**3.3.2 The ionic LbL assembly**

18 Biomedicine

Fig. 5. (a) Chemical structure of biotin–PEG-conjugate (biotin–PEG–lipid). (b) Schematic illustration of the interaction between streptavidin and biotin–PEG–lipid at the lipid bilayer cell membrane. Biotin–PEG–lipid has hydrophobic acyl chains and is incorporated into the cell surface by anchoring into the lipid bilayer. Streptavidin is immobilized on the cell surface by anchoring to biotin–PEG–lipid. (c) Scheme for the immobilization of streptavidin-immobilized HEK293 cells on the surface of biotin–PEG–lipid-modified islets. After mixing streptavidinimmobilized HEK293 cells and biotin–PEG–lipid-modified islets, they were cultured in medium at 37°C on a culture dish. During culture, HEK293 cells were spread and grown on the cell surface to cover the whole surface. (d) Hamster islets modified with biotin–PEG–lipid and immobilized with streptavidin-immobilized HEK293 cells. The HEK293 cells were labeled with CellTracker. Reprinted from Teramura & Iwata, 2009 with permission from Elsevier.

soluble polymers on surfaces from aqueous solutions which results in nano-thin coatings of controllable thickness and composition (Decher & Schlenoff, 2002; Kharlampieva & Sukhishvili, 2006; Tang et al., 2006). The ultrathin conformal coating affords a faster response to stimulation and the possibility to bind factors or protective molecules to the protective ultrathin shell with the later slow triggered release of these molecules (Chluba et al., 2001). By selecting specific polyelectrolytes, a defined cutoff of the coating (Kozlovskaya & Sukhishvili, 2006) is possible, as is inhibitor binding to prevent graft rejection, microphage attacks, or antibody recognition (Kim & Park, 2006). Modification of the last coating layer can be used to support functionality of islets and reduce the immune response from a host system. The cutoff of the polyelectrolyte multilayer (PEM) is defined by polyelectrolytes

used in coating formation (Krol et al., 2006).

To promote a multilayer film formation on the cell surfaces, the negatively charged cell surface is treated with a cationic polymer solution and the cell surface is further exposed to an anionic polymer solution to form an electrostatically-paired polyelectrolyte complex film (Fig. 3, A4). Effect of molecular weight of polyelectrolytes and the charge of outermost layer was demonstrated in case of the LBL encapsulation of human islets into poly(allylamine hydrochloride)/poly(styrenesulfonate sodium salt) (PAH)/(PSS) and poly- (diallyldimethylammonium chloride) (PDADMAC)/PSS layers. Islets encapsulated into PAH/PSS and PDADMAC/PSS multilayers using a higher polycation molecular weight demonstrated a limited insulin release due to a lowered permeability of insulin through the polyelectrolyte membrane. A decrease in a polycation molecular weight resulted in larger pores of the polyelectrolyte membrane and restored responsive relationship between glucose stimulation and insulin response of the coated islets (Krol et al., 2006).

Most cationic polymers widely used in the LbL modification of surfaces such as poly(Llysine) (PLL) and poly(ethylene imine) (PEI) are extremely cytotoxic and cells treated with the polycations can be severely damaged. Their cytotoxic effect though has been observed to be dependent on polycation concentration and exposure time (De Koker et al., 2007). The overall cytotoxicity of the polyelectrolytes originates from positive charge of polycations which can induce pore formation within the cell membrane causing its damage and, eventually, cell death (Bieber et al., 2002; Godbey et al., 1999). The high toxicity of the PAH/PSS LbL film was confirmed by Wilson et al (Wilson et al., 2008). They demonstrated that coating the murine islets with only 3 layers of PAH/PSS/PAH led to the reduction of islet viability by 70%. Similar effect was found for islets coated with 3 layers of PLL/alginate LbL film. Even 15 minutes of islets incubation with low concentration of PLL results in ~60% decrease in cell viability. Menger et al showed that PLL was able to pass through the lipid bilayer if it was previously allowed to form complex with anionic lipids (Menger et al., 2003). PEI was found extremely toxic to the islets. This polycation destroys the cell membrane immediately after its interactions with the membrane surface (Teramura et al., 2008b). The overall charge arrangement of a polycation and its interaction with the cell membrane strongly depends on the three-dimensional structure and flexibility of the polymer chains. It has been shown that polymers with highly flexible chains and a high cationic density will exert tremendous cytotoxicity. Thus, the polycations with globular structures demonstrated good biocompatibility, whereas polymers with more linear and flexible structure such as PLL and PEI showed higher cytotoxicity (Teramura et al., 2008b).

