**3. Approaches to prevent immunogenic reactions on the islet surfaces**

The immune reactions against encapsulated islets can be divided into non-specific immune activation initiated by surgery; a host response against the encapsulating materials and implanted islets provokes the immune response by releasing the bioactive molecules.

The instant blood-mediated inflammatory reaction (IBMIR) is an inflammatory reaction that occurs when isolated islets come in contact with human blood. This process is responsible for islets destruction together with overall failing of transplantation. One of the major

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 7

islets. Another advantage is the possibility to employ different coating techniques. The major requirement for the materials used for islets encapsulation is biocompatibility which can be evaluated by a degree of fibrotic overgrowth (Liu et al., 2010). Overgrowth of fibrous tissue upon microcapsule surface would affect the oxygen and nutrition diffusion by clogging microcapsule pores (Nafea et al., 2011). Indeed, insufficient supply of oxygen is the major reason for necrosis of microencapsulated islets. The biocompatibility of the materials used for microcapsules formation strongly depends on chemical composition of materials

Also, biocompatible materials must have selective permeability to promote the survival of encapsulated islets. Selective permeability should allow fluxes of oxygen, nutrients and metabolism products freely in and out of the protective membrane. At the same time the microcoating should prevent the immune system compounds such as antibodies and cytokines to reach the encapsulated cells. The viability and functioning of encapsulated islets is in majority determined by the molecular weight cutoff of the microcapsules. The cutoff determines the upper size limit of molecular weight that is allowed to go through coating. However, diffusion of the molecules very often depends on size, shape and charge of the molecules. Diffusion coefficient and permeability can be considered the more useful and informative characteristics of the cutoff. Control over the permeability can be achieved by varying polymer molecular weight, increasing or decreasing functional group and crosslinking densities and polymerization conditions (Dembczynski & Jankowski, 2001). Molecular weight and concentration of polymer can drastically change the permeability of the hydrogel. It has been shown that poly(vinyl alcohol) (PVA) hydrogel with a high molecular weight of PVA backbone had a higher swelling ratio due to the decrease in crosslink density (Martens et al., 2007). In radical polymerization, an increase in polymer concentration leads to the increase in cross-link density and decrease in the swelling ability. The chemical structure of hydrogel has a significant effect on hydrogel permeability. Presence of electrostatic interactions between charged hydrogel groups and small molecules will slow down the diffusion rates over time. Moreover, during time, changes in the diffusion coefficient occur because of changes in the hydrogel network due to physical or

The microcapsule shape (Sakai et al., 2006) morphology such as roughness (Bunger et al., 2003), mechanical properties and especially stiffness of the hydrogel (Berg et al., 2004), play an important role in provoking immune response or fibrotic overgrowths. The importance of hydrogel roughness was demonstrated for implanted rough poly(L-lysine) (PLL)/Alginate microcapsules and only smoothing the surface by adding poly(acrylic acid) completely abolished the tissue response (Bünger et al., 2003). In most cases, smooth round surfaces had the lowest fibrosis promoting effect (Zhang et al., 2008). The most commonly applied materials for microencapsulation are alginate (Lim et al., 1980), agarose (Iwata et al., 1992), PEG (Weber & Anseth, 2008) and poly(hydroxyethylmetacrylate-methyl

Alginate microcapsule production can be made under physiological conditions and provide

an environment that allows maintaining the islets functionality and viability.

and applied purification techniques.

chemical absorption of proteins.

methacrylate) (Dawson et al., 1987).

**3.1.1 Alginate microgels** 

triggers for the IBMIR reaction is a secretion of tissue factors by islets surfaces. Disruption of host tissue by surgery leads to release of bioactive molecules such as fibrinogen, histamine and fibronectin. Release of serum components and presence of extracellular matrix and cell debris attract tissue macrophages to the surgery site to clean up and start the process of wound healing. The immune cells have the ability to produce various small bioactive compounds such as interleukins, tissue necrosis factors (TNF) and histamine. There are several extravascular approaches to prevent IBMIR reaction against pancreatic islets (Nafea et al., 2011).

#### **3.1 Macro and microencapsulation**

'Macroencapsulation' can be defined as encapsulation of large numbers of islets together in one device. The shape of the devices can be a hollow fiber, planar membranes or macrocapsules. Macroencapsulation provides immune-isolation of islets within semipermeable membranes. The major advantage of macrocapsules is the possibility to easily retrieve the islets from an implantation site in case of surgery complications. Capsules reduce the risk of the IBMIR reaction from occuring, therefore, allowing the use of not only allogenic but also xenogenic materials and avoiding the use of immunosuppressive medications. However, macroencapsulation has not found a broad use in islet encapsulation and transplantation due to a large volume of protective devices. Relatively large sizes will cause limited oxygen and nutrition access and, as a result, cell necrosis (De Vos et al., 1999). Such macrocapsules do not allow for tuning the molecular weight cutoff (or semi-permeable properties) to prevent recognition by antibodies, and cytokines cannot be sufficiently excluded either (Cui et al., 2004). Similarly, recently developed poly(ethylene glycol) (PEG) hydrogels although demonstrated facile control over porosity but formed microbeads are large and present a barrier for rapid molecular transport (Weber et al., 2007). The capsules with larger diameters than an islet itself are also expected to plug blood vessels. This can exert harmful effects on the patient's liver. The diameter of encapsulated islets must be much smaller than that currently attained to allow transplantation of the islets into portal veins. Thus, new methods for the microencapsulation of islets without increasing the diameter of the implant are required. In this respect, modification of islet surfaces would be a powerful tool that can provide an artificial nurturing environment and preserve islet viability and function (Raymond et al., 2004; Wilson & Chaikof, 2008; Ricordi & Strom, 2004; Lim et al., 2011).

