Assessment of Molecular Weight and Drug Loading on Release From Hydrogel Therapeutics
thesisposted on 09.12.2012, 00:00 by Amy E. Ross
Hydrogels are hydrophilic crosslinked polymers that have been demonstrated to be useful in controlled drug delivery. This work pertains to an implantable polyethylene glycol diacrylate (PEGDA) hydrogel for controlled drug delivery triggered by the enzyme matrix metalloproteinase-2 (MMP-2). MMP-2 is one of a family of enzymes responsible for degrading and remodeling the extracellular matrix. MMP-2 is also overactivated in many forms of cancer. An active pharmaceutical ingredient (API), conjugated to the hydrogel matrix via an MMP-2 sensitive peptide, is released when MMP-2 cleaves the peptide. By attaching the API-peptide conjugate to the hydrogel backbone, drug release occurs in the presence of MMP-2. This work explored optimization of this release and the ability of MMP-2 to enter the hydrogel. Mesh sizes for different PEGDA molecular weights were measured by using swelling and tensile testing. PEGDA 3,400 had a mesh size smaller than the dimensions of MMP-2. PEGDA 10,000 and PEGDA 20,000 had a mesh size larger than MMP-2, with PEGDA 20,000 being larger than PEGDA 10,000. Using the fluorescent molecule tetramethyl rhodamine (TAMRA) as a model drug, release studies with purified MMP-2 showed an increased ratio of release with MMP-2 compared to buffer for PEGDA 20,000 compared to PEGDA 3,400 at three different loading concentrations. PEGDA 10,000 had a higher ratio of release compared to PEGDA 3,400 at two of the three loading concentrations tested. Alternate MMP sensitive peptide sequences TAMRA-PAGLLGC and TAMRA-IPVSLRSGC showed more consistent release than GPLGVRG as indicated by a smaller standard deviation in release amounts. TAMRA-IPVSLRSGC had the greatest absolute amount of release among all three peptides. U-87 MG cells embedded in collagen released peptide as confirmed by HPLC. GM6001, an MMP inhibitor, prevented release. MMP-2 secretion and activation was confirmed by gelatin zymography. Fractionated HPLC peaks were analyzed using mass spectrometry, and showed cleavage was occurring at TAMRA-GPLG, the expected site. The increase in ratio of release with PEGDA 10,000 and PEGDA 20,000 comapred to PEGDA 3,400 suggests MMP-2 enters the hydrogel. Optimization can be attained by using PEGDA 20,000 and the peptide sequence IPVSLRSGC. Further optimization can be attained by further release studies with IPVSLRSGC.