Since the polycations toxicity partially depends on the polymer charge density, it can be attenuated by conjugating neutral molecules, such as PEG, to the critical number of amino groups along the polycation backbones. PEGylation of PLL is carried out through grafting of N-hydroxysuccinimide-PEG (NHS-PEG) chains to amino groups on PLL backbone to produce PLL-g-PEG. The grafted PEGs are unbranched, hydrophilic, discrete-length molecules in the form of Methyl-PEGn-NHS ester, where the subscript "n" denotes a number of the ethylene glycol units. The NHS ester end group is spontaneously reactive with primary amines, providing for efficient PEGylation of amine-containing molecules or surfaces. The methoxy(ethylene glycol) grafts were conjugated to PLL backbone through a covalent attachment to lysine residues (Wilson et al., 2009). Forty percent of PEG substitutes on the PLL chain allowed for attenuation of the PLL positive charges without any

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 21

deleterious effect on the islet viability (Fig. 6). The modification of PEG grafts with various functional molecules, such as biotin, hydrazide and azide, can extend the functional capabilities of the PLL-g-PEG-based LbL islet coatings. For example, the deposition of the PLL-g-PEG-biotin outmost layer on top of the PLL-g-PEG/Alginate multilayer film can generate surface densities of biotin functional groups comparable with that obtained by the treatment of islet surfaces with only NHS-PEG-biotin molecules (PEGylation/biotinylation through the NHS-ester coupling). Unlike the latter, the former approach is advantageous as it does not alter cell surface morphology and allows for controlled densities of the biotin on the modified islet surfaces (Krishnamurthy et al., 2010). The first successful *in vivo* transplantation of PEM engineered islets was demonstrated for the murine islets coated

High toxicity of most polyelectrolyte polycations limits their use in biomedical applications. However, natural biopolymers chitosan and alginate have more similarities with the extracellular matrix, are chemically versatile and have a good biocompatibility. These linear polysaccharides carry opposite charge and can be electrostatically bound in a PEM. The (Chitosan/Alginate)3 islet coating was achieved *via* alternate deposition starting from positively charged chitosan (Zhi et al., 2010). The deposition conditions had been shown to greatly influence the islets viability, which well correlates with difference in charge density and toxicity of the polycation at high and neutral pH values. Additional protective outermost coating layer of phosphorylcholine (PC)-modified chondroitin-4-sulfate was introduced to reduce non-specific protein adsorption of the film. PC-moieties demonstrated remarkable repelling and hemocompatible properties. Increase in the coating thickness by adding additional layers of (Chitosan/Alginate) up to 5 bilayers did not adversely affect islets viability and insulin release, and the coated islets were viable up to 5 weeks of post-

with conformal PLL-g-PEG/Alginate LbL films (Fig. 6g) (Wilson et al., 2011).

**3.3.3 The LbL assembly based on covalent and/or specific interactions** 

A multilayer PVA membrane formed via the layer-by-layer assembly was investigated for islet immunoprotective capabilities (Fig. 3, A3) (Teramura et al., 2007). In this approach maleimide-PEG-conjugated phospholipids were used to first modify the cell membrane surface to promote further interactions with PVA derivatives. It is well-known that PEGconjugated phospholipids can be immobilized on the cell membrane through incorporation of the lipid chains into the cell membrane due to their hydrophobic interactions with the lipid bilayers of the membrane (Iwata et al., 1992). Moreover, PEG-phospholipids are more compatible with cells compared to polycations used in the ionic LbL surface modification of islets. A layer of PVA with introduced thiol groups (PVA-SH) was covalently attached to the maleimide-PEG anchors via thiol-maleimide reaction. The LbL multilayer of PVA was then deposited by alternating immersion of the islets into PVA-SH and PVA-pyridyl disulfide (PVA-PD). The driving force for the multilayer formation was a thiol/disulfide exchange reaction between the PVA derivatives. This ultra-thin PVA membrane affected neither cell

The LbL assembly of heparin multilayers was investigated for suppression of instant bloodmediated inflammatory reactions for the case when islets are to be transplanted through the portal vein to liver (Luan et al., 2011). The heparin well-known for its anti-thrombogenic properties was co-assembled with human soluble form complement receptor 1 (sCR1) which

encapsulation.

viability nor insulin release function.

