Protein release from alginate matrices

Advanced Drug Delivery Reviews 31 (1998) 267–285

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Protein release from alginate matrices Wayne R. Gombotz*, Siow Fong Wee Department of Analytical Chemistry and Formulation, Immunex Corporation, 51 University Street, Seattle, WA 98101, USA

Abstract There are a variety of both natural and synthetic polymeric systems that have been investigated for the controlled release of proteins. Many of the procedures employed to incorporate proteins into a polymeric matrix can be harsh and often cause denaturation of the active agent. Alginate, a naturally occurring biopolymer extracted from brown algae (kelp), has several unique properties that have enabled it to be used as a matrix for the entrapment and / or delivery of a variety of biological agents. Alginate polymers are a family of linear unbranched polysaccharides which contain varying amounts of 1,49-linked b-D-mannuronic acid and a-L-guluronic acid residues. The residues may vary widely in composition and sequence and are arranged in a pattern of blocks along the chain. Alginate can be ionically crosslinked by the addition of divalent cations in aqueous solution. The relatively mild gelation process has enabled not only proteins, but cells and DNA to be incorporated into alginate matrices with retention of full biological activity. Furthermore, by selection of the type of alginate and coating agent, the pore size, degradation rate, and ultimately release kinetics can be controlled. Gels of different morphologies can be prepared including large block matrices, large beads ( . 1 mm in diameter) and microbeads ( , 0.2 mm in diameter). In situ gelling systems have also been made by the application of alginate to the cornea, or on the surfaces of wounds. Alginate is a bioadhesive polymer which can be advantageous for the site specific delivery to mucosal tissues. All of these properties, in addition to the nonimmunogenicity of alginate, have led to an increased use of this polymer as a protein delivery system. This review will discuss the chemistry of alginate, its gelation mechanisms, and the physical properties of alginate gels. Emphasis will be placed on applications in which biomolecules have been incorporated into and released from alginate systems.  1998 Elsevier Science B.V. Keywords: Bioadhesion; Biodegradable polymer; Cell encapsulation; Diffusion controlled release; DNA encapsulation; Hydrogel; Mucosal delivery; Natural polymer; Vaccine delivery

Contents 1. Introduction ............................................................................................................................................................................ 2. Alginate chemistry .................................................................................................................................................................. 2.1. Sources of alginate ........................................................................................................................................................... 2.2. Extraction and preparation ................................................................................................................................................ 2.3. Chemical structure ........................................................................................................................................................... 3. Gel formation and dissolution chemistry ................................................................................................................................... 3.1. General formation mechanism........................................................................................................................................... 3.2. Large bead preparation ..................................................................................................................................................... 3.3. Microbead preparation...................................................................................................................................................... 3.4. Matrix / block gels, fibers and in situ gelling systems........................................................................................................... *Corresponding author. Tel: 1 1 206 3894085; Fax: 1 1 206 6247496; email: [email protected]

0169-409X / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII S0169-409X( 97 )00124-5

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4. Physical properties .................................................................................................................................................................. 4.1. Chemical reactivity .......................................................................................................................................................... 4.2. Porosity and macromolecular diffusion .............................................................................................................................. 4.3. Chemical stability / degradation ......................................................................................................................................... 5. Biological properties ............................................................................................................................................................... 5.1. Immunogenicity ............................................................................................................................................................... 5.2. Bioadhesion..................................................................................................................................................................... 6. Protein encapsulation............................................................................................................................................................... 6.1. Targeting to mucosal tissues ............................................................................................................................................. 6.1.1. TGF-b 1 ...................................................................................................................................... 6.1.2. Vaccines ................................................................................................................................................................ 6.2. Slow release applications .................................................................................................................................................. 6.2.1. Basic fibroblast growth factor (bFGF) ..................................................................................................................... 6.2.2. CD40 Ligand (CD40L)........................................................................................................................................... 6.2.3. Interleukin-17 receptor (IL-17R) ............................................................................................................................. 6.2.4. Tumor necrosis factor receptor (TNFR:Fc), interleukin-1 receptor (IL-1R), interleukin-4 receptor (IL-4R) and Granulocyte Macrophage–Colony Stimulating Factor (GM-CSF) .............................................................................. 6.2.5. Leukaemia inhibiting factor (LIF) ........................................................................................................................... 6.2.6. Nerve growth factor (NGF) .................................................................................................................................... 6.2.7. Interleukin-2 (IL-2) ................................................................................................................................................ 6.2.8. Bovine serum albumin (BSA) ................................................................................................................................. 6.2.9. Angiogenic factors ................................................................................................................................................. 6.3. Cell encapsulation ............................................................................................................................................................ 6.3.1. Islet cells ............................................................................................................................................................... 6.3.2. Chromaffin cells..................................................................................................................................................... 6.3.3. Hybridoma cells..................................................................................................................................................... 7. DNA encapsulation ................................................................................................................................................................. 8. Microsphere and liposome encapsulation .................................................................................................................................. 9. Summary ................................................................................................................................................................................ References ..................................................................................................................................................................................

1. Introduction Alginate is a naturally occurring biopolymer that is finding increasing applications in the biotechnology industry. Alginate has been used successfully for many years in the food and beverage industry as a thickening agent, a gelling agent and a colloidal stabilizer. Alginate also has several unique properties that have enabled it to be used as a matrix for the entrapment and / or delivery of a variety of proteins and cells. These properties include: (i) a relatively inert aqueous environment within the matrix; (ii) a mild room temperature encapsulation process free of organic solvents; (iii) a high gel porosity which allows for high diffusion rates of macromolecules; (iv) the ability to control this porosity with simple coating procedures and (v) dissolution and biodegradation of the system under normal physiological conditions. This review will first describe the preparation, chemical structure and characterization of alginate. The different methods of gel formation and

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physical properties of the gels will then be discussed. Finally, specific examples of alginate systems and their application to protein delivery, nucleic acid delivery and cell encapsulation will be given.

2. Alginate chemistry

2.1. Sources of alginate Commercial alginates are extracted primarily from three species of brown algae (kelp). These include Laminaria hyperborea, Ascophyllum nodosum, and Macrocystis pyrifera. Other sources include Laminaria japonica, Eclonia maxima, Lesonia negrescens and Sargassum species [1]. In all of these algae, alginate is the primary polysaccharide present and it may comprise up to 40% of the dry weight [2]. Alginate is found in the intracellular matrix where it exists as a mixed salt of various cations found in sea water such as Mg 21 , Ca 21 , Sr 21 , Ba 21 , and Na 1 .

