**3.2.2 Incorporation of molecules in the cell membrane**

Islets surface modification can be achieved via hydrophobic interactions of amphiphilic polymers, such as PEG-phospholipids and PVA with long alkyl chains, with lipids in cell membranes (Takemoto et al., 2011; Chen et al., 2011). Presence of hydrophobic parts, phospholipids and alkyl chains, in polymer and cell membranes are responsible for spontaneous incorporation of co-polymers into the lipid layers of cell membrane. The spontaneous incorporation is greatly affected by the length and hydrophobicity of alkyl chains (Rabuka et al., 2008; Teramura et al., 2007). Using this approach, the PEGphospholipid-based coating was modified with fibrinolytical enzyme urokinase and with a soluble domain of the anticoagulant, thrombomodulin (Fig. 3, B) (Chen et al., 2011). The immobilization of heparin, urokinase, or thrombomodulin can improve the graft survival after the transplantation. The maleimide-PEG-lipids were utilized to immobilize proteins on the islets surface through reaction of maleimide and thiol-modified proteins. However, thiol groups can easily form disulfide bonds under physiological conditions and that decreased the efficiency of the conjugation.

Urokinase is a serine protease that activates plasminogen into plasmin, which dissolves fibrin blood clots. Urokinase can be conjugated onto the surface of islets to dissolve blood clots surrounding islets in the liver for inhibition of the cascade reactions (Takemoto et al., 2011). Hybridization between urokinase and DNA-PEG-lipids was made due to complementary sequences on the protein and single strain DNA (Fig. 4). DNA hybridization method is versatile for conjugation of various bioactive molecules. Using different sequences, it was possible to conjugate different molecules on the cell surfaces. The cell morphology was not affected by such modification, although, urokinase activity disappeared after 4 days in culture.

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 17

Heparin has an affinity to a number of plasma proteins and growth factors including endothelium and vascular endothelium growth factors. Employing this property of heparin, the synthetic heparin-binding peptide amphiphile was used to immobilize heparin and, subsequently, growth factors on islet surfaces (Chow et al., 2010). Initial immobilization of the amphiphile on the islet surfaces can be achieved *via* electrostatic interactions of positively charged amphiphile and negatively charge cell membrane (Fig. 3, A4). Later, negatively charged heparin can be bound to the amphiphile. The so-formed amphiphileheparin complex coating on the islets surface was able to bind and retain growth factors up to 48 hours. The complex nanostructures provide extracellular matrix-like scaffold for the encapsulated islets for supporting their viability. Such immobilization of growth factors does not affect viability or function, yet, they are able to stimulate an angiogenic response in

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

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

**3.2.3 Surface modification through electrostatic interactions** 

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

the islets.

presence on islets surfaces.

**3.3 Conformal coating of islets** 

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

Overall, surface modification achieved via hydrophobic interaction and insertion of hydrophobic chains into the cell membrane demonstrated a limited retention time. For PEGlipids this time depends on the alkyl chain length of lipid chains (Inui et al., 2010). The longer alkyl chains allowed PEG-lipid molecules to retain longer on the cell membrane. However, the overall retention time was no longer than 48 hours before the all molecules disappeared from the islets surfaces. Incorporation of hydrophobic molecules into the cell membrane leads to their uptake into the cell cytoplasm. Adding amphiphilic part to the structures prevents the uptake but molecules are released into the surrounding media.

Fig. 4. Top: (a) Chemical structure of oligo(dT)20-conjugated PEG-lipid (oligo(dT)20-PEGlipid). (b) Urokinase is modified with Sulfo-EMCS to introduce maleimide groups, and then oligo(dA)20-SH is conjugated on urokinase through the thiol/maleimide reaction. (c) Oligo(dT)20-PEG-lipid is incorporated into the cell surface by the hydrophobic interaction between alkyl chains and the lipid bilayer of the cell membrane. Oligo(dA)20-urokinase is applied to cells carrying oligo(dT)20. Urokinase is conjugated on the cell surface through oligo(dT)20 -oligo(dA)20 hybridization. Bottom: Confocal laser scanning microscopic images of urokinase--modified-islets which were subjected to immunostaining for urokinase. Islets were modified with oligo(dT)20-PEG-lipids and then oligo(dA)20-urokinase. The urokinasemodified-islets just after treatment, after 1 and 2 days of culture. Reprinted with permission from Takemoto et al., 2011. Copyright 2011 American Chemical Society.
