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Biocompatibility Evaluation of Different Alginates and Alginate-Based Microcapsules

G. Orive, A. M. Carcaboso, R. M. Hernandez, A. R. Gascon, and J. L. Pedraz*
Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of the Basque Country, Vitoria-Gasteiz, Spain Received October 1, 2004; Revised Manuscript Received December 10, 2004

Biocompatibility of biomaterials and biomaterial-based medical devices is a critical issue for the long-term function on multiple therapeutic systems. In the past few years, there has been an increasing interest in producing more biocompatible biomaterials and in developing novel assays to analyze the quality of the products. In this study, a battery of in vitro techniques to assess the biocompatibility of alginates with different compositions and purities and alginate-based microcapsules is presented. Study of the protein and polyphenol content of the alginates revealed clear differences between the nonpurified and the purified alginates. A similar behavior was observed when the mitogenic activity and the tumor necrosis factor-R secretion induced by the alginates were assessed. Interestingly, when the latter two techniques were adapted to evaluate the different alginate microcapsules, a correlation with the results obtained for the alginate samples was observed. These results reinforce the idea of using the full battery of assays here reported to screen alginates and alginate-based microcapsules before implantation. 1. Introduction Biomaterials are already having an enormous impact on medicine. The use of both natural and synthetic polymers involves multiple scientific disciplines including wound dressings,1 dental implants, tissue engineering,2 and microencapsulation of drugs, peptides, and cells as controlled drug delivery systems.3 However, to ensure the future clinical use of all these biomaterials, the current critical problems in purity, biocompatibility, and batch-to-batch reproducibility, especially in the case of naturally occurring polymers, must be addressed. To succeed, more efforts in developing fast, sensitive, and simple assays to validate the purification regimes and analyze the quality of the products are needed. Furthermore, these techniques should be used not only to study the biocompatibility properties of the biomaterials but also to evaluate the medical systems elaborated with them. In the present research, we present a battery of easy and fast in vitro techniques to assess the biocompatibility of natural and synthetic biomaterials. Interestingly, these assays can be also easily applied for the study of microcapsules elaborated with the same polymers, an approach that has not been addressed by other assays described in the field so far. Among all the possible biomaterials, we selected alginate as a model polymer, in part because purity and biocompatibility of alginates are obligatory requirements if their therapeutic use in human beings is intended.3 In addition, alginate-based microcapsules can be easily prepared by the extrusion of alginate droplets into millimolar concentrations of calcium or barium ions. The immobilization of cells and tissues within alginate-poly(L-lysine)-alginate (APA) micro* Corresponding author. Phone: +34 945-013091. Fax: +34 945013040. E-mail: knppemuj@vc.ehu.es.

capsules allows the correction of a wide range of hormone deficiency diseases without the need of immunosuppressants.3 However, as observed in small and large animal experiments, impurities in the alginates as well as irregularly shape microcapsules could favor the overgrowth of fibroblasts and macrophages over the capsules, leading to graft failure.4-7 Assuming this knowledge, five alginates with different compositions (guluronic/mannuronic acid ratio), purities, and viscosities were selected and their polyphenol and protein content were determined. The tumor necrosis factor-R (TNFR) production and the splenocyte proliferation induced by the different alginates was assessed using macrophages and splenocytes isolated from Balb/c mice. More interestingly, the latter two techniques were also employed in the biocompatibility study of APA microcapsules elaborated with each type of alginate. The results obtained corroborate the importance of selecting totally purified biomaterials if the development of biocompatible microcapsules is intended. The data presented here demonstrate the efficacy of using such a battery of assays to validate and analyze the quality and biocompatibility of both biomaterials and biomaterialbased encapsulation systems. 2. Materials and Methods 2.1. Materials. All tissue culture media and serum were purchased from Gibco BRL (Invitrogen S.A., Spain). All chemicals were purchased from Sigma Chemicals (St. Louis, MO) unless otherwise indicated. 2.2. Alginates. Five types of alginates were used in this study, a nonbiomedical grade, low viscosity, high mannuronic acid content alginate (LVM) obtained from Sigma

