Cross-Linked Alginate Film Pore Size Determination Using Atomic Force Microscopy and Validation Using Diffusivity Determinations

Cheryl Simpliciano, Larissa Clark, Behrokh Asi, Nathan Chu, Maria Mercado, Steven Diaz, Michel Goedert, Maryam Mobed-Miremadi

Department of Biomedical, Chemical and Materials Engineering, San Jose State University, San Jose, USA..

DOI: 10.4236/jsemat.2013.34A1001                

Abstract

The deficit of organ donors has fueled the need for advances in tissue engineering and regenerative medicine. Microencapsulation in alginate immuno-isolation membranes has been used to treat many disabling metabolic disorders, namely, phenylketonuria, kidney failure and diabetes mellitus. Systematic nutrient flux determinations are hindered by the lack of experimental data on alginate-based membrane topography and the pore size thus preventing the full therapeutic potential of the bio-membranes to be reached. In this study, samples of cross-linked alginate membranes were subjected to the following analytical characterization: 1) pore size characterization using atomic force microscopy operated in contact mode to detect and measure pore size; 2) differential scanning calorimetry to confirm biopolymer cross-linking; and 3) diffusivity measurements using spectrophotometry and fluorescence microscopy to confirm the presence of through pores and to calculate reflection coefficients. The pore sizes for the pre-clinical standard formulation of 1.5% (w/v) medium viscosity alginate cross-linked with 1.5% CaCl₂ and 0.5% (w/v) alginate and chitosan cross-linked with 20% CaCl₂ are 5.2 nm ± 0.9 nm and 7.0 nm ± 3.1 nm, respectively. An increase in the glass transition temperatures as a function of cross-linker concentration was observed. Diffusivity values obtained from the inward diffusivity of creatinine into macrocapsules (d = 1000 μm ± 75 μm) and the outward diffusivity of FITC dextrans from macrocapsules (d = 1000 μm ± 75 μm) and microcapsules (d = 40 μm ± 5 μm) were shown to correlate strongly (R² = 0.9835) with the ratio of solute to pore sizes, confirming the presence of through pores. Reflection coefficients approaching and exceeding unity correlate with the lack of permeability of the membranes to MW markers that are 70 kDa and greater.

Keywords

Alginate; Atomic Force Microscopy; Pore Size; Stokes’ Radius; Diffusivity; Cross-linking; Differential Scanning Calorimetry; Reflection Coefficient

