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

Title from PDF of title page viewed August 28, 2018Dissertation advisor: Ashim K. MitraVitaIncludes bibliographical references (pages 208-225)Thesis (Ph.,D.)--School of Pharmacy and Department of Chemistry. University of Missouri--Kansas City, 2017Pentablock (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.Literature review -- Part I. Composite nanoformulation of macromolecules for back of the eye diseases ; Introduction -- Biodegradable and biocompatible thermosensitive gelling pentablock copolymers for long term ocular delivery of macromolecules -- Composite anoformulation for long term ocular delivery of macromolecules: effect of molecular weight on drug release -- Composite nanoformulation for long term ocular delivery of IgG-FAB and ranibizumab -- Summary and recommendations -- Part II. Dexamethasone nanoformulation to develop an in vitro model glaucoma -- Introduction -- Pentablock copolymer based dexamethasone nonoformulations elevate NYOC: in vitro liberation, activity and safety in human trabecular meshwork cells and in vivo histology -- Summary and future prospective -- Appendi

An Assessment of Subject coverage of Scientometrics from 2001 to 2010

The study has categorised 1241 articles into research and non-research articles, for analysing subject coverage, publication trend and prolific author of the journal Scientrometrics from the period 2001 to 2010. All articles were classified under 22 subjects using non-parametric method. The study found a sharp positive growth in research articles over the period while a negative growth has been observed in non-research articles only. Most of the research articles were devoted to Bibliometric Study, Citation Studies, Research Output, Collaboration Analysis, while the major topic for non-research articles were Bibliometric study, patent analysis, statistics and research assessment over the period. The most prolific author of the period (2001-2010) was Glanzel, W. (Wolfgang Glänzel, Katholieke University Leuven, Belgium)

Novel Approaches for Overcoming Biological Barriers

The human body poses a spectrum of biological mechanisms operating at different levels that are important for its normal functioning and development [...

Division of labour and its regulation in a primitively eusocial wasp

Social insect species that exhibit overlap of generations, cooperative brood care and reproductive caste differentiation have been termed eusocial. Among these, those that also exhibit morphological caste differentiation are termed highly eusocial while those that have morphologically identical queen and worker castes are termed primitively eusocial. Because queens and workers are not morphologically differentiated in primitively eusocial species, caste differentiation is often post-imaginal. The processes by which some individuals succeed in becoming reproductive queens while others end up as sterile workers in primitively eusocial species is of obvious interest. Such post-imaginal reproductive caste differentiation is absent in highly eusocial species because reproductive caste differentiation in these species is usually pre-imaginal. On the other hand, further differentiation of the workers into different kinds of task specialists is a phenomenon that has been extensively studied in highly eusocial species (Wilson, 1971). There has seldom been an opportunity to study both post-imaginal reproductive caste differentiation as well as post-imaginal division of non-reproductive labour in the same species because these two phenomena usually do not occur in the same species. Ropalidia marginata is an old world tropical primitively eusocial wasp widely distributed in southern India. Although classified as primitively eusocial by the traditional criteria of overlap of generations, cooperative brood care, reproductive caste differentiation and absence of morphological differentiation between queens and workers (Gadagkar, 2001a,b), R. marginata appears to have acquired some features resembling highly eusocial species. One such feature concerns the mode of division of non-reproductive labour among the adult wasps. R. marginata exhibits a remarkably well-developed honeybee like age polyethism. Workers show a strong tendency to feed larvae, build the nest, forage for pulp and forage for food, in that order, as they age. The frequency and probability of performance of different tasks is strongly influenced by the age of the individual (Naug and Gadagkar, 1998a). Like in the honeybees, age polyethism in R. marginata is very flexible and this flexibility comes from the fact that workers are allocated tasks based more on their relative age rather than their absolute age. This can be demonstrated experimentally by creating artificial colonies containing only young individuals (young cohort colonies) or containing only old individuals (old cohort colonies). In young cohort colonies, precocious foragers, i.e., some individuals who begin to forage at an early age when they would not do so in normal colonies, compensate for the absence of older individuals (Naug and Gadagkar, 1998b). In old cohort colonies, hard working nurses, i.e., some individuals who feed larvae at rates higher than they would in normal colonies, compensate for the absence of younger individuals (Agrahari and Gadagkar, submitted). Juvenile hormone modulates post-imaginal reproductive division of labor in primitively eusocial species and promotes the production of queens (e.g., Polistes) while it modulates age polyethism and promotes the production of foragers in highly eusocial species (e.g., the honeybee). Since R. marginata shows both post-imaginal regulation of reproductive division onabor as well as age polyethism, it is a particularly interesting model system to study the effect of juvenile hormone. A single, topical application of 100 ~g. of juvenile hormone-III per female wasp accelerates ovarian development of wasps held in isolation. Similar application to wasps released back on to their natal nests has no effect on their rate of behavioral development as witnessed from the age of first performance of feed larva, build, bring pulp and bring food. It appears, therefore, that in R. marginata, juvenile hormone has retained its function of modulating reproductive division of labor and has not acquired the function of modulating age polyethism (Agrahari and Gadagkar, 2003)

Juvenile hormone accelerates ovarian development and does not affect age polyethism in the primitively eusocial wasp, Ropalidia marginata

Juvenile hormone modulates post-imaginal reproductive division of labor in primitively eusocial species and promotes the production of queens (e.g., Polistes) while it modulates age polyethism and promotes the production of foragers in highly eusocial species (e.g., the honey bee). Ropalidia marginata is a primitively eusocial wasp that shows both post-imaginal regulation of reproductive division of labor as well as age polyethism. Hence, R. marginata is a particularly interesting model system to study the effect of juvenile hormone. We demonstrate here that a single, topical application of 100 µg of juvenile hormone-III per female wasp accelerates ovarian development of wasps held in isolation. Similar application to wasps released back on to their natal nests has no effect on their rate of behavioral development as witnessed from the age of first performance of feed larva, build, bring pulp and bring food. We conclude therefore that in R.marginata, juvenile hormone has retained its function of modulating reproductive division of labor and has not acquired the function of modulating age polyethism

Crystal structure of 9-methacryloylanthracene

The authors would like to thank the Graduate College and Chemistry Department at Cleveland State University for support, the Ohio Supercomputing Center for a grant of computer time, and the National Science Foundation (CHE-0840446) for funds used to purchase the Bruker APEXII DUO X-ray diffractometer used in this research.Peer reviewedPublisher PD