Cell Microencapsulation Technologies for Sustained Drug Delivery: Latest Advances in Efficacy and Biosafety

Journal of Controlled Release 335 (2021) 619–636 Available online 9 June 2021 0168-3659/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Review article Cell microencapsulation technologies for sustained drug delivery: Latest advances in efficacy and biosafety Tania B. Lopez-Mendez a , b , Edorta Santos-Vizcaino a , b , c , Jose Luis Pedraz a , b , c , Gorka Orive a , b , c , d , e , * , Rosa Maria Hernandez a , b , c , * a NanoBioCel Research Group, School of Pharmacy, University of the Basque Country (UPV/EHU), Paseo de la Universidad, 7, 01006 Vitoria-Gasteiz, Spain b Biomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Instituto de Salud Carlos III, C/Monforte de Lemos 3-5, 28029 Madrid, Spain c Bioaraba, NanoBioCel Research Group, Vitoria-Gasteiz, Spain d University Institute for Regenerative Medicine and Oral Implantology - UIRMI (UPV/EHU-Fundaci´ on Eduardo Anitua), BTI Biotechnology Institute, Vitoria-Gasteiz, Spain e Singapore Eye Research Institute, The Academia, 20 College Road, Discovery Tower, Singapore ARTICLE INFO Keywords: Cell encapsulation Efficacy Biosafety Biocompatibility Biomaterial Alginate ABSTRACT The development of cell microencapsulation systems began several decades ago. However, today few systems have been tested in clinical trials. For this reason, in the last years, researchers have directed efforts towards trying to solve some of the key aspects that still limit efficacy and biosafety, the two major criteria that must be satisfied to reach the clinical practice. Regarding the efficacy, which is closely related to biocompatibility, substantial improvements have been made, such as the purification or chemical modification of the alginates that normally form the microspheres. Each of the components that make up the microcapsules has been carefully selected to avoid toxicities that can damage the encapsulated cells or generate an immune response leading to pericapsular fibrosis. As for the biosafety, researchers have developed biological circuits capable of actively responding to the needs of the patients to precisely and accurately release the demanded drug dose. Furthermore, the structure of the devices has been subject of study to adequately protect the encapsulated cells and prevent their spread in the body. The objective of this review is to describe the latest advances made by scientist to improve the efficacy and biosafety of cell microencapsulation systems for sustained drug delivery, also highlighting those points that still need to be optimized. 1. Introduction For more than four decades, different materials, both of natural and synthetic origin, have been used to manufacture sustained drug delivery systems. Among them, we find those that allow the sustained release of encapsulated growth factors, proteins or drugs; but also others, of greater complexity, that are capable of immobilizing and protecting living cells, selectively isolating them from their environment while they secrete the therapeutic molecules of interest. Cell encapsulation systems have shown wide applicability in pathologies with very diverse characteristics, such as diabetes mellitus (DM), anemia, hemophilia B or pathologies of the central nervous system (CNS), among others [1]. They are especially convenient for pathologies in which maintaining a strict control over the release of the therapeutic molecule is essential. Cell encapsulation can be classified based on the size of the system. On the one hand, we find cell macroencapsulation systems, in which the cells are immobilized in relatively large diffusion chambers, with semipermeable properties. They can have different shapes, such as discs, flat sheets or hollow fibers. The application of cell macroencapsulation devices have shown very good results in vivo demonstrating their undeniable therapeutic potential. However, macrocapsules are characterized by a relatively small surface/volume ratio, which is probably their worst disadvantage, since this implies the need for large amounts of nutrients and oxygen to achieve an adequate diffusion into the chamber and limits the amount of cells that can be encapsulated without creating necrotic nuclei in the innermost and inaccessible areas [2]. * Corresponding authors at: NanoBioCel Research Group, School of Pharmacy, University of the Basque Country (UPV/EHU), Paseo de la Universidad, 7, 01006 Vitoria-Gasteiz, Spain. E-mail addresses: [email protected] (G. Orive), [email protected] (R.M. Hernandez). Contents lists available at ScienceDirect Journal of Controlled Release journal homepage: www.elsevier.com/locate/jconrel https://doi.org/10.1016/j.jconrel.2021.06.006 Received 14 November 2020; Received in revised form 4 June 2021; Accepted 6 June 2021 Journal of Controlled Release 335 (2021) 619–636 620 Cell microencapsulation represents a very interesting alternative, greatly improving the surface/volume ratio and increasing the diffusion of nutrients and oxygen inside the capsules. Cell microencapsulation strategy is based on the immobilization of cells that produce therapeutically relevant molecules in spherical particles between 100 and 1500 μ m in diameter, approximately. The particles are elaborated with biocompatible materials and usually surrounded by a semi-permeable polymeric membrane that prevents the passage of high molecular weight molecules — antibodies and other components of the immune system —, protecting these cells from the host's immune response and from the mechanical stress that may occur when the implant is placed in the selected tissue [3]. In addition, the microcapsule must exert a tight control over the bidirectional diffusion of molecules — entrance of nutrients and oxygen; and release of de novo synthesized therapeutic factors and metabolic subproducts —, and provide cells with a suitable environment to enhance and modulate their function. This technology also suppresses, or at least reduces, the chronic administration of immunosuppressive agents, thus avoiding some of the adverse events associated with organ and tissue transplantation. On the other hand, the constant improvements in imaging techniques and robotic surgery procedures allow the access to difficult to reach areas for implantation [4]. Today, the results obtained in the various clinical trials carried out to date, make clear the advantages and potential applications of this promising technology. However, there are still aspects that need to be improved so that cell microencapsulation systems can be applied routinely in clinical practice. For this reason, in the last years, researchers have directed efforts towards trying to solve some of the key aspects that still limit efficacy and biosafety, the two major criteria that must be satisfied to reach the clinical practice. Those two concepts are closely related to each other and must be carefully defined and regulated due to their implications regarding patient well-being. The objective of this review is, therefore, to group and describe the extensive work carried out with the aim to improve these criteria, emphasizing the points that still need to be optimized. 