In contrast to macroencapsulation, 'microencapsulation' can be defined as encapsulation of single islets or a small group of islets inside the polymer gel coating. Microencapsulation can be achieved via the formation of a gel shell around the islets by polymerization of a precursor solution around islet surfaces. Emulsification is one of the microbead formation methods (Iwata et al., 1992; Yang et al., 1994). PEG/Alginate was used for islets microencapsulation through their mixing with two-phase aqueous emulsion. The islets contained in emulsion microdroplets underwent cross-linking with calcium ions (Calafiore et al., 2006).

During the microencapsulation process, a bio-inert coating with minimal host response and cell toxicity should be created. Individual islet coating offers a number of advantages over the macroencapsulation. An individual coating provides a better surface:volume ratio that allows faster diffusion of oxygen and nutrients which supports the viability of encapsulated

triggers for the IBMIR reaction is a secretion of tissue factors by islets surfaces. Disruption of host tissue by surgery leads to release of bioactive molecules such as fibrinogen, histamine and fibronectin. Release of serum components and presence of extracellular matrix and cell debris attract tissue macrophages to the surgery site to clean up and start the process of wound healing. The immune cells have the ability to produce various small bioactive compounds such as interleukins, tissue necrosis factors (TNF) and histamine. There are several extravascular approaches to prevent IBMIR reaction against pancreatic islets (Nafea

'Macroencapsulation' can be defined as encapsulation of large numbers of islets together in one device. The shape of the devices can be a hollow fiber, planar membranes or macrocapsules. Macroencapsulation provides immune-isolation of islets within semipermeable membranes. The major advantage of macrocapsules is the possibility to easily retrieve the islets from an implantation site in case of surgery complications. Capsules reduce the risk of the IBMIR reaction from occuring, therefore, allowing the use of not only allogenic but also xenogenic materials and avoiding the use of immunosuppressive medications. However, macroencapsulation has not found a broad use in islet encapsulation and transplantation due to a large volume of protective devices. Relatively large sizes will cause limited oxygen and nutrition access and, as a result, cell necrosis (De Vos et al., 1999). Such macrocapsules do not allow for tuning the molecular weight cutoff (or semi-permeable properties) to prevent recognition by antibodies, and cytokines cannot be sufficiently excluded either (Cui et al., 2004). Similarly, recently developed poly(ethylene glycol) (PEG) hydrogels although demonstrated facile control over porosity but formed microbeads are large and present a barrier for rapid molecular transport (Weber et al., 2007). The capsules with larger diameters than an islet itself are also expected to plug blood vessels. This can exert harmful effects on the patient's liver. The diameter of encapsulated islets must be much smaller than that currently attained to allow transplantation of the islets into portal veins. Thus, new methods for the microencapsulation of islets without increasing the diameter of the implant are required. In this respect, modification of islet surfaces would be a powerful tool that can provide an artificial nurturing environment and preserve islet viability and function (Raymond et al., 2004; Wilson & Chaikof, 2008; Ricordi & Strom, 2004;

In contrast to macroencapsulation, 'microencapsulation' can be defined as encapsulation of single islets or a small group of islets inside the polymer gel coating. Microencapsulation can be achieved via the formation of a gel shell around the islets by polymerization of a precursor solution around islet surfaces. Emulsification is one of the microbead formation methods (Iwata et al., 1992; Yang et al., 1994). PEG/Alginate was used for islets microencapsulation through their mixing with two-phase aqueous emulsion. The islets contained in emulsion microdroplets underwent cross-linking with calcium ions (Calafiore

During the microencapsulation process, a bio-inert coating with minimal host response and cell toxicity should be created. Individual islet coating offers a number of advantages over the macroencapsulation. An individual coating provides a better surface:volume ratio that allows faster diffusion of oxygen and nutrients which supports the viability of encapsulated

et al., 2011).

Lim et al., 2011).

et al., 2006).

**3.1 Macro and microencapsulation** 

islets. Another advantage is the possibility to employ different coating techniques. The major requirement for the materials used for islets encapsulation is biocompatibility which can be evaluated by a degree of fibrotic overgrowth (Liu et al., 2010). Overgrowth of fibrous tissue upon microcapsule surface would affect the oxygen and nutrition diffusion by clogging microcapsule pores (Nafea et al., 2011). Indeed, insufficient supply of oxygen is the major reason for necrosis of microencapsulated islets. The biocompatibility of the materials used for microcapsules formation strongly depends on chemical composition of materials and applied purification techniques.