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The native alginate is mainly present as an insoluble Ca 21 crosslinked gel [2]. Bacterial alginates have also been isolated form Azotobacter vinelandii and several Pseudomonas species [3].

2.2. Extraction and preparation To commercially prepare alginates, the algae is mechanically harvested and dried before further processing except for M. pyrifera which is processed when wet. Alginate is then extracted from dried and milled algal material after treatment with dilute mineral acid to remove or degrade associated neutral homopolysaccharides such as laminarin and fucoidin. Concurrently, the alkaline earth cations are exchanged for H 1 . The alginate is then converted from the insoluble protonated form to the soluble sodium salt by addition of sodium carbonate at a pH below 10. After extraction, the alginate can be further purified and then converted to either a salt or acid [2]. Since alginates are obtained from a natural source, a variety of impurities may potentially be present. These include heavy metals, endotoxin, proteins, other carbohydrates and polyphenols present in the kelp [4]. For applications in the food and beverage industry, low levels of these impurities do not pose a problem, but for pharmaceutical applications, particularly when alginate will be administered via the parenteral route, these impurities should be removed. Alginates of a pharmaceutical grade can now be obtained from several manufacturers including Kelco (Surrey, UK), ProNova Biopolymer (Drammen, Norway), Chemical MFG Corp. (Gardena, CA, USA) and Junsei (Tokyo, Japan).

2.3. Chemical structure Alginates are a family of linear unbranched polysaccharides which contain varying amounts of 1,49linked b-D-mannuronic acid and a-L-guluronic acid residues (Fig. 1). The residues may vary widely in composition and sequence and are arranged in a pattern of blocks along the chain. These homopolymeric regions of b-D-mannuronic acid blocks and a-L-guluronic acid blocks are interdispersed with regions of alternating structure (b-D-mannuronic

Fig. 1. Structure of alginate showing both b-D-mannuronic acid and a-L-guluronic acid residues. Reprinted from Ref. [129], with kind permission from Wiley, New York.

acid–a-L-guluronic acid blocks) [5,6]. The composition and extent of the sequences and the molecular weight determine the physical properties of the alginates. The molecular variability is dependent on the organism and tissue from which the alginates are isolated. For example, alginates prepared from the stipes of old L. hyperborea kelp contain the highest content of a-L-guluronic acid residues while alginates from A. nodosum and L. japonica have a low content of a-L-guluronic acid blocks. Alginates do not have a regular repeating unit and the distribution of monomers along the polymer chain cannot be described by Bernoullian statistics. Analytical characterization of alginates is more difficult than for other polysaccharides since acid hydrolysis can lead to destruction of the uronic acids. Circular dichroism spectroscopy has been used to match the linear spectra of the alginate to model samples of well characterized homopolymeric blocks [7]. NMR spectroscopy has contributed significantly to our understanding of alginate structure [8]. This technique can determine the monomer composition as well as the frequencies of the four possible diad (nearest neighbor) structures FGG , FMG , FMM and FGM (G 5 a-L-guluronic acid; M 5 b-D-mannuronic acid). NMR can also provide an estimate of the eight possible triad frequencies and the average block length. The viscosity of alginate solutions depends primarily on the molecular weight of the material. Characterization of purified alginate samples by gel permeation chromatography indicates a polydisperse size distribution [9]. Light scattering has been used to determine the average molecular weights of several alginate samples which have been shown to range from 80 kilodaltons (kDa) to 290 kDa for Azotobacter vinelandii and Pseudomonas aeruginosa, respectively [10].

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3. Gel formation and dissolution chemistry

3.1. General formation mechanism Alginate beads can be prepared by extruding a solution of sodium alginate containing the desired protein, as droplets, into a divalent crosslinking solution such as Ca 21 , Sr 21 , or Ba 21 . Monovalent cations and Mg 21 ions do not induce gelation [2] while Ba 21 and Sr 21 ions produce stronger alginate gels than Ca 21 [11]. Other divalent cations such as Pb 21 , Cu 21 , Cd 21 , Co 21 , Ni 21 , Zn 21 and Mn 21 will also crosslink alginate gels but their use is limited due to their toxicity. The gelation and crosslinking of the polymers are mainly achieved by the exchange of sodium ions from the guluronic acids with the divalent cations, and the stacking of these guluronic groups to form the characteristic egg-box structure shown in Fig. 2 [12]. The divalent cations bind to the a-L-guluronic acid blocks in a highly cooperative manner and the size of the cooperative unit is more than 20 monomers [1]. Each alginate chain can dimerize to form junctions with many other chains and as a result gel networks are formed rather than insoluble precipitates [13].

3.2. Large bead preparation In general, beads greater than 1.0 mm in diameter can be prepared by using a syringe with a needle or a pipette [14–19]. Sodium alginate solution that contains the solubilized protein is transferred dropwise into a gently agitated divalent crosslinking solution. The diameter of the beads formed is dependent on the size of the needle used and the viscosity of the alginate solution. A larger diameter needle and higher viscosity solutions will produce larger diameter beads. The viscosity of sodium alginate can also influence the shape of the microbeads produced. The beads become more spherical as the concentration of the sodium alginate solution is increased [20]. However, in general, sodium alginate solutions of greater than 5% (w / v) are difficult to prepare. The beads that are formed are allowed to be fully cured in the crosslinking solution for a short period of time, usually in minutes, before they are rinsed with distilled water. A poly-L-lysine coating can be per-

Fig. 2. Schematic representation of the egg-box association of the poly-L-guluronate sequences of alginate crosslinked by calcium ions. The upper section of the figure shows conversion of random coils to buckled ribbonlike structures which contain arrays of Ca 21 ions. The bottom section shows the proposed stereochemistry of Ca 21 ion complexation. The oxygen atoms involved in the coordination sphere are shown as filled circles. Reprinted from Ref. [12], with kind permission from Elsevier Science, Amsterdam.

formed at this stage, followed by a final external alginate coating. Alginates with high b-D-mannuronic acid content have been shown by Fourier transform infrared spectroscopy to link more strongly to the poly-L-lysine coating [21]. These beads are typically stored in 0.9% NaCl solution.

