Long Term Ocular Drug Delivery with Novel Pentablock Copolymers; Part I: Composite Nanoformulation of Macromolecules For Back of the Eye Diseases, Part II: Dexamethasone Nanoparticle to Develop an In Vitro Model for Glaucoma

Abstract

Pentablock (PB) copolymers have been successfully synthesized for long term delivery in the treatment of posterior segment ocular diseases. PB copolymers are comprised of FDA approved biodegradable polymer such as polyethylene glycol (PEG), polycaprolactone (PCL), polylactic acid (PLA) and polyglycolic acid (PGA). PB copolymers of different composition, molecular weights and block arrangements were synthesized by ring opening bulk copolymerization method and analyzed by NMR, GPC FT-IR and XRD analyses. Further, these PB copolymers have been utilized to develop the macromolecule embedded thermosensitive gels or nanoparticles (NPs) or composite nanoformulation (NPs suspended in gel) for a sustained drug delivery. PBG (PLA-PCL-PEG-PCL-PLA; PBG-1 and PEG-PCL-PLA-PCL-PEG; PBG-2) gelling copolymers were evaluated for their utility as injectable in situ hydrogel forming depot for controlled ocular delivery of macromolecules (proteins, peptides and Fab fragments). A wide variety of macromolecules (Octreotide, IgG-Fab, IgG-Fab‘ and IgG) with molecular weights ranging from 1 - 150 kDa have been used for this purpose. The kinematic viscosity of the copolymer solution was studied at different polymer concentration with different block arrangment. It was observed that viscosity of hydrophobic polymer (PBG-1) was considerably higher relative to PBG-2 copolymer. Sol-gel transition curves for PBG-1 and PBG-2 copolymer was compared to understand the effect of hydrophobicity and effect of block arrangement on the sol-gel behavior of block copolymers. Sol-gel transition and rheology revealed that PBG block arrangements were easy to handle at room temperature and easy to administer through small gauge needle. Cell viability and cytotoxicity studies confirmed that PBG copolymers are superior biomaterials for ocular delivery. It was observed that the in vitro release pattern was depended on the molecular weight of the macromolecules and amphiphilic nature of the PBG copolymers. It is anticipated that much longer release can be obtained by altering block composition or change in hydrophobicity and/or hydrophilicity of the gelling polymer. The in vitro release pattern was in conjunction with the facts that amorphous and hydrophilic polymer degrades fast. CD spectroscopy results revealed no changes in the secondary structure of macromoelcules (studied for IgG as a model macromoelcule). The in vitro degradation study for PBG-2 copolymer was performed under four different conditions; (i) in pH 7.4 PBS at 37°C, (ii) in presence of enzymes acetylcholinesterase (14.7 mU/mL) and butyrylcholinesterase (5.9 mU/mL), (iii) in pH 9.0 borate buffer at 37°C and (iv) in pH 7.4 PBS at 40°C. The samples were analyzed by XRD and GPC to determine the weight loss of the PBG-2 copolymer. It was observed that accelerated conditions such as pH 9.0 (37°C) and high temperature (40°C) exhibited weight loss of ~45% and ~40%, respectively which were significantly higher than weight loss observed under normal condition (pH - 7.4, 37°C) i.e., ~35%. No significant effect of enzymes was observed on polymer degradation. Besides, in vivo assessment of PBG-2 copolymer provided a safe environment and was well tolerated in the rabbit eyes analyzed up to 33 weeks. Further, PB-NPs were formulated with different molecular weights of PB copolymer (PCL-PLA-PEG-PLA-PCL) to study the release pattern of macromolecules (lysozyme, IgG-Fab, ranibizumab and IgG). The macromolecules encapsulated in PB NPs were prepared by W1/O/W2 double emulsion solvent evaporation method. The macromolecules were optimized to achieve a high drug loading (~17%) and entrapment efficiency (~66%) in the NPs. PB-NPs alone exhibited significant burst release in the first few days however, the dual approach i.e., composite nanoformulations (macromolecules encapsulated PB-NPs dispersed in thermosensitive gel) eliminated the burst release effect and exhibited nearly zero-order protein release for significantly longer durations (~3-6 months). In order to compare the duration of in vitro release, PB copolymers with different molecular weight have been studied. The enzymatic activity of lysozyme with its respective enzymatic assays was used to investigate the activity of released macromolecule. Anti-VEGF activity of ranibizumab released from composite nanoformulation was analyzed by indirect ELISA. It was observed that macromolecules maintained their structural integrity and bioactivity during the preparation of the nanoformulation and also during the drug release process. The mean particle size distribution of NPs in PBS was found in the range of ~150 nm and was consistent throughout the study in different media analyzed up to 10 days. The results confirmed the higher stability of NPs in different cell culture media. In vitro cell viability, cytotoxicity and biocompatibility studies performed on various ocular cells, confirmed the safety of PB copolymers for ocular applications.