2. Efficacy - biocompatibility When talking about efficacy of cell microencapsulation, biocompatibility is one of the most important aspects to be considered. It will determine implant’s viability, functionality and durability, becoming in many cases a limiting factor to succeed. The biocompatibility of the implant must be given in 2 directions (Fig. 1). From outside to inside, the materials used must protect the immobilized cells, avoiding direct toxicity or the blockage of nutrients and oxygen diffusion. From inside to outside, none of the system components — cells, biomaterials, crosslinking agents, etc. — or procedures used must be toxic for the patient or elicit an immune response in the host. When this occurs, the foreign body reaction (Box 1) can eventually isolate the implant within a fibrotic capsule, thereby preventing the access of essential molecules and leading to graft failure. In addition, the biocompatibility must last over time, since live cell therapies are normally used for long-term treatments. In this sense, several experts in the field of cell encapsulation have decided to define the term "biotolerability", considering it more appropriate than "biocompatibility" [5]. Despite the undeniable improvement occurred in recent decades, the biomaterials and cells that are used today continue to produce, to a greater or lesser extent, an inflammatory response by the host, so searching for suitable components remains a priority. The final performance of the device will depend not only on the biomaterials and cells used, but also on the site of implant, the local application of immunosuppressive drugs, or even the size and shape of the implant. 2.1. Biomaterials, crosslinkers and coatings 2.1.1. Biomaterials and cross-linkers On the one hand, the elaborated devices must present a suitable structure, resistant to unwanted degradation that avoids contact between the encapsulated cells and the host immune system. Furthermore, the biomaterials must guide the processes of proliferation and differentiation of encapsulated cells, enhancing their viability and functionality. On the other hand, the choice of all the materials must be made taking into account possible toxicities. The latter includes, in addition to the main materials, cross-linkers, physicochemical modifications and possible degradation byproducts. Today, the materials used include ceramics, plastics and various polymers, among others. The latter can be classified as natural (polysaccharides, polypeptides and polynucleotides) or synthetic. Among natural polymers, polysaccharides are the most used because they allow relatively smooth encapsulation processes that are compatible with cell viability. Examples of natural polymers are alginate, agarose, collagen, or cellulose. On the other hand, polyethylene glycol (PEG) continues to be the most widely used option among synthetic polymers, along with poly(lactic-co-glycolic acid) (PLGA) and polyvinyl alcohol (PVA) [11,12]. Among all the available polymers, alginate is by far the most widely Fig. 1. Factors that may compromise implant biocompatibility/biotolerability. Regarding the biomaterials and coatings used, factors such as the permeability of the microcapsule to proinflammatory molecules, the characteristics of the implant surface, the size and shape of the sphere or structural deficiencies can trigger an immune response against the capsules. The encapsulated cells can also release pro-inflammatory molecules or protrude outside the implant. Futhermore, the characteristics of the implant site should also be considered in detail when trying to improve the biocompatibility/biotolerability of the system. T.B. Lopez-Mendez et al. Journal of Controlled Release 335 (2021) 619–636 621 used biomaterial in cell microencapsulation systems, due to its excellent biocompatibility and easy handling [13]. Alginate is a natural anionic polysaccharide that creates three-dimensional structures, going from sol to gel, when it reacts with divalent ions. It is made up of different proportions of residues of β-D-manuronic acid (M) and α -L-guluronic acid (G) that create different structures according to the ratio of G and M. Determining and standardizing this proportion is essential since it has a great influence on some of alginate hydrogels properties, such as their biocompatibility, stability, mechanical resistance and permeability, among others [13]. In general, alginates with a higher proportion of G blocks are stiffer, compared to those with a higher proportion of M blocks that have better elastic properties, due to the greater affinity of guluronic acid for divalent ions, and these physical-mechanical differences affect the way the immune system reacts against the implant [14]. On the other hand, the purity degree of the alginate is directly related to its biocompatibility. Low purity alginates contain endotoxins, proteins and polyphenols that reduce the biocompatibility of the implants and can damage the encapsulated cells [15]. Several commercial alginates have been described to contain pathogen-associated molecular patterns (PAMPs). These are potent initiators of inflammatory responses [16] and produce the release of small proinflammatory cytokines — such as interleukin 1β (IL-1β), the tumor necrosis factor α (TNFα ) or interleukin 6 (IL-6) —, which can come into contact with the encapsulated cells and cause damage. The most common endotoxin that can be found in alginate are lipopolysaccharides (LPS), which can bind to tolllike receptors 4 (TLR-4) [17], producing an inflammatory response mediated by a variety of cells of the immune system [18]. Therefore, in