Also, biocompatible materials must have selective permeability to promote the survival of encapsulated islets. Selective permeability should allow fluxes of oxygen, nutrients and metabolism products freely in and out of the protective membrane. At the same time the microcoating should prevent the immune system compounds such as antibodies and cytokines to reach the encapsulated cells. The viability and functioning of encapsulated islets is in majority determined by the molecular weight cutoff of the microcapsules. The cutoff determines the upper size limit of molecular weight that is allowed to go through coating. However, diffusion of the molecules very often depends on size, shape and charge of the molecules. Diffusion coefficient and permeability can be considered the more useful and informative characteristics of the cutoff. Control over the permeability can be achieved by varying polymer molecular weight, increasing or decreasing functional group and crosslinking densities and polymerization conditions (Dembczynski & Jankowski, 2001). Molecular weight and concentration of polymer can drastically change the permeability of the hydrogel. It has been shown that poly(vinyl alcohol) (PVA) hydrogel with a high molecular weight of PVA backbone had a higher swelling ratio due to the decrease in crosslink density (Martens et al., 2007). In radical polymerization, an increase in polymer concentration leads to the increase in cross-link density and decrease in the swelling ability. The chemical structure of hydrogel has a significant effect on hydrogel permeability. Presence of electrostatic interactions between charged hydrogel groups and small molecules will slow down the diffusion rates over time. Moreover, during time, changes in the diffusion coefficient occur because of changes in the hydrogel network due to physical or chemical absorption of proteins.

The microcapsule shape (Sakai et al., 2006) morphology such as roughness (Bunger et al., 2003), mechanical properties and especially stiffness of the hydrogel (Berg et al., 2004), play an important role in provoking immune response or fibrotic overgrowths. The importance of hydrogel roughness was demonstrated for implanted rough poly(L-lysine) (PLL)/Alginate microcapsules and only smoothing the surface by adding poly(acrylic acid) completely abolished the tissue response (Bünger et al., 2003). In most cases, smooth round surfaces had the lowest fibrosis promoting effect (Zhang et al., 2008). The most commonly applied materials for microencapsulation are alginate (Lim et al., 1980), agarose (Iwata et al., 1992), PEG (Weber & Anseth, 2008) and poly(hydroxyethylmetacrylate-methyl methacrylate) (Dawson et al., 1987).

#### **3.1.1 Alginate microgels**

Alginate microcapsule production can be made under physiological conditions and provide an environment that allows maintaining the islets functionality and viability.

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 9

volume of the formed capsules. Mixture, containing PEG diacrylate, photoinitator accelerator and islets were mixed in a chamber. Once the islets were trapped into droplets, they were drawn into the spout. PEG polymerization was done by the exposure of the droplets to a green light. The free-radical polymerization of PEG diacrylate produce branched polymer chains. The encapsulated islets passed through the withdrawing tube with the same inner diameter that allowed for the same size capsules. Islets encapsulation required two encapsulation rounds. Despite this double coating produced capsules with mean thickness of 20.5 μm, islets were capable to respond to high glucose stimulation

Hydrogel network diffusion properties can be adjusted by altering the cross-link density or polymer molecular weight. The PEG microgel cross-linked by photoinitiated polymerization is a well studied system for islet encapsulation. An average pore size of the PEG gel formed by chain polymerization is dependent on the length of the polymer chains between crosslinks and can be used to predict and calculate the diffusion coefficient within the gel. Studies revealed that the molecular weight of PEG can not only affect the cross-link density, but also can change the concentration profile of radicals in time. Changes in the concentration of free radicals affect both a number of cross-linkable double bonds and diffusion limitation introduced during gel formation (Weber et al., 2008). More importantly, microcapsules produced from PEG with Mw 2000–10,000 g/mol via photopolymerization did not affect the viability of encapsulated islets or alter their insulin secretion. The level of glucose secretion observed during 1 hour of glucose stimulation was at the same level as for unmodified islets. Insulin secretion from islets encapsulated into 1 mm thick PEG gel was delayed at specific time point within stimulation period. The diffusion-related delay can be minimized through reducing the distance between encapsulated islets and surrounding environment

The created hydrogel environment is extremely different from the natural surroundings of islets. Indeed, cell viability is strongly dependent on cell-cell or cell-matrix interactions. For example, the viability of MIN6 pancreatic β-cells encapsulated in just PEG-based matrix reduced to 17% within 10 days (Weber et al., 2007b). Mimicking the extracellular environment in an artificial matrix can provide additional enhancement of islets viability and functioning. For that, adhesive peptides or various biological components for promoting cell-matrix interactions can be co-polymerized within hydrogels. Thus, physical incorporation of laminin and collagen type IV, the most abundant proteins present in the extracellular matrix, was performed during formation of hydrogel from PEG dimethacrylate block co-polymers via photoinitiated polymerization (Weber and Anseth, 2008; Weber et al., 2006). Such introduction of the proteins to the PEG matrix preserved the viability and functioning of the microencapsulated islets up to 28 days in culture. Interestingly, the ratio of the proteins had an effect on the level of insulin secretion from the encapsulated islets. For example, equal amounts of laminin and collagen IV in the hydrogel did not have any synergetic effect on the insulin production, while enhanced insulin secretion was observed

Isolation and purification of matrix proteins is, however, complex and cost inefficient way to recreate extracellular matrix environment. The substitution of natural proteins by artificial sequences is a promising approach to resolve this issue. Recently, several therapeutic agents (Drucker, 2001; Drucker, 2002; Holz & Chepurny, 2003) have been proposed to increase

by reducing the thickness of the gel (Weber et al., 2008).

for the increasing amount of laminin.

without a delay.

