Gorka Orive, Rosa María Hernández, Alicia Rodríguez Gascón, and José Luis Pedraz
The aim of cell microencapsulation technology is to treat multiple diseases in the absence of immunosuppression. For this purpose, cells have been immobilized experimentally within carefully designed capsules that allow the long-term function of the graft. Recently, several advances have brought the whole technology much closer to a realistic proposal for clinical application. This chapter reviews the potential impact of this alternative approach for the long-term delivery of therapeutic products and discusses the main limitations, advantages, and challenges in order to assure the same quality standards as those associated with approved drug delivery systems.
Key Words: alginate; cell encapsulation; drug delivery; immobilization; microcapsules. 1. Introduction
Many approaches have been developed over the past decades that have set the stage for tissue and organ replacement as well as for the continuous and controlled release of therapeutic agents to the host. Encapsulation of living cells is one of these technologies in which cells are enclosed within semipermeable encapsulation systems fabricated both by natural or artificial polymers designed to circumvent immune rejection (see Fig. 1) (1). In general, this biotechnology strategy has two important therapeutic potentials: on the one hand the long-term transplantation of biologically active molecules to restore or improve native tissue function and on the other hand the development and optimization of novel drug delivery systems that would allow the long-term secretion of the therapeutic products. Both approaches have significant impact from a therapeutic and economic standpoint. In fact, as the technologically optimized encapsulation system would prevent antibodies and/or other immune cells from entering and destroying the encapsulated cells, chronic administration of immunosuppressant drugs might be reduced, thereby facilitating a better quality of life for patients undergoing this treatment.
From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ
The initial attempts in transplanting cells embedded in polymers dates back to 1933, when Bisceglie decided to introduce tumor cells in a polymer structure and transplant it into a pig's abdominal cavity (2). In the 1960s, the concept of "artificial cell" was invoked to refer the immuno-isolation of cells and enzymes within semipermeable microcapsules (3). Since then, tremendous efforts have been made all around the world to advance the understanding of biology, genetics, polymer science, and pharmaceutical technology. More sophisticated and technologically optimized microcapsules have been fabricated to improve the viability and functionality of the enclosed cells as well as to resist the handling, transplantation, and stress inside the host. In addition, many different cell sources have been immobilized and assayed as potential drug-secreting factories. From all these studies, we now know that clearly not all cells are suitable for microencapsulation. Cells that proliferate following encapsulation could eventually fill the entire capsular space and lead to diminished efficacy of therapeutic diffusion, which could compromise the long-term viability of immobilized cells. In contrast, cells that do not proliferate after encapsulation, such as myoblasts, have the potential to deliver therapeutic products for longer periods of time.
Interestingly, since the pioneering approaches put in practice in the 1980s to immobilize xenograft islet cells in alginate-poly-L-lysine (PLL) microcapsules to aid in glucose control for diabetes in small animal models (4), the use of this bio-technological strategy has extended to novel therapeutic objectives. In fact, its proof of principle as a drug delivery sytem and allogeneic transplantation has been successfully demonstrated in animal models for a wide range of diseases, including cancer (5), hemophilia (6), and renal failure (7).
2. Microencapsulation Process: Polymers and Cells
In cell microencapsulation technology the correct combination of the two main components, cells and polymers, is totally necessary if the success of this strategy is pretended. Many natural and synthetic polymers have been assayed both for capsule matrix and outer membrane. All these materials must be biocompatible, which means that they must not interfere with enclosed cell homeostasis nor induce a host immune response that will reduce the long-term functionality of the transplanted microcapsules (8). Furthermore, the microcapsules fabricated with these polymers must present enough stability to resist mechanical stress and osmotic stress once implanted. Therefore, it is essential to select correctly the materials that will take part in the final structure of the immobilization device.
