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Title: Thermoresponsive Insitu Gelling Depot Formulations for Controlled Localized and Systemic Drug Delivery, Development, Invitro and Invivo Evaluation
Authors: Khan, Sami Ullah
Keywords: Pharmaceutics
Issue Date: 2019
Publisher: Islamia University, Bahawalpur.
Abstract: Abstract Background and Objective 5-Fluorouracil and Curcumin are widely used in clinics against many types of solid cancer. However owing to their short half-life, high toxicity, rapidly clearance from circulation and damage of healthy tissues has limited their applications. Many studies reported their encapsulation in various types of controlled release delivery devices in order to control their release and overcome the limitations. However the results obtained are substantial and the therapy need to be modified. The present study was aimed to develop insitu formed thermoresponsive injectable hydrogels within body temperature range through subcutaneous route. The hydrogels will act as drug loaded depot at body temperature and will provide the prolonged release of the encapsulated drug in a controlled manner. Moreover the limitations associated with 5-Fluorouracil and Curcumin will be controlled to great extent and will be present in the general circulation for longer time. Methodology In this study insitu formed thermoresponsive injectable hydrogels were prepared though physical self-assembled and insitu polymerization chemical crosslinking approaches and characterized. The thermoresponsive hydrogels were prepared by cold method using three different thermoresponsive polymers and monomers i.e. N-isopropylacrylamide (NIPAAm), Pluronic®-127 (PF-127) and N-vinylcaprolactam. The thermoresponsive hydrogels were prepared in various ratios alone and in combination with other natural and synthetic materials such as β-cyclodextrin, ethylene glycol, carboxymethyl chitosan, sodium alginate and 2-acrylamido-2-methylpropane sulfonic acid. For preparation of chemically cross-linked injectable hydrogels, glutaraldehyde and N, N-methylene-bis-acrylamide (MBA) were used as crosslinking agent and ammonium persulphate as initiator respectively. 5-Fluorouracil and Curcumin were used as model drugs in this study. Drug loaded injectable hydrogels were prepared either by insitu loading or post synthesis loading techniques. All the developed formulations were characterized for clarity and physical appearance, phase transition from sol-gel state at body temperature via tube titling method, rheological analysis, optical transmittance study, swelling study (at variable pH and temperature values) and invitro drug release study in buffer solutions of different pH values at variable temperature programs. The safety of blank and cytotoxic potential of drug loaded injectable hydrogels was confirmed through methyl thiazolyl tetrazolium (MTT) assay against various cell lines. Structural analysis was carried out through nuclear magnetic resonance spectroscopy (NMR), Fourier xxix transformed infrared spectroscopy (FTIR) and X-ray diffraction analysis (XRD). The phase transition and thermal properties were confirmed through thermogravimetric analysis (TGA) and differential scanning calorimetric analysis (DSC). While the surface and cross-sectional morphology of the hydrogel samples was analyzed through scanning electron microscopy (SEM). Based on invitro results, one formulation of thermoresponsive hydrogels was selected for invivo evaluation. The invivo analysis was carried out in albino rabbits by administering drug loaded injectable hydrogels in comparison to free drug solution through subcutaneous route. Moreover acute toxicity study was also conducted in rabbits after administering drug loaded injectable hydrogels in comparison to free drug solution through subcutaneous route. Results The hydrogel formulations developed and reported in this study showed response to temperature change depending upon the ratio of thermoresponsive material. All the optimized hydrogel formulations exhibited phase transition from solution to gel state within body temperature range (32oC-37oC) confirmed through tube titling, time sweep, temperature ramp test and optical transmittance study. The injectability of the formulation was analyzed by conducting continuous ramp test which confirmed its viscoelastic nature. The self-assembled physically cross-linked hydrogels showed a thermoreversible nature while chemically cross-linked hydrogels exhibited good mechanical strength confirmed through frequency sweep test. All the hydrogel samples subjected to swelling experiments showed highest swelling below the lower critical solution temperature (LCST) of the formulation (25oC) owing to the relaxed gel state and faster diffusion of solvent. Moreover depending upon the nature of copolymer, the respective hydrogel formulation also exhibited its pH responsive behavior. The invitro release experiments showed that drug release from injectable hydrogels are pH and temperature dependent and maximum release (> 90%) was observed at lower temperature (25oC) attributed to relaxed gel state. Moreover the release from these injectable hydrogels was controlled for 48 hours invitro. MTT assay confirmed that blank hydrogels are safe and biocompatible tested against mouse fibroblast (L929) and Vero cell lines respectively. Moreover it was also confirmed from MTT assay that drug encapsulated in these hydrogels can cause cells killing tested against Human cervical (HeLa) and breast (MCF-7) cancer cell lines in controlled fashion in comparison to free drug solution and positive control. NMR and FTIR analysis confirmed the formation of new copolymer structure, TG and DSC analysis confirmed the phase transition around body temperature and thermal stability of the formulations. XRD analysis showed the amorphous nature of the formulations while SEM xxx analysis confirmed the porous nature of the formulations. Invivo analysis in albino rabbits showed that depot formation occur at body temperature. Additionally low drug availability in rabbit plasma in initial phase and retarding up to 144 hours confirmed that drug release from insitu depot occur in controlled manner. Moreover acute toxicity study in rabbits also confirmed that cytotoxic drug encapsulated in injectable hydrogels cause no toxicity to normal tissues and organs and the blood chemistry remain normal. Conclusion It was concluded from the results that all the developed formulations showed thermoresponsive behavior and exhibited phase transition within physiologic temperature range. Moreover the developed injectable formulations loaded with anticancer agents (5-FU and Curcumin) are safe and can be effectively used as drug depot after subcutaneous administration invivo for controlled and prolonged release
Gov't Doc #: 19265
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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