Live immobilised cells as new therapeutics

J. DRUG DEL. SCI. TECH., 18 (1) 3-14 2008

Live immobilised cells as new therapeutics S. Prakash*, J. Bhathena Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering and Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, 3775 University Street, Montreal, Québec, Canada H3A 2B4 *Correspondence: [email protected] Clinical trials have increasingly provided adequate data for the use of live cells in medicinal practice especially in diarrhea, inflammatory bowel disease including Crohn’s disease, reduction of serum cholesterol, prevention of allergies, cancer, and numerous other diseases. Oral delivery of live cells has met with limited success; chiefly due to viability losses on passage through the gastrointestinal tract. Conflicting reports exist on the effectiveness of the protection afforded by traditional immobilization of live cells in gel matrices such as calcium alginate and kappa-carrageenan. An alternative approach, microencapsulation, builds on immobilization technologies by combining enhanced mechanical stability of the capsule membrane with improved mass transport, increased cell loading and greater control of parameters. This review abridges recent developments in the therapeutic use of live cells, addresses the promises and challenges of current immobilization technologies and provides insights into the concept of artificial cells for the effective delivery of therapeutic live cells. Key words: Live cell therapy – Clinical trials – Immobilization – Microencapsulation – Oral delivery – Artificial cells.

The concept of live cell therapy was established near the beginning of the last century by the Russian physiologist Metchnikoff [1], who hypothesized that the ingestion of fermented milk products containing lactic acid-producing bacteria had a beneficial impact on health and human longevity. In the last few decades, the field of controlled biotherapeutic delivery has developed rapidly. Many delivery approaches are being optimized for the long-term secretion of therapeutic products, especially in the field of biotechnology, where most of the drugs used are proteins or peptides. More recently, live therapeutic cells have been the focus of much scientific and commercial interest due to a myriad of preventative and curative effects and there is ample evidence that oral ingestion of live cells, especially bacteria, alleviates or prevents various disorders [2-4]. The live cell concept has most promisingly been associated with bacteria for regulating a variety of gastrointestinal functions yet remains controversial partly as a result of viability losses in traditional probiotic products. Beyond strain selection, a number of approaches are currently being explored to increase cell viability [5]. Amongst new technologies, cell immobilization approaches represent a strategy in which bacterial or mammalian cells working as drug-factories are immobilized and immunoprotected within polymeric and biocompatible devices. For example, immobilization of live microbial cells in gel matrices, most notably calcium alginate and kappa-carrageenan, is a mild process that has been used to protect cells from storage and gastrointestinal transit [6, 7]. However, many current gel-based entrapment carriers are acid sensitive and provide a sub-optimal environment for cell proliferation and metabolic activity [8, 9]. Cell immobilization technology has been full of accomplishments and also failures. On the one hand, the functional applicability of mammalian cell immobilization for continuous therapeutic delivery has been demonstrated successfully in several animal models of diseases such as hormone-based deficiencies, hemophilia, central nervous system (CNS) diseases or cancer and in several clinical trials [10, 11]. However, the general feeling is that the field has not lived up to expectations [12]. Although many efforts have been focused on the field, the reality is that, to date, no product is on the market [13]. Some possible explanations for this are the lack of reproducible results in animal models, the requirement of a standardized technology and the urgent need for reproducible and biocompatible materials that provide stable and

immunocompatible devices. In addition, the long-term secretion of the therapeutic products by the enclosed cells also represents another important consideration if the promise of cell immobilization technology is to be realized [14]. An alternative approach for live cell protection utilizes semipermeable microcapsules with strong and thin multi-layer membrane components with specific mass transport properties. The idea of microencapsulation in “artificial cells” was first developed by Chang in the early 1960s for the encasement of biological materials such as transplanted cells, enzymes and adsorbents [15]. Based on this concept, a wide spectrum of cells may be encapsulated, avoiding or at least reducing the administration of immunosuppressive drugs and the implementation of strict and tedious immunosuppressive protocols. Indeed, the potential impact of this technology conjures up visions of optimized cell immobilization devices which could fulfill the exigent requirements applicable to any other pharmaceutical drug, including performance, biosecurity, tolerance, retrievability, scale-up and cost [14]. The review that follows focuses on immobilization technologies for the entrapment and protection of viable cells for biotherapeutic purposes, the applications and current status of live immobilized cell therapy, and the introduction of microencapsulation in artificial cells as a biotherapeutic delivery vehicle for future applications.

I. IMMOBILIZATION TECHNOLOGIES FOR USE IN LIVE CELL THERAPY

Immobilization of whole cells has been defined as the physical confinement or localization of intact cells to a defined region of space without appreciable loss of catalytic activity [16]. While free live cells show significant therapeutic potential, there are limitations precluding their use in clinical or supplemental therapy. To provide functional properties, a minimum level of viable cells is required [5]. However, the stability of free live cell cultures is often significantly diminished prior to administration. Many live cell cultures are fastidious, noncompetitive and very sensitive to environmental parameters such as oxygen and low pH. In addition, free live cells in storage cultures encounter a higher level of inhibitory metabolic by-products such as lactic and acetic acid, hydrogen peroxide and bacteriocins [5, 17]. Tablets incorporating freeze-dried microorganisms prolong shelf life especially with added protectants, however viability losses are 3