recent years, different purification methods have been developed in order to obtain ultra-pure alginates with less immunogenicity in vivo [19–22]. However, there is great variability between the procedures used in the different research groups and it is still necessary to improve the tools for the screening and elimination of these and other impurities, such as peptidoglycans and lipoteicoic acid [23–27]. In this sense, there are divided opinions on whether or not it will be possible to achieve an adequate and sufficient level of purification of the alginate — so that it becomes nearly inert to the immune system — or whether it will also be necessary to chemically modify its structure. Indeed, pericapsular fibrosis has been one of the major drawbacks in clinical studies carried out to date with alginate as the main material. However, the composition of the alginate and the variability between administration protocols, cell types or the concomitant use of different coating materials, among others, make the comparison complicated. In recent years, alginate purification protocols have been refined [27], while some groups have begun to include chemical modifications in the alginates [28,29]. A few years ago, Paredes-Juarez et al. created a platform that allows the identification of pattern recognition receptor (PRR) activating polymers, in order to identify contaminants in the biomaterials [16]. On the other hand, in a study carried out by Vegas et al., a combinatorial approach was used to generate a wide range of alginate variants with the aim of finding those that were able to decrease the foreign body response [28]. After a first selection, the most promising hydrogel spheres were evaluated in vivo, in rodents and non-human primates (cynomolgus macaques). Three triazole-containing analogues (Z2-Y12, Z1-Y15 and Z1-Y19) were identified, which significantly reduced the foreign body response, when compared to conventional SLG20 alginate spheres inhibiting macrophage recognition and fibrosis formation (Fig. 2). When implanted intraperitoneally into non-human primates (n =3 each alginate variant), Z2-Y12, Z1-Y15 and Z1-Y19 spheres with 1.5-mm diameters displayed substantially reduced fibrotic responses after 4 weeks compared to 1.5-mm SLG20 spheres. SLG20 spheres had more extensive immune macrophage and fibrosisassociated activated myofibroblast coverage, consistent with the visible fibrotic overgrowth seen in the phase contrast imaging. Z1-Y19 spheres displayed more coverage by macrophage and myofibroblasts than either Z2-Y12 or Z1-Y15, but less compared to SLG20. Z2-Y12 and Z1-Y15 spheres had markedly lower levels of smooth muscle actin (SMA) protein, the major morphological characteristic of myofibroblasts, as compared to SLG20 spheres, but Z1-Y19 spheres did not, despite the lower average SMA measured. Hydroxyproline quantification revealed lower collagen levels for all three lead formulations compared to SLG20. In an interesting and more recent study, Liu et al. proposed a group of zwitterionic sulfobetaine (SB) and carboxybetaine (CB) modifications of alginate (SB-SLG20 and CB-SLG20) to reduce cell accumulation and Box 1 Foreign body reaction against biomaterials. Although the materials and therapeutic applications differ, the process by which the body produces rejection against implants has many points in common. This is known as "foreign body reaction" and consists of the following phases: Immediately after the implantation and depending on the characteristics of its surface —material, shape, roughness, electrostatic charge, etc. — and the injury caused during the surgical process, various host proteins — such as albumin, fibronectin or complement molecules — will start to adhere to the surface of the implant. This creates a chemoattractive gradient for the innate immune response [6]. Neutrophils are the first cell type present at the implant site and their function is to engulf the microorganism remains and dead cells. Neutrophils also secrete proteases, lysozymes, reactive species and other enzymes to eliminate any type of biodegradable material. At the same time, they secrete cytokines and other factors that cause the activation of macrophages (differentiated from the recruited monocytes), which will be the predominant cell type in the following phases of the foreign body response. This acute phase of the inflammatory reaction would end with a return to homeostasis if the material recognized as foreign disappears completely. On the contrary, if the host cannot destroy the implant, its continued presence can lead to a second phase of chronic inflammation. The “frustrated” macrophages start to fuse into multinucleated cells around the implant, giving rise to foreign-body giant cells [7]. At the molecular level, pro-inflammatory cytokines, such as IL-6, TNFα , interleukin 4 (IL-4), and interleukin 13 (IL-13), have been reported to be overexpressed [8]. In this phase, there is a continuous presence of monocytes and lymphocytes and a constant activation of macrophages and neutrophils, which secrete enzymes and reactive species. At the same time, neovascularization phenomena are observed, with the appearance of functional capillaries. In the final phase, fibroblasts, activated by macrophages, deposit collagen fibers to form a dense and fibrous acellular capsule that isolates the implant from the surrounding tissue [9]. This prevents the passage of nutrients and oxygen, and eventually leads to compromising the viability of the encapsulated cells. In vivo the chronology varies depending on the organism, ranging from the appearance of fibrosis in just 7 days, in the case of mini-pigs, to 14 days in rats [10]. T.B. Lopez-Mendez et al. Journal of Controlled Release 335 (2021) 619–636 622 Fig. 2. Modified hydrogels mitigate foreign body response in non-human primates. Z2-Y12, Z1-Y15 and Z1-Y19 alginate spheres significantly reduce fibrosis in cynomolgus macaques, while conventional SLG20 alginate spheres become fibrotic. a Phase contrast imaging of spheres retrieved after 4 weeks in the intraperitoneal space show less fibrosis on Z2-Y12, Z1-Y15 and Z1-Y19 spheres than on SLG20. Scale bars, 2,000 μ m; n =3. b Confocal imaging of retrieved spheres from a after 4 weeks in the intraperitoneal space show significantly less macrophage (CD68, CD11b), myofibroblast (SMA) and general cellular deposition (DAPI) on Z2-Y12 spheres. Scale bars, 200 μ m; n =3. Brightfield images of the stained spheres are inset; scale bars, 100 μ m. c Western-blot analysis of protein extracted from the top three alginate analog spheres and control spheres in a; n =3. Blots were stained for SMA and loading was normalized to β-actin. Dots represent measurements from individual biological replicates, and lines show the average of the three replicates. One-way ANOVA with Bonferroni correction was used to allow for statistical comparison of multiple means. #P <0.05; **P <0.001; ns, not significant. d Collagen content using a hydroxyproline quantification assay of protein extracted from the top three alginate analog spheres and control spheres in a. n =3. Dots represent measurements from individual biological replicates and lines show the average of the three replicates. One-way ANOVA with Bonferroni correction was used to allow for statistical comparison of multiple means. #P <0.05; **P <0.001; ns, not significant. Reprinted from ref. [28], with permission from Springer Nature. T.B. Lopez-Mendez et al. Journal of Controlled Release 335 (2021) 619–636 623 fibrotic processes around the capsules [30]. Studies in mice, dogs, and pigs showed a significant reduction in these processes. Finally, rat pancreatic islets immobilized in SB-SLG20 microbeads (control group with SLG20), were transplanted, for 200 days, into the peritoneal cavity of streptozotocin (STZ)-induced C57BL/6J diabetic mice and four out of six mice maintained normoglycemia by the end of the study (the shortest duration of glycemic control was ~135 days) (Fig. 3). An intraperitoneal glucose tolerance test (IPGTT) 200 days after transplantation, showed that the mice (cured ones, n=3) in the SB-SLG20 group cleared blood glucose (BG) and restored normoglycemia at a rate comparable to that of non-diabetic mice. An ex vivo glucose-stimulated insulin secretion (GSIS) of islets retrieved from cured mice (n=3) indicated again the normal function of islets. Dark-field microscopic images and hematoxylin-eosin (H&E) histological analysis of retrieved SB-SLG20 microcapsules from normoglycemic mice after 200 days revealed no or minimal cellular deposition on the microcapsules and the presence of numerous functional islets inside. Microencapsulation systems based on the gelation of alginate mainly use barium and calcium as crosslinking agents and, in some cases, strontium [31]. Today there is no clear preference between calcium or barium and its use depends mainly on the protocol adopted in the different research groups. The arguments in favor of using calcium as a crosslinking ion revolve around the lower toxicity [32]. However, the resistance of the spheres cross-linked with calcium is lower than that achieved with barium, and that is why other groups opt for this option when developing their systems [28,29]. Some authors argue that, barium-cross-linked beads could be significantly less immunogenic as they do not need subsequent coatings to increase the immunoisolation, which are usually necessary when calcium is selected as the crosslinker [33]. This last statement is still in doubt, since some studies seem to indicate that the level of immunoisolation would not be sufficient in the barium beads if they do not have posterior coatings. In addition, the in vivo implantation of alginate and barium beads have originated a fibrotic response to the implant in different administration routes [26]. On the other hand, the release of cross-linking ions must also be taken into account, especially when barium is selected, due to its toxicity [34]. However, despite the advantages of alginate, there are still aspects that need to be optimized. Among them its mechanical properties, since the systems made with alginate and different ions tend to undergo changes in size due to the osmotic processes that occur in the physiological environment, increasing the permeability of the capsule, weakening its structure and finally causing rupture of the system [31]. Ion concentration, the selected crosslinking agent or the alginate composition are determining factors in obtaining adequate and homogeneous gelation. Simply varying the gelling conditions, the spatial distribution of the alginate chains in the microsphere can vary from homogeneous to very heterogeneous, with up to 10 times more concentration on the surface than in the nucleus [31]. Some studies have suggested that a truly homogeneous distribution of alginate chains can only be achieved by internal and external gelation applied simultaneously [35]. Release of components from the microcapsules can also stimulate an inflammatory response. This includes degradation products that may arise from reactions occurring under physiological conditions, detachments of parts of the system or ion exchange, among others. Alginate is subjected to hydrolytic and enzymatic degradation. It has been described to have a very low rate of hydrolysis at physiological pH and the low molecular weight chains released are excreted via the urinary tract. These degradation processes have been extensively studied, in vitro and in vivo [36,37], in the subcutaneous space, the peritoneum and in Fig. 3. Sulfobetaine-alginate (SB-SLG20) microcapsules improve diabetes correction in mice in a 200-day study. a Blood glucose concentrations of mice (n=6 mice per treatment group). b Intraperitoneal glucose tolerance test (IPGTT) before retrieval (n=3). c Ex vivo glucose-stimulated insulin secretion test (GSIS) of the retrieved rat islets from SB-SLG20 microcapsules, n=3, Mean±SEM, *P<0.05. d A dark-field phase contrast image of retrieved islet-containing SB-SLG20 microcapsules. (n=6; scale bar, 2mm). e An hematoxylin-eosin (H&E) stained cross-sectional image of retrieved islet-containing SB-SLG20 microcapsules. Scale bar, 500 μ m. f Immunohistochemical staining of rat islets in retrieved SB-SLG20 microcapsules. Insulin is stained red and nuclei are stained blue (Scale bar: 50 μ m). g A dark-field phase contrast image of retrieved islet-containing SLG20 microcapsules. (n=6; scale bar, 2mm). h An H&E stained cross-sectional image of retrieved isletcontaining SLG20 microcapsules. Scale bar, 500 μ m. i Immunohistochemical staining of rat islets in retrieved SLG20 microcapsules. Insulin staining is negative and nuclei are stained blue. Scale bar, 500 μ m. Reprinted from ref. [30] http://creativecommons.org/licenses/by/4.0/. T.B. Lopez-Mendez et al. Journal of