Engineering processive cellulase of Clostridium thermocellum to divulge the role of the carbohydrate-binding module.

The processive cellulase (CelO) is an important modular enzyme of Clostridium thermocellum. To study the effect of the carbohydrate-binding module (CBM3b) on the catalytic domain of CelO (GH5), four engineered derivatives of CelO were designed by truncation and terminal fusion of CBM3b. These are CBM at the N-terminus, native form (CelO-BC, 62 kDa); catalytic domain only (CelO-C, 42 kDa); CBM at the C-terminus (CelO-CB, 54 kDa) and CBM attached at both termini (CelO-BCB, 73 kDa). All constructs were cloned into pET22b (+) and expressed in Escherichia coli BL21 (DE3) star. The expression levels of CelO-C, CelO-CB, CelO-BC, and CelO-BCB were 35%, 35%, 30%, and 20%, respectively. The enzyme activities of CelO-C, CelO-CB, CelO-BC, and CelO-BCB against 1% regenerated amorphous cellulose (RAC) were 860, 758, 985, and 1208 units per µmole of the enzyme, respectively. The enzymes were partially purified from the lysate of E. coli cells by heat treatment followed by anion exchange FPLC purification. Against RAC, CelO-C, CelO-CB, CelO-BC, and CelO-BCB showed KM values of 32, 33, 45, and 43 mg⋅mL-1 and Vmax values of 3571, 3846, 3571, and 4545 U⋅min-1 , respectively. CBM3b at the N-terminus of GH5 linked through a P/T-rich linker was found to enhance the catalytic activity and thermostability of the enzyme.

Novel benzothiazole hydrazine carboxamide hybrid scaffolds as potential in vitro GABA AT enzyme inhibitors: Synthesis, molecular docking and antiepileptic evaluation.

In the present study, a series of newer benzothiazole derivatives containing thiazolidin-4-one (5a-g) and azetidin-2-one (6a-g), were synthesized by the cyclization of benzothiazolyl arylidene hydrazine carboxamide derivatives with thioglycolic acid and chloroacetyl chloride, respectively. Results of in vivo anticonvulsant screening revealed that compounds having 2,4-dicholoro (5c and 6c) and 4-nitro substituent (5g) at the phenyl ring have promising anticonvulsant activities without any neurotoxicity. Selected compounds were also evaluated for their in vitro GABA AT inhibition. The results indicated that compound 5c (IC50 15.26 µM) exhibited excellent activity as compared to the standard drug vigabatrin (IC50 39.72 µM) suggesting the potential of these benzothiazole analogues as new anticonvulsant agents.

Environment friendly green synthesis method based natural bioactive functional "catechin and gingerol" loaded nanomedicine for the management of obesity.

Obesity is one of the major health problems across the world and the leading cause of death. Natural bioactive functional compounds (catechin, Cat and gingerol, Gin) have been reported to control obesity. The application of a nanoparticulate (NPs) delivery system has shown greater efficacy in the treatment of different diseases. The present study was designed to prepare the Cat-Gin silver nanoparticles (AgNPs) using the microwave method. The prepared Cat-Gin-AgNPs were characterized for particle size, zeta potential, encapsulation efficiency and drug release. Further, it was evaluated for anti-obesity activity (body weight, total abdominal fat, lipid profile and liver profile) using a high-fed diet (HFD) induced obesity model. The formulated Cat-Gin-AGNPs showed nano-metric size (110 ± 4.65 nm), polydispersity index (PDI) of 0.27 ± 0.03, and surface charge (-17.6 ± 2.6 mV) with a spherical shape. It also depicted high encapsulation efficiency (<80 %) with prolonged drug release behaviour (Cat - 84.34 ± 4.12 % and Gin - 72.33 ± 3.87 %). The in vivo study parameters revealed a significant (p ≤ 0.001) reduction in body weight, food intake and total abdominal fat. The biochemical results displayed a reversal in altered biochemical parameters to the normal range (Group 4) as compared to HFD control (Group 2). It also helps restored the liver enzymes in obese rats. The results of the study highlighted the applicability of Cat-Gin-AGNPs as a novel delivery system in the treatment of obesity.

Formulation of intranasal surface engineered nanostructured lipid carriers of rotigotine: Full factorial design optimization, in vitro characterization, and pharmacokinetic evaluation.