Alginate (composed by mannuronic and guluronic dimers) is the most frequently employed material reported in the scientific literature, in part because of its excellent biocompatible and biodegradable properties. A large number of experiments have focused on identifying the effects of alginate composition, purity, and concentration on the viability and peptide production of the enclosed cells (9,10). In general, it is assummed that purification of the alginates is a priority for ensuring their biocompatibility, while the suitability of monomer composition of the capsules is still a matter of debate. Recently, novel materials have come under development, including oligochitosans, cellulose sulfate, pectins, and different synthetic polymers. Coating of the fabricated capsule matrix with a suitable semipermeable membrane is another emerging area of research. This outer membrane will allow the bidirectional diffusion of nutrients, oxygen, and waste, while the entrance of immune cells, antibodies, and other components of the immune response will be prevented. Alginate-PLL complex is the most studied membrane chemistry but other polycations such as poly(ethylene glycol) (PEG), polyvinylalcohol (PVA), and poly-L-ornithine (PLO) have also been evaluated (11).
A suitable choice of encapsulated cells is key for the success of any biomedical application. A large number of cells have been studied as potential candidates for long-term secretion of therapeutic products as shown in Table 1.
After the polymers and therapeutic cells have been selected, technologically optimized microcapsules must be fabricated in order to obtain a functional drug delivery system. The choice of the microencapsulation technology as well as the microencapsulation system will rely on the selected biomaterials. Therefore, totally different fabrication procedures will be used if natural or synthetic polymers are chosen. In the case of alginate-PLL-alginate microcapsules, the most widely used immobilization devices for cell entrapment, polymeric devices are produced at room temperature and sterile conditions by an ionic gelation method using an extrusion system. Initially, therapeutic products secreting cells are suspended with the sodium alginate (usually 1.5% [w/v] or 2% [w/v]). This cell-gel suspension is extruded into a gellifying solution such as calcium chloride or barium chlo-
Examples of Cells Selected for Immobilization Purposes
Parathyroid cells Hepatocytes Chondrocytes Leydig cells
Adrenal chromaffin cells Stem cells
Fibroblasts Myoblasts Kidney cells Hybridoma Tumour cells Virus producer cells Bacteria Neurons ride. The contact of the sodium alginate with the gellifying solution provokes the polymerization of the matrix because of the ionic change leading to calcium or barium alginate microbeads (12).
Upon total gelation, cell loaded microbeads are chemically cross-linked with PLL and after a washing step, the beads are coated with another layer of alginate (see Fig. 2A). The latter is necessary to address the problem of immune rejection because the polycations used to form the semipermeable membrane attracts inflammatory cells inducing necrosis of the encapsulated cells (13). Once fabricated, the microcapsules can be used in the treatment of several diseases (see Fig. 2B).
One priority in the field of cell microencapsulation is the validation and optimization of the biomaterials and technology used in the fabrication of the immobilization systems as well as the assays and protocols needed to analyze and reproduce the devices. The large number of techniques and assays that have recently been refined have opened new avenues in the systematization of this therapeutic strategy. The purity and biocompatibility of the materials is a matter of much research. For example, several assays have been validated for purification of alginates, including the use of chemical regents and dialysis (14), the induction of apoptosis in Jurkat cells, and the lymphocyte proliferation assay. The latter is a rapid and sensitive screening for any fibrosis-inducing impurities in alginate samples (15). Production of high levels of cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6 from human monocytes stimulated by alginates of different composition has also been reported (16).
The preliminary study of the biomaterials must be followed by a morphological evaluation and in vivo biocompatibility of the microcapsules. To address these issues of techniques across a wide spectrum have been optimized, such as confo-cal laser scanning microscopy (CLSM), advanced nuclear magnetic resonance (NMR), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), and the X-ray photoelectron spectroscopy analysis. All these approaches allow for a detailed analysis of the microcapsules and their components. The CLSM method is employed to study the distribution of the alginate and PLL in
intact microcapsules (17), while the combination of NMR and AFM is used to give clear-cut evidence of the capsular surface topography and the cross-linkage characteristics between alginate and the gellifying ion (18). Finally, FT-IR and the X-ray photoelectron spectroscopy are used to evaluate the reaction against the microcapsules in the immediate posttransplant period and to determine the pivotal factors for producing biocompatible capsules (19,20). In addition, the detailed histological and immunopathological analysis of the implanted microcapsules (21) as well as their in vivo antigenicity evaluation (10) are exciting tools to study the biocompatibility of the immobilization devices.
