSimone, 83. Antonietti, M. and Förster, S. (2003) J.M. (2007) Nanofabricated particles for Vesicles and liposomes: a self-assembly engineered drug therapies: a prelimi- principle beyond lipids. Adv. Mater., 15 nary biodistribution study of PRINT™ (16), 1323–1333. nanoparticles. J. Controlled Release, 121 84. Discher, B.M., Won, Y.-Y., Ege, D.S., (1–2), 10–18. Lee, J.C.-M., Bates, F.S., Discher, 88. Morton, S.W., Herlihy, K.P., Shopsowitz, D.E., and Hammer, D.A. (1999) Poly- K.E., Deng, Z.J., Chu, K.S., Bowerman, mersomes: tough vesicles made from C.J., DeSimone, J.M., and Hammond, diblock copolymers. Science, 284 (5417), P.T. (2013) Scalable manufacture of 1143–1146. built-to-order nanomedicine: spray- 85. Lomas, H., Johnston, A.P.R., Such, G.K., assisted layer-by-layer functionalization Zhu, Z., Liang, K., Van Koeverden, M.P., of PRINT nanoparticles. Adv. Mater., 25 Alongkornchotikul, S., and Caruso, F. (34), 4707–4713. (2011) Polymersome-loaded capsules for 89. Kim, E., Song, I.T., Lee, S., Kim, J.-S., controlled release of DNA. Small, 7 (14), Lee, H., and Jang, J.-H. (2012) Draw- 2109–2119. ing sticky adeno-associated viruses on 86. Boudou, T., Kharkar, P., Jing, J., Guillot, surfaces for spatially patterned gene R., Pignot-Paintrand, I., Auzely-Velty, expression. Angew. Chem. Int. Ed., 51, R., and Picart, C. (2012) Polyelectrolyte 5598–5601. multilayer nanoshells with hydrophobic nanodomains for delivery of Paclitaxel. J. Controlled Release, 159 (3), 403–412.