3.3. Microbead preparation There are three widely-known methods used to prepare alginate microbeads that are less than 0.2 mm in diameter; atomization, emulsification and coacervation. The most commonly used technique is an atomization or spraying method using an extrusion device with a small orifice. There are numerous variations of this method which have been reported by different research groups [22–27]. A general overview of alginate microbead preparation is as

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follows. Solutions containing the alginate and protein, as described above in the preparation of large beads, are well mixed and loaded into a syringe mounted on a syringe pump. The mixture of alginate and protein solution is then delivered through an atomization device with a defined diameter ( | 1 mm) orifice at the tip. Much smaller diameter orifices can be used but may run the risk of orifice clogging / plugging by the high viscosity alginate solution. The sizes of these beads can be controlled by either the pressure of the infusing nitrogen gas, the flow-rate of the syringe pump or the distance between the orifice and the surface of the crosslinking solution. Fine droplets of sodium alginate and protein solution will form the microbeads when crosslinked with the divalent solution. Outer coatings of poly-L-lysine and alginate can then be performed. This method is a simple technique which involves only aqueous solutions. Abraham et al. have reported the successful production of sterile alginate poly-L-lysine microbeads using the above atomization method [28]. The authors described the use of a modified Bellco Bioreactor (Bellco Glass, Vineland, NJ, USA) attached to a Turbotak atomizer (Waterloo, Ontario, Canada) to produce these sterile microbeads. Aseptic protocols were used along with sterilized equipment and reagents. The sterility of these microbeads was verified by performing a modified United States Pharmacopia USP sterility test in which the microbeads were ‘‘plated’’ on agar plates and the growth of bacteria colonies on the plates was monitored. The authors concluded that the use of a modified Bellco Bioreactor is a novel approach for producing laboratory scale sterile alginate poly-Llysine microbeads. The second method of microbead preparation involves protein encapsulation by an oil-in-water emulsification technique [29–32]. This encapsulation method may work better for stable peptides and proteins or synthetic low molecular weight drugs since it involves the use of harsher chemical reagents such as ethyl ether to remove the oil at the end of the process. The size of the microbeads formed by this technique is highly dependent on the stirring speed and the rate of the addition of the crosslinking solution. Complex coacervation of oppositely charged polyelectrolytes has been commonly used as a method for

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preparing microbeads. Under specific conditions of polyion concentration, pH and ionic strength, the polyelectrolyte mixture can separate into two distinct phases; a dense coacervate phase which contains the microbeads and a dilute equilibrium phase [33]. Complex coacervation between alginic acid, gelatin [34], chitosan [35], and albumin [36] has been reported. In the alginate–chitosan system, the complex is formed by spraying a sodium alginate solution into the chitosan solution. The resultant alginate–chitosan microbeads are mechanically strong and stable over a wide pH range. With the alginate– albumin system, coacervation is found to be limited compared to other polypeptide–polysaccharide systems due to the high viscosity of the albumin–alginic acid complex and a propensity to precipitate. The optimum conditions for maximum coacervate yield are a pH of 3.9, an ionic strength of 1 mM and a 0.15% w / v total polyion concentration.

3.4. Matrix /block gels, fibers and in situ gelling systems Alginate gels of different morphologies than spherical beads have also been prepared. Protein loading can be conducted in two different ways, imbibition [37,38] or incorporation of the protein into the alginate solution prior to crosslinking. To form a ‘‘block’’ gel, alginate solution with or without the protein, is poured into a container, (i.e. petri dish or perspex box template), and leveled. The container is then placed in the crosslinking solution for gelation to occur. Gelation of this ‘‘block’’ matrix can take several hours. Once gelation has occurred, gels can be cut into the desired shapes, e.g., disc or blocks. Protein loading using the imbibition process involves the immersion of the block gel into a solution of the protein of interest. This loading technique requires more protein and time, and can be dependent on both the size of the protein and the size of the pores in the gel. An alternative method of alginate film preparation has been described by Aslani and Kennedy [39]. They poured a 3% (w / w) aqueous solution of sodium alginate into a glass plate and allowed the solution to completely dry to a film at 208C. The dry sodium alginate film was then removed and crosslinked in a solution of either calcium or zinc acetate.

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Alginate fibers can be prepared by extruding solutions of sodium alginate into a bath of calcium ions. The resultant calcium alginate hydrated fibers are then dried to give tough fibers that can be collected on spools for knitted fabrics or directly chopped for use in nonwoven materials. Several commercially available wound dressings contain calcium alginate fibers [40]. Sodium ions in wound fluids slowly convert the fibers into a viscous sodium alginate solution which soothes and protects the wounds. One can easily envision incorporating a drug into the fibers which would be released when applied to the wounds surface. The in situ gelation properties of alginate were reported as early as 1947 by Major George Blaine [41]. Several surgical applications of alginate gels were proposed including the prevention of adhesion formation in the course of tissue repair, the arrest of capillary hemorrhage and the treatment of burns. Alginate solutions have also been shown to form gels in situ when placed on the surface of the eye [42]. A high a-L-guluronic acid content alginate is crosslinked by calcium ions present in the tear fluid. This system was suggested as a carrier for the prolonged delivery of drugs to the eye. An oral controlled release system has been described which contains sodium alginate and a calcium ion donor (CaHPO 3 ) [43]. A dry mixture of these substances and a solid drug are compressed into tablets. Upon administration, the gastrointestinal (GI) fluid dissolves the alginate and the calcium complex. The alginate and calcium ions react immediately and a spongelike matrix is formed progressively from the outside to the inside of the tablet. Release of the drug is dependent on both dissolution of the gel and diffusion of the drug into the GI fluid. In another report sodium alginate was used as a sustained release oral drug delivery system with a potential for prolonged gastric residence [44]. Although the studies were performed with low molecular weight drugs, the system is applicable to proteins. Formulations containing sodium alginate, calcium phosphate, sodium bicarbonate, lactose and the drug were filled in gelatin capsules. Upon dissolution of the gelatin capsule and contact of the formulation with an acid medium, the alginate was hydrated and crosslinked by the calcium, creating a gel barrier at the surface. The sodium bicarbonate effervesced

releasing carbon dioxide which was trapped in the gel producing a buoyant capsule.