Controlled Release 335 (2021) 619–636 624 some areas of the brain. Some groups have devised strategies to improve the mechanical stability of alginate hydrogels, covalently crosslinking it with different polymers, via photocrosslinking solutions or enzymatic reactions, for example [38–40]. In a recent study, sodium alginate was functionalized, with cross-reactive PEG derivatives presenting a terminal thiol and carbon electrophile functionalities, and the spheres formed by a combination of Ca-alginate interactions and sulfur-carbon covalent bonds. The resulting spheres showed greater mechanical resistance and better preserved shape, compared to the simple alginate and calcium beads. When these spheres were implanted in the intraperitoneal space of immunocompetent mice, tissue adherence was not observed and integrity was not compromised in the 30 days of the study [41]. In another interesting study, it was shown that modifying the alginate with 2-aminomethyl methacrylate hydrochloride can decrease immune reactions against the implant. The authors performed a first ionic crosslinking, followed by the application of UV light to form the covalent bonds. This showed greater mechanical stability when it was evaluated in vivo for 3 weeks [42]. Material selection is especially demanding in this type of system since the resulting particles must not only have a high durability after implantation, they must also be capable of responding to the biological needs of the immobilized cells for long periods of time. However, many materials, such as alginate, do not have cellular signaling motifs and must be biofunctionalized to improve their interaction with encapsulated cells. In this regard, in recent years, different proteins — such as collagen, laminin or fibronectin — or small short peptides — such as RGD (arginine-glycine-aspartic acid) — have been incorporated into microcapsules, trying to imitate the physical and biomechanical characteristics of the native environment of the encapsulated cells to improve and control cellular behavior [43–46]. In this regard, there are divided opinions on whether it is more appropriate to use complete extracellular matrix proteins, such as fibronectin or collagen, or small synthetic peptides, such as RGD [47,48]. Interestingly, the best strategy in every case seem to be strongly cell-dependent. For example, Garate et al. evaluated the influence of RGD functionalization of alginate encapsulating C 2 C 12 myoblasts, baby hamster kidney (BHK) fibroblast or stromal mesenchymal cells (MSCs) and the results showed different optimal concentrations of RGD in every case [47,49,50]. In this sense, Gonzalez-Pujana et al. designed a sensitive analytical tool that permits the evaluation of different cell adhesion kinetics, but also the integrin profiling and their contribution to cell attachment and adhesion strengthening via clustering, which allows the design of specific biofunctionalization strategies depending on the cell type [51]. Other components of the extracellular matrix (ECM), such as hyaluronic acid (HA) have also been added to the alginate matrix of microcapsules. Recently, pancreatic pseudo-islets derived from MSCs were immobilized in alginate-HA microcapsules and the results showed better cell viability, with lower levels of initial apoptosis [52]. Moreover, the inclusion of HA in the alginate matrix, enhanced the differentiation of the MSCs towards pancreatic progenitors and increased the insulin release [53,54]. 2.1.2. Coating materials In some cases, and depending on the application, the microbeads composed of different biomaterials and cells, are the final product to be administered. However, obtained pore size in most cases is too large and does not present a real barrier against the threats that the implant will face once implanted. Therefore, many groups coat these beads with different polymers to elaborate microcapsules that control the molecules and cells that can come into contact with the immobilized cells. Currently, this filtering is carried out by defining a minimum molecular weight — molecular weight cut-off (MWCO) — of solute that is totally excluded by the semipermeable membrane [55]. This definition can be misleading since molecules of similar molecular weight can have very different sizes, as is the case with proteins and polysaccharides. Today, there is still no consensus regarding what should be the most optimal criterion for the exclusion of molecules that can access the interior of the microcapsule, despite Chick et al. already named the concept of the immuno-barrier in 1977 [56]. Adequately defining this concept is essential to develop biocompatible and biotolerable systems and it should be a priority issue. To create a biocompatible and biotolerable environment, the semipermeable membrane must first avoid contact of the encapsulated cells with the cellular components of the immune system and the antibodies. A MWCO of around 70 kDa seem to be adequate for many drug delivery applications, but it has been found that this is not enough if bidirectional flow of antigenic, chemotactic and cytotoxic molecules — such as reactive oxygen species or pro-inflammatory cytokines — is allowed. With the classic approach of size-exclusion, low molecular weight molecules such as IL-1β (17.5 kDa) or TNFα (51 kDa) will be able to easily access the interior of the capsule and cause damage to the encapsulated cells, as they are even smaller than some of the therapeutic molecules that are usually released from these systems. On the other hand, encapsulated cells secrete antigens — e.g. chemokines as low as 813 kDa in molecular weight — to the exterior of the microcapsules that are responsible for recruiting cells from the host immune system. For the elaboration of the semi-permeable membrane, different polymers have been used, such as chitosan, oligo-chitosan or poly (methylene-co-guanidine) (PMCG), but both in preclinical studies and in human trials, the most used molecules are poly-L-lysine (PLL) and poly-L-ornithine (PLO) [57–59]. However, both molecules are known to be immunogenic, so many groups choose to add a last layer of alginate on the particles to mask the positive charges that would otherwise be exposed to the components of the immune system. Resulting microcapsules are known as APA (alginate-poly-L-lysine-alginate or alginatepoly-L-ornithine-alginate) [60,61]. This strategy has been intensely debated