10.1021/bm049380x CCC: $30.25 2005 American Chemical Society Published on Web 02/25/2005

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Table 1. Chemical Compositions and Sequences Obtained by 1H NMR Spectra and SEC-MALLS Measurementsa alginate type LVGP LVMP MVGP MVMP
b

FG
0.68 0.44 0.69 0.43

FM
0.32 0.56 0.31 0.57

FGG FGM FMM

0.57 0.23 0.58 0.23 0.11 0.2 0.11 0.2 0.21 0.36 0.2 0.37

Mwb Mnb -3) ( 10-3) M /M b ( 10 w n

89 133 263 215 56 78 141 134 1.59 1.71 1.87 1.61

a Data was provided by NovaMatrix, FMC BioPolymer (Oslo, Norway). SEC-MALLS measurements.

Chemicals (St. Louis, MO), which has been used as the positive control, and four purified and biomedical grade alginates bought from FMC BioPolymers (Drammen, Norway): a low viscosity, high guluronic acid content alginate (LVGP), a low viscosity, high mannuronic acid content alginate (LVMP), a medium viscosity, high guluronic acid content alginate (MVGP), and a medium viscosity, high mannuronic acid content alginate (MVMP). Analytical data and polymer sequence distribution of the four purified alginates are presented in Table 1. 2.3. Microcapsule Elaboration. Five types of APA microcapsules were prepared for this study employing the different alginates. Each microcapsule was elaborated using the same type of alginate both for the matrix and for the outer membrane. Briefly, a 2% (w/v) solution of each alginate was extruded using an electrostatic droplet generator into a 0.05 M solution of CaCl2. Once gelled and washed, alginate particles were coated with a 0.05% (w/v) solution of poly(L-lysine) (PLL) for 5 min and then with another layer of the same alginate (0.1% w/v) for another 5 min. Finally, the microcapsules were placed in a saline solution. 2.4. Microscopic Evaluation of the Microcapsules. The diameter of the microcapsules prepared with each type of alginate was quantified, and their morphology and uniformity were assessed using an inverted optical microscope (Nikon TMS) equipped with a camera (Sony CCD-Iris). 2.5. Protein Determination. The protein content of the alginates was determined by the method of Bradford.8 A total of 100 L of Bradford reagent was added to 100 L of alginate solution (0.5% w/v). After 10 min of incubation, the absorption of the samples was photometrically measured at 560 nm using a microplate reader. For the quantitative evaluation of the protein content, bovine serum albumin (Sigma Chemicals, St. Louis, MO) was used for calibration. Results are expressed as protein weight versus total alginate weight (w/w) for five replicates. 2.6, Fluorescense Spectroscopic Analysis of Alginates. Phenolic compounds, the usual contaminants of alginates, were detected by their specific fluorescence with a SFM25 fluorimeter (Kontron Instruments, Zurich, Germany) follow ing the protocol of Skjk-Brk et al.9 The alginate samples were dissolved in ultrapure water (2% w/v). Employing 366nm fluorescence excitation, emission spectra was recorded at 450 nm (which represents the maximum fluorescence emission as we observed previously).10 Results are expressed as mean ( standard deviation for five replicates. 2.7. Mitogenic Activity of the Alginates and Alginate Microcapsules. The mitogenic activity of the alginates in vitro was determined by their impact on murine splenocyte