J. DRUG DEL. SCI. TECH., 18 (1) 3-14 2008

Live immobilised cells as new therapeutics S. Prakash, J. Bhathena

nevertheless observed [18]. Orally administered live cell therapeutics must also circumvent passage from the mouth to the intestine. Physiological conditions such as acidic pH, mechanical stresses, digestive enzymes (e.g. pepsin and pancreatin), bile acids and oxygen provide an effective barrier against entry [19, 20]. Moreover, novel cells may present a risk of systemic infections, deleterious metabolic activities, adjuvant side-effects, immunomodulation and gene transfer [21]. Beyond strain selection, a number of approaches are currently being explored to increase the viability of live cells in commercial or experimental products [5]. In addition, the use of immobilization and microencapsulation technologies, whereby the live cell is separated from its environment by a protective coating, has been proposed to retain higher cell concentrations during storage and during treatment in a human body. There are currently a myriad of techniques available for immobilization of live cells on a variety of natural and synthetic supports. The selection for a particular application is dependent on the nature of the cells being immobilized as well as the availability, nature and cost of the carrier material. The search for matrices which provide facile, secure immobilization with good interaction with substrates, and which conform in shape, size, density to the use for which they are intended still continues. Support materials have usually been chosen for their properties, low cost, non-toxicity, maximum biocatalyst loading while retaining desirable characteristics, durability, ease of availability, and ease of immobilization [22]. A variety of techniques and supports investigated for immobilization have been reviewed [23-26]. Hydrogels have been investigated for cell immobilization in medicine and biotechnology. They chemically or physically hold the cells to provide stability, structural support, or immunoisolation. Both synthetic and naturally derived hydrogels have been explored for immobilization of a variety of cell types. For successful immobilization, the hydrogel must be conducive to cell viability and function (biocompatible). It must also have proper permeability to allow sufficient diffusion and transport of oxygen and essential nutrients, metabolic waste, and secretory products across the hydrogel network [27]. Chemical and mechanical stability are two more demands of hydrogels as biomaterials. The ability to engineer the bulk and surface properties of hydrogels to satisfy these requirements makes hydrogels attractive for cell immobilization. Biocompatibility is necessary for successful cultivation of cells on or within the polymer. The hydrogel must be nontoxic; it should be relatively inert and not interfere with cell function [28]. The hydrophilicity of hydrogels renders most of them biocompatible For in vivo applications, host tissue must accept the cell-hydrogel transplant with only minor inflammatory response [29].Sufficient supply of oxygen and essential nutrients through the hydrogel network, plus adequate removal of metabolic waste and phenotypic secretions are vital for sustaining the immobilized cells in the hydrogel complexes. The ability of the hydrogel to facilitate diffusion and transport via media or body fluid limit the size and shape of the hydrogel device feasible for immobilization. Proper design of the hydrogel network structure, pore size, and chemical composition, which affect the interaction between the diffusing species and the molecular mesh, is essential otherwise cell morbidity results. Three main methods for immobilizing cells in hydrogels are available: adhesion, matrix entrapment and microencapsulation [30]. The three main methods may be combined to achieve complex goals. Each immobilization method accomplishes different objectives; therefore, the criteria imposed for cell immobilization techniques usually determine the nature of the application. Adhesion, as the name suggests, is based on the attachment of cells to the polymer substrate. This method is effective for surface binding, either on top of gel films or within hydrogel foams. Immobilization by adhesion is generally used to stabilize cells for culture or analytical procedures, provide a structural template directing cell growth and differentiation, or both.

The primary distinction between adhesion and matrix entrapment is how the cells are held by the hydrogel. Unlike adhesion, matrix entrapment relies on physical constraint of the cells within the hydrogel network. Matrix entrapment is sometimes called macroencapsulation when the hydrogel isolates the entrapped cells and provides immunoprotection. Hydrogels are ideal for matrix entrapment because the crosslinks of both synthetic and naturally derived hydrogels provide the essential three-dimensional mesh and porous network to prevent the cells from diffusing into surrounding medium while allowing the transport of substrates, wastes, and other essential molecules via the bulk fluid. In addition to in vitro applications shared with the adhesion technique, matrix entrapment can be used with in vivo studies to protect transplanted cell-hydrogel complexes from mechanical and immunological damage. As a result, the physical retention of live cells has far outnumbered attempts at binding cells to carriers in food and medical based applications. Microencapsulation involves utilizing a spherical, semipermeable, thin and strong membrane to surround a liquid or solid matrix containing biological material, including living cells. Its primary purpose is to protect the encapsulated cells from the host’s immune system, allowing cell survival and function [30]. Of these, the most widely used and studied immobilization technique is the entrapment of cells within a polymeric matrix. Entrapment techniques based on the formation of thermal and ionotropic gels have proven useful for live cell applications. An array of polymers, such as agarose, alginate, chitosan, cellulose, gellan and kappa-carrageenan, has been chosen as the support material for the gel matrix. These are generally non-toxic, readily available and acceptable for use as additives in the food and dairy industries. Recognizing the complexities inherent to the process, a variety of materials have been investigated for use in matrix entrapment of cells. The major polymers currently under investigation for the encapsulation of live cells include alginate, HEMA-MMA and other hydrogels, PAN-PVC, (HFMs), siliceous encapsulates, cellulose membranes, and molecular variations on each of the above (Table I). However conflicting reports exist on the protection offered by matrix entrapment techniques from gastric conditions when used for oral delivery. Shah and Ravula reported that probiotic bacteria immobilized in calcium alginate were able to survive for 3 h at pH 2.5 [31]. In contrast, when B. longum entrapped in calcium alginate beads were exposed to simulated gastric juice and bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and the bead size [32]. Other studies, however, indicate that calcium alginate beads are acid sensitive and unable to prevent cell death at low pH [8]. In contrast, kappa-carrageenan-locust bean gum gel beads have shown to be more resistant to acidic conditions, however, potassium ions required during processing are damaging to certain probiotic strains [33;34]. Sun et al. reported that gellan gum -xanthan gum form spherical beads in the presence of calcium ions at room temperature and effectively protect bifidobacteria in yogurt storage and gastric juice [35]. However, survival at acidic pH was strongly dependent on the strain employed. Among other formulations, gelatin and polymer coated gelatin capsules have been studied for oral delivery of probiotic cells [36;37]. The latter, with 20% w/v of the polymer, has shown promising results in vitro [37]. Attempts have been made at adhering probiotic bacteria to prebiotics [38]. The presence of high-amylase maize resistant starch was shown to increase survival of bifidobacteria at low pH, in bile and during intestinal transit in mice [39]. However, Crittenden et al. reported that adhesion of bifidobacteria to starch is sensitive to acid and protease activity and would be unlikely to survive through the stomach [38]. A host of other formulations have been proposed for matrix entrapment, however, general limitations persist. Furthermore, while live bacterial cell survival in the gel entrapped matrix has been shown to be enhanced in response to environmental stresses (freezing, storage and simulated gastric transit), reports are 4