The microcapsule fabrication technique is based on the entrapment of islets within the spherical droplets that are produced by extrusion of solution containing polymer and islets through a needle. Two forces are usually used to control the size of droplets: an air flow that builds around the tip of the needle (Wolters et al., 1992), and a high voltage pulse around the needle tip (Halle et al., 1994; Hsu et al., 1994), the formed droplets very often require an additional stabilization via gelation and beads formation (Stabler et al., 2001). Cross-linking with metal ions, calcium and barium, and chemical or covalent cross-linking are the two methods generally used in microbead or microcapsule formation. Calcium cross-linked alginate microcapsules very often require an additional stabilization with PLL.

Alginate is a linear polysaccharide extracted from algae with a chemical structure combining different ratios of α-L-guluronic (G) and β-D-mannuronic acids (M) and saccharides (Haug et al., 1974). Chemical composition of alginate may affect the biocompatibility (De Vos et al., 1997) or/and function, and the activity of incorporated cells (Stabler et al., 2001). Guluronic acids and alternating mannuronic-guluronic acids have a high ability to cooperatively bind with divalent metal ions and form a cross-linked gel. Barium ion cross-linked gels have a higher stability compared to calcium ion cross-linked gels, though barium ions produce a more stable cross-linking only for alginate with a high ratio of guluronic acid (> 60%). However, ionically cross-linked alginate hydrogels undergo slow degradation under physiological conditions. During this process, alginate microcapsules with metal ion crosslinking undergo slow exchange of divalent cations with sodium ions, which leads to the microgel degradation. Leakage of barium ions is not desirable due to their toxicity which is based on their ability to inhibit K+ channels at concentration greater than 5-10 mM (Zimmermann et al., 2000). Variations in alginate structures obtained from different alginate sources introduce an additional limitation for the use of the barium cations for alginate cross-linking. Permeability and swelling of alginate-based capsules also strongly depend on the ratio of the acids in alginate. Barium cross-linked G-rich alginate beads had lower permeability to IgG than Ca2+ cross-linked microgel. However, cross-linking M-rich alginate beads with Ba2+ ions leads to the permeability increase and an overall higher gel swelling (Mørch et al., 2006). Applying an outer coating of polycations such as PLL, or poly-Lornithine can readily stabilize the alginate capsules with low G-ratio (Thu et al., 1996). Thus, the alginate must be chosen according to a specific application. Moreover, polycations, such as PLL, are proinflammatory molecules responsible for the fibrotic overgrowth (King et al., 2001) and the soluble PLL induces the cytokine production in monocytes and can be a reason for cellular necrosis (Strand et al., 2001).

#### **3.1.2 Poly(ethylene glycol)-based gels**

PEG is a less immunogenic material that is generally studied for use in islet encapsulation. PEG is a non-ionic hydrophilic polymer stable at physiological conditions with the highly hydrated polymer coils. The variation in its molecular weight can be used to control protein adsorption and the permeability of PEG gels by changing their porosity unlike alginate (Chen et al., 1998). However, the formed microbeads are larger and diffusion of molecules is slower (Weber et al., 2007a).

Selective-withdrawal coating technique was used to encapsulate rat pancreatic islets within PEG thin coating (Wyman et al., 2007). Geometry of the capsules is determined by selective withdrawal throughout the bulk solution and allows the cross-linking throughout the

The microcapsule fabrication technique is based on the entrapment of islets within the spherical droplets that are produced by extrusion of solution containing polymer and islets through a needle. Two forces are usually used to control the size of droplets: an air flow that builds around the tip of the needle (Wolters et al., 1992), and a high voltage pulse around the needle tip (Halle et al., 1994; Hsu et al., 1994), the formed droplets very often require an additional stabilization via gelation and beads formation (Stabler et al., 2001). Cross-linking with metal ions, calcium and barium, and chemical or covalent cross-linking are the two methods generally used in microbead or microcapsule formation. Calcium cross-linked

Alginate is a linear polysaccharide extracted from algae with a chemical structure combining different ratios of α-L-guluronic (G) and β-D-mannuronic acids (M) and saccharides (Haug et al., 1974). Chemical composition of alginate may affect the biocompatibility (De Vos et al., 1997) or/and function, and the activity of incorporated cells (Stabler et al., 2001). Guluronic acids and alternating mannuronic-guluronic acids have a high ability to cooperatively bind with divalent metal ions and form a cross-linked gel. Barium ion cross-linked gels have a higher stability compared to calcium ion cross-linked gels, though barium ions produce a more stable cross-linking only for alginate with a high ratio of guluronic acid (> 60%). However, ionically cross-linked alginate hydrogels undergo slow degradation under physiological conditions. During this process, alginate microcapsules with metal ion crosslinking undergo slow exchange of divalent cations with sodium ions, which leads to the microgel degradation. Leakage of barium ions is not desirable due to their toxicity which is based on their ability to inhibit K+ channels at concentration greater than 5-10 mM (Zimmermann et al., 2000). Variations in alginate structures obtained from different alginate sources introduce an additional limitation for the use of the barium cations for alginate cross-linking. Permeability and swelling of alginate-based capsules also strongly depend on the ratio of the acids in alginate. Barium cross-linked G-rich alginate beads had lower permeability to IgG than Ca2+ cross-linked microgel. However, cross-linking M-rich alginate beads with Ba2+ ions leads to the permeability increase and an overall higher gel swelling (Mørch et al., 2006). Applying an outer coating of polycations such as PLL, or poly-Lornithine can readily stabilize the alginate capsules with low G-ratio (Thu et al., 1996). Thus, the alginate must be chosen according to a specific application. Moreover, polycations, such as PLL, are proinflammatory molecules responsible for the fibrotic overgrowth (King et al., 2001) and the soluble PLL induces the cytokine production in monocytes and can be a