proliferation. Splenocytes were obtained from Balb/c mice as explained elsewhere.11 The cells were cultured at a suspension density of 1.5 106 cell/mL in RPMI medium supplemented with 10% fetal bovine serum, 2 mM Lglutamate, and 50 M -mercaptoethanol. A total of 100 L of this cell suspension and 25 L of each stimulus (medium as the negative control, concanavalin A as the positive control, and the alginate solutions or alginate microcapsules) were added to the wells of a 96-well plate. The cells were grown for 36 h at 37 C in a 5% CO2supplemented atmosphere. Lymphocyte stimulation was determined by a colorimetric assay for growth and viability [3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), Sigma Chemicals, St. Louis, MO].12 A total of 30 L of the 5 mg/mL MTT staining solution was added to each well of the cultured lymphocytes. After 4 h at 37 C, the plates were carefully centrifugated (10 min, 2400 rpm) and the supernatant was removed. Finally, 75 L of dimethyl sulfoxide was added to each well, and the plate was incubated for 1 min at 37 C. The purple solution was read on a microplate reader at a 560-nm test wavelength with a reference at 690 nm. The results are expressed as mean ( standard deviation for five replicates. 2.8. TNF-r Secretion Assay. Macrophages were obtained from the peritoneum of Balb/c mice. Briefly, 7 mL of cold RPMI culture medium was administered into the peritoneum of the mice. By washing the peritoneum carefully it is possible to obtain a rich macrophage suspension. This suspension was concentrated, and the macrophages were cultured in a 96-well plate. A total of 25 L of each stimulus (medium as the negative control, 5 g/mL LPS as the positive control, and the alginate solutions or alginate microcapsules) was added to the wells of a 96-well plate. The cells were grown for 12 h at 37 C in a 5% CO2-supplemented atmosphere. Finally, the supernatant was removed, and the TNF-R content was quantified using an ELISA. 3. Results 3.1. Protein and Polyphenol Content Determination. As observed in Figure 1A, the Bradford protein determination assay showed that the total protein content in relation with the alginate weight was clearly higher for the nonpurified LVM alginate. In fact, all the purified alginates (LVGP, LVMP, MVGP, and MVMP) presented a reduction of more than 40% in their protein content in comparison with the nonbiomedical grade alginate. Similarly, the fluorescence determination assay revealed that polyphenolic-like compounds, proteins, and other impurities in purified alginates were lower in relation to nonbiomedical grade LVM alginates (Figure 1B). The presence of residues, which exhibited emission at 450 nm (in arbitrary fluorescence units), in LVMP and MVGP was 79 and 86% lower in comparison with that of the nonbiomedical grade LVM alginates, while in the case of LVGP and MVMP the polyphenol content was decreased by 96 and 97%, respectively. These results are consistent with those of other research groups,13 reflecting that the purification process of the alginates reduces considerably the impurity content of the alginates.

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Figure 3. Release of TNF-R from murine macrophages stimulated by the alginates at two different concentrations. Mean values ((SD) of five replicates are shown.

Figure 1. Protein (A) and polyphenol (B) content of alginates with different compositions, purities, and viscosities.

Figure 2. Proliferation of alginate-stimulated splenocytes calculated by the MTT assay. Results were standardized by dividing the absorbance values obtained for alginate-stimulated cultures (increments with reference to the absorbances of nonstimulated cultures) by those of the ConA-stimulated controls as the maximum value. Mean values ((SD) of five replicates are shown. N.D.: not detectable.

3.2. Activation of Splenocyte Proliferation by the Alginates. The mitogenic activity of all the alginates at the different concentrations was studied by their impact on Balb/c splenocyte proliferation. The peak responses of splenocytes to contaminants of the alginates are shown in Figure 2. Interestingly, nonbiomedical grade alginate showed the highest splenocyte proliferation as a result of its higher impurity content, a feature that has also been described by others.14 Regarding the low viscosity purified alginates, no detectable mitogenic response was obtained when the lowest concentration was tested (0.1% w/v). LVMP and MVGP purified alginates induced certain splenocyte proliferation which could be related with their higher content in polyphenolic-like compounds (Figure 1B). Furthermore, we observed that the purity of individual alginate preparation, rather than its chemical composition (mannuronic and