J. DRUG DEL. SCI. TECH., 18 (1) 3-14 2008

Live immobilised cells as new therapeutics S. Prakash, J. Bhathena

Table I - Polymeric matrices evaluated for cell immobilization. Microcapsule membrane polymer type

Microcapsule membrane features Strengths

Limitations

APA [alginate-poly(l-lysine)-alginate] + variants (/PEG, /Ba+2, /Ca+2)

Acceptable food additive Cell and tissue compatible Mild reaction conditions Low cost Ease of control over parameters Starch useful as prebiotic probiotic Short/medium-term Mechanical stability Flexible permselectivity Established synthesis protocols Low immunogenicity when PEGylated

Susceptible to acid Reduced mechanical stability during lactic fermentation Insufficient immunoprotection Susceptible to long-term Ca+2 loss, consequent mechanical instability Structurally rigid Must be PEGylated to prevent fibrotic overgrowth

A-PMCG-A [alginatepoly(methylene-co-guanidine)alginate]

Better mechanical stability than Ca+2/A, Ba+2/A, BAPA, PMCG Cheaper than PLL Capsule size/permeability independently adjustable

Immunogenicity Long-term mechanical stability yet to be determined

KC/LBG [kappa-carrageenan/locust bean gum gel]

Strong, rigid gel Acid resistant Thermoreversible Good results in cryopreservation studies

Less biocompatible than alginate Potassium ions damaging to cells and potentially host electrolyte composition Insufficient immunoprotection

CAP [cellulose acetate phthalate]

Resistant to gastric acid conditions Established enteric coating material for controlled release Readily dissolves in mildly acidic to neutral environment of small intestine

Harsh reaction conditions (HCl) limits probiotic viability during membrane formation Membrane is non-porous Limited access to substrate during storage

CS/A/PMCG [cellulose sulphate/ sodium alginate/poly(methylene-coguanidine)]

Short-term applications negate long-term mechanical stability and biocompatibility concerns

Encapsulated cells sensitive to alginate purity

Gellan gum/xanthan gum

Acid resistant Stabilized by calcium ions Easy to mix bacterial suspension with gum prior to gelation Economical processing Retention of cellular viability in pasteurized yogurt No shrinkage in lactic and acetic acid solutions

Gellan gum requires high setting temperature Acid survival dependent on strain Some reports indicate poor viability in storage Insufficient Immunoprotection

Agarose

Prolonged stability in storage Cell and tissue compatible Mild reaction conditions Narrow size distribution of beads Low cost/readily available

Limited mechanical stability Insufficient immunoprotection due to cellular protrusion and absence of permselective layer

Artificial sesame oil

Acceptable food additive Lactic acid viability retained in storage Acid and bile resistant

Sensitive to mechanical stress Wide size distribution Processing requires high temperatures

Starch

Natural adhesion (prebiotic) Exhibits good spray drying properties allowing for easy scale-up and economical processing Small sized microparticles with excellent cell coverage

Limited protection against acid stress, protease activity and pancreatin Poor survival in foods Nature of adhesion dependent on strain (presence of cell surface protein)

HEMA-MMA (hydroxymethylacrylate-methyl methacrylate)

Insolubility in aqueous solutions confers greater mechanical stability

Non-adherent membrane properties require coencapsulation with matrix to facilitate anchoragedependent cell adhesion/growth

Multi-layered HEMA-MMA-MAA

Exceptional design flexibility Independent adjustment of mechanical stability, permselectivity Promising compatibility with blood-contact applications

Single-layered capsules possess insufficient mechanical stability Immunogenicity yet to be determined Synthesis protocol more complex than other designs

PAN-PVC [poly(acrylonitrilevinylchloride)]

Established mechanical stability, permselectivity Good biocompatibility

Molecular-weight cut-offs currently in question Long-term immunogenicity not yet established

AN-69 (acrylonitrile/sodium methallylsulfonate)

Good mechanical stability, permselectivity Amitogenic Large-scale encapsulation (~50 million cells/minute) now possible

Immunogenicity not well established

5

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Live immobilised cells as new therapeutics S. Prakash, J. Bhathena

Table I - Polymeric matrices evaluated for cell immobilization (continued). Microcapsule membrane polymer type

Microcapsule membrane features Strengths

Limitations

PDMAAm [poly (N,N-dimethyl acrylamide)]