PART II: DEXAMETHASONE NANOPARTICLE TO DEVELOP AN IN VITRO MODEL FOR GLAUCOMA The aim of the present study was to examine the elevation of myocillin (MYOC); one of the extra cellular matrix related proteins whose expression is altered in presence of long-term treatment of Glucocorticoids. In this study, dexamethasone (DEX) was selected as model drug. The different strains of primary cultures of human trabecular meshwork (HTM) cell line (HTM120, 136, 126, 134 and 141) were used to develop the in vitro cell culture model of glaucoma. To obtain a long-term delivery of DEX, pentablock (PB) copolymer was synthesize using the ring opening bulk copolymerization method and characterized by NMR, GPC and XRD analyses. PB copolymer was used to formulate the DEX encapsulated nanoparticles (NPs) with entrapment efficiency of ~63% and drug loading of ~11% w/w. The mean particle size distribution of NPs was analyzed by NTA in PBS was found in the range of ~109 nm. The biomaterial was further studied for in vitro cytotoxicity and cell viability. Results showed that neither cell viability nor cytotoxicity was affected up to 12 weeks of treatment. DEX-PB-NPs or control NPs treatments were given to the HTM cells and cell culture supernatant was collected/replaced with fresh 1% DMEM once/week for 12 weeks. DEX or vehicle was used as controls to compare MYOC secretion levels by Western blot (WB). Four HTM cell strains tested showed similar MYOC secretion patterns, having robust responses for the entire monitoring period. In contrast, one cell strain responded only for a few weeks. Quantitation of WB data from five HTM cell strains showed that MYOC increased by 5.2 ± 1.3, 7.4 ± 4.3, and 2.8 ± 1.1 fold at 4, 8, and 12 weeks in the presence of DEX-PB-NPs compared to 9.2 ± 3.8, 2.2 ± 0.5, and 1.5 ± 0.3 fold at 4, 8, and 12 weeks in control DEX treatment group. Based on the decline in MYOC levels after withdrawal of DEX from control wells, results indicate that DEX-PB-NPs released biologically active DEX for at least 10 weeks. By comparison, MYOC levels in vehicle treated control wells remained unchanged. Moreover, PB copolymers were biocompatible and didn‘t modifying the cellular functions of HTM cells. Although the PB copolymers did not show any sign of cytotoxicity to HTM cells in this long-term study, they did modify HTM cell morphology. HTM cell elongation was present in all cell strains after both Con-NPs and DEX-PB-NPs treatment. Morphological modification of HTM cells by the polymers may accompany functional changes those were not measured in the present study, but needs further investigations. Meanwhile, this study provides the evidence that our in vitro system developed in this study is a valuable tool for analyzing the safety of the polymers and biological effects of steroids released from the polymers. In addition, histological observations in the C57BL/6 mice showed normal phenomenon in ocular tissue morphology.