**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 viability nor insulin release function.

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

cells.

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 23

cytocompatible compared to acrylate-PEG and demonstrated protective effects against T

Fig. 7. Immobilization of sCR1and heparin on the islet cell surface (top). Relative thrombin inactivation activities of multiple sCR1-heparin layers (1 double-layer (1DL), three-double layer (3 DL) on glass plates. The activities were determined after the glass plates were maintained in culture medium (medium RPMI-1640 containing 10% FBS) for the indicated

Thus, despite the advantages the ultrathin coating can present, chemical modification of the cell membrane using covalent conjugation of molecules, for example, covalent PEG binding via NHS-ester conjugation to the cell membrane proteins, can result in damaging of the membrane proteins and, consequently, in cell physiology disturbance. Moreover, the immunoisolation capabilities of ultra-thin hydrogel-based multilayers are still limited because of poor control over selective permeability towards small cytotoxic cytokines, and hypoxia within the gel environments. Current investigations are therefore focused on synthesis of hydrogels containing bioactive moieties, rather than unmodified coatings, aiming at "smart" protection of encapsulated islets from the damage originating from T-

Though pancreatic islet transplantation has emerged as a promising treatment for diabetes, its clinical application, however, remains limited due to serious side effects of immunosuppressive therapy necessary to prevent host rejection of transplanted islets.

periods (bottom). Reprinted from Luan et al., 2011 with permission from Elsevier.

cells and small free radical species.

**4. Outlook and perspective** 

is a potent inhibitor of the classical and alternative complement activation pathways (Pratt et al., 1996). The islets surfaces were first modified with PEG-phospholipid conjugates bearing maleimide groups (Fig. 7). The thiol-modified sCR1 molecules were then covalently bound to islets through the maleimide-PEG-lipid anchors via thiol-maleimide reaction. Following heparin immobilization with sCR1 was possible due to its strong affinity to sCR1. It is known though that covalent immobilization does not allow for longterm stability of the immobilized molecules at the cell surfaces due to fast degradation of the conjugation bonds. For instance, PEG-lipids gradually disappeared from the cell surface without uptake into the inside of cells dissociating from the cell surface into the medium (Teramura et al., 2008b). However, increase in the number of heparin /sCR1 layers upto 3 bilayers allowed subsequently increase the heparin and sCR1 retention time on islets surfaces up to 8 days *in vitro* with the gradual release from the islet surfaces over longer time periods. As the severe IBMIR reactions, especially the activation of the coagulation system and the complement cascade, occur in the first 2 days after transplantation, the heparin/sCR1 LBL approach can be a promising method for protecting the transplanted islets. Nevertheless, the protective effects and anti-thrombin activity of the LbL coatings are to be yet evaluated *in vivo*.

The LbL coating of islets based on streptavidin/biotin anchoring as a driving force for the LBL was also developed (Dai et al., 2007; Kizilel et al., 2010). PEGylated multilayers were grown using LbL assembly of biotinylated PLL and streptavidin. PEG was incorporated into the LbL architectures by assembly with biotin-derivatized PLL-g-PEG (Dai et al., 2007). In another example, the rat islet cell membranes were first biotinylated through covalent reaction of the membrane proteins and NHS-ester groups of NHS-PEG-biotin. The second layer of streptavidin was followed by a layer of a biotin-PEG conjugate with the insulinotropic Glucagon-like Ppetide 1 (GLP-1) (Kizilel et al., 2010). GLP-1 is a 30-amino acid peptide hormone produced in the intestinal epithelial endocrine L-cells. The main action of GLP-1 is to stimulate insulin secretion, acting as an incretin hormone, and to stimulate islet growth and neogenesis. Insulinotropic activity of GLP-1 is glucose-dependent and exerted via interaction with the GLP-1 receptor located on the cell membrane of the βcells. Stimulation of insulin secretion due to GLP-1 immobilization at the islet surfaces can result in reducing the number of islets necessary for normalization of blood glucose of a patient. GLP1-PEG-Biotin was immobilized as an outermost layer to enhance insulin secretion. Islets coated with PEG-GLP-1 demonstrated enhanced insulin secretion and no time-delay in response to high glucose level. Streptavidin, however, is an immunogenic protein derived from bacteria *Streptomyces avidinii* and in most cases is not suitable for use in clinical studies.

