In-vitro and in-vivo cytotoxicity and efficacy evaluation of novel glycyl-glycine and alanyl-alanine conjugates of chitosan and trimethyl chitosan nano-particles as carriers for oral insulin delivery.

PURPOSE: The aim of this research work was to explore the possibility of providing multifunctional oral insulin delivery system by conjugating several types of dipeptides on chitosan and trimethyl chitosan to be used as drug carriers. METHOD: Conjugates of Glycyl-glycine and alanyl-alanine of chitosan and trimethyl chitosan (on primary alcohol group of polymer located on carbon 6) were synthesized and nanoparticles containing insulin were prepared for oral delivery. Preparation conditions of nanoparticles were optimized and their performance to enhance the permeability of insulin as well as cytotoxicity of nanoparticles in Caco-2 cell line was evaluated. To evaluate the efficacy of orally administered nanoparticles, nanoparticles with the most permeability enhancing ability were studied in male Wistar rats as animal model by measuring insulin and glucose Serum levels. RESULT: Structural study of all the conjugates by infrared spectroscopy and nuclear magnetic resonance confirmed the successful formation of the conjugates with the desirable substitution degree. By optimizing preparation conditions, nanoparticles with expected size (157.3-197.7 nm), Zeta potential (24.35-34.37 mV), polydispersity index (0.365-0.512), entrapment efficiency (70.60-86.52%) and loading capacity (30.92-56.81%), proper morphology and desirable release pattern were obtained. Glycyl-glycine and alanyl-alanine conjugate nanoparticles of trimethyl chitosan showed 2.5-3.3 folds more effective insulin permeability in Caco-2 cell line than their chitosan counterparts. In animal model, oral administration of glycyl-glycine and alanyl-alanine conjugate nanoparticles of trimethyl chitosan demonstrated reasonable increase in Serum insulin level with relative bioavailability of 17.19% and 15.46% for glycyl-glycine and alanyl-alanine conjugate nanoparticles, respectively, and reduction in Serum glucose level compared with trimethyl chitosan nanoparticles (p < 0.05). CONCLUSION: It seems that glycyl-glycine and alanyl-alanine conjugate nanoparticles of trimethyl chitosan have met the aim of this research work and have been able to orally deliver insulin with more than one mechanism in animal model. Hence, they are promising candidates for further research studies.

Preparation, characterization and in vivo evaluation of a combination delivery system based on hyaluronic acid/jeffamine hydrogel loaded with PHBV/PLGA blend nanoparticles for prolonged delivery of Teriparatide.

In the current study, biodegradable PHBV/PLGA blend nanoparticles (NPs) containing Teriparatide were loaded in hyaluronic acid/jeffamine (HA-JEF ED-600) hydrogel to prepare a combination delivery system (CDS) for prolonged delivery of Teriparatide. The principal purpose of the present study was to formulate an effective and prolonged Teriparatide delivery system in order to reduce the frequency of injection and thus enhance patient's compliance. Morphological properties, swelling behaviour, crosslinking efficiency and rheological characterization of HA-JEF ED-600 hydrogel were evaluated. The CDS was acquired by adding PHBV/PLGA NPs to HA-JEF ED-600 hydrogel simultaneously with crosslinking reaction. The percentage of NPs incorporation within the hydrogel as well as the loading capacity and morphology of Teriparatide loaded CDS were examined. Intrinsic fluorescence and circular dichroism spectroscopy proved that Teriparatide remains stable after processing. The release profile represented 63% Teriparatide release from CDS within 50days with lower burst release compared to NPs and hydrogel. MTT assay was conducted by using NIH3T3 cell line and no sign of reduction in cell viability was observed. Based on Miller and Tainter method, LD50 of Teriparatide loaded CDS was 131.8mg/kg. In vivo studies demonstrated that Teriparatide loaded CDS could effectively increase serum calcium level after subcutaneous injection in mice. Favourable results in the current study introduced CDS as a promising candidate for controlled delivery of Teriparatide and pave the way for future investigations in the field of designing prolonged delivery systems for other peptides and proteins.

Preparation, statistical optimisation and in vitro characterisation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate)/poly (lactic-co-glycolic acid) blend nanoparticles for prolonged delivery of teriparatide.

The purpose of this study was the preparation, optimisation and in vitro characterisation of PHBV and PLGA blend nanoparticles (NPs) for prolonged delivery of Teriparatide. Double emulsion solvent evaporation technique was employed for the fabrication of NPs. The nanoformulation was optimised using the Box-Behnken methodology. The morphological properties of NPs were assessed by both SEM and transmission electron microscopy (TEM). Encapsulation of Teriparatide within the NPs and lacking of chemical bonds between drug and copolymers were proved by XRPD, FTIR and DSC. The structural stability of Teriparatide after processing was confirmed by fluorescence spectrometry. The average size of optimised NPs was 250.0 nm with entrapment efficiency (EE) of 89.5% and drug loading (DL) of 5.0%. Teriparatide release from optimised NPs led to 64.4% release over 30 days and it showed a diffusion-based mechanism. Based on the favourable results, PHBV/PLGA blend NPs could be a promising candidate for designing a controlled release formulation of Teriparatide.

Development of Molecularly Imprinted Olanzapine Nano-particles: In Vitro Characterization and In Vivo Evaluation.

Molecularly imprinted nano-particles (MINPs) selective for olanzapine were prepared using methacrylic acid (MA) as monomer, ethylene glycol dimethacrylate (EGDMA) as a cross-linker, and 2,2-azobis (2-isobutyronitrile) (AIBN) as the initiator in 36 different ratios. The reaction runs with considerable fine powder formation were selected for further binding and selectivity studies. The MINP with the best selectivity (MINP-32) was chosen for further structural characterization by Fourier transform infrared spectroscopy (FT-IR), thermal gravimetric analysis (TGA), scanning electron microscopy (SEM), adsorption-desorption isotherm for specific surface area, volume and average pore diameter determination. All characterization methods confirmed the successful formation of MINP. The optimum conditions for maximum template loading on the MINP-32 were found by experimental design using response surface methodology (RSM) and choosing absorbent amount, pH, and time as the main factors. MINPs with maximum template loading also indicated significant selectivity between template and its analog (clozapine). The release profile demonstrated a maximum release of about 95% after 288 h for MINP-32 in comparison with about 94% after 120 h for non-MINP-32. The same slow release of drug from MINP-32 was also observed during animal study of the plasma level of template, 20-28 µg/ml versus 5-10 µg/ml. The MINP-32 of this study represents a desirable ability to keep the memory of the template with significant selectivity and good capability to control the release of template in vitro and in vivo and hence could be a promising drug delivery system.