since there are studies showing that this second layer of alginate may not be sufficient to inactivate all the exposed positive charges [62–64]. In fact, the studies carried out to analyze the surface of the microcapsules coated with these polycations, showed that these molecules are exposed — and in great quantity — in the outermost 1-2 monolayers of the membrane, thus the outer alginate layer appears to overlap with the PLL layer, rather than form an additional outer membrane [65,66]. In addition, both polycations show limited physicochemical properties but most works chose PLO for apparently having greater mechanical stability, biocompatibility and permeability [63]. The increased immunogenicity of microcapsules coated with this type of polycations is mainly due to the physicochemical changes that affect protein adhesion on the surface of the microcapsules, such as zeta potential, hydrophobicity or roughness. On the one hand, the zeta potential of this type of implant must be negative and similar to that of the membranes of adjacent cells. In a study carried out by De Vos et al. [67], it was found that the zeta potential of APA-PLL microcapsules showed a more negative value before they were implanted. Although all the values were less negative than those described in other studies for the uncoated alginate microbeads [68]. On the other hand, in a study carried out by Lekka et al. [69], a lower surface roughness, of less than 1 nm deep, was associated with the uncoated alginate microspheres and with the PLLcoated microcapsules, compared to much higher values, of up to 14.4 nm, of PMCG-sulfate coated microcapsules. Finally, the addition of coatings to the alginate microbeads increases the hydrophobicity of the system [63], although the type of alginate used in each case also influences the final result. In a very interesting study by Rokstad et al. [62] a lepirudine-based human whole blood model was used as a tool for measuring the biocompatibility of different microcapsules. The results showed that alginate polycation (AP) or APA capsules trigger the complement activation, whereas Ca/Ba alginate do not. Fig. 4 shows that the deposition of complement component 3 (C3) on the bead surface is higher in AP or APA beads than Ca/Ba beads. T.B. Lopez-Mendez et al. Journal of Controlled Release 335 (2021) 619–636 625 Taking into account the obvious need to improve the coatings of these particles, in recent years several groups have analyzed other molecules that may be appropriate, both to substitute the PLL/PLO molecules or in combination with them [70–72]. In a recent study, genipin was used in association with PLL [73]. Using force spectroscopybased simultaneous topographical and mechanical characterization to study polymer to polymer interaction, the study concluded that genipin crosslinking avoided membrane detachment in alginate microspheres with double polycation coatings. Attempts have also been made to improve the biocompatibility of the microcapsules by coating them with polymers capable of reducing protein adsorption and the fibrotic response to the implant. By coating the alginate microcapsules with hydrophilic polymers such as PEG [74–76], the biocompatibility of the implant can be improved, although the level of protein adsorption will depend on the density, length and conformation of its chains. In one study, alginate-PEG microcapsules containing allogenic islets were evaluated and their biocompatibility was improved when transplanted into the intraperitoneal space, but not into the epididymal fat pad [77]. The strategy of coating the alginate microcapsules with PEG and rapamycin, evaluated by another group, was also able to reduce macrophage proliferation and fibrotic response [78]. Modifying the surface of the microcapsules with a patented macromolecular heparin conjugate has also been shown to improve biocompatibility and significantly reduce the fibrotic response against the implant, in syngeneic and allogeneic transplant models [79]. With a similar strategy, but coating the alginate microcapsules with the C-X-C motif chemokine 12 (CXCL12) (also known as "stroma-derived factor 1", SDF-1), the biocompatibility was also improved but, in this case, a longterm improvement in xenogeneic pancreatic islet survival and Fig. 4. Deposition of C3 on the microsphere surface after incubation in human lepirudin anti-coagulated whole blood. A–L 3D projections made by sectioning entire microspheres after incubation for 30, 120 and 360 min. M–P Projections through the equator overlaid with transmitted light images after 360 min. Q–T Controls are given in the lower panels as projections (black pictures). The inserts show transmitted light equatorial sections for visualization. Bars are 100 μ m. Reprinted from ref. [62] with permission from Elsevier. T.B. Lopez-Mendez et al. Journal of Controlled Release 335 (2021) 619–636 626 functionality was also achieved, due to the recruitment of immunosuppressive regulatory T cells to the implant site [80]. Recently, in another study, coating the alginate spheres with chitosan also significantly reduced the fibrotic response against the implant, improving its biocompatibility, while maintaining glucose levels for one year, in a canine allogeneic transplant model and in a xenotransplant in rodents [81]. Another strategy that may be very interesting is to incorporate motifs that have anti-inflammatory properties into the design of the microcapsule. Sulfated alginates [82] or the interleukin-1 receptor (IL-1R) [83] are good examples of this, as they decrease the production of some cytokines and improve the viability of encapsulated cells. In some systems, such as those made by the alginate-PLL combination, it is not possible to independently adjust the mechanical stability and the permeability of the microcapsules, which is a notable limitation [84]. However, there are studies in which this permeability-stiffness relationship has been divided using various polymers [85]. The possibility of independently modifying critical parameters for cell encapsulation, such as capsule size, thickness, mechanical resistance and membrane permeability, offers great advantages in the design of this type of system. Poor mechanical resistance can lead to protrusion of encapsulated cells, a phenomenon that needs to be fixed when designing these systems [86]. In this sense, Johnson et al. carried out an analysis quantifying cell protrusion in alginate microcapsules, coated with PLL and 50% hydrolyzed poly(methylvinylether-alt-maleic anhydride) (PMM). According to the results obtained, around 30% of the encapsulated INS-1E β cells were located in the last 20 μ m of the alginate-PLL-PMM50 layer, with 7% of the cells protruding [87]. Reinforcing the capsules with crosslinked shells may help preventing cell exposure and scape. Lastly, in some cases, for example if the secreted molecule is especially large, it will be necessary to optimize the system so that it allows the passage of the therapeutic molecule out of the capsule, without compromising the protection of the encapsulated cells. In this sense, recently Montanucci et al. modified the permeability of alginate microcapsules to allow the continuous secretion of immunoglobulin M (IgM), with no signs of inflammation [88]. 