The objective of the present research was to develop, optimize, and evaluate rotigotine (RT)-loaded chitosan (CH) coated nanostructured lipid carriers (RT-CH-NLCs) for nose-to-brain delivery. The NLCs were prepared by homogenization and sonication technique as well as optimized by using three factors at three-level Box-Behnken design. The prepared NLCs were evaluated for particle size, zeta potential, entrapment efficiency, drug release, and ex vivo permeation. The pharmacokinetic study was conducted on albino Wistar rats to evaluate the bioavailability and neuropharmacokinetic parameters after intranasal administration of the optimized formulation (RT-CH-NLCs-OPT). The optimized formulation showed the particle size (170.48 ± 8.37 nm), PDI (0.19 ± 0.03), zeta potential (+26.73 mV), and entrapment efficiency (82.37 ± 2.48 %). In vitro drug release study displayed a sustained drug release pattern from RT-CH-NLCs-OPT (86.73 ± 8.58 % in 24 h) in comparison to RT-Dis (98.61 ± 7.24 % in 16 h). The permeability coefficient (PC) was found to be 11.39 ± 1.08 × 10-4 cm.h-1 and 2.34 folds higher than RT-Dis (4.85 ± 1.53 × 10-4 cm.h-1). The relative bioavailability of RT from RT-CH-NLCs-OPT was 3.2-fold greater as compared to RT-Dis. The absolute bioavailability of RT after intranasal administration of RT-CH-NLCs-OPT was 2.1-fold higher than RT-CH-NLCs-OPT administered intravenously. The brain targeting and targeting potential was displayed by DTE (422.03 %) and DTP (76.03 %) after intranasal administration of RT-CH-NLCs-OPT as compared to RT-Dis (DTE 173.91 % and DTP 59.97 %). Furthermore, confocal laser scanning microscopy results confirmed better brain targeting for RT-CH-NLCs-OPT as compared to RT-Dis. From these findings, it could be concluded that RT-CH-NLCs could serve as a promising strategy for targeting RT through the intranasal route.

Novel therapeutic interventions for combating Parkinson's disease and prospects of Nose-to-Brain drug delivery.

Parkinson disease (PD) is a progressive neurodegenerative disorder prevalent mainly in geriatric population. While, L-DOPA remains one of the major choices for the therapeutic management of PD, various motor and non-motor manifestations complicate the management of PD. In the last two decades, exhaustive research has been carried out to explore novel therapeutic approaches for mitigating motor and non-motor symptoms of PD. These approaches majorly include receptor-based, anti-inflammatory, stem-cell and nucleic acid based. The major limitations of existing therapeutic interventions (of commonly oral route) are low efficacy due to low brain bioavailability and associated side effects. Nanotechnology has been exploited and has gained wide attention in the recent years as an approach for enhancement of bioavailability of various small molecule drugs in the brain. To address the challenges associated with PD therapy, nose-to-brain delivery utilizing nanomedicine-based approaches has been found to be encouraging in published evidence. Therefore, the present work summarises the major challenges and limitations with antiparkinsonian drugs, novel therapeutic interventions, and scope of nanomedicine-based nose-to-brain delivery in addressing the current challenges of antiparkinsonian therapy. The manuscript tries to sensitize the researchers for designing brain-targeted nanomedicine loaded with natural/synthetic scaffolds, biosimilars, and nucleic acids that can bypass the first-pass effect for the effective management of PD.

Formulation of sitagliptin-loaded oral polymeric nano scaffold: process parameters evaluation and enhanced anti-diabetic performance.

PURPOSE: The aim of the study to formulate and statistically optimize sitagliptin-loaded eudragit nanoparticles (SIT-NPs) and evaluate the in-vitro pharmaceutical quality and in-vivo anti-diabetic assessment. METHOD: SIT-NPs were prepared by using combination method of solvent evaporation and nano-precipitation techniques. The influence of different independent variables as eudragit RL100 concentration (%), tween 80 concentration (%) and sonication time (min) were evaluated on dependent variables like particle size (nm), drug loading (%) and in-vitro drug release (%). Further, the optimized formulation was evaluated for surface morphology, CLSM, ex-vivo permeation study and in-vivo anti-diabetic activity and stability study. RESULTS: The developed SIT-NPs formulations showed particle size range (135.86-193.45 nm), drug loading (6.36-8.76%) and prolonged drug release over 24 h. The prepared SIT-NPs were found to be nearly spherical with smooth surface. The comparative in-vitro release study and CLSM study results revealed that SIT-NPopt showed significantly (p < .05) enhanced release and permeation as compared to SIT free solution (SIT-Fs). The in-vivo anti-diabetic assessment revealed that SIT-NPopt