In the quest for stronger microcapsules, several tests that measure the mechanical stability of the particles have been designed. The resistance of the microcapsules against compression can be measured using a texture analyzer system (22), while a more complete idea of the mechanical properties of the polymer systems might be obtained by exposing the particles to destabilizing forces including swelling solutions and shear forces (23). A novel osmotic pressure test has been developed to quantify the strength of the microcapsules by exposing them to a graded series of hypotonic solutions and quantifying the percentage of the broken capsules (24). Additionally, high-frequency scanning acoustic microscopy (SAM) has been successfully employed to characterize the mechano-elastic parameters and surface structure and topography of alginate microcapsules (25).
Another focus of interest is the evaluation of the permeability properties (ingress and egress behavior) of the semipermeable microcapsules. The feasibility of a membrane depends on how appropriate is the control over both the size-based exclusion and the rate of diffusion of the molecules, which either have to or must not permeate the membrane to control the survival as well as the metabolic efficacy of the graft. Generally, the process of transport of species across a membrane, characterized as the membrane permeability, is governed by both the ther-modynamic parameter known as equilibrium partition coefficient and the kinetic parameter known as diffusion coefficient. A number of assays have been refined to characterize the permeability properties, including the permeability to hemoglobin and immunoglobulin G (IgG) (26), of the immobilization devices: size exclusion chromatography using dextran standards of different molecular weights (27) and the determination of the mass transfer coefficient value (28). The latter has been succesfully employed in the preliminary screening of microcapsules composed of three different membrane chemistries: PLL, poly-L-ornithine (PLO), and poly-methylene-co-guanidine hydrochloride (PMCG) (29).
In the future, It seems to be advisable that the optimization of encapsulated cells will involve assessing the mechanical stability, diffusion, and permeability properties in vivo. These challenges will be crucial for the standardization of immuno-isolation devices for the pre-Food and Drug Administration stage.
The wide range of therapeutic applications of cell microencapsulation technology can be classified in five groups: (1) treatment of classical Mendelian disorders, (2) cancer treatment, (3) central nervous system (CNS) diseases, (4) artificial organs and, (5) others. Some of the most interesting approaches are described in Table 2.
In light of the current desperate shortage of donor organs, both allogeneic and xenogeneic cells and tissues have been tested as potential living drugs. Xenografts, however, have become a major cause of concern because of the possible transmission of infectious agents, particularly the porcine endogeneous retrovirus (PERV), from the donor to recipient (45). Additionally, the construction of genetically modified cells has opened new avenues for the treatment of classical Mendelian disorders and cancer. In essence, genes can now be used as templates, cells as reactors to secrete the final product and capsules as immunoisolation vehicles for drug delivery in vivo. Another important consideration in using genetically manipulated cells is biosafety. In fact, safety and stability of gene expression will have to be balanced in the use of genetically modified cells.
The results achieved from small and large animal models have provided the scientific basis for several clinical trials, including the encapsulation of allogeneic islets for the treatment of diabetes (46), the encapsulation of cytochrome P450 enzyme expressing cells for the eradication of pancreatic cancer (47), or the immobilization of retinal pigmented epithelial (hRPE) cells on gelatin microcarriers to treat patients with advanced Parkinson's disease.
Because the fields of gene therapy, cell biology, pharmaceutical technology, and chemical engineering are developing rapidly, scientists are continually report
Therapeutic Applications of Cell Microencapsulation Technology
Therapeutic Applications of Cell Microencapsulation Technology
Spinar cord injury
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