4. Physical properties The functional and physical properties of cation crosslinked alginate beads are dependent on the composition, sequential structure, and molecular size of the polymers [4,10]. The flexibility of the alginate polymers in solution increases in the order MG . MM . GG (G 5 a-L-guluronic acid; M 5 b-D-mannuronic acid) [4]. Beads with the lowest shrinkage, highest mechanical strength, highest porosity, and best stability towards monovalent cations are made from alginate with an a-L-guluronic acid content greater than 70% and an average length of the a-L-guluronic acid blocks higher than 15. These polymers are called ‘‘high G’’ alginates and for molecular weights higher than 2.4 3 10 5 , the gel strength is independent of the molecular weight [10]. For lower molecular weight alginates however, there is a certain critical molecular weight below which the gel forming properties of alginates are reduced [19]. While a gel made from a high a-L-guluronic acid alginate may be rigid and brittle, gels produced from alginates with a low a-L-guluronic acid content are more elastic [45]. Alginate forms stable gels over the temperature range of 0–1008C, although the modulus of rigidity of the gels decreases with an increase in temperature [46]. The gels can be prepared in both hot or cold water [45].

4.1. Chemical reactivity Although the microenvironment in an alginate bead can be relatively inert to protein drugs and cells (alginate beads typically contain up to 95% water) a positively charged protein can potentially compete with calcium ions for available carboxylic acid sites on the alginate. This has been observed with small drugs by several investigators [47,44] and has been shown to result in protein inactivation in the case of the protein transforming growth factor-beta (TGFb 1 ) [48]. Since alginates can form coacervates with cationic proteins, it may be necessary to include

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additives which protect the active agent from the alginate polymer. This was reported with TGF-b 1 which forms an irreversible complex with the alginate [48]. The addition of the anionic polymer poly(acrylic acid) shields the TGF-b 1 from interaction with the alginate and allows its activity to be retained.

4.2. Porosity and macromolecular diffusion Proteins encapsulated in alginate matrices are released by two mechanisms: (i) diffusion of the protein through the pores of the polymer network and (ii) degradation of the polymer network. Analysis of calcium alginate gels microbeads by electron microscopy has shown that the pore size ranges from 5 nm to 200 nm in diameter [49]. In a different approach the porosity was determined by packing alginate beads in a column and recording the exclusion volumes for macromolecular standards [50]. A cut off value of 12–16 nm was determined which is smaller than the pore size distribution obtained by electron microscopy. This difference suggests that there is a more constricted polymer network on the bead surface than in the gel core [4]. Diffusion of small molecules such as glucose and ethanol is unaffected by the alginate matrix while diffusion of larger proteins from the gels has been shown to be dependent on their molecular weight [10]. The diffusion of several proteins from alginate beads has been reported including immunoglobulin G (IgG) [51,52], fibrinogen [51] and insulin [17]. Increasing the concentration of alginate in the beads decreases the rate of diffusion of the proteins from the gel. Gels made from high a-L-guluronic acid alginates have the most open pore structure and exhibit the highest diffusion rates for proteins which may be related to the lower shrinkage of this type of gel or to a difference in the diffusion barrier at the surface of the spheres [10]. The charge on a protein can also influence its rate of diffusion from an alginate matrix. A protein with a high pH and overall net positive charge can potentially interact with the negatively charged alginate polymer, thus inhibiting diffusion from the gel [1,48]. Conversely a protein with a low pI may be released more rapidly from the matrix than would be

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expected from free molecular diffusion. This is also the case with low molecular weight drugs. In one study the cationic drug chlorpheniramine maleate was shown to have a slower release rate from an alginate gel than the anionic drug, sodium salicylate [44]. In some cases interactions of a cationic drug with alginate can lead to dramatic changes in bead morphology. This was shown in a study with propranolol which was loaded into preformed alginate beads by passive diffusion [47]. As the propranolol concentration in the loading solution is increased, the resultant bead loading capacity also increases. The highest loadings in the beads cause a contraction in diameter and a change in color from translucent to a whitish color. The color change is attributed to precipitation of the drug–polymer complex in the beads. The porosity of an alginate gel can be significantly reduced by partially drying the beads [4]. Beads made from a high a-L-guluronic acid alginate will reswell only slightly upon rehydration resulting in an increased alginate concentration in the bead and a reduced pore size. Complete dehydration of alginate beads, however, can result in surface cracking which can facilitate the surface erosion of the beads upon rehydration. Blue dextran (molecular weight 2 000 000), a model for a macromolecular drug, was incorporated into alginate beads and dried to completion. Upon rehydration the release of the dextran is more rapid from the dried beads compared to the undried samples [53]. A reduction in pore size of an alginate matrix can also be achieved by exposing the gel to low pH. In a 0.1 N HCl solution at pH 1.0, alginate beads undergo a decrease in diameter [48]. Release of macromolecules from alginate beads in low pH solutions is also significantly reduced which could be advantageous in the development of an oral delivery system [48,53– 55].

4.3. Chemical stability /degradation Degradation of a Ca 21 crosslinked alginate gel can occur by removal of the Ca 21 ions. This can be accomplished by the use of a chelating agent such as ethylene glycol-bis (b-aminoethyl ether)-N,N,N9,N9 tetraacetic acid (EGTA), lactate, citrate and phos-

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phate or by a high concentration of ions such as Na 1 or Mg 21 [2]. As Ca 21 ions are removed, the crosslinking in the gel decreases and the gels are destabilized. This can lead to leakage of entrapped material and solubilization of the high molecular weight alginate polymers. Alginate gels will also degrade and precipitate in a 0.1 M phosphate buffer solution and will completely dissolve in 0.1 M sodium citrate at pH 7.8 [56]. If Ca 21 is used in the crosslinking solution and phosphate is used as the dissolution medium, the dissolution medium will turn turbid due to the Ca 21 dissociating from the polymer network and forming calcium phosphate precipitate. This phenomenon is more evident when a high guluronic content alginate is used. Low a-Lguluronic acid content alginate and lower molecular weight alginate are known to release encapsulated proteins at a much faster rate [57]. Degradation of the gel can be prevented by storing the gel beads in a medium that contains free Ca 21 ions and to keep the Na 1 :Ca 21 ratio less than 25:1 for high a-L-guluronic acid alginates and 3:1 for low a-L-guluronic acid alginates [10]. Alginates have been reported to undergo proton catalyzed hydrolysis which is dependent on time, pH, and temperature [5,6,58,59]. A crosslinked alginate matrix delivery system when exposed to low pH can therefore undergo a reduction in alginate molecular weight which results in faster degradation and release of a molecule when the gel is reequilibrated in a neutral pH solution [48]. Alginate forms strong complexes with polycations including chitosan, polypeptides such as polylysine and synthetic polymers such as polyethyleneimine. These complexes do not dissolve in the presence of Ca 21 chelators and can be used to both stabilize the gel and reduce its porosity [4]. Sawhney and Hubbell described a system in which the poly-L-lysine chain is modified with monomethoxy poly(ethyleneglycol) (MPEG) [60]. The authors reported that alginate microbeads coated with layers of modified poly-Llysine-MPEG and poly-L-lysine show reduced protein adsorption, complement binding and cell adhesion, and display greater mechanical stability. The decreased cellular interaction with the surrounding environment also lessened the immunogenicity that the normal alginate–poly-L-lysine–alginate microbeads produced upon intraperitoneal implantation.