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	T. R. Hoare and D. S. Kohane, “Hydrogels in Drug Delivery: Progress and Challenges,” Polymer, Vol. 49, No. 8, 2008, pp. 1993-2007. http://dx.doi.org/10.1016/j.polymer.2008.01.027
[2]	T. M. S. Chang, “Artificial Cells: Biotechnology, Nanomedicine, Regenerative Medicine, Blood Substitutes, Bioencapsulation and Cell/Stem Cell Therapy—Vol. 1,” World Scientific Publishing Co. Pte. Ltd., Singapore City, 2007.
[3]	H. Li, “Smart Hydrogel Modelling,” Springer, Singapore City, 2009. http://dx.doi.org/10.1007/978-3-642-02368-2
[4]	A. Paul, G. Chen, A Khan, V. T. Rao, D. Shum-Tim and S. Prakash, “Genipin-cross-linked Microencapsulated Human Adipose Stem Cells Augment Transplant Retention Resulting in Attenuation of Chronically Infarcted Rat Heart Fibrosis and Cardiac Dysfunction,” Cell Transplantation, Vol. 21, No. 12, 2012, pp. 2735-2751. http://dx.doi.org/10.3727/096368912X637497
[5]	M. Sridhar-Keralapura, S. Thirumalai and M. MobedMiremadi, “Structural Changes and Imaging Signatures of Acoustically Sensitive Microcapsules under Ultrasound,” Ultrasonics, Vol. 53, No. 25, 2013, pp. 1044-1057. http://dx.doi.org/10.1016/j.ultras.2013.02.001
[6]	J. Zhu and R. E. Marchant, “Design Properties of Hydrogel Tissue-engineering Scaffolds,” Expert Review of Medical Devices, Vol. 8, No. 5, 2011, pp. 607-626. http://dx.doi.org/10.1586/erd.11.27
[7]	N. Wang, G. Adams, L. Buttery, F. H. Falcone and S. Stolnik, “Alginate Encapsulation Technology Supports Embryonic Stem Cell,” Journal of Biotechnology, Vol. 144, No. 4, 2009, pp. 304-312. http://dx.doi.org/10.1016/j.jbiotec.2009.08.008
[8]	I. Braccini, R. P. Grasso and S. Pérez, “Conformational and Configurational Features of Acidic Polysaccharides and Their Interactions with Calcium Ions: A Molecular Modeling Investigation,” Carbohydrate Research, Vol. 317, No. 1, 1999, pp. 119-130. http://dx.doi.org/10.1016/S0008-6215(99)00062-2
[9]	H. Uludag, P. De Vos and P. A. Tresco, “Technology of Mammalian Cell Encapsulation,” Advanced Drug Delivery Reviews, Vol. 42, No. 1, 2000, pp. 29-64. http://dx.doi.org/10.1016/S0169-409X(00)00053-3
[10]	H. Zimmermann, F. Wahlisch, C. Baier, N. Westhoff, R. Reuss, D. Zimmermann, M. Behringer, F. Ehrhart, A. Katsen-Globa, C. Giese, U. Marx, V. L. Sukhorukov, J. A. Vásquez, P. Jakob, S. G. Shirley and U. Zimmermann, “Physical and Biological Properties of Barium Crosslinked Alginate Membranes,” Biomaterials, Vol. 28, No. 7, 2006, pp. 1327-1345. http://dx.doi.org/10.1016/j.biomaterials.2006.11.032
[11]	H. Chen, W. Ouyang, B. Lawuyi and S. Prakash, “Genipin Cross-Linked Alginate-Chitosan Microcapsules: Membrane Characterization and Optimization of Cross-Linking Reaction,” Biomacromolecules, Vol. 7, No. 7, 2006, pp. 2091-2098. http://dx.doi.org/10.1021/bm050862y
[12]	A. Tamayol, M. Akbari, N. Annabi, A. Paul, A. Khadamhosseini and D. Juncker, “Fiber-Based Tissue Engineering: Progress, Challenges, and Opportunities,” Biotechnology Advances, Vol. 31, No. 5, 2012, pp. 669-687. http://dx.doi.org/10.1016/j.biotechadv.2012.11.007
[13]	M. Mobed-Miremadi, “High Throughput Methods for Miniaturization of Implantable Artificial Cells,” In: T. M. S. Chan, Ed., Selected Topics in Nanomedicine: Regenerative Medicine, Artificial Cells and Nanomedicine—Vol. 3, World Scientific Publishing Co. Pte. Ltd., Singapore City, in Press.
[14]	Y. K. Demir, Z. Akan and O. Kerimoglu, “Sodium Alginate Microneedle Arrays Mediate the Transdermal Delivery of Bovine Serum Albumin,” PLoS ONE, Vol. 8, No. 5, 2013, Article ID: e63819. http://dx.doi.org/10.1371/journal.pone.0063819
[15]	W. R. Gombotz and S. Wee, “Protein Release from Alginate Matrices,” Advanced Drug Delivery Reviews, Vol. 31, No. 3, 1998, pp. 267-285. http://dx.doi.org/10.1016/S0169-409X(97)00124-5
[16]	M. A. J. Mazumder, N. A. D. Burke, F. Shen, T. Chu, M. A. Potter and H. D. H. Stover, “Synthetic Reactive Polyelectrolytes for Cell Encapsulation,” ACS Symposium Series, Oxford University Press, Cary, 2010, pp. 131-159. http://dx.doi.org/10.1021/bk-2010-1053.ch007