guluronic acid content), is probably of greater importance in determining the mitogenic acitivity. 3.3. TNF-r Production Induced by the Alginates. The release of TNF-R from murine macrophages stimulated by the different alginates was analyzed as described above. The cytokine data obtained revealed important differences between the purified and the nonbiomedical grade alginates (Figure 3). In fact, the production of the proinflammatory cytokine TNF-R was approximately 100 times higher in the case of nonbiomedical grade alginate in comparison with the purified ones. The in vivo induction of proinflammatory cytokines by the alginates may be advantageous in some clinical situation such as the fabrication of wound dressings1 but contra-indicated in other clinical approaches including the design of cell microencapsulation systems for the controlled and continuous delivery of therapeutic products.16 In the latter situation, the induction of an inflammatory signal by the alginates could promote fibroblast growth over the capsules which will impair effective secretion of the therapeutic protein, while also causing a metabolic obstacle to implant by decreasing diffusion of oxygen, leading to graft failure.17 Additionally, we did not observe differences in TNF-R stimulation among the purified alginates as a consequence of their viscosity or composition. As can be observed in Figure 3, all the purified alginates were weak inducers of TNF-R production because none of them produced concentrations higher than 10 pg/mL. 3.4. Microcapsule Elaboration and Characterization. Five types of APA microcapsules were prepared using the different alginates. Each type of capsule was prepared employing the same alginate both for the matrix and for the outer membrane. Photographs of each type of microcapsule are shown in Figure 4. Results showed that all types of microcapsules appeared totally spherical and uniform with a mean diameter ranging from 514 m for LVGP and 529 m for LVMP to 541 m for nonbiomedical grade LVM alginates. Finally, alginates of higher viscosity generated microcapsules with increased diameters: 574 m and 602 m for MVGP and MVMP, respectively. 3.5. Splenocyte Proliferation and TNF-R Production Induced by the Alginate Capsules. The principal goal of this work was to adapt techniques and assays usually employed in alginate characterization to the biocompatibility

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Figure 6. TNF-R secretion from murine macrophages induced by the APA microcapsules elaborated with the different alginates. Mean values ((SD) of five replicates are shown. Figure 4. Photographs of the microcapsules elaborated with the five types of alginates: the nonpurified LVM and the purified LVGP, LVMP, MVGP, and MVMP. Photographs were taken using an inverted optical microscope equipped with a camera. Magnification: 200. The scale bar is 210 m.

4. Discussion In the past few years, researchers have understood the importance of using totally biocompatible polymers, which do not interfere with cell homeostasis, in the elaboration of polymer-based devices for therapeutic purposes. In essence, it is necessary to produce purified and reproducible biomaterials and polymer-based devices. Furthermore, we need sensitive in vitro assays and protocols to validate and screen both the biomaterials and the medical systems elaborated with them. Recently, several assays have been tested for purification of alginates including the use of chemical reagents and dialysis,11 the induction of apoptosis in Jurkat cells, and the lymphocyte proliferation assay.13 Furthermore, a wide spectrum of techniques have been optimized to analyze the morphology of polymer-based microcapsules including confocal laser scanning microscopy to study the distribution of the alginate and PLL in intact microcapsules,18 advanced nuclear magnetic resonance (NMR) and atomic force microscopy to give clear-cut evidence of the capsular surface topography,19 and Fourier transform infrared spectroscopy and the X-ray photoelectron to analyze the reaction against the microcapsules in the immediate posttransplant period.6,7 Here, we report a battery of assays useful to test the in vitro biocompatibility of biomaterials and biomaterial-based devices. For that purpose, alginates from different sources and with different compositions, purities, and viscosities were selected and screened because these polymers have gained the attention of scientists as a result of their multiple therapeutic applications.22 In particular, alginates can be used as carriers for cell immobilization and subsequent implantation. The entrapment of cells in APA microcapsules represents an alternative drug delivery system in which cells are immunoisolated from the hosts immunoresponse while they maintain a controlled and continuous delivery of therapeutic products. However, this therapeutic strategy might be limited due to the poor biocompatibility of the capsule-forming materials, leading to an overgrowth of collagen-secreting fibroblasts and activated macrophages secreting growth regulatory cytokines over the capsules.23 In this research, a splenocyte proliferation test and a TNF-R production assay have been adapted to evaluate alginates and alginate-based microcapsules. By measuring the mitogenic activity and TNF-R release induced by the

Figure 5. Mitogenic activity induced by APAmicrocapsules elaborated with the different alginates (the nonpurified LVM and the purified LVGP, LVMP, MVGP, and MVMP). Represented values are the percentage of absorbance for alginate-stimulated cultures relative to the absorbance of ConA-stimulated ones. Mean values ((SD) of five replicates are shown.