5. Biological properties

5.1. Immunogenicity There are many factors involved in determining the successful application of polymers as drug delivery carriers in humans, with polymer biocompatibility or / and immunogenicity being two of the more important issues. There are numerous reports addressing the fibrotic reaction of implanted alginates [61–65]. Most authors agree that the chemical composition and the mitogenic contaminants found in alginates are the two main contributors to alginate immunogenicity. Alginates can be readily purchased in several different grades namely, ultra pure, food or research grade. One of the major distributors of sodium alginate, Kelco has conducted studies comparing the immunogenicity of different alginates [64]. Commercial research grade alginate and ultra pure alginate have been tested for their endotoxin levels and their ability to activate lymphocytes. The study showed that mitogenic impurities which are found in commercial alginate but not in purified alginate, are solely responsible for the side effects observed. Side effects included cytokine release and inflammatory reactions. Other groups have also shown that alginate rich in mannuronic acid seem to activate cytokine production more than guluronic-rich alginate [61– 63]. It is therefore strongly recommended by these investigators that ultra pure alginate with low b-Dmannuronic acid and high a-L-guluronic acid contents should be considered for any in vivo research if inflammatory reactions are to be avoided. High a-L-guluronic acid alginate implants are also reported to have lower immunological responses at the implant sites when compared to polyvinyl alcohol and agarose gels [63]. Studies employing alginates as surgical gauzes or films have also demonstrated that not only is alginate completely absorbed (biodegradable) in animal tissues but the tissue reaction is found to be very minimal [41]. Studies in our laboratory have also documented that little or no specific inflammatory response was associated with the upper nasopharynx when high a-L-guluronic acid alginate microbeads were intranasally instilled in mice [66]. In another study, the physical imperfections of the

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individual capsules, rather than the chemical composition of alginate, was suggested to be the main underlying cause of immunogenicity [67]. The paper reported that inadequately encapsulated rat pancreatic islets are associated with graft rejection. The authors concluded that high a-L-guluronic acid content alginates produce microbeads with a defined size. This reduces the number of improperly formed microbeads and ultimately decreases the amount of fibrotic reactions. It is very conceivable that with all the contradictory reports, more than one factor can be attributed to alginate immunogenicity. Cappai et al. have summarized this concern very well by stating that factors such as sphericity, strength and volume of the implanted beads, smoothness of the membrane, viscosity, composition and purity of the alginate solution, are all contributing factors in preventing cell over growth [65].

jejunum. The force required to detach the beads from the jejunum’s surface was recorded and compared with the values obtained from other types of polymer beads. These studies showed that alginate has the highest mucoadhesive strength when compared to polymers such as polystyrene, chitosan, carboxymethylcellulose and poly(lactic acid). Mucoadhesive drug delivery systems work by increasing the drug residence time at the site of activity or resorption. This mucoadhesive feature of alginate may aid in its utility as a potential delivery vehicle for drugs to mucosal tissues such as the GI tract or the nasopharynx. The adherence of these microbeads to the mucosal tissues localizes the drug and delays the protein transit time, therefore potentially improving the overall drug effectiveness and bioavailability.

5.2. Bioadhesion

6. Protein encapsulation

Alginate possesses, among other features, a bioadhesive property which could serve as a potential advantage in mucosal drug delivery. The term bioadhesion can be generally defined as the adhesion or contact between two surfaces, with one being a biological substratum [68]. If one of the surfaces involved is a mucosal layer, the term mucoadhesion is then used [69]. Studies have shown that polymers with charge density can serve as good mucoadhesive agents [70–73]. Peppas and colleagues believed that mucoadhesion is achieved by chain penetration across a polymer–mucosa interface [74,75]. It has been reported that polyanion polymers are more effective bioadhesives than polycation polymers or nonionic polymers [70]. Alginate, with its carboxyl end groups, is classified as an anionic mucoadhesive polymer. Alginate mucoadhesion studies, conducted by Chickering et al., were performed with a tensile testing apparatus (Cahn Dynamic Contact Angle Analyzer; CAHN Instruments, Cerritos, CA, USA) in which the adhesive forces between different polymers and living intestinal epithelium were evaluated [71,72]. The intestinal epithelium used in these experiments was from excised rat jejunum. In brief, individual polymer beads were placed on an inverted

Numerous reports have been published on the encapsulation and release of proteins from alginate matrices. These are summarized in Table 1.

Table 1 Summary of proteins encapsulated in alginate microbeads Types of proteins

Refs.

Albumin Bovine serum albumin (BSA) CD40L Endothelial cell growth factor (ECGF) Epidermal growth factor Acidic FGF Basic FGF (bFGF) Fibrinogen Gamma globulin Horse-radish peroxidase IgG Insulin Leukaemia inhibitory factor (LIF) Myoglobin Nerve growth factor (NGF) Ovalbumin TGF alpha (TGF-a) TGF beta (TGF-b) Tumour necrosis factor receptor (TNFR:Fc) Interleukin-2 (IL-2)

[36] [37,121,122,96,19] [98,99] [104] [103] [103] [97,96,103] [51] [51,19] [121] [51,52] [17] [113] [96] [30] [92,24,23,66] [103] [48,76] [14] [37]

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6.1. Targeting to mucosal tissues 6.1.1. TGF-b1 The rapidly proliferating epithelium of the intestinal mucosa is often adversely affected by cytotoxic drugs. TGF-b 1 which is known to inhibit the growth of many cells of epithelial origin, was incorporated into alginate beads and tested in a rat model to determine its effect on in vivo stem cells [76]. The alginate beads contained polyacrylic acid as an excipient which is necessary to protect the TGF-b 1 from irreversibly binding to the alginate. In vitro studies showed that the protein is not released from the alginate microbeads when incubated in 0.1 N HCl, pH 1.0. However, when the beads were transferred to phosphate-buffered saline (PBS) at a pH of 7.4, all of the TGF-b 1 is released within 2 h in an active form (Fig. 3) [48]. The acid incubation of the delivery system increases both the release rate of the TGF-b 1 and the degradation rate of the alginate beads. Both of these effects are attributed to hydrolysis of the alginate in the low pH solution. The in vitro studies established that the alginate delivery system is theoretically capable of protecting the entrapped protein from the harsh environment of the stomach and later releasing it at its potential site of action in the small intestine. Rats were treated with TGF-b 1 (25 mg total each day for 5 days) in alginate beads perorally, with TGF-b 1 in PBS perorally or

Fig. 3. Cumulative percent in vitro release of 125 I-TGF-b 1 at 378C in (j) (square) PBS, pH 7.4 and (d) 0.1 N HCl, pH 1.0 transferred to PBS after 24 h. Reprinted from Ref. [48], with kind permission of Elsevier Science, Amsterdam.

intraperitoneally or with PBS alone perorally. Histomorphometrical analyses show a marked reduction in villus height (50–70%) in the intestinal mucosa of animals treated with TGF-b 1 in the alginate beads. The proliferating and mitotic indices are also significantly reduced in these animals when compared to the controls and other routes of administration (Fig. 4).