Improved mechanical stability when cross-linked with telechelic stars

Oxygen permeability inferior to copolymers with PDMS

Siliceous encapsulates

Simple synthesis mechanism confers high design flexibility

Questionable toxicity, immunogenicity

while simultaneously allowing for the metabolism of selected solutes capable of passing into and out of the microcapsule. Studies show that artificial cell microcapsules can be used for oral administration of live cells that can be useful for therapeutic functions (Figure 1). The cells are retained inside, and excreted with the intact microcapsules, addressing many of the major safety concerns associated with the use of live bacterial cells for various clinical applications. The membranes of the microcapsules are permeable to smaller molecules, and thus the cells inside the microcapsules metabolize small molecules found within the gut during passage through the intestine. The artificial cell concept has immediate application for microencapsulation-based oral therapy in renal failure and liver failure, physiologically responsive gene therapy and somatic gene therapy [30;45]. The principle of mammalian cell encapsulation with hydrogels is that the permeability of the membrane is engineered to allow the passage of oxygen, important nutrients, and cellular products, but it stops the ingress of immunoglobulins or immune cells responsible for transplant rejection [46]. The primary application of encapsulation is immunoprotection of healthy xenogeneic or allogeneic cells for transplantation into a recipient in need of functional replacement of a metabolic tissue. The protected cells then function and secrete an effective supply of the needed hormone, protein, or other bioactive secretory product for the host. Perhaps the most well-investigated clinical application of microencapsulation and subsequent transplantation of cells is in the treatment of diabetes. The semipermeable hydrogel membrane allows the passage of oxygen, nutrients, and cell products including insulin as needed, but it protects the transplanted cells from the patient’s immune system. In addition to diabetes, microencapsulation with hydrogels has

often conflicting and dependent on the strain employed. It has therefore become difficult to isolate a particular matrix entrapment procedure as a candidate for rigorous optimization studies and eventual scaleup. Support materials for matrix entrapment are also insufficiently immunoprotective for novel microorganisms as well as traditional live cells that have shown in certain cases to instigate an inflammatory response [21]. In addition, diffusional properties and inadequate mechanical strength limit the proliferation of the entrapped cells. Encased cells have been reported to leak, escape from the gel matrix and as a result grow in the surrounding solution [9, 40]. Growth in gel beads is restricted, especially in larger beads where proliferation occurs only in the periphery due to substrate limitation. In many cases, the maximum cell loading of entrapped gel beads is limited to 25% by volume due to weak mechanical strength [9]. Furthermore, diffusional limitations of both substrates and metabolic by-products can lead to the development of steep gradients regarding pH and inhibitory products that can hinder the viability and metabolic activity of the entrapped live cell. Therefore, despite reported advantages, the associated drawbacks of immobilizing live cells by entrapment in gel matrices have limited the influence of this technology in commercial and experimental products.

II. ARTIFICIAL CELLS FOR LIVE CELL THERAPEUTIC DELIVERY

Microencapsulation of live cells is currently being studied to confront limitations with traditional immobilization technologies. Orive et al. demonstrated that microencapsulated hybridoma cells presented a better growth pattern and improved their viability and antibody production [41]. To date, although little is known about the physiological alteration of microencapsulation cells, the data about cellular physiological alteration of other immobilization methods can provide us clues to study the microencapsulated cells. Doran and Bailey demonstrated that yeast cells immobilized on glutaraldehyde cross-linked, gelatin-coated glass beads showed a pattern of DNA, RNA, protein, and polysaccharide content different from that of freely suspended cells [42]. In addition, it has been reported that cell immobilization can lead to modification of cell wall and cell membrane compositions [43]. The physiological alteration may also have a profound impact on cell stress resistance. As a general technique, microencapsulation offers certain advantages over more conventional methods of cell immobilization. These include: i) improved mass transfer of the substrate and product between the external environment and the aqueous cell-containing phase due to the smaller size of the microcapsules and higher surface-to-volume ratio; ii) chemical and mechanical stability of microcapsules in a wide range of solvents and under various experimental conditions; iii) the possibility of incorporating water-soluble nutrients and/or cosubstrates to enhance the viability of cells and the rate of biotransformation [44]. The idea of microencapsulation in “artificial cells” was first developed by T.M.S. Chang in the early 1960s for the encasement of biological materials such as transplanted cells, enzymes and adsorbents [15]. The concept behind microencapsulation involves utilizing a spherical, semipermeable, thin and strong membrane to surround a liquid or solid core containing biological material. The polymer membrane can protect encapsulated materials from harsh external environments

Figure 1 - Principle for oral administration of microencapsulated live bacterial cells for therapy. Live bacterial cells can be encased in a polymeric matrix which allows diffusion of nutrients, wastes and protein products while acting as a barrier to the conduits of the immune system once the cells are ingested by a patient. Also, small molecules (including some peptides) produced by the enclosed bacterial cells can be designed to diffuse out into the body for therapeutic purposes.

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Live immobilised cells as new therapeutics S. Prakash, J. Bhathena