study of alginate-based microcapsules. To verify this, the splenocyte proliferation and TNF-R production induced by each type of alginate microcapsule was determined and compared with the alginate solution. One of the main findings we observed was that nonpurified microcapsules induced the highest mitogenic activity (Figure 5) and TNF-R secretion (Figure 6). These results are similar to those obtained with the alginate solutions (Figures 2 and 3). Although the relative splenocyte proliferation stimulated by the capsules was lower than that of the alginate solutions, the mitogenic activity of nonbiomedical grade capsules was more than 50% higher than that of purified alginates (Figure 5). Similarly, nonbiomedical grade microcapsules induced the strongest TNF-R release compared with the purified alginates. Interestingly, we did not find important differences among the purified alginates, confirming the idea that impurity content more than alginate composition is also the major factor in determining the biocompatibility of the microcapsules.10 These results raise the possibility of using both techniques for the complementary study of both polymers and polymer-based immobilization devices.

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(6) De Vos, P.; Van Hoogmoed, C. G.; De Haan, B. J.; Busscher, H. J. Tissue responses against immunoisolating alginate-PLL capsules in the immediate posttransplant period. J. Biomed. Mater. Res. 2002, 62, 430. (7) Van Hoogmoed, C. G.; Busscher, H. J.; De Vos, P. Fourier transform infrared spectroscopy studies of alginate-PLL capsules with varying compositions. J. Biomed. Mater. Res. 2003, 67, 172. (8) Bradford, M. M. A rapid and sensitive method for the quantification of microgram quantities of protein, utilizing the principle of proteindye binding. Anal. Biochem. 1976, 248. (9) Skjk-Brk, G.; Murano, E.; Paoletti, S. Alginate as immobilization material. II: determination of polyphenol conatminants by fluorescence spectroscopy, and evaluation of methods for their removal. Biotechnol. Bioeng. 1989, 33, 90. (10) Orive, G.; Ponce, S.; Hernandez, R. M.; Gascon, A. R.; Igartua, M.; Pedraz, J. L. Biocompatibility of microcapsules for cell immobilization elaborated with different type of alginates. Biomaterials 2002, 23, 3825. (11) Klock, G.; Pfeffermann, A.; Ryser, C.; Grohn, P.; Kuttler, B.; Hahn, H. J.; Zimmermann, U. Biocompatibility of mannuronic acid-rich alginates. Biomaterials 1997, 18, 707. (12) Uludag, H.; Sefton, M. V. Colorimetric assay for cellular activity in microcapsules. Biomaterials 1990, 11, 708. (13) Leinfelder, U.; Brunnenmeier, F.; Cramer, H.; Schiller, J.; Arnold, K.; Vasquez, J. A.; Zimmermann, U. A highly sensitive cell assay for validation of purification regimes of alginates. Biomaterials 2003, 24, 4161. (14) Klock, G.; Frank, H.; Houben, R.; Zekorn, T.; Horcher, A.; Siebers, U.; Wohrle, M.; Federlin, K.; Zimmermann, U. Production of purified alginate suitable for use in immunoisolated transplantation. Appl. Microbiol. Biotechnol. 1994, 40, 638. (15) Deleted in proof. (16) Orive, G.; Hernandez, R. M.; Gascon, A. R.; Igartua, M.; Pedraz, J. L. Cell microencapsulation technology for biomedical purposes: novel insights and challenges. Trends Pharm. Sci. 2003, 24, 207. (17) Cole, D. R.; Waterfall, M.; McIntyre, M.; Baird, J. D. Microencapsulated islet grafts in the BB/E rat: a possible role for cytokines in graft failure. Diabetologia 1992, 35, 231. (18) Strand, B. L.; Mrch, Y. A.; Espevik, T.; Skjk-Brk, G. Visualization of alginate-poly-L-lysine alginate microcapsules by confocal laser scanning microscopy. Biotechnol. Bioeng. 2003, 82, 386. (19) Zimmermann, H.; Hillgartner, M.; Manz, B.; Feilen, P.; Brunnen meineier, F.; Leinfelder, U.; Weber, M.; Cramer, H.; Schneider, S.; Hendrich, C.; Volke, F.; Zimmermann, U. Fabrication of homogeously cross-linked, functional alginate microcapsules validated by NMR-, CLSM- and AFM-imaging. Biomaterials 2003, 24, 2083. (20) Deleted in proof. (21) Deleted in proof. (22) Drury, J. L.; Mooney, D. J. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 2003, 24, 4337. (23) Rihova, B. Immunocompatibility and biocompatibility of cell delivery systems. AdV. Drug DeliVery ReV. 2000, 42, 65. (24) Kulseng, B.; Skjk-Brk, G.; Flling, I.; Espevik, T. TNF production from peripheral blood mononuclear cells in diabetic patients after stimulation with alginate and lipopolysaccharide. Scand. J. Immunol. 1996, 43, 335. (25) Tracey, K. J.; Cerami, A. Tumor necrosis factor: a pleiotropic cytokine and therapeutic target. Annu. ReV. Med. 1994, 45, 491. (26) Zimmermann, U.; Klock, G.; Federlin, K.; Haning, K.; Kowaslski, M.; Bretzel, R. G.; Horcher, A.; Entenmann, H.; Siebers, U.; Zekorn, T. Production of mitogen contamination free alginates with variable rations of mannuronic to guluronic acid by free flow electrophoresis. Electrophoresis 1992, 13, 269.