Obviously, multilayer PEGylation technique provides an advantage over the single layer via enhancing immune-protective barrier but, on the other hand, the LbL method based on subsequent immersion of islets in alternating polymer solutions can be time-consuming and thus inappropriate for clinical use. The ultra-thin multilayer build-up process was further modified by Hume et al to overcome this issue (Hume et al., 2011). In their approach, the hydrogel was formed via photoinitiated polymerization of PEG diacrylate (PEGDA) or methacrylate pre-cursor solutions. Proteins such as immunoglobulins, and intercellular adhesion molecule-2 (ICAM-2) were immobilized on the coating surface via thiol- (meth)acrylate co-polymerization. The methacrylate-PEG was found to be more

is a potent inhibitor of the classical and alternative complement activation pathways (Pratt et al., 1996). The islets surfaces were first modified with PEG-phospholipid conjugates bearing maleimide groups (Fig. 7). The thiol-modified sCR1 molecules were then covalently bound to islets through the maleimide-PEG-lipid anchors via thiol-maleimide reaction. Following heparin immobilization with sCR1 was possible due to its strong affinity to sCR1. It is known though that covalent immobilization does not allow for longterm stability of the immobilized molecules at the cell surfaces due to fast degradation of the conjugation bonds. For instance, PEG-lipids gradually disappeared from the cell surface without uptake into the inside of cells dissociating from the cell surface into the medium (Teramura et al., 2008b). However, increase in the number of heparin /sCR1 layers upto 3 bilayers allowed subsequently increase the heparin and sCR1 retention time on islets surfaces up to 8 days *in vitro* with the gradual release from the islet surfaces over longer time periods. As the severe IBMIR reactions, especially the activation of the coagulation system and the complement cascade, occur in the first 2 days after transplantation, the heparin/sCR1 LBL approach can be a promising method for protecting the transplanted islets. Nevertheless, the protective effects and anti-thrombin

The LbL coating of islets based on streptavidin/biotin anchoring as a driving force for the LBL was also developed (Dai et al., 2007; Kizilel et al., 2010). PEGylated multilayers were grown using LbL assembly of biotinylated PLL and streptavidin. PEG was incorporated into the LbL architectures by assembly with biotin-derivatized PLL-g-PEG (Dai et al., 2007). In another example, the rat islet cell membranes were first biotinylated through covalent reaction of the membrane proteins and NHS-ester groups of NHS-PEG-biotin. The second layer of streptavidin was followed by a layer of a biotin-PEG conjugate with the insulinotropic Glucagon-like Ppetide 1 (GLP-1) (Kizilel et al., 2010). GLP-1 is a 30-amino acid peptide hormone produced in the intestinal epithelial endocrine L-cells. The main action of GLP-1 is to stimulate insulin secretion, acting as an incretin hormone, and to stimulate islet growth and neogenesis. Insulinotropic activity of GLP-1 is glucose-dependent and exerted via interaction with the GLP-1 receptor located on the cell membrane of the βcells. Stimulation of insulin secretion due to GLP-1 immobilization at the islet surfaces can result in reducing the number of islets necessary for normalization of blood glucose of a patient. GLP1-PEG-Biotin was immobilized as an outermost layer to enhance insulin secretion. Islets coated with PEG-GLP-1 demonstrated enhanced insulin secretion and no time-delay in response to high glucose level. Streptavidin, however, is an immunogenic protein derived from bacteria *Streptomyces avidinii* and in most cases is not suitable for use in

Obviously, multilayer PEGylation technique provides an advantage over the single layer via enhancing immune-protective barrier but, on the other hand, the LbL method based on subsequent immersion of islets in alternating polymer solutions can be time-consuming and thus inappropriate for clinical use. The ultra-thin multilayer build-up process was further modified by Hume et al to overcome this issue (Hume et al., 2011). In their approach, the hydrogel was formed via photoinitiated polymerization of PEG diacrylate (PEGDA) or methacrylate pre-cursor solutions. Proteins such as immunoglobulins, and intercellular adhesion molecule-2 (ICAM-2) were immobilized on the coating surface via thiol- (meth)acrylate co-polymerization. The methacrylate-PEG was found to be more

activity of the LbL coatings are to be yet evaluated *in vivo*.

clinical studies.

cytocompatible compared to acrylate-PEG and demonstrated protective effects against T cells.

Thus, despite the advantages the ultrathin coating can present, chemical modification of the cell membrane using covalent conjugation of molecules, for example, covalent PEG binding via NHS-ester conjugation to the cell membrane proteins, can result in damaging of the membrane proteins and, consequently, in cell physiology disturbance. Moreover, the immunoisolation capabilities of ultra-thin hydrogel-based multilayers are still limited because of poor control over selective permeability towards small cytotoxic cytokines, and hypoxia within the gel environments. Current investigations are therefore focused on synthesis of hydrogels containing bioactive moieties, rather than unmodified coatings, aiming at "smart" protection of encapsulated islets from the damage originating from Tcells and small free radical species.