PEG is a less immunogenic material that is generally studied for use in islet encapsulation. PEG is a non-ionic hydrophilic polymer stable at physiological conditions with the highly hydrated polymer coils. The variation in its molecular weight can be used to control protein adsorption and the permeability of PEG gels by changing their porosity unlike alginate (Chen et al., 1998). However, the formed microbeads are larger and diffusion of molecules is

Selective-withdrawal coating technique was used to encapsulate rat pancreatic islets within PEG thin coating (Wyman et al., 2007). Geometry of the capsules is determined by selective withdrawal throughout the bulk solution and allows the cross-linking throughout the

alginate microcapsules very often require an additional stabilization with PLL.

reason for cellular necrosis (Strand et al., 2001).

**3.1.2 Poly(ethylene glycol)-based gels** 

slower (Weber et al., 2007a).

volume of the formed capsules. Mixture, containing PEG diacrylate, photoinitator accelerator and islets were mixed in a chamber. Once the islets were trapped into droplets, they were drawn into the spout. PEG polymerization was done by the exposure of the droplets to a green light. The free-radical polymerization of PEG diacrylate produce branched polymer chains. The encapsulated islets passed through the withdrawing tube with the same inner diameter that allowed for the same size capsules. Islets encapsulation required two encapsulation rounds. Despite this double coating produced capsules with mean thickness of 20.5 μm, islets were capable to respond to high glucose stimulation without a delay.

Hydrogel network diffusion properties can be adjusted by altering the cross-link density or polymer molecular weight. The PEG microgel cross-linked by photoinitiated polymerization is a well studied system for islet encapsulation. An average pore size of the PEG gel formed by chain polymerization is dependent on the length of the polymer chains between crosslinks and can be used to predict and calculate the diffusion coefficient within the gel. Studies revealed that the molecular weight of PEG can not only affect the cross-link density, but also can change the concentration profile of radicals in time. Changes in the concentration of free radicals affect both a number of cross-linkable double bonds and diffusion limitation introduced during gel formation (Weber et al., 2008). More importantly, microcapsules produced from PEG with Mw 2000–10,000 g/mol via photopolymerization did not affect the viability of encapsulated islets or alter their insulin secretion. The level of glucose secretion observed during 1 hour of glucose stimulation was at the same level as for unmodified islets. Insulin secretion from islets encapsulated into 1 mm thick PEG gel was delayed at specific time point within stimulation period. The diffusion-related delay can be minimized through reducing the distance between encapsulated islets and surrounding environment by reducing the thickness of the gel (Weber et al., 2008).

The created hydrogel environment is extremely different from the natural surroundings of islets. Indeed, cell viability is strongly dependent on cell-cell or cell-matrix interactions. For example, the viability of MIN6 pancreatic β-cells encapsulated in just PEG-based matrix reduced to 17% within 10 days (Weber et al., 2007b). Mimicking the extracellular environment in an artificial matrix can provide additional enhancement of islets viability and functioning. For that, adhesive peptides or various biological components for promoting cell-matrix interactions can be co-polymerized within hydrogels. Thus, physical incorporation of laminin and collagen type IV, the most abundant proteins present in the extracellular matrix, was performed during formation of hydrogel from PEG dimethacrylate block co-polymers via photoinitiated polymerization (Weber and Anseth, 2008; Weber et al., 2006). Such introduction of the proteins to the PEG matrix preserved the viability and functioning of the microencapsulated islets up to 28 days in culture. Interestingly, the ratio of the proteins had an effect on the level of insulin secretion from the encapsulated islets. For example, equal amounts of laminin and collagen IV in the hydrogel did not have any synergetic effect on the insulin production, while enhanced insulin secretion was observed for the increasing amount of laminin.

Isolation and purification of matrix proteins is, however, complex and cost inefficient way to recreate extracellular matrix environment. The substitution of natural proteins by artificial sequences is a promising approach to resolve this issue. Recently, several therapeutic agents (Drucker, 2001; Drucker, 2002; Holz & Chepurny, 2003) have been proposed to increase

TNF-α.

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 11

precursor solution, encapsulated islets had higher insulin secretion even in the presence of

One of the common applications of microgel beads is cryopreservation of islets. Cryopreservation can be achieved through freezing to preserve the cells within the polymer matrix. However, during freezing islets can still be damaged by extracellular ice formation and cell shrinkage due to dehydration nature of freezing. Recently proposed vitrification approach allowed avoiding these issues. In this process, the presence of certain compounds, e.g., sugars (Doxastakis et al., 2005), stabilizes cells through a direct substitution of water molecules in the outer hydrophilic part of the membrane which leads to the formation of a glass that prevents fusion and reduces dehydration-induced stresses (Koster et al., 2000). Cryopreservation by freezing had been shown to have a significant negative effect on the structure of agarose gel microcapsules (Agudelo & Iwata, 2008). Cryopreservation changed the molecular network structure of agarose gel. Structural changes affected the diffusion of molecules within the gel. The small molecules, such as glucose, were still able to freely diffuse through porous gel, while, molecules of a medium size, such as insulin, had much lower diffusion coefficient after gel freezing. In contrast, vitrification process applied to the same materials had not introduced any changes in scaffolds. Islets preserved by vitrification do not suffer from ice formation and shrinkage and islets viability is drastically increased. Rat pancreatic islets were successfully cryopreserved by freezing in alginate beads (Schneider & Klein, 2011) or in poly(vinyl alcohol) macro-scaffolds (Qi et al., 2010). In the former case, islets were mixed with alginate solution of ultra-high viscosity prior to freezing. Beads then were formed using an air jet droplet generator to create a certain flow rate and finally cross-linked with barium ions. After 7 days of cryopreservation islets were able to maintain normal function *in vitro* and normalized non-fasting blood glucose in diabetic mice *in vivo*. However, since vitrification procedure requires the presence of low molecular weight molecules, some of them, such as poly(vinyl pyrrolidone) and PEG of a low molecular weight, can exert some toxicity to the vitrification solution. For example, insulin release ability of the islets vitrified in the presence of those compounds decreased by 45%