potential as a treatment for several other diseases and disorders such as Parkinson’s disease or liver failure [47, 48]. Microencapsulation therapy with hydrogels has been applied to humans; however, there are extensive documented successes with animal models. In 1980, Lim developed the alginate-poly-L-lysine-alginate (APA) membrane system for islet encapsulation[49], a protocol that is widely regarded for providing the impetus for the cell encapsulation field and has been applied to effectively encapsulate microbial cells.[50-52] APA microcapsules have been used to encase probiotic bacteria with several advantages noted over traditional immobilization technologies. The aqueous core provides minimal mass transfer resistance which, coupled with the large surface area-to-volume ratio of the semi-permeable membrane, allows permeant substrates and products to diffuse rapidly [9]. In addition, cells have a larger accessible volume to grow and proliferate with no compromise of membrane stability. Poly-Llysine provides a permselective layer that can be quantified for mass transport, which in turn can be controlled by adjusting reaction time and poly-L-lysine concentration [53]. For oral delivery, alginate is perhaps the best suited polymer for capsular design, in part because of its excellent biocompatibility and status as an FDA approved food additive [54;55]. Reports on the mechanical stability of APA, however, are conflicting. Reports have found APA microcapsules unstable in simulated GI conditions [56] and susceptible to enzymatic hydrolysis [57]. To overcome this, researchers have used a higher concentration of alginate cross-linked with barium instead of calcium [58]. This modification prolonged the stability of the microcapsule for systemic delivery applications in canine models, but not for oral delivery. Chitosan and poly-L-ornithine have been employed as polycationic replacements for poly-L-lysine with noted improvements in biocompatibility [59, 60]. Alginate-chitosan (AC) microcapsules were found to have superior strength, flexibility and biocompatibility when coated with glutaraldehyde. In addition, cross-linking of AC microcapsules with naturally-derived genipin improved performance in membrane strength tests. A novel design utilizing alginate, cellulose sulfate and polymethyline co-guanidine introduced both weak (alginate) and strong (cellulose sulfate) interactions with the polycation (polymethyline co-guanidine) and was found to have greatly enhanced mechanical strength and capsule durability over traditional APA capsules [61]. Many other intricate membrane systems utilizing polyelectrolyte complexation have been proposed for encapsulating live cells (Figure 2). For instance, alginate-polylysine-pectin-polylysine-alginate (APPPA) membranes have been prepared and tested for stability in simulated GI fluid [62]. Results indicate increased resistance to acidic and basic conditions, as well as in the presence of ion chelators, while allowing for more precise control over membrane permeability than traditional APA capsules. Synthetic polymer systems such as HEMAMMA processed by interfacial polymerization have been shown to have easily adjustable parameters and improved mass transfer, stability and durability [63]. However, the reaction conditions required for its formation are damaging to cells.

A

B

C

D

Figure 2 - Photomicrographs of: (A) HepG2 mammalian cells encapsulated in alginate-chitosan- polyethylene glycol (PEG) - poly-L-lysine-alginate [ACPPA] microcapsules in MEM media 5 days after coating, empty ACPPA microcapsules in physiological solution. Capsules Size: 450 ± 30 µm. (Magnification: 6.5 X) [82]; (B) alginate-poly-L-lysine-alginate [APA] membrane system for encapsulation and oral delivery of empty microcapsules and microcapsules containing E. coli DH5 cells [52, 71]; (C) encapsulated HepG2 cells in alginate -poly-L-lysine-PEG-alginate [APPA] microcapsules 1 day after encapsulating; magnification:10X, size: 450 µm and alginate-chitosan-PEG [ACP] microcapsules 1 week after encapsulating; magnification:10X, size: 450 µm [109]; (D) CLSM images of genipin cross inked microcapsules under normal and fluorescent light. Bars represent 200 µm [110].

gous proteins. In this way, it is possible to control gene expression in engineered lactic acid bacteria by an inductor, a repressor or by environmental factors such as temperature, pH or ion concentrations [64]. Additionally, one may expand the range of possible active components beyond protein therapeutics by integrating foreign enzymes. To accomplish this end, Prakash and Chang developed nonpathogenic Escherichia coli strain DH5 expressing Klebsiella aerogenes urease for use as an oral artificial kidney [52]. The oral administration of genetically engineered E. coli encased in APA artificial cells decomposes urea that diffuses into the capsule from the GI tract into ammonia, which the bacteria can utilize for its biosynthesis [52]. As a result, pathological plasma urea levels in patients are alleviated. Jones et al. examined the potential of artificial cell, microencapsulated, genetically engineered Lactobacillus plantarum 80 (pCBH1)

III. APPLICATIONS OF ARTIFICIAL CELLS CONTAINING LIVE CELLS FOR THERAPY

Live cell enclosed material is retained inside the microcapsule and separated from the external environment, making microencapsulation particularly useful for biomedical and clinical applications (Figure 3). There is considerable evidence supporting the importance of oral feeding on live normal or genetically engineered bacterial cells for diverse therapeutic applications highlighting the underlying potential of this approach to therapy. Table II summarizes the potential of the use of bacterial cells and various gel entrapment matrices to therapy. Early developments of microencapsulated live bacterial cell therapy have utilized genetically engineered microorganisms with novel or enhanced probiotic properties. For instance, researchers have developed inducible expression promoters for high-level production of heterolo7

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Live immobilised cells as new therapeutics S. Prakash, J. Bhathena

Table II - Immobilised (gel entrapped) bacteria for use in live cell therapy. Material

Probiotic

Strengths

Limitations

Ref.

Calcium-alginate

L. acidophilus L. casei L. lactis B. bifidum B. longum B. infantis

Acceptable food additive Cell and tissue compatible Mild reaction conditions Low cost Ease of control over parameters

Susceptible to acid Reduced mechanical stability during lactic fermentation Insufficient immunoprotection

[6, 8, 111, 112]

Kappa-carrageenan/ locust bean gum gel

L. casei L. lactis B. bifidum B. infantis B. longum

Strong, rigid gel Acid resistant Thermoreversible Good results in cryo­preservation studies

Less biocompatible than alginate Potassium ions damaging to cells and potentially host electrolyte composition Insufficient immunoprotection

[7, 33, 34]

Cellulose acetate phthalate

L. acidophilus B. lactis B. pseudolongum

Resistant to gastric acid conditions Established enteric coating material for controlled release Readily dissolves in mildly acidic to neutral environment of small intestine

Harsh reaction conditions (HCl) limits probiotic viability during membrane formation Membrane is non-porous Limited access to substrate during storage

[113, 114]

Gellan gum/xanthan gum

B. adolescentis B. bifidum B. breve B. infantis B. longum

Acid resistant Stabilized by calcium ions Easy to mix bacterial suspension with gum prior to gelation Economical processing Retention of cellular viability in pasteurized yogurt No shrinkage in lactic and acetic acid solutions

