(121e) Drug Release Properties of the Enzyme Chondroitinase Cabc from Plga Nanospheres into the Spinal Cord Injury Area

The Spinal Cord Injury has severe impact on millions of people around the world. It has been historically believed that finding a cure for the SCI to be nearly impossible. However, increasing number of research has been revealed that there is a great hope for a cure for patients with SCI. It is currently known that neurons have ability to regenerate their own injured axons. However, the local environment in the lesion is not hospitable for regeneration due to the inhibitory molecules derived from several cell types in the glial scar. After the Injury, the Chondroitin Sulfate Proteoglycan (CSPG), an extracellular matrix produced by various cells in the area, is the major type of inhibitory molecule in the glial scar.

The axonal regeneration may be enhanced if the amount of CSPG produced after the SCI can be decreased. Since the enzyme chondroitinase (cABC) can degrades CSPG molecules, cABC's controlled release in the region of the spinal cord injury may be of considerable therapeutic value.

The enzyme cABC loaded PLGA nanospheres are fabricated using double emulsion technique. Then, in vitro release experiments are performed. Concentrations of cABC and CS are measured using an improved dimetylmethylene blue (DMMB) method.

If cABC is added to a solution of CS or CSPG containing DMMB, the enzyme degrades the CS chains, and the course of the degradation can be traced by following the decrease in absorbance at 525 nm. If the enzymatic degradation follows traditional Michaelis-Menten kinetics, the rate of degradation is expected to be proportional to the concentration of cABC and independent of the CS or CSPG concentration, if these concentrations are much greater than the saturation constant, K_s:

dS/dt=kE₀(S/(K_s+S)) (1) dS/dt=kE₀ if S>>Ks (2)

This relationship can be used to measure the cABC concentration, since a calibration curve can be prepared relating the rate of degradation of an unknown concentration of CS or CSPG and the concentration of cABC.

Enzyme loaded PLGA nanospheres are fabricated with two different initial concentrations of cABC. (Set-1): 300 microliters of 5 unit cABC and (Set-2): 150 microliters of 10 units of cABC. A typical release experiment was done by adding 5 mg of nanospheres to 1000 microliters of Phosphate Buffer Solution (PBS) and incubating at 37 ^oC. Each set of experiments was done in triplicates (total 6 tubes). Periodically, 100 microliters of sample from each tube was taken and mixed with 1 ml of 50 mg/ml CS to perform degradation experiments as previously explained. 100 microliters of fresh PBS were added to the release tubes to compensate the lost volume. This routine procedure was continued about 30 days and total of 72 digestion experiments was carried out.

Cumulative cABC release was calculated for both two sets of experiments. The amount of enzyme released for the first set of experiments is 7.03x10^-03 units and for the second set of experiments is 7.16x10^-03 units. The maximum amount of enzyme that can be possibly released was calculated from the total enzyme added during the nanosphere fabrication 6.25x10-02 units, indicating that approximately 11% of the original cABC is incorporated into the nanospheres and is active.

In summary, it is shown that the enzyme cABC can be incorporated into PLGA nanospheres and cABC's prolonged release is possible. Furthermore, the DMMB method can be used effectively to measure concentrations of cABC and CS.

Development of a mathematical model which describes the drug release mechanism of cABC from PLGA nanospheres is underway.