2.2. Cell source and target pathology Both allogenic and xenogeneic cells have been incorporated into the microencapsulation systems. In case of human origin cells, their acquisition can be complicated and expensive. Besides, they can be subject to biological, ethical and legal limitations. Therefore, the use of xenogeneic cells has spread in the field of cell microencapsulation, thanks to the immunoisolation produced by the semipermeable membrane [89]. However, the systems used to date to encapsulate both cell types have been practically identical, without taking into account that the different immunological responses caused by allogenic or xenogenic cells require capsular configurations capable of protecting the cellular content against variable immunological environments. In the case of allogeneic transplants, it is probably sufficient to avoid contact between the encapsulated cells and the cells of the host's immune system [90]. Therefore, the simplest microcapsules of cations and alginate, without great limitations in the diffusion of molecules, may be suitable. When a xenotransplantation is performed, the scenario is more complex and the simplest systems may not be effective in avoiding immune rejection (Fig. 5). These cells produce xenogenic epitopes, such as galactosyl (Gal) residues, that are secreted outside the capsule and are recognized by the immune system of higher mammals, including humans. In addition, in recent years the role of N-glycolyl neuraminic acid (Neu5Gc), another pig xenoantigen, is being studied as a possible obstacle in xenotransplantation [91]. The complexes formed by Gal residues and the antibodies linked to them, are powerful activators of the classical complement pathway. As these complexes begin to accumulate on the capsular surface, chemotaxis of different cell types, such as neutrophils [92] occurs, which initiate powerful inflammatory reactions. During this first phase, many Fig. 5. Pro-inflamatory molecule secretion from the microcapsules activates the immune response in different scenarios. Xenogeneic transplant. Some antibodies are able to enter the capsule or contact the encapsulated cells located in the most superficial layers of the implant. These antibodies recognize sequences that are not present in the host species, such as the Gal carbohydrate in the case of primates, and this leads to the activation of complement pathways. This activation produces direct cell lysis and the release of molecules that promote inflammation and the recruitment of immune cells, such as neutrophils, to the implant site. The recruited cells release small cytokines that can cross the microcapsule membrane and damage the encapsulated cells. In more advanced phases, fibrotic processes appear, which could end up isolating the implant and compromising the supply of nutrients and oxygen to the interior of the capsule. Intracapsular necrosis. Necrotic cells release the so-called DAMPs or alarmins, such as high-mobility group box-1 (HMGB1), heat shock proteins (HSPs), S100 proteins, DNA/RNA fragments etc., to the extracellular fluid. These small molecules can diffuse outside the microcapsules and activate cells of the immune system, such as macrophages, dendritic cells, neutrophils or lymphocytes, binding PRRs, such as TLRs. These cells, in response, will secrete pro-inflammatory cytokines, such as IL-1β, IL-6, interleukin-8 (IL8), or TNFα , which will produce inflammation and recruit more immune cells to the area. These molecules can enter the microcapsules and damage the encapsulated cells. If the situation persists over time, the adaptive immune response may be activated. T.B. Lopez-Mendez et al. Journal of Controlled Release 335 (2021) 619–636 627 small cytokines are able to cross the semipermeable membrane of the microcapsules, causing damage to the encapsulated cells. Following the first innate response, a second IgM-mediated [92] delayed hypersensitivity response to xenogeneic epitopes begins, promoting the recruitment of new cells of the immune system to the implant site and the secretion of more chemokines and cytokines. After these events, the microcapsules are usually surrounded by inflammatory cells and fibroblasts that hinder the passage of nutrients and oxygen, compromising the survival of the encapsulated cells. Finally, the appearance of fibrosis can lead to total isolation of the implant. Due to these differences between allo and xenografts, the latter require systems that protect encapsulated cells against more potent threats. The membranes must be less permeable and, ideally, prevent the passage of molecules produced by the immune system, while preventing the exit of hyperinflammatory xenogeneic epitopes, such as Gal residues. Another way of activating the immune response may occur when cell necrosis appears inside the microcapsules [93] (Fig. 5). Unfortunately, this is still quite common, when there are problems in the diffusion of nutrients and oxygen, due to insufficient permeability of the biomaterials, fibrotic processes associated with foreign body reaction or an excess of encapsulated cell mass [94]. Necrotic phenomena are directly related to damage-associated molecular patterns (DAMPs). These molecules are normally found inside the cells, but are released outside when cell damage occurs [95]. Some examples are heat shock proteins or DNA/RNA fragments. The mammalian immune system has specific receptors for this type of signals, the PRRs, such as Toll-like receptors (TLRs). The DAMPs released from the microcapsules are powerful activators of the immune