New approach for islets microencapsulation has been introduced by Gimi et al. (Fig. 2) (Gimi et al., 2009). They proposed to use microcontainers made by polymerization of epoxybased polymer. The microcontainer has a membrane containing nano-slots with the size of 25 nm that permit the bidirectional transport of metabolites and nutritional ingredients. Highly cross-linked epoxy-based polymer SU-8 with high glass transition temperature, thermal stability and high Young's modulus was used in fabrication of the microcontainer structure. The containers are capable of holding islets with dimensions upto 200x200x200μm3. Overall device consists of two major components: a hollow cubical base and a nano-porous lid. The cubical base is intended for islets encapsulation, and the lid is sealed after an islet is placed inside the base. The base and the lid have a similar multilayer structure and a complicated forming procedure involving metal sputtering, polymer coating and nano-slot fabrication steps. Murine islets encapsulated into the microcontainers were viable after 48 hours in culture. The main advantage of this microcontainer approach is the

**3.1.3 Cryopreservation of islets using microencapsulation** 

after a vitrification/warming cycle (Agudelo & Iwata, 2008).

**3.1.4 Encapsulation in epoxy-based microcontainers** 

insulinotropic effect of pancreatic cells and protect them from apoptosis. The most promising agent is glucagon-like peptide 1 (GLP-1) that has an extremely short half-life time (less than 2 min) *in vivo* (Lee et al., 2005). Synthesis of artificial peptides with the same biological activities as a substitution for the natural peptide can be one of the solutions for this problem. Synthesis and evaluation of the synthetic glucagon like peptide functionality had been made by Lin et al (Lin & Anseth, 2009). The biological activity of the synthetic peptide physically entrapped in photopolymerized PEG microgel was not affected by photopolymerization. Presence of the synthetic substitute of GLP-1 in the hydrogel had a positive effect on insulin secretion and viability of rat islets encapsulated into the PEG hydrogel.

Many cytotoxic molecules of a low molecular weight are able to easily diffuse inside the microgel capsules *in vivo*. Early islet graft failing and islet damaging occurs due to infiltration of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), tissue necrosis factor-alfa (TNF-α), and interferon-gamma (IFN-γ). The size of these molecules allows them to freely diffuse within the matrixes commonly used in islets encapsulation. A reasonable approach to resolve this issue would be a further reduction of microcapsule permeability. However, the microcapsule pore size cannot be reduced much and still has to allow the permeability of insulin. The decrease in capsule size generally leads to a higher permeability towards cytotoxic molecules. For example, the islets encapsulated into 400-500 μm capsules had minor damages from cytokines, while the islets encapsulated into capsules with smaller dimensions become more susceptible to damage by cytokines (Basta et al., 2004). Moreover, a combination of cytokines and their concentration are also important factors (King et al., 2000; De Vos et al., 2003a).

Incorporation of molecules that can locally inhibit interactions between the small cytotoxic molecules and islets can be used to increase the life-time of encapsulated islets. Indeed, when anti-inflammatory peptide, the cytokine-inhibitory peptide IL-1RIP, had been introduced into PEG hydrogel even in low molar ratio, the survival of islets challenged with the combination of three pro-inflammatory cytokines (IL-1β, TNF-α and IFN-γ) reached up to 21 days with 75 % of cells surviving while less than 25% cells survived by day 4 in the absence of the anti-inflammatory peptide (Su et al., 2010). Furthermore, a synergetic protective effect had been observed for the IL-1RIP within the microgel, with even slightly enhanced insulin secretion in the presence of IL-1RIP in the microgel. Importantly, the hydrogel was produced by mixing 4-arm PEG macromers modified with cysteine and 4-arm PEG with thiol-end groups. The formed hydrogel network possessed the thiol groups that exhibited mild reductive properties and could protect encapsulated cells from oxidative stress (Robertson & Harmon, 2007; Pi et al., 2007).

Immobilization of cytokine antagonizing antibody within the hydrogel is complicated due to the large size of such a molecule (antibodies are usually of hundreds kDa), and protein stability and functioning can be altered during the immobilization process. Additionally, the presence of foreign antibodies in microgel structures can provoke host immune system response. Employing small molecules with the similar function can be one of the solutions such an issue. For example, small WP9QY peptide has been identified as a potential antagonist peptide that binds to human TNF-α. This peptide was used to produce the TNFα-antagonizing PEG hydrogel (Lin et al., 2009). When the PEG hydrogel was formed via photoinitiated polymerization in presence of acryl-WP9QY and adhesive peptides in a precursor solution, encapsulated islets had higher insulin secretion even in the presence of TNF-α.