Fig. 4. The proliferating index (A) and mitotic index (B) of intestinal cells in rats after administration of (h) control vehicle, ( ) TGF-b 1 intraperitoneally, ( ) TGF-b 1 in phosphate-buffered saline perorally, and (j) TGF-b 1 in alginate beads perorally. The * indicates a statistically significant reduction (P , 0.01) in proliferating or mitotic indices. Reprinted from Gastroenterology, Vol 107, Puolakkainen, P.A., et al., Novel delivery system for inducing quiescence in intestinal stem cells in rats by transforming growth factor b 1 , pages 1319–1326, (1994) with kind permission of W.B. Saunders Company, Philadelphia, P.A.

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6.1.2. Vaccines The market for effective vaccines against pathogens is large. Most commercial vaccines to date, such as mumps, childhood measles, and rubella, are currently administered via the parenteral route. Even though the conventional parenteral route of vaccine administration has proven to be ineffective in protecting individuals from airborne or mucosal-related respiratory infectious diseases [77], nonparenteral routes are still infrequently used. However, immunologists are now recognizing the significance of immunization at the mucosal surfaces which produces the so-called protective antibody, secretory immunoglobulin A (IgA) [78,79]. Development of effective delivery systems for presentation of antigens to mucosal surfaces is critical to the success of these vaccines. The use of polymers to microencapsulate antigens has increased in recent years [80–83]. The most widely published microparticle vaccine delivery systems used to date are liposomes [84,85] and poly(lactide-co-glycolide) microspheres [86–88]. The use of other microencapsulation vehicles such as immunostimulating complexes [89], cochleates [90] protenoids [91] are also rapidly progressing. Due to its excellent bioadhesive property and mild encapsulation conditions, alginate would seem to be an ideal mucosal delivery system for protein antigens. Ongoing studies on alginate as a vaccine delivery system in our laboratory showed that strong antibody responses were effectively produced when soluble antigens were encapsulated and released from polyL-lysine coated alginate microbeads [23]. Intranasal administration of these ovalbumin-containing beads in mice induced high serum levels of antigen specific antibodies of all subclasses except immunoglobulin E. Intranasal administration of unencapsulated soluble antigen mounted no antibody responses. The data also showed that administration of empty alginate microbeads evoked no immune responses, suggesting that alginate does not possess any adjuvancy properties on its own. Further experiments showed that high levels of IgA were detected in the bronchial alveolar lavage fluids of mice when both primary and secondary immunizations were conducted intranasally but not when the administration of the antigen was done subcutaneously. The experiments described here have provided evidence that alginate could be

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successfully used as a mucosal drug delivery vehicle for the delivery of vaccines or drugs to the upper respiratory tract. Kwok et al. have reported the encapsulation of Bacillus Camette Guerin (BCG) virus in alginate microbeads [26]. The paper reported the potential feasibility of delivering live BCG vaccine to the lung by either inhalation or intravenous injection. In this paper, the authors described the successful encapsulation of heat-killed BCG virus into 5–15 mm diameter alginate microbeads using an atomization technique. The sizes of alginate microbeads reported here will work well if delivery is by intravenous injection. The microbeads, however, would have to be smaller (1–5 mm) if delivery by inhalation to the lung is desired. In the area of veterinary vaccines, Bowersock et al. have evaluated the use of alginate hydrogels to deliver oral vaccines to different species of animals [92,24]. Studies from his group have indicated that alginate microbeads show great promise in delivering vaccine antigens orally to several species of animals including rodents and cattle. Results showed that the release of the model protein ovalbumin from alginate microbeads is capable of inducing immunity at mucosal sites. The character of the immune response varied depending on antigen and vaccination protocol. The advantage of using this oral administration technology is that a large number of animals can be vaccinated very conveniently.

6.2. Slow release applications The controlled release of proteins from a variety of polymeric matrices has been reported [93–95]. These systems are generally utilized for prolonging the circulation half-lives of proteins or for targeted delivery of proteins to specific tissues. Alginate matrices have proven to be useful for the slow release of several potential therapeutic proteins and several studies have demonstrated the in vitro and in vivo efficacy of these systems.

6.2.1. Basic fibroblast growth factor ( bFGF) bFGF plays a multifunctional role in stimulation of cell growth and tissue repair. This protein has a very short half-life when administered by the parenteral route and is unstable in solution. A stable slow

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release system for bFGF was developed by first binding the factor to heparin–Sepharose beads [96]. This permitted prolonged storage and repeated handling of the growth factor and enabled it to be encapsulated in alginate microbeads with an efficiency of 77%. Continuous release of the bFGF is demonstrated in vitro for more than 14 days. The release of the bFGF from the system is enhanced by the addition of heparinase to the alginate microbeads. Alginate beads containing the bFGF heparin–Sepharose complex without the heparinase were evaluated in vivo in a rat model. The beads are found to effectively deliver bFGF to the extravascular space without transendothelial transport [97]. The amount of bFGF deposited in arteries adjacent to the release devices is 40 times that deposited in similar arteries in animals that received a single intravenous bolus of bFGF.