[17]	R. Russo, M. Malinconico and G. Santagata, “Effect of Cross-Linking with Calcium Ions on the Physical Properties of Alginate Films,” Biomacromolecules, Vol. 8, No. 10, 2007, pp. 3193-3197. http://dx.doi.org/10.1021/bm700565h
[18]	S. Ahn, H. Lee, L. J. Bonassar and G. Kim, “Cells (MC3T3E1)-Laden Alginate Scaffolds Fabricated by a Modified Solid-Freeform Fabrication Process Supplemented with an Aerosol Spraying,” Biomacromolecues, Vol. 13, No. 9, 2012, pp. 2997-3003. http://dx.doi.org/10.1021/bm3011352
[19]	R. L. Fournier, “Basic Transport Phenomena in Biomedical Engineering,” 3rd Edition, Taylor & Francis, Philadelphia, 2011.
[20]	S. Amin, S. Rajabnezhad and K. Kohli, “Hydrogels as Potential Drug Delivery Systems,” Scientific Research and Essay, Vol. 3, No. 11, 2011, pp. 1175-1183.
[21]	N. W. Choi, M. Cabodi, B. Held, J. P. Gleghorn, L. J. Bonassar and A. D. Strook, “Microfluidic Scaffolds for Tissue Engineering,” Nature Materials, Vol. 6, No. 11, 2007, pp. 908-915. http://dx.doi.org/10.1038/nmat2022
[22]	A. W. Chan and R. J. Neufeld, “Tuneable Semi-Synthetic Network Alginate for Absorptive Encapsulation and Controlled Release of Protein Therapeutics,” Biomaterials, Vol. 31, No. 34, 2010, pp. 9040-9047. http://dx.doi.org/10.1016/j.biomaterials.2010.07.111
[23]	R. H. Li, D. H. Altreuter and F. T. Gentile, “Transport Characterization of Hydrogel Matrices for Cell Encapsulation,” Biotechnology and Bioengineering, Vol. 50, No. 4, 1996, pp. 365-373. http://dx.doi.org/10.1002/(SICI)1097-0290(19960520)50:4<365::AID-BIT3>3.0.CO;2-J
[24]	B. Wright, R. A. Cave, J. P. Cook, V. V. Khutoryansky, S. Mi, B. Chen, M. Leyland and C. J. Connon, “Enhanced Viability of Corneal Epithelial Cells for Efficient Transport/storage Using a Structurally Modified Calcium Alginate Hydrogel,” Regenerative Medicine, Vol. 7, No. 3, 2012, pp. 295-307. http://dx.doi.org/10.2217/rme.12.7
[25]	A. Jejurikar, G. Lawrie, D. Martin and L. Grøndahl, “A Novel Strategy for Preparing Mechanically Robust Ionically Cross-Linked Alginate Hydrogels,” Biomedical Materials, Vol. 6., No. 2, 2011, Article ID: 025010. http://dx.doi.org/10.1088/1748-6041/6/2/025010
[26]	S. X. Hsiong, P. H. Cooke, H. Kong, M. L. Fishman, M. Ericsson and D. J. Mooney, “AFM Imaging of RGD Presenting Synthetic Extracellular Matrix Using Gold Nanoparticles,” Macromolecular Bioscience, Vol. 8, No. 6, 2008, pp. 469-477. http://dx.doi.org/10.1002/mabi.200700313
[27]	T. Schmid, J. Burkhard, B. Yeo, W. Zhang and R. Zenobi, “Towards Chemical Analysis of Nanostructures in Biofilms I: Imaging of Biological Nanostructures,” Analytical and Bioanalytical Chemistry, Vol. 391, No. 5, pp 1899-1905. http://dx.doi.org/10.1007/s00216-008-2100-2
[28]	A. Leal-Egana, U. Braumann, A. Díaz-Cuenca, M. Nowicki and A. Bader, “Determination of Pore Size Distribution at the Cell-hydrogel Interface,” Journal of Nanobiotechnology, Vol. 9, No. 24, 2011, pp. 1-7. http://dx.doi.org/10.1186/1477-3155-9-24
[29]	M. Mobed-Miremadi, B. Asi, J. Parasseril, E. Wong, M. Tat and Y. Shan, “Comparative Diffusivity Measurements for Alginate-Based Atomized and Inkjet-Bioprinted Artificial Cells Using Fluorescence Microscopy,” Artificial Cells, Nanomedicine, and Biotechnology, Vol. 41 No. 3, 2013, pp. 196-201. http://dx.doi.org/10.3109/10731199.2012.716064
[30]	M. Mobed-Miremadi and S. Dharba, “Immobilization of R. Erythropolis in Alginate-Based Artificial Cells for Simulated Plaque Degradation in Aqueous Media,” Taylor & Francis, Philadelphia. http://dx.doi.org/10.3109/02652048.2013.814726
[31]	S. Prakash and T. M. S. Chang, “Preparation and In-Vitro Analysis of Genetically Engineered E. coli DH5 Cells Microencapsulated in Artificial Cells for Urea and Ammonia Removal,” Biotechnology and Bioengineering, Vol. 46, No. 6, 1995, pp. 621-626. http://dx.doi.org/10.1002/bit.260460615