polymer solutions and the microcapsules, a valid tool to test and compare the biomaterials and capsules before implantation could be obtained. In fact, the TNF-R is such a potent proinflammatory cytokine that it is known to activate leucocytes, stimulate fibroblast proliferation, promote migration of inflammatory cells into the intercellular matrix, and trigger local secretion of other proinflammatory cytokines.24,25 According to the results obtained, the protein and polyphenol content assays as well as the splenocyte proliferation and TNF-R production tests are valid and sensitive techniques for the in vitro biocompatibility assessment of alginates. In all the cases, the nonbiomedical grade alginate was less biocompatible because of its higher impurity content. In addition, we did not observe biocompatibility differences as a consequence of the alginate composition or purity, reinforcing the idea that impurity content is the major factor in determining the biocompatibility properties of the alginates. This conclusion agrees perfectly with that expressed by others26 and by our group in a previous work.10 One major observation in this work is that the splenocyte proliferation test and the TNF-R production assay can be applied to the analysis of both alginates and alginate microcapsules. More interestingly, the data obtained from these experiments confirmed the idea that selection of purified alginates is essential if the elaboration of biocompatible microcapsules is desired. In summary, a full battery of assays to screen alginates and alginate-based microcapsules have been reported. This could shed light on the urgent need for sensitive and fast assays to validate and ensure the reproducibility of biomaterials and biomaterial-based medical devices. The combination of these approaches could allow the identification of clinical grade alginates and capsules suitable for implantation. Acknowledgment. This project was partially supported by the Ministry of Education, Culture and Sports of Spain (CYCYT SAF2002-02268) and by the University of the Basque Country (UPV/EHU; 9/UPV 00101.125-13496/ 2001). References and Notes
(1) Thomas, A.; Harding, K. G.; Moore, K. Alginates from wound dressings activate human macrophages to secrete tumour necrosis factor-R. Biomaterials 2000, 21, 1797. (2) Vacanti, J. P.; Langer, R. Tissue engineering: the design and fabrication of living replacement devices for surgical reconstruction and transplantation. Lancet 1999, 354, 32. (3) Orive, G.; Hernandez, R. M.; Gascon, A. R.; Calafiore, R.; Chang, T. M. S.; De Vos, P.; Hortelano, G.; Hunkeler, D.; Lack, I.; Shapiro, A. M. J.; Pedraz, J. L. Cell encapsulation: promise and progress. Nat. Med. 2003, 2, 104. (4) Van Schilfgaarde, R.; De Vos, P. Factors influencing the properties and performance of microcapsules for immunoprotection of pancreatic islets. J. Mol. Med. 1999, 77, 199. (5) De Vos, P.; De Haan, B.; Van Schilfgaarde, R. Effect of the alginate composition on the biocompatibility of alginate-polylysine microcapsules. Biomaterials 1999, 18, 2273.

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