#### **3.1.3 Cryopreservation of islets using microencapsulation**

10 Biomedicine

insulinotropic effect of pancreatic cells and protect them from apoptosis. The most promising agent is glucagon-like peptide 1 (GLP-1) that has an extremely short half-life time (less than 2 min) *in vivo* (Lee et al., 2005). Synthesis of artificial peptides with the same biological activities as a substitution for the natural peptide can be one of the solutions for this problem. Synthesis and evaluation of the synthetic glucagon like peptide functionality had been made by Lin et al (Lin & Anseth, 2009). The biological activity of the synthetic peptide physically entrapped in photopolymerized PEG microgel was not affected by photopolymerization. Presence of the synthetic substitute of GLP-1 in the hydrogel had a positive effect on insulin secretion and viability of rat islets encapsulated into the PEG

Many cytotoxic molecules of a low molecular weight are able to easily diffuse inside the microgel capsules *in vivo*. Early islet graft failing and islet damaging occurs due to infiltration of pro-inflammatory cytokines, such as interleukin-1β (IL-1β), tissue necrosis factor-alfa (TNF-α), and interferon-gamma (IFN-γ). The size of these molecules allows them to freely diffuse within the matrixes commonly used in islets encapsulation. A reasonable approach to resolve this issue would be a further reduction of microcapsule permeability. However, the microcapsule pore size cannot be reduced much and still has to allow the permeability of insulin. The decrease in capsule size generally leads to a higher permeability towards cytotoxic molecules. For example, the islets encapsulated into 400-500 μm capsules had minor damages from cytokines, while the islets encapsulated into capsules with smaller dimensions become more susceptible to damage by cytokines (Basta et al., 2004). Moreover, a combination of cytokines and their concentration are also important factors (King et al.,

Incorporation of molecules that can locally inhibit interactions between the small cytotoxic molecules and islets can be used to increase the life-time of encapsulated islets. Indeed, when anti-inflammatory peptide, the cytokine-inhibitory peptide IL-1RIP, had been introduced into PEG hydrogel even in low molar ratio, the survival of islets challenged with the combination of three pro-inflammatory cytokines (IL-1β, TNF-α and IFN-γ) reached up to 21 days with 75 % of cells surviving while less than 25% cells survived by day 4 in the absence of the anti-inflammatory peptide (Su et al., 2010). Furthermore, a synergetic protective effect had been observed for the IL-1RIP within the microgel, with even slightly enhanced insulin secretion in the presence of IL-1RIP in the microgel. Importantly, the hydrogel was produced by mixing 4-arm PEG macromers modified with cysteine and 4-arm PEG with thiol-end groups. The formed hydrogel network possessed the thiol groups that exhibited mild reductive properties and could protect encapsulated cells from oxidative

Immobilization of cytokine antagonizing antibody within the hydrogel is complicated due to the large size of such a molecule (antibodies are usually of hundreds kDa), and protein stability and functioning can be altered during the immobilization process. Additionally, the presence of foreign antibodies in microgel structures can provoke host immune system response. Employing small molecules with the similar function can be one of the solutions such an issue. For example, small WP9QY peptide has been identified as a potential antagonist peptide that binds to human TNF-α. This peptide was used to produce the TNFα-antagonizing PEG hydrogel (Lin et al., 2009). When the PEG hydrogel was formed via photoinitiated polymerization in presence of acryl-WP9QY and adhesive peptides in a

hydrogel.

2000; De Vos et al., 2003a).

stress (Robertson & Harmon, 2007; Pi et al., 2007).

One of the common applications of microgel beads is cryopreservation of islets. Cryopreservation can be achieved through freezing to preserve the cells within the polymer matrix. However, during freezing islets can still be damaged by extracellular ice formation and cell shrinkage due to dehydration nature of freezing. Recently proposed vitrification approach allowed avoiding these issues. In this process, the presence of certain compounds, e.g., sugars (Doxastakis et al., 2005), stabilizes cells through a direct substitution of water molecules in the outer hydrophilic part of the membrane which leads to the formation of a glass that prevents fusion and reduces dehydration-induced stresses (Koster et al., 2000). Cryopreservation by freezing had been shown to have a significant negative effect on the structure of agarose gel microcapsules (Agudelo & Iwata, 2008). Cryopreservation changed the molecular network structure of agarose gel. Structural changes affected the diffusion of molecules within the gel. The small molecules, such as glucose, were still able to freely diffuse through porous gel, while, molecules of a medium size, such as insulin, had much lower diffusion coefficient after gel freezing. In contrast, vitrification process applied to the same materials had not introduced any changes in scaffolds. Islets preserved by vitrification do not suffer from ice formation and shrinkage and islets viability is drastically increased. Rat pancreatic islets were successfully cryopreserved by freezing in alginate beads (Schneider & Klein, 2011) or in poly(vinyl alcohol) macro-scaffolds (Qi et al., 2010). In the former case, islets were mixed with alginate solution of ultra-high viscosity prior to freezing. Beads then were formed using an air jet droplet generator to create a certain flow rate and finally cross-linked with barium ions. After 7 days of cryopreservation islets were able to maintain normal function *in vitro* and normalized non-fasting blood glucose in diabetic mice *in vivo*. However, since vitrification procedure requires the presence of low molecular weight molecules, some of them, such as poly(vinyl pyrrolidone) and PEG of a low molecular weight, can exert some toxicity to the vitrification solution. For example, insulin release ability of the islets vitrified in the presence of those compounds decreased by 45% after a vitrification/warming cycle (Agudelo & Iwata, 2008).

