**3.2.4 Immobilization of living cells on islet surfaces**

Endothelial cells on the islets surfaces have a good tolerance towards blood presence and can provide a protection against IBMIR. Introduction of such protective cells around islets can be used as an effective protection strategy. The human aortic endothelial cells were introduced onto isolated human islets of Langerhans by mixing of both cell types and incubation for several hours (Johansson et al., 2005). The clotting can be significantly reduced with the 90%-cell-coating present on islet surfaces. The consistency of the cell amounts in such coating can be improved using the PEG-phospholipid-based approach (Teramura & Iwata, 2009; Teramura & Iwata, 2008a). In that case, islets and human endoderm kidney cells were first separately biotinylated through biotin-PEG-lipid anchors to cell membranes with further streptavidin coating on the kidney cells (Fig. 5). Immobilization of the endothelium cells around islets was then made via streptavidin-biotin reaction. The cell enclosure was stable on the islet surfaces within 3-5 days *in vitro*. To overcome the streptavidin immunogenicity, the cell deposition was also made via the PolyDNA-PEG-lipid conjugate (Teramura & Chen, 2010; Teramura & Minch, 2010). Human endoderm kidney cells were able to rapidly proliferate forming a cell multilayer on the islets surfaces protecting the encapsulated islets from the host immune response. However, the cell oxygen consumption can result in lowered oxygen available for the encased islets. Thus, additional studies are necessary to clarify the short- and long-term effects of the cell presence on islets surfaces.

## **3.3 Conformal coating of islets**

### **3.3.1 Layer-by-layer (LbL) approach**

The layer-by-layer (LBL) assembly of polymers based on sequential adsorption of oppositely charged components is one of the established methods for the preparation of thin polyelectrolyte multilayer films with controlled properties. The LBL represents a universal surface modification approach that allows for producing surface-attached films with controlled thickness, permeability, mechanical properties and surface chemistry. The technique has been recently applied to modify islet surfaces (Krol et al., 2006; Wilson et al., 2008). The LbL modification of islet surfaces is based on alternating deposition of water

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 19

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

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

glucose stimulation and insulin response of the coated islets (Krol et al., 2006).

**3.3.2 The ionic LbL assembly** 

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).
