**3.2.1 Covalent conjugation of molecules to islet surfaces**

Thin conformal coatings based on PEG are highly attractive for cell surface modification due to its biocompatibility and protein-resistant properties based on PEG low interfacial free energy with water, its unique properties in aqueous solutions, its high surface mobility, and its substantial steric stabilization effects (Amiji & Park, 1993). PEGylation has been used for camouflaging islet surfaces to immunologically protect transplanted islets from the immune system (Panza et al., 2000; Lee et al., 2002; Lee et al., 2004). Methoxy-PEG-succinimidyl propionic acid molecules conjugated with amino groups of collagen matrix at the islet surface through a stable amide bond protected islets from immune cell attack (Fig. 3, A1). The conjugated PEG molecules have been shown to block immune cells from diffusing to the transplanted islets, which allowed islets to function in the diabetic recipients for several weeks (Lee et al., 2006a; Lee et al., 2006b; Lee et al., 2007). Another advantage of the nanothin PEG layer is the small size of the produced shell compared to the size of encapsulated islets. The small shell size and the demonstrated protection against the host immune system should allow transplantation into the portal vein by catheter injection and still prevent immune response of the host.

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 15

which can deactivate procoagulant–proinflammatory properties of thrombin through its binding (Esmon et al., 2004). The formed thrombomodulin–thrombin complex leads to rapid inactivation of clotting factors Va and VIIIa and reduction of new thrombin generation (Esmon et al., 1982). This approach of thrombomodulin conjugation allowed for the substantial delay of clot formation upon incubation of islets in human plasma with

Despite the immunoprotective capabilities, the camouflaging layer of PEG cannot provide a long-term protection against small cytotoxic molecules produced by the immune system in response to metabolic activity of encapsulated islets after the transplantation. A combination of a protective coating and a low dose of an immunosuppressive drug can be a way to provide a suppression of host immune system without causing highly toxic effects. A low dose of Cyclosporine A has been shown to effectively prevent rejection of PEG-modified islets and support their functioning and survival up to 1 year (Lee at al., 2007). Similar effect was demonstrated by employing 6-arm-PEG-catechol that allowed for a coating with a higher density and a lower amount of immunosuppressive drug Tacrolimus (Jeong et al., 2011). Catechol is a surface independent anchor molecule which ensured conjugation of PEG-catechol to collagen matrix around the islet. The survival time of 6-arm-PEG-catechol grafted islets was similar to that of unmodified islets. However, the administration of the drug increased the survival time of 6-arm-PEG-catechol grafted islet almost two times.

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

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

preserved islet viability and glucose-stimulated insulin secretion capability.

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

the efficiency of the conjugation.

disappeared after 4 days in culture.

To achieve grafting of the end-functionalized PEG polymer onto the islet surfaces, islets are cultured in media in the presence of various concentrations of the polymer. To increase the amount of a grafted polymer on surfaces, the incubation time can be increased or the incubation step can be repeated. The grafting of methoxy-PEG (mPEG) onto the islets surface is usually carried out via the succinimidyl ester end groups introduced in mPEG. These groups couple to amino groups present in the collagen matrix around islets. Controlling the grafting time is important since longer grafting times can lead to diffusion of PEG inside the islets which may increase a chance of swelling and exposure of islets to outside environment. Furthermore, long grafting time increases the probability of islet damaging (Lee et al., 2002).

The molecular weight of the grafted PEG is another important parameter for islet surface modification. Barani et al., showed that grafted mPEG of 5 and 10 kDa onto islets isolated from Wistar rats had a different effect on cell functioning and viability (Barani et al., 2010). Insulin secretion for the islets grafted with 5 kDa mPEG was at the same level as for unmodified islets, while overall insulin secretion from islets modified with 10 kDa mPEG decreased. However, its protective ability was higher which correlates well with the decrease in mesh size and thus less exposure to immune system. The effect of PEG molecular weight was also studied on porcine islets (Xie et al., 2005). The highest protection ability without affecting islets functional capability was found for 5-6 kDa PEG that allowed complete coverage of islets surface.

In the case of two reactive succinimidyl ester end groups, additional functional molecules such as albumin can be brought to the islet surfaces (Xie et al., 2005). Introducing albumin and PEG on the surface can be used to suppress the immunogenic reactions (Hortin et al., 1997). Presence of human albumin not only increased cytoprotection of islets but also significantly increased the insulin production which was possible due to increasing density of protective coverage with albumin presence. The so-modified islets maintained their functionality *in vivo* up to 15 days.

Heparin is a highly sulfated glycosaminoglycan and is very often used as an injectable anticoagulant. Systemic delivery of heparin at therapeutic doses, however, substantially increases the risk of bleeding. Furthermore, the effect of soluble heparin is limited to 2-3 hours *in vivo* and has no significant effect on long-term IBMIR reaction (Bennet et al., 1999). At the same time, heparin, immobilized on artificial surfaces which mimic the protective ability of the endothelial cells, demonstrated inhibition of coagulation and complement activation (Bennet et al., 1999). Immobilized heparin coating was successfully made on human, porcine and mouse islets by step-by-step incubation procedure (Cabric et al., 2007). Islet surfaces were first biotinylated through covalent attachment of succinimidyl esterbiotin, then incubated with avidin and finally, heparin conjugate was covalently attached on the modified islets surface (Fig. 3, B). The heparin coating produced by this method was present of the islets surfaces at least 72 hours, though not detectable after 4-5 weeks of transplantation.