#### **3.1.4 Encapsulation in epoxy-based microcontainers**

New approach for islets microencapsulation has been introduced by Gimi et al. (Fig. 2) (Gimi et al., 2009). They proposed to use microcontainers made by polymerization of epoxybased polymer. The microcontainer has a membrane containing nano-slots with the size of 25 nm that permit the bidirectional transport of metabolites and nutritional ingredients. Highly cross-linked epoxy-based polymer SU-8 with high glass transition temperature, thermal stability and high Young's modulus was used in fabrication of the microcontainer structure. The containers are capable of holding islets with dimensions upto 200x200x200μm3. Overall device consists of two major components: a hollow cubical base and a nano-porous lid. The cubical base is intended for islets encapsulation, and the lid is sealed after an islet is placed inside the base. The base and the lid have a similar multilayer structure and a complicated forming procedure involving metal sputtering, polymer coating and nano-slot fabrication steps. Murine islets encapsulated into the microcontainers were viable after 48 hours in culture. The main advantage of this microcontainer approach is the

Encapsulation and Surface Engineering of Pancreatic Islets: Advances and Challenges 13

hydrophobic interactions between a lipid bilayer of the cell membrane are general approaches employed in cell surface modification (Fig. 3). The covalent binding of protective layer to islet cell surfaces is expected to be stable and be present at the islet

Fig. 3. (A) Modification of cell surfaces by covalent conjugation to cell surfaces (1), by incorporation of molecules into the cell membrane via hydrophobic interactions (2, 3), via electrostatic interactions of polyions with the cell membrane (4). Immobilization of bioactive molecules to cell surfaces via interactions of the cell membrane with polymers (B). Adapted

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

from Teramura & Iwata, 2010 with permission from Elsevier.

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

immune response of the host.

surface for certain periods of time.

possibility to directly visualize the encapsulated islets due to optical transparency of the microcontainer material. However, apart from complex fabrication, this approach might require additional steps for biocompatibility adjustment of the material and needs additional studies on islet long-term survival and functioning.

Fig. 2. Scanning electron microscope images of the microcontainer base (A) and lid (B) showing the 500-nm thin-membrane island structures. The thin membrane recessed within each island was milled with 25-nm slots using a focused ion beam. For encapsulated islets, the intracellular Ca2+ changes of an encapsulated islet in response to glucose and tetraethylammonium stimulations were measured using a spectral two-photon confocal microscope (C), and changes in the concentration of intracellular Ca2+ in response to glucose and tetraethylammonium were quantified as seen in this representative trace (D). Reprinted with permission from Gimi, et al., 2009 with permission from Journal of Diabetes Science and Technology.

#### **3.2 Surface modification**

Surface modification of living cells is a new approach in islet encapsulation. The main idea of surface modification is to reduce the size of implanted islets. This can be achieved by forming a conformal protective layer on the islet surfaces using various surface modification techniques. Both synthetic and natural polymers can be used to introduce various functional groups on the islets surfaces. Covalent binding to amino groups of cell membrane proteins, insertion of amphiphilic polymers into a cell membrane via

possibility to directly visualize the encapsulated islets due to optical transparency of the microcontainer material. However, apart from complex fabrication, this approach might require additional steps for biocompatibility adjustment of the material and needs

Fig. 2. Scanning electron microscope images of the microcontainer base (A) and lid (B) showing the 500-nm thin-membrane island structures. The thin membrane recessed within each island was milled with 25-nm slots using a focused ion beam. For encapsulated islets,

the intracellular Ca2+ changes of an encapsulated islet in response to glucose and tetraethylammonium stimulations were measured using a spectral two-photon confocal microscope (C), and changes in the concentration of intracellular Ca2+ in response to glucose

and tetraethylammonium were quantified as seen in this representative trace (D).

Surface modification of living cells is a new approach in islet encapsulation. The main idea of surface modification is to reduce the size of implanted islets. This can be achieved by forming a conformal protective layer on the islet surfaces using various surface modification techniques. Both synthetic and natural polymers can be used to introduce various functional groups on the islets surfaces. Covalent binding to amino groups of cell membrane proteins, insertion of amphiphilic polymers into a cell membrane via

Reprinted with permission from Gimi, et al., 2009 with permission

from Journal of Diabetes Science and Technology.

**3.2 Surface modification** 

additional studies on islet long-term survival and functioning.

hydrophobic interactions between a lipid bilayer of the cell membrane are general approaches employed in cell surface modification (Fig. 3). The covalent binding of protective layer to islet cell surfaces is expected to be stable and be present at the islet surface for certain periods of time.

Fig. 3. (A) Modification of cell surfaces by covalent conjugation to cell surfaces (1), by incorporation of molecules into the cell membrane via hydrophobic interactions (2, 3), via electrostatic interactions of polyions with the cell membrane (4). Immobilization of bioactive molecules to cell surfaces via interactions of the cell membrane with polymers (B). Adapted from Teramura & Iwata, 2010 with permission from Elsevier.