[32]	M. Mobed-Miremadi, E. Acks, S. Polsaward and D. Chen, “High Throughput Miniaturization of Artificial Cells,” Artificial Cells, Nanomedicine, and Biotechnology, Vol. 39, No. 5, 2011, pp. 310-316. http://dx.doi.org/10.3109/10731199.2011.574637
[33]	M. F. A. Goosen, “Fundamentals of Animal Cell: Encapsulation and Immobilization,” CRC Press, Boca Raton, 1993.
[34]	Y. Shan, “Viability of Microencapsulation of E. coli DH5 for Urea Removal Using Inkjet Bio-Printing,” Masters Project Report, San Jose State University, San Jose, 2011. http://generalengineering.sjsu.edu/docs/pdf/mse_prj_rpts/fall2011/VIABILITY_OF_MICROENCAPSULATION_OF_E.COLI_DH5_FOR_UREA.pdf
[35]	L. Muscariello, F. Rosso, G. Marino, A. Giordano, M. Barbarisi, G. Cafiero and A. Barbarisi, “A Critical Overview of ESEM Applications in the Biological Field,” Journal of Cellular Physiology, Vol. 205, No. 3, 2005, pp. 328-334. http://dx.doi.org/10.1002/jcp.20444
[36]	M. Rubert, M. Monjo, S. P. Lyngstadaas and J. M. Ramis, “Effect of Alginate Hydrogel Containing PolyprolineRich Peptides on Osteoblast Differentiation,” Biomedical Materials, Vol. 7, No. 5, 2012, Article ID: 055003. http://dx.doi.org/10.1088/1748-6041/7/5/055003
[37]	Z. Keresztes, T. Rigo, J. Telegdi and E. Kalman, “Investigation of Biopolymer Networks by Means of AFM,” Applied Physics A, Materials Science & Processing, Vol. 72, Suppl. 1, 2001, pp. S113-S116. http://dx.doi.org/10.1007/s003390100680
[38]	S. K. H. Gulrez, S. Al-Assaf and G. O. Phillips, “Hydrogels: Methods of Preparation, Characterisation and Appliation,” In: A. Carpi, Ed., Progress in Molecular and Environmental Bioengineering—From Analysis and Modeling to Technology Applications, Intech Europe, Croatia, 2011, pp. 117-150. http://www.intechopen.com/books/progress-in-molecularand-environmental-bioengineering-from-analysis-and-modelng-to-technology-applications/hydrogels-methods-of-preparation-characterisation-and-applications
[39]	V. Urquiza, K. Turner, O. Perales Pérez and M. Gálvez Saldana, “Effect of the Cross-Linking with Calcium Ions on the Structural and Thermo-Mechanical Properties of Alginate Films,” MRS Proceedings, Vol. 1355, No. 1, Cambridge University Press, Cambridge, 2011. http://dx.doi.org/10.1557/opl.2011.1136
[40]	S. J. Kim, S. G. Yoon and S. I. Kim, “Synthesis and Characteristics of Interpenetrating Polymer Network Hydrogels Composed of Alginate and Poly (Diallydimethylammonium Chloride),” Journal of Applied Polymer Science, Vol. 91, No. 6, 2004, pp. 3705-3709. http://dx.doi.org/10.1002/app.13615
[41]	K. Nakamura, T. Hatakeyama and H. Hatakeyama, “Relationship between Hydrogen Bonding and Bound Water in Polyhydroxystyrene Derivatives,” Polymer, Vol. 24, No. 7, 1983, pp. 871-876. http://dx.doi.org/10.1016/0032-3861(83)90206-9
[42]	T. S. Pathak, J. H. Yun, J. Lee and K. J. Paeng, “Effect of Calcium Ion (Cross-Linker) Concentration on Porosity, Surface Morphology and Thermal Behavior of Calcium Alginates Prepared from Algae (Undaria pinnatifida),” Carbohydrate Polymers, Vol. 81, No. 3, 2010, pp. 633-639. http://dx.doi.org/10.1016/j.carbpol.2010.03.025
[43]	S. Shabbear, S. Ramanamurthy and K. V. Ramanamurthy, “Formulation and Evaluation of Chitosan Sodium Alginate Microcapsules of 5-Fluorouracil for Colorectal Cancer,” International Journal of Research in Pharmacy and Chemistry, Vol. 2, No. 1, 2012, pp. 7-19. http://dx.doi.org/10.1186/2194-0517-2-1
[44]	S. J. Florczyk, D. J. Kim, D. L. Wood and M. Zhang, “Influence of Processing Parameters on Pore Structure of 3D Porous Chitosan-Alginate Polyelectrolyte Complex Scaffolds,” Journal of Biomedical Materials Research Part A, Vol. 98, No. 4, 2011, pp. 614-620. http://dx.doi.org/10.1002/jbm.a.33153
[45]	J. Shaji, V. Jain and S. Lodha, “Chitosan: A Novel Pharmaceutical Excipient,” International Journal of Pharmaceutical Excipient and Applied Sciences, Vol. 1, No. 1, 2010, pp. 11-28.
[46]	S. Prakash and C. Martoni, “Toward a New Generation of Therapeutics,” Applied Biochemistry and Biotechnology, Vol. 128, No. 1, 2006, pp. 1-22. http://dx.doi.org/10.1385/ABAB:128:1:001