(646c) Comparison of Ionically and Novel Covalently Crosslinked Alginate Microspheres

Breger, J. C. - Presenter, University of Maryland, College Park
Isayeva, I. - Presenter, Food and Drug Administration, Center of Devices and Radiological Health
Langone, J. J. - Presenter, Food and Drug Administration, Center of Devices and Radiological Health
Pollack, S. K. - Presenter, Food and Drug Administration, Center of Devices and Radiological Health
Wang, N. S. - Presenter, University of Maryland-College Park

Alginate is a popular biomaterial that is used in a number of applications including: cell encapsulation, drug delivery, and other combination devices. Alginate is a natural biopolymer composed of (1-->4)-linked β-D-mannuronate (M) and α-L-guluronate (G) units sequenced as block or alternating copolymers. One of the main reasons why alginate is popularly used in many tissue engineering applications is its ability to instantaneously gel in the presence of divalent cations such as calcium; providing a nontoxic environment to encapsulate therapeutic drugs or cells. Calcium crosslinked alginate has been most commonly used to encapsulate islets of Langerhans to produce a bioartificial pancreas as a treatment for Type I Diabetes. The crosslinked alginate serves as an immunoprotective barrier between the donor islets and the host's immune system; eliminating the need for immunosuppressive drugs. Alginate allows the diffusion of nutrients, hormone signals, and insulin in and out of the gel while blocking immune reactive species. However, one the problems in the development of the bioartificial pancreas via calcium alginate encapsulation is the questionable long term stability and durability of the microspheres (e.g., the potential exchange of calcium with sodium ions in the body). Over time, this ion exchange would cause the alginate microspheres to degrade; exposing donor islets to the host immune system.

?Click' chemistry was a term coined by Sharpless et al. to describe a class of chemical reactions that are modular, have high yields, generate inoffensive byproducts, are stereospecific, and use benign solvents. The ?cream of the crop' ?click' reaction is the Cu (I) catalyzed Huisgen 1,3-dipolar cycloaddition of azides and alkynes to form five membered 1,2,3-triazole rings. Since this reaction yields a chemical species that is impervious to hydrolysis or dimerization, it has been used in a number of applications including creating drug libraries, cellular labeling, and more. Currently, ?click' chemistry has not been applied to create covalently crosslinked alginate hydrogels for tissue engineering constructs.

The aim of this study is to synthesize covalently crosslinked alginate hydrogels for cell encapsulation using the ?click' chemistry approach. A covalent crosslink made via ?click' chemistry would not be susceptible to enzymatic hydrolysis or ion leaching; creating a new, longer lasting hydrogel. Select carboxylate groups of alginate were modified with either azide or alkyne functional groups. This was qualitatively confirmed using IR spectroscopy and NMR techniques.

Ionically and ?click' crosslinked microspheres of different diameters were created using an electrostatic microencapsulator. Microsphere size was controlled using the following parameters on the microencapsulator: voltage, pump speed, distance form gelation bath, and needle diameter. A few water swelling properties, i.e., volumetric swelling ratio and initial water content were studied to compare the degree of ionic and covalent crosslinking. The presence of covalent crosslinking was confirmed by potentiometric titration.

The volumetric swelling ratio and initial water content data suggest that ?click' and ionically crosslinked hydrogels exhibited a comparable degree of crosslinking under similar processing conditions. However, only ?click' crosslinked hydrogels were able to be rehydrated with d.i.H2O. EDTA potentiometric titration of ionically or ?click' crosslinked microspheres confirmed the presence of covalent crosslinks in ?click' microspheres compared to ionic crosslinks. ?Click' microsphere stored in EDTA maintained their integrity and shape during several months of storage and did not disintegrate, while ionically crosslinked microspheres dissolved in EDTA within minutes.

Insulin diffusion and cell survival were also employed to compare ionic versus ?click' crosslinked microspheres. RIN-5F is a rat insulinoma cell line that continuously produces insulin even in the absence of stimuli. This ability to continuously produce insulin was used to study the diffusion characteristics of ionic and ?click' crosslinked alginate microspheres. RIN-5F was encapsulated in either calcium or ?click' crosslinked alginate using a microencapsulator. Insulin produced by encapsulated RIN-5F and released through the capsule was measured over a period of time and compared to insulin produced by nonencapuslated RIN-5F cells grown at a similar cell density.

Further work is being done to quantify the degree of functionalization of alginate. Rheological measurements are planned to compare the elasticity and strength of ?click' and ionically crosslinked alginate hydrogels. The possibility of ionic and ?click' alginate co-crosslinking to achieve the optimal microsphere performance is also under investigation.


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