system, activating inflammatory and angiogenesis processes, which mediate the release of large amounts of cytokines that jeopardize the survival of the encapsulated cells [96]. In this sense, there are different studies that tried to improve the viability of microencapsulated cells, incorporating chemical compounds capable of generating oxygen [97,98] or through strategies that promote the vascularization of the implant. For the latter, several strategies have been tested. On the one hand, the ability of different angiogenesisinducing growth factors, such as fibroblast growth factor (FGF) [99,100] or vascular endothelial growth factor (VEGF) [101,102] has been exploited to promote the neovascularization of the implant, thus improving the results of the therapy. On the other hand, the implantation of the microcapsules in pre-vascularized spaces is also considered as a beneficial option, either generated in the host’s organism or in macrodevices [103,104]. Even if the risks associated with xenotransplantation are being reduced, the advances in the field of stem cell use have unlocked an unthinkable potential. The ability to differentiate human stem cells, from different sources, to obtain the desired cell type or the possibility of reprogramming adult cells to induced pluripotent stem cells (iPSCs) [105,106] have defined the path to a sufficient source of human cells. Furthermore, in the particular case of iPSCs, there are not ethical restrictions [107]. In this vein, the studies carried out to date have shown that it is possible to obtain fully functional beta cells or pancreatic progenitors, starting from human pluripotent stem cells (hPSCs) — either human embryonic stem cells (hESCs) [108–113] or iPSC [114]. In recent years, these cells, known as human stem cell derived β cells (SC-β), have been seen as an excellent source of unlimited pancreatic cells [30,111,115]. For example, Vegas et al. implanted human SC-β, immobilized on alginate beads, in the intraperitoneal space of immunocompetent C57BL/6J mice previously treated with streptozocin. C-peptide levels and blood glucose concentration showed therapeutically relevant results up to 174 days, without the need for immunosuppressive treatment [116]. In another recent study, the maturation of SC-β was stimulated by forming aggregates, similar in size to pancreatic islets, which make them respond to glucose stimulation in just 3 days after transplantation [117]. Likewise, stem cells from other origins, such as amniotic fluid or adipose tissue, can also be transformed into insulin-producing cells, which can be encapsulated and transplanted in diabetic animal models to normalize blood glucose values [118–120]. For example, Montanucci et al. managed to remit hyperglycemia in diabetic mice, implanting human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS), immobilized in alginate and PLO microcapsules [121]. Moreover, in a more recent study by the same authors [122], hUCMS cells were co-encapsulated with human pancreatic islet-derived progenitor cells (hIDC) and implanted into non-obese diabetic mice. The objective of this synergy was to maintain tracer insulin output by hIDC, while exploiting the immunoregulatory properties of hUCMS. A decline of blood glucose levels was observed in vivo. MSCs have demonstrated to be very suitable for their use in cell encapsulation systems, due to their hypoimmunogenic and immunomodulatory characteristics [123–125]. These cells inhibit immune responses by secreting cytokines and soluble growth factors that produce a local immunosuppressive effect in the surrounding cells [126]. In recent studies, efforts have focused on analyzing the behavior of immortalized MSCs, genetically modified to secrete erythropoietin (EPO), for the treatment of anemia [50,127–129]. In addition, their benefits have also been evaluated in hepatic pathologies [130,131], as an alternative to porcine hepatocytes [132,133]. Moreover, MSCs not only are a very interesting option as a secretory cell [50,128], but also as a coencapsulated auxiliary cell [134–137]. In a recent study, pancreatic islets and MSCs were co-encapsulated in alginate and PEG microcapsules and implanted into the intraperitoneal space of a diabetic mouse model [138]. The results showed that MSCs interact with N-cadherin and increase insulin secretion, in addition to providing structural support to the islets, improving their viability and functionality. It is also important to highlight that the customizable environment generated in these 3D structures can notably improve cell viability and cellular function. In this regard, cell microencapsulation technologies are used far beyond sustained release purposes, for example, for enhanced cell culture [139] or for recapitulating tumor microenvironment or in vitro disease models [140], among others. 2.3. Microcapsule size and shape The optimal size for cell microencapsulation systems remains a matter of debate. On the one hand, it is evident that a larger capsule size could be an obstacle for the diffusion of nutrients and oxygen to the nucleus of the system. This would lead the encapsulated cells to situations of hypoxia and cell death, as well as to slower responses to the stimuli from their environment [141]. In fact, in a recent study, it was suggested that, in the case of pancreatic islets, the maximum distance between these and the extracapsular fluid should not be more than 100 μ m, to allow adequate exchange of nutrients and oxygen [142]. Therefore, many have been the studies aimed at obtaining smaller capsules. Coaxial air flow and flow focusing technologies were presented as attractive alternatives to the usual methods of making microcapsules by means of electrostatic dripping, making it possible to manufacture capsules of 100-200 μ m in diameter, that allow for more complicated routes of administration, such as intracranial administration (in the case of CNS pathologies) or even intravitreal [143,144]. Furthermore, trying to reduce the size of the capsules as much as possible, in recent years nanoencapsulation strategies have also been evaluated — such as conformal coating or layer-by-layer coating —, especially for the immunoprotection of the islets of Langerhans [124]. Conformal coating is a form of non-spherical encapsulation that reduces the diffusion distance and the volume of the implant [145]. 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