Gellan gum requires high setting temperature Acid survival dependent on strain Some reports indicate poor viability in storage Insufficient immunoprotection

[35, 115]

Agarose

Bacillus CalmetteGuérin (BCG)

Prolonged stability in storage Cell and tissue compatible Mild reaction conditions Narrow size distribution of beads Low cost/readily available

Limited mechanical stability Insufficient immunoprotection due to cellular protrusion and absence of permselective layer

[116, 117]

Artificial sesame oil

L. delbrueckii ssp. bulgaricus

Acceptable food additive Lactic acid viability retained in storage Acid and bile resistant

Sensitive to mechanical stress Wide size distribution Processing requires high temperatures

Starch

B. adolescentis B. bifidum B. breve B. lactis B. longum B. pseudolongum

Natural adhesion (prebiotic) Exhibits good spray drying properties allowing for easy scale-up and economical processing Small sized microparticles with excellent cell coverage

Limited protection against acid stress, protease activity and pancreatin Poor survival in foods Nature of adhesion dependent on strain (presence of cell surface protein)

[118]

[38, 39, 119]

therapy [69]. The possibility of safely implanting xenogeneic cells has brought about new treatment methods for an ever widening range of diseases, including parathyroidism, chronic pain, spinal-cord damage, CNS disorders and also some aggressive cancers [70]. Beginning in the late 1970s, pancreatic islet cells and hepatocytes were effectively encapsulated into millimeter size artificial cells and implanted for treatment of diabetes mellitus and liver failure, respectively [71]. Bio-artificial pancreas (BAP), composed of insulin producing cells that are protected from the host’s immune reaction by biocompatible, selective permeable and chemically stable artificial membranes, provide for continuous insulin delivery under strict regulation by the extra-cellular glucose levels and would ideally apply to the potential cure of type1 diabetes mellitus by transplantation [72]. In recent years, microencapsulation has been shown to allow for temporary survival of transplanted islets in diabetic humans [73]. Encapsulation regimes for liver cell encapsulates have utilized relatively inert substances such as alginate, agarose and cellulose on the one hand and extremely bioactive substances such as collagen gels on the other. A recent report has attempted to modify the collagen

cells for bile acids deconjugation to lower cholesterol and showed that microencapsulated LP80 (pCBH1) may prove to be an excellent choice of cholesterol-lowering agents for use in combination therapy with statins and other lipid-lowering therapies [65]. Recent interest in characterizing oxalate-degrading enzymes in Bifidobacterium has given rise to microencapsulating such bacteria for oral probiotic therapy. Oxalate accumulation in humans can prove toxic and result in a number of pathological conditions including hyperoxaluria, calcium oxalate nephrolithiasis and cardiomyopathy [66, 67]. The degradation of oxalate is dependent on the key enzyme, oxalyl-CoA decarboxylase which has been characterized and heterologously expressed in Bifidobacterium lactis by Federici et al. [68]. The potential exists to microencapsulate B. lactis with heterologous expression of oxalyl-CoA decarboxylase for colon targeting oral delivery. Artificial cells containing live cells may thus well prove to be very good candidates for oral live bacterial cell therapy (Table III). The strategy of cell microencapsulation for cell therapy has been extensively developed by many groups especially using artificial cells containing endocrine tissues, hepatocytes and other cells for cell 8

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Live immobilised cells as new therapeutics S. Prakash, J. Bhathena

seen in animals receiving daily injections and the dose required was 8-10 fold lower [90]. Tumor growth has also been reduced by the use of an alginate microcapsule delivery system containing IL-2 secreting cells [91]. Significant tumor regression has shown to be also induced by the microencapsulation of recombinant tumor cells secreting fusion protein [92]. Thus, biologically active molecules administered locally to animals may selectively inhibit growth of malignant tumors, including gliomas. Further, embryonic kidney cells secreting CYP2B1 were encapsulated in cellulose sulfate microcapsules and implanted in mice with pancreatic cancer in direct contact with the tumor. Lowdose ifosfamide given to tumor bearing mice was converted to the toxic compound phosphoramide mustard by CYP2B1, and in so doing killed the adjacent cancer cells [93]. This strategy has been the basis of a human trial with promising results in which researchers have used encapsulated genetically modified allogeneic cells expressing pro-drug-activating enzymes such as cytochrome P450 as a possible treatment for inoperable pancreatic carcinoma [94-96]. The delivery of various neurotrophic factors to the central nervous system (CNS) is a novel treatment for neurological disorders. Encapsulated fibroblasts secreting adenosine, an inhibitor of neuronal activity in the brain, offered a nearly complete protection against seizures in a rat model of partial epilepsy [97]. Encapsulated cells secreting glial cell line-derived neurotrophic factor (GDNF) have shown to be efficacious in protecting the nigral dopaminergic neurons against lesion-induced cell death in rodent as well as in primate models of Parkinson’s disease [98]. An encapsulated xenogeneic system was found to allow the release of therapeutically effective amounts of neurotrophic factors, alone or in combination, to many sites within the CNS. The sustained release of human nerve growth factor (hNGF) observed from a retrieved capsule for more than six months, is relevant for chronic neurodegenerative diseases such as Alzheimer’s disease [99]. The potential of cell encapsulation in gene therapy is reflected in the initiation of a phase I/II clinical trial of amyotrophic lateral sclerosis (or Lou Gehrig’s disease) and other similar applications of implantable devices containing recombinant cells secreting therapeutic products for the CNS such as in an animal model of Huntington’s disease [98, 100]. In another study, researchers gave cynomolgus monkeys intrastriatal implants of polymer-encapsulated baby hamster kidney fibroblasts that had been genetically modified to secrete human ciliary neurotrophic factor (CNTF). The results showed that human CNTF has a trophic influence on degenerating striatal neurons as well as on critical non-striatal regions such as the cerebral cortex, supporting the idea that human CNTF may help to prevent the degeneration of vulnerable striatal populations and cortical-striatal basal ganglia circuits in Huntington’s disease [101]. Implantable polymeric devices containing bovine chromaffin cells that naturally secrete a mixture of analgesic compounds have been used in various pre-clinical and human clinical studies of chronic pain that failed to respond to standard treatment [102, 103]. Erythropoietin (epo) is responsible for the production of red blood cells. As such, it is widely used to treat anemia. Transgenic mice with severe chronic anemia received subcutaneous implantation of encapsulated C2C12 myoblasts secreting murine epo. The hematocrit in the treated mice rose, indicating the delivery of functional epo [104]. Patients suffering from b-thalassemia also show signs of anemia. The implantation of encapsulated cells secreting epo resulted in the clinical benefit of a murine model of b-thalassemia [105]. The implantation of encapsulated cells is a particularly suitable strategy to treat metabolic deficiencies [106]. Promising results have been obtained in pre-clinical studies of growth hormone deficiency in dwarf mice [107], or b-glucuronidase deficiency in a mucopolysaccharidosis type VII mouse model [108], among others. As described in an excellent review by Hortelano, encapsulated cell therapy has also proven to be a very popular gene therapy approach for the treatment of hemophilia A and B, and has been used with varying degrees of success in pre-clinical and clinical studies [106].

