Controlled Delivery of (-)-Epigallocatechin 3-Gallate as a Therapeutic Strategy for the Degenerative Disc Disease

Abstract

Degenerative disc disease (DDD) is the accelerated process of intervertebral disc decay (decrease of disc thickness, enhanced cell death, ECM damage and collagen loss etc.), accompanied with pain, as well as structural and functional failure. So far, there are no therapies that can inhibit or even reverse the degenerative progress of this disease. Regimens and treatments that eliminate the pathological symptoms of DDD are mainly used in patients suffering from DDD. Among the promising treatments of DDD in preclinical development is the use of natural bioactive compounds, such as (-) -Epigallocatechin 3-Gallate (EGCG). EGCG is a compound of green tea leaves, indicating beneficial effects against several diseases, however it is rather degradable in its free form. Hence, on-target EGCG facilitation in human body, in a manner providing protection to EGCG against degradation and dose control over time, is necessary. It was hypothesized that encapsulation could protect EGCG and attenuate DDD features, once replaced locally and released long-term. Therefore, the aim of this master thesis was the design and synthesis of a drug delivery system (DDS) for EGCG encapsulation, stabilization and protection against unrestrained release, for potential local delivery as a DDD therapy.
EGCG was encapsulated in gelatin particles via electrospraying. These particles were then embedded into HA-pNIPAM, a thermo-responsive hydrogel for further stabilization and release control. The hydrogel-particles mixture, consisting the DDS, would then be injected into degenerative disc tissue. The DDS was evaluated regarding its biocompatibility, EGCG protection and prolonged release rates. Firstly, an electrospraying protocol was established for EGCG encapsulation in gelatin microparticles. These particles were tested for their morphology and cytotoxicity. EGCG-loaded microparticles were combined with HA-pNIPAM hydrogel and tested in vitro (t=7days) for their ability to release EGCG and determine the activity of encapsulated EGCG via colorimetric assays and absorbance measurements.
The electrospraying conditions of 2μL/min (flow rate), 20kV (voltage), 4% Gelatin solution and cross-linking with 37.5μg/mL glutaraldehyde (GA) gave spherical, dispersed particles with average size of 669.72 nm, facilitating injectability. Their effect on cells indicated 83-88% metabolic activity, compared to control cells (100% viability), regarding cytotoxicity. Encapsulated EGCG was released up to 50% slower than free EGCG, but kept the same activity (80%). Overall, it was demonstrated that the designed DDS is a promising approach, that can limit EGCG release and possibly protect its activity.