Fig. 6. Cell-surface-supported PEMs were assembled on individual pancreatic islets through LbL deposition of PLL-g-PEG copolymers and alginate. (a) Method to assemble PEMs on islets. (b) Representative confocal micrographs overlaid on bright-field images of coated islets coated using flourescein-labeled alginate (F-Alg) with eight bilayers (8x), a single bilayer (1x), or treated only with F-Alg (8x, w/o cation). (c) F-Alg is localized on the extracellular surface of cells, confirming the cell-surface-supported nature of films. (d) Deposition of a single PLL/F-Alg bilayer resulted in intracellular internalization of alginate by peripheral cells. (e) Chemistry and reactivity of cell-surface-supported films can be tailored through integration of biotin- and azide-functionalized PLL-g-PEG copolymers. (f) Insulin secretion by islets coated with a (PLL-g-PEG /alginate)8 film (gray) and untreated islets (black) in response to a step-change in glucose. (g) Confocal (left) overlaid on brightfield micrographs (right) of frozen sections of liver (L) after intraportal transplantation of islets (I) engineered with PEM films labeled with streptavidinCy3. Scale bars: b,e (top), g=50 μm; c,d,e (bottom)=10 μm. Reprinted with permission from Wilson et al., 2011. Copyright 2011 American Chemical Society.

Fig. 6. Cell-surface-supported PEMs were assembled on individual pancreatic islets through LbL deposition of PLL-g-PEG copolymers and alginate. (a) Method to assemble PEMs on islets. (b) Representative confocal micrographs overlaid on bright-field images of coated islets coated using flourescein-labeled alginate (F-Alg) with eight bilayers (8x), a single bilayer (1x), or treated only with F-Alg (8x, w/o cation). (c) F-Alg is localized on the extracellular surface of cells, confirming the cell-surface-supported nature of films. (d) Deposition of a single PLL/F-Alg bilayer resulted in intracellular internalization of alginate by peripheral cells. (e) Chemistry and reactivity of cell-surface-supported films can be tailored through integration of biotin- and azide-functionalized PLL-g-PEG copolymers. (f) Insulin secretion by islets coated with a (PLL-g-PEG /alginate)8 film (gray) and untreated islets (black) in response to a step-change in glucose. (g) Confocal (left) overlaid on brightfield micrographs (right) of frozen sections of liver (L) after intraportal transplantation of islets (I) engineered with PEM films labeled with streptavidinCy3. Scale bars: b,e (top), g=50 μm; c,d,e (bottom)=10 μm. Reprinted with permission from Wilson et al., 2011.

Copyright 2011 American Chemical Society.

deleterious effect on the islet viability (Fig. 6). The modification of PEG grafts with various functional molecules, such as biotin, hydrazide and azide, can extend the functional capabilities of the PLL-g-PEG-based LbL islet coatings. For example, the deposition of the PLL-g-PEG-biotin outmost layer on top of the PLL-g-PEG/Alginate multilayer film can generate surface densities of biotin functional groups comparable with that obtained by the treatment of islet surfaces with only NHS-PEG-biotin molecules (PEGylation/biotinylation through the NHS-ester coupling). Unlike the latter, the former approach is advantageous as it does not alter cell surface morphology and allows for controlled densities of the biotin on the modified islet surfaces (Krishnamurthy et al., 2010). The first successful *in vivo* transplantation of PEM engineered islets was demonstrated for the murine islets coated with conformal PLL-g-PEG/Alginate LbL films (Fig. 6g) (Wilson et al., 2011).

High toxicity of most polyelectrolyte polycations limits their use in biomedical applications. However, natural biopolymers chitosan and alginate have more similarities with the extracellular matrix, are chemically versatile and have a good biocompatibility. These linear polysaccharides carry opposite charge and can be electrostatically bound in a PEM. The (Chitosan/Alginate)3 islet coating was achieved *via* alternate deposition starting from positively charged chitosan (Zhi et al., 2010). The deposition conditions had been shown to greatly influence the islets viability, which well correlates with difference in charge density and toxicity of the polycation at high and neutral pH values. Additional protective outermost coating layer of phosphorylcholine (PC)-modified chondroitin-4-sulfate was introduced to reduce non-specific protein adsorption of the film. PC-moieties demonstrated remarkable repelling and hemocompatible properties. Increase in the coating thickness by adding additional layers of (Chitosan/Alginate) up to 5 bilayers did not adversely affect islets viability and insulin release, and the coated islets were viable up to 5 weeks of postencapsulation.