Applications of microencapsulated live cells Pharmaceutical: Live biodrug delivery systems (rationally designed macromolecular drugs, polymer-drug and polymer-protein conjugates, polymeric micelles containing covalently bound drug, polyplexes for DNA delivery) Biotechnological: Single enzymes, multienzymes, proteins, protein-enzyme conjugates, Conversion of wastes (e.g. urea and ammonium) into useful products (e.g. essential amino acids), Monoclonal antibodies production from hybridomas Medical: Routine clinical treatments (acute poisoning, aluminium and iron overload, end-stage kidney failure supplement to dialysis, limited types of acute liver failure, bioadsorbents for detoxification). Clinical trials (red blood cell substitutes for transfusion, enzyme defects in inborn errors of metabolism, bioencapsulated pancreatic islets for feedback controlled secretion of insulin for diabetic mellitus, artificial kidney, encapsulated kidney cells to secrete erythropoeitin to treat anaemia, bioencapsulated hepatocytes for bioartificial liver to support liver function in liver failure and as a model for cell and gene therapy). Experimental therapy using bioencapsulated cells or microorganisms (conversion of cholesterol into carbon dioxide, bilirubin removal, encapsulation of erythropoietin secreting renal cells to treat anaemia, parathyroid cells for secreting parathyroid hormone to treat hypoparathyroidism, mouse myoblasts to secrete and deliver human factor IX for haemophilia B, growth hormone for dwarfism, monoclonal antibodies for IgG1 plasmacytosis and erythropoietin for b-thalassaemia, hamster kidney fibroblasts that secrete human nerve growth factor (hNGF) for Parkinsonism, baby hamster kidney cells engineered to synthesize and release human or mouse ciliary neurotrophic factor (CNTF) for treating the neurodegenerative disease amyotrophic lateral sclerosis and to treat Huntington’s disease, encapsulated transfected cells that secrete endostatin for malignant brain tumour suppression, stem cell therapy, removal of unwanted metabolites from the body (phenylalanine in phenylketonuria (PKU), hypoxanthine in Lesch–Nyhan disease, tyrosine in melanoma and uraemic waste metabolites in kidney failure, catalase into animals with a congenital deficiency in catalase). Figure 3 - Probable uses for live cell microencapsulation technology.

gel system by combining the biological advantages of collagen with the more bio-inert capabilities of sol-gel SiO2 coated as a superficial microlayer over the collagen gel [74, 75]. Other work has focused on the use of alginate as an encapsulation medium for human hepatocyte cell lines for use in bioartificial livers [76-82]. Two groups have used porcine hepatocytes in calcium alginate beads in a fluidized bed reactor to treat experimental liver failure induced by devascularisation in the pig [83, 84]. The use of implantation of encapsulated hepatocytes for liver support including acute liver failure and hyperbilirubinemia in Gunn rats has been studied [69]. A new concept which has not yet reached clinical trials is that alginate encapsulated cells secreting molecules with anti-tumor properties can be implanted into the tumor resection cavity of malignant glioblastomas resulting in tumor necrosis and prolonged survival in mice and rats [85-89]. A substantial body of data indicate that the encapsulated cells may be kept alive secreting recombinant proteins for months in rat brain parenchyma [86, 87]. Continuous release of such substances ensures a stable local effect. Anti-tumor effects of endostatin administered continuously by local delivery exceeded those 9

J. DRUG DEL. SCI. TECH., 18 (1) 3-14 2008

Live immobilised cells as new therapeutics S. Prakash, J. Bhathena

Table III - Therapies based on the delivery of microencapsulated live bacterial cells. Targeted therapy

Encapsulated bacterial cells

Proposed mode of action

Ref.