6.2.2. CD40 Ligand ( CD40 L) Studies in our laboratory [98,99] showed that alginate encapsulated murine CD40L (mCD40L) administration to transgenic CD40L-deficient mice restored both humoral and cell-mediated immunity in mice previously lacking these immune responses. Mice lacking the CD40L gene develop a similar phenotype to human individuals with hyperimmunoglobulin M (Hyper IgM) syndrome [100]. Hyper IgM syndrome is characterized by severe hypogammaglobulinemia and opportunistic infections. Using CD40L-deficient mice as an animal model for this disease, alginate encapsulated mCD40L was implanted once subcutaneously. These mice were later immunized with sheep red blood cells. Foot pad swelling was measured as an indicator of an antigen-specific delayed-type hypersensitivity (DTH) response. Only CD40L-deficient mice treated with encapsulated mCD40L mounted a strong DTH response whereas mice given the control encapsulated ovalbumin did not mount an immune response. These experiments suggest that the controlled release of CD40L can be of great therapeutic value in the treatment of immunodeficiency disorders in which CD40L deficiencies have been implicated such as Hyper IgM syndrome and Common Variable Immunodeficiency.

6.2.3. Interleukin-17 receptor ( IL-17 R) IL-17R, a newly-discovered molecule [101,102] has potential applications in the treatment of inflammatory diseases such as osteoarthritis. An allogeneic cell model was used to assess the effectiveness of the sustained release of IL-17R from implanted alginate beads. In this model, irradiated spleen cells from B6 mice were injected into the foot pads of Balb /c mice. The weight increase of lymph nodes from these Balb /c mice were used as an indicator in this model. Inflamed animals will have higher a lymph nodes weight increase than noninflamed animals. Unpublished studies in our laboratory showed that 1 mm diameter alginate beads containing IL-17R, when implanted subcutaneously caused a decrease in lymph node weight which was comparable to that seen in animals receiving subcutaneous injections of IL-17R, more importantly, only one third of the dose was required with the alginate system. The single administration of alginate beads was also more convenient than the three subcutaneous injections required of the unencapsulated protein. 6.2.4. Tumor necrosis factor receptor ( TNFR: Fc), interleukin-1 receptor ( IL-1 R), interleukin-4 receptor ( IL-4 R) and granulocyte macrophagecolony stimulating factor ( GM-CSF) Our laboratory has evaluated the in vitro release of several other recombinant proteins from alginate beads (Fig. 5). Martinsen et. al. [19] have reported that the release rate of acidic proteins from alginate beads is inversely proportional to the protein molecular weight. This is due to the fact that the polymer network has pore sizes ranging from 5 to 200 nm [49]. Acidic proteins are less likely to interact with the anionic alginate polymer and will therefore be more likely to release by diffusion through the network pores. Basic proteins however, such as TGF-b 1 and bFGF, interact with the alginate polymer network and hence, diffusion through the pores is greatly hindered. In cases such as this, the majority of the protein is released by network disintegration (erosion). In this group of acidic proteins tested, GM-CSF with the lowest protein molecular weight of 16 kDa, was released to the dissolution medium the fastest, followed by both IL-4R and IL-1R both

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observed are comparable with animals which received NGF via implanted Alza (Palo Alto, CA) mini-osmotic pumps for 1 week.

Fig. 5. The cumulative percent in vitro release profiles of rhuTNFR:Fc (– j –), IL-1R (– d –), IL-4R (– m –) and GMCSF (– ♦ –) from 70% G content alginate beads in PBS at 378C.

having a molecular weight of 50 kDa, and finally TNFR:Fc with a molecular weight of 180 kDa. The order of the release of these proteins from the alginate beads is consistent with the above diffusion mechanism theory proposed for acidic proteins.

6.2.5. Leukaemia inhibiting factor ( LIF) Austin et al. have shown that the cytokine, LIF, can potentially be used for the treatment of a variety of muscle diseases [15]. The paper described the release of LIF from alginate beads for up to 80 days in vitro. Slow release of LIF could be advantageous because of the protein’s short biological half-life. 6.2.6. Nerve growth factor ( NGF) Reduced production of NGF has been implicated in age-dependent cholinergic neuronal atrophy and neuronal degeneration of the forebrain. Maysinger et al. tested the suitability of alginate for the microencapsulation of NGF [30]. The authors described the advantages associated with using this alginate technology which include ease of administration and the protection of NGF from hydrolytic cleavage. The study indicated that the release of encapsulated NGF can prevent neuronal degeneration in the rat model for central cholinergic degeneration. Also, the effects

6.2.7. Interleukin-2 ( IL-2) Recently alginate microspheres have been used as a matrix for the delivery of IL-2 [37]. Three types of microspheres were prepared by first dissolving sodium alginate in distilled water at a concentration of 2.0% (w / v). The solution was then spray-dried into a 0.5% CaCl 2 solution. After curing for 10 min the microspheres were placed in coating solutions of: (i) chitosan hydrochloride; (ii) poly-L-lysine or (iii) CaCl 2 . The IL-2 was incorporated into the preformed microspheres by diffusion from an external aqueous solution of IL-2. In vitro sustained release of IL-2 from the alginate–chitosan system is found to last for 5 days and IL-2 is completely recovered from the matrix. The in vitro activity of the released IL-2 was investigated by determining the induction of cytotoxic T lymphocytes (CTL) when incubated with tumor cells and lymphocytes. The IL-2 remains active in the alginate–chitosan microspheres and is more efficient in triggering the induction of CTL than free IL-2.

6.2.8. Bovine serum albumin ( BSA) Fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) was incorporated into the three different types of alginate microspheres described above by mixing the protein with the sodium alginate solution prior to gelation in the CaCl 2 [37]. In vitro release studies showed that in all cases a large initial burst is observed. After the burst, the release is sustained for 4 days from the alginate–chitosan system. The alginate–CaCl 2 and alginate–polylysine systems exhibit a sustained release of FITC-BSA for 6 h and 24 h respectively. The authors attributed the short release time from the alginate–CaCl 2 microspheres to the low stability of the chelating junction in a phosphate buffer above pH 5. The longer release times from the chitosan coated systems may be the result of a strong interaction between the two polymers and a stabilization of the polycation salt by the phosphate ions.