Islets surface modification with other inhibitors of the coagulation cascade through bifunctional PEG linkers can provide another strategy to reduce IBMIR. Human recombinant thrombomodulin was conjugated to the islet surface through bifunctional PEG grafts (Stabler et al., 2007). Thrombomodulin is an endothelial cell transmembrane protein

To achieve grafting of the end-functionalized PEG polymer onto the islet surfaces, islets are cultured in media in the presence of various concentrations of the polymer. To increase the amount of a grafted polymer on surfaces, the incubation time can be increased or the incubation step can be repeated. The grafting of methoxy-PEG (mPEG) onto the islets surface is usually carried out via the succinimidyl ester end groups introduced in mPEG. These groups couple to amino groups present in the collagen matrix around islets. Controlling the grafting time is important since longer grafting times can lead to diffusion of PEG inside the islets which may increase a chance of swelling and exposure of islets to outside environment. Furthermore, long grafting time increases the probability of islet

The molecular weight of the grafted PEG is another important parameter for islet surface modification. Barani et al., showed that grafted mPEG of 5 and 10 kDa onto islets isolated from Wistar rats had a different effect on cell functioning and viability (Barani et al., 2010). Insulin secretion for the islets grafted with 5 kDa mPEG was at the same level as for unmodified islets, while overall insulin secretion from islets modified with 10 kDa mPEG decreased. However, its protective ability was higher which correlates well with the decrease in mesh size and thus less exposure to immune system. The effect of PEG molecular weight was also studied on porcine islets (Xie et al., 2005). The highest protection ability without affecting islets functional capability was found for 5-6 kDa PEG that allowed

In the case of two reactive succinimidyl ester end groups, additional functional molecules such as albumin can be brought to the islet surfaces (Xie et al., 2005). Introducing albumin and PEG on the surface can be used to suppress the immunogenic reactions (Hortin et al., 1997). Presence of human albumin not only increased cytoprotection of islets but also significantly increased the insulin production which was possible due to increasing density of protective coverage with albumin presence. The so-modified islets maintained their

Heparin is a highly sulfated glycosaminoglycan and is very often used as an injectable anticoagulant. Systemic delivery of heparin at therapeutic doses, however, substantially increases the risk of bleeding. Furthermore, the effect of soluble heparin is limited to 2-3 hours *in vivo* and has no significant effect on long-term IBMIR reaction (Bennet et al., 1999). At the same time, heparin, immobilized on artificial surfaces which mimic the protective ability of the endothelial cells, demonstrated inhibition of coagulation and complement activation (Bennet et al., 1999). Immobilized heparin coating was successfully made on human, porcine and mouse islets by step-by-step incubation procedure (Cabric et al., 2007). Islet surfaces were first biotinylated through covalent attachment of succinimidyl esterbiotin, then incubated with avidin and finally, heparin conjugate was covalently attached on the modified islets surface (Fig. 3, B). The heparin coating produced by this method was present of the islets surfaces at least 72 hours, though not detectable after 4-5 weeks of

Islets surface modification with other inhibitors of the coagulation cascade through bifunctional PEG linkers can provide another strategy to reduce IBMIR. Human recombinant thrombomodulin was conjugated to the islet surface through bifunctional PEG grafts (Stabler et al., 2007). Thrombomodulin is an endothelial cell transmembrane protein

damaging (Lee et al., 2002).

complete coverage of islets surface.

functionality *in vivo* up to 15 days.

transplantation.

which can deactivate procoagulant–proinflammatory properties of thrombin through its binding (Esmon et al., 2004). The formed thrombomodulin–thrombin complex leads to rapid inactivation of clotting factors Va and VIIIa and reduction of new thrombin generation (Esmon et al., 1982). This approach of thrombomodulin conjugation allowed for the substantial delay of clot formation upon incubation of islets in human plasma with preserved islet viability and glucose-stimulated insulin secretion capability.

Despite the immunoprotective capabilities, the camouflaging layer of PEG cannot provide a long-term protection against small cytotoxic molecules produced by the immune system in response to metabolic activity of encapsulated islets after the transplantation. A combination of a protective coating and a low dose of an immunosuppressive drug can be a way to provide a suppression of host immune system without causing highly toxic effects. A low dose of Cyclosporine A has been shown to effectively prevent rejection of PEG-modified islets and support their functioning and survival up to 1 year (Lee at al., 2007). Similar effect was demonstrated by employing 6-arm-PEG-catechol that allowed for a coating with a higher density and a lower amount of immunosuppressive drug Tacrolimus (Jeong et al., 2011). Catechol is a surface independent anchor molecule which ensured conjugation of PEG-catechol to collagen matrix around the islet. The survival time of 6-arm-PEG-catechol grafted islets was similar to that of unmodified islets. However, the administration of the drug increased the survival time of 6-arm-PEG-catechol grafted islet almost two times.