Tuberculosis

M. bovis BCG

Tuberculin-specific cell-mediated immune responses, system-level lymphocyte sensitization evidenced by in vitro tuberculin-specific IFN-g production in splenocyte cultures, establishment of protective immunity against TB

[120-122]

Kidney dialysis

E. coli DH5

Inserted Klebsiella aerogenes urease gene, in genetically engineered E. coli DH5 cells, causes over-expression of the urease enzyme, subsequent lowering of elevated blood levels of urea and ammonia, thus E. coli DH5 cells normalize elevated levels of several metabolites during renal failure

[52, 123]

Kidney stones

O. formigenes

Oxalate-degrading enzymes produced by Oxalobacter formigenes break down unwanted oxalate, a major risk factor for renal stone formation and growth in patients with idiopathic calcium-oxalate urolithiasis. Can be used to prevent subsequent evolution of kidney stones

[124, 125]

Immunomodulation

B. bifidum Bb-11

Significantly induces total IgA and IgM synthesis by both mesenteric lymph nodes (MLN) and Peyer’s patch (PP) cells, regulates the synthesis of IgA by mucosal lymphoid cells, significantly increases the number of Ig (IgM, IgG, and IgA) secreting cells

[126]

Elevated blood levels of cholesterol

Genetically engineered Lactobacillus plantarum LP80 (pCBH1) and L. reuteri

Overproduced BSH enzyme deconjugates intraluminal bile acids making them less likely to be reabsorbed into the ECH, causing de novo synthesis of bile acids in the liver from blood serum cholesterol. L. reuteri can be microencapsulated in combination with LP80 (pCBH1), as it has been shown to precipitate and bind bile acids, making them less bioavailable which may be important to their carcinogenic potential

[127]

Preventative therapy for colon cancer

Lactobacillus plantarum LP80 (pCBH1) and L. reuteri

The BSH enzyme is overproduced by LP80 (pCBH1) cells and hydrolyzes available conjugated bile acids in the intestinal lumen. L reuteri, shown to precipitate and bind bile acids, then binds the deconjugated bile acids making them incapable of leaving the microcapsule and thus less bioavailable for exfoliation of the GI and any potential carcinogenic damage

[127]

Disease of the bowel (elevated intraluminal levels of bile acids)

Lactobacillus plantarum LP80 (pCBH1) and L. reuteri

LP80 (pCBH1) and L. reuteri deconjugate, precipitate, and then bind conjugated bile acids within microcapsules, mitigating the problems associated with excessive electrolyte and water secretion associated clinically with diarrhea and dehydration

[127]

Probiotics

Lactobacillus, Bifidobacterium

Live micro-organisms used as dietary supplements with the aim of benefiting the health of the consumer by positively influencing their intestinal microbial balance. Microencapsulated probiotic bacteria should help alleviate diarrhea, lower cholesterol, modulate immunity, and prevent colon cancer

[114, 119, 128-130]

Urogenital infections (urinary tract infection (UTI), bacterial vaginosis (BV), or yeast vaginitis (YV)

L. rhamnosus GR-1 L. fermentum RC-14 L. acidophilus

Restoration and maintenance of a healthy urogenital tract possibly by competition with pathogenic microbes, protect the host from recurrence of UTI, Creation of an environment better able to support indigenous lactobacilli growth

[131]

Elevated blood levels of nitrogen and hydrogen gas

Hydrogen metabolizing (Msmithii) and N2 fixing (Enterobacteriaceae)

Live micro-organisms delivered orally to a diver’s large intestine during hyperbaric exposure to a gas mixture containing H2 or N2 metabolizes the H2 or N2 gas to other compounds such as methane or water for hydrogen and ammonia for nitrogen to prevent decompression sickness or reduce decompression time

[132]

Several additional live cell therapies have been microencapsulated in artificial cells and quantified both in-vitro and in-vivo. Others remain prime candidates. On account of advances in heterologous protein expression, metabolic induction, genetic engineering and newly isolated probiotic strains, it is expected that a number of novel applications will soon emerge.

ched something of an impasse. For example, even though progress continues to be made, drug development is slowing and antimicrobial resistance is relentlessly increasing. Thus, new therapies are being continuously explored. Live cell therapy is increasingly studied and used in humans. Although there is sufficient evidence, principally from European studies showing that live cell formulations may be beneficial in some cases to warrant careful exploration of the therapeutic value of proposed various live cell cultures, the hallmark of live cell research to date has been the variability of experimental results and contradictory findings. As a result, at present it must be concluded that the value of

* Conventional drugs have continuously offered exciting breakthroughs in disease treatments. However, in certain cases they have rea10

J. DRUG DEL. SCI. TECH., 18 (1) 3-14 2008

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beneficial live cells has not been adequately established by rigorous scientific research to support their use in all but a few cases. Globally there is an urgent need for inexpensive alternative formulations for disease prevention and therapy. The future of live cells as therapeutic alternatives, or adjuncts, to existing and emerging lifestyle and clinical practice requires the adoption of this principle of evidence-based legitimacy. The early adopters of this principle are now emerging. And the emergences in innovative methods of using live cells (such as artificial cell microencapsulation) for the treatment of diseases are set to generate a totally new drug sector. With the aid of genetic engineering, cell encapsulation technology is poised to offer innovative solutions for the treatment of both genetic and acquired diseases. The versatility of this strategy is such that it can be used in a myriad of settings. The success of this strategy depends largely on a judicious choice of a suitable cell type for encapsulation, and the appropriate selection of immunoisolation polymer for the desired application. It is worth reinforcing the notion that each medical application presents unique characteristics and challenges to be solved. Therefore, it is not always possible to extrapolate a particular set of results to all medical conditions. There seems little doubt that live immobilized cell therapeutics will become part of a global clinical arsenal for prevention and treatment of disease or maintenance of health.

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Acknowledgements This work was supported by research grants from the Canadian Institute of Health Research (CIHR). Jasmine Bhathena acknowledges support from the Canadian Liver Foundation for a Graduate Studentship.

Manuscript Received 30 May 2007, accepted for publication 1 August 2007.

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