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6.2.9. Angiogenic factors Several angiogenic factors have been incorporated into alginate beads including acidic FGF and bFGF, epidermal growth factor, and transforming growth factor alpha (TGF-a) [103]. In all cases the biological activity of the proteins is retained. The bFGF, which has the highest pI of the proteins evaluated (i.e. 9.6) has the highest entrapment efficiency. In an in vivo murine model, all angiogenic factors in the alginate beads cause quantifiable neovascularization when injected subcutaneously. Injection of the nonencapsulated purified angiogenic factors does not cause neovascularization. In another study the in vitro release of endothelial cell growth factor (ECGF) from alginate microbeads was determined [104]. The rate of release is found to vary inversely with time. There is an initial substantial release in the first 2 h, followed by a controlled slow release for 4–5 days, and then a much slower release for approximately 14 days. The rate of growth factor release in the first 96 h and the amount released over the first 300 h are both found to be dependent on the initial ECGF concentration in the microbeads. The rate of release appears to be independent of the concentration of alginate used except for an initial rate difference in the first 2 h. 6.3. Cell encapsulation Cells encapsulated in the alginate matrix have numerous potential applications in biotechnology [105]. Encapsulation of hormone, neurotransmitter producing cells or recombinant cells for the treatment of diabetes mellitus [106–111], liver diseases [112], parathyroid disorders [113] and most recently neurological disorders [114], have been successfully performed. In this review, we will be describing the encapsulation of islet cells for the treatment of diabetes, the encapsulation of chromaffin cells for the treatment of Parkinson disease and the entrapment of hybridoma cells for antibody production. A more comprehensive list of different cell types that have been encapsulated in alginate matrices is shown in Table 2.

6.3.1. Islet cells Animal models of transplanted islets of Langerhans encapsulated in alginate have been reported as

Table 2 Summary of cells and DNA encapsulated in alginate microbeads Types of cell / others

Refs.

Algae Bacillus Calmette Guerin Bacteria Chromaffin DNA Fibroblasts Fungi Hepatocytes Hybridoma Islets of Langerhans Lymphoma cells Plant cells

[123] [56] [124] [114] [120,31] [125] [126] [112] [116,117,119] [107–111] [127] [128]

early as in 1980 [110]. But, it was only recently that the technology was used in humans. The first human clinical trial which utilizes alginate as the encapsulation polymer is accomplished with the encapsulation of pancreatic islet cells to treat patients with insulindependent diabetes. The transplantation was successfully performed without any adverse reactions by Dr. Soon-Shiong at St. Vincent Medical Center (Los Angeles, CA, USA) in 1993 [107,108]. The operation which is categorized as minimal invasive surgery, is a relatively simple procedure. Liquid containing the alginate encapsulated pancreatic cells is poured through a funnel into the patient’s abdominal cavity. Insulin secretion is observed within 24 h after transplantation. The success of this transplantation has given much impetus for further clinical research in this area. Ongoing investigations involve optimizing the doses of encapsulated islet cells, generation of chemically-stable crosslinked alginate, and an assessment of the safety issues governing the administration of encapsulated cells to humans.

6.3.2. Chromaffin cells The work conducted by Tsang et al. [114] shows great promise in the application of the alginate microbead technology in an animal model of Parkinson’s disease. Dopamine, which is produced by chromaffin cells, has been shown to reverse the behavioral deficits observed in animal models of Parkinson [115]. Rats with substantial nigra lesions were first induced with the drug apomorphine to exhibit the characteristic rotational behavior. Alginate microbeads containing chromaffin cells were

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then transplanted in the rat’s striatum. The authors reported a reduction by 68.2% in the numbers of rotations with the animals receiving the encapsulated chromaffin cells. Long-term recovery is also reported in these animals receiving encapsulated chromaffin cells.

6.3.3. Hybridoma cells The first successful industrial productions of monoclonal antibodies, (mAbs), from alginate polyL-lysine encapsulated hybridoma cells was reported in 1985 and 1986 by Rupp [116] and Posillico [117], respectively. The method used by these two groups is an adaptation to the original work performed by Lim [118] under the trade name of ENCAPSEL TM . The advantages associated with using the ENCAPSEL TM approach over conventional cell suspension cultures are higher starting purity of intracapsular antibody, a greater than 98% final purity level and an overall lower cost in manufacturing production. Multigrams of high purity mAbs from hybridoma cells can be efficiently produced using this alginate microencapsulation technology. Lee and Palsson have attributed the success of mAb production in alginate to the microenvironment milieu created by the defined cellular entrapment [119]. They postulated that this enclosed and restricted microenvironment prevents cells from genetic drift, and hence results in an improved stability in mAb production.

7. DNA encapsulation With recent advances in the field of gene therapy, new methods to efficiently deliver DNA oligonucleotides are being evaluated. There are two studies to date that report the potential application of alginate as an enteric delivery vehicle for DNA [120,31]. The encapsulation of DNA and its derivatives may be used in enteric targeting of nucleic acids as gene transfer agents, modified oligonucleotides and carriers for DNA-intercalators. In vitro studies showed that DNA can be successfully encapsulated and released at pH 6.5 without any denaturation of the DNA molecule [120]. DNA depurination, however, is not easily assessed by the technique the investigator used. Depurination of the molecule which

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occurs at low pH is desirable because it may assist in the release of drug in the stomach and duodenum. In another study the in vivo evaluation of encapsulated calf thymus DNA in chitosan–alginate microbeads through the GI tract is discussed [31]. The aim of this study was to demonstrate the feasibility of using alginate–chitosan encapsulated DNA as a target or carrier for evaluating intestinal carcinogens. High recoveries of intact chitosan–alginate microbeads are obtained from rat feces following gavage and GI transit. Needless to say, DNA encapsulation in alginate microbeads is still a relatively new field of research and we will expect to see more of this application in the near future.

8. Microsphere and liposome encapsulation Alginate gels have been used to encapsulate other delivery systems including microspheres and liposomes. Ethylcellulose microspheres were dispersed into an aqueous solution of sodium alginate which was subsequently dropped into a CaCl 2 solution [18]. The authors suggested that the beads could potentially be useful as an oral delivery system for micro- or nanoparticles. Liposomes that contained the model proteins BSA or horse-radish peroxidase were incorporated into alginate spheres with a diameter of 500–800 mm [121,122]. Prior to their entrapment, the liposomes were coated with either phospholipase C, D, or A 2 . The alginate microbeads that contained the liposomes remain stable at 108C. Upon heating to 378C, release of the protein is triggered by the enzymatic degradation of the phospholipids by the phospholipases. By selecting the appropriate phospholipase the duration of protein release could be controlled.

9. Summary The chemistry and relatively mild crosslinking conditions of alginate have enabled this naturally occurring biopolymer to be used for the encapsulation of a wide variety of biologically active agents including proteins, cells and DNA oligonucleotides. By selection of the appropriate alginate type, gelation conditions, added excipients, and coating agents,

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matrices of various morphologies, pore size, water content and dehydration rates can be fabricated. This high degree of flexibility can result in delivery of active agents over time periods ranging from minutes to months. As research and development continues with alginate polymeric delivery systems, we expect to see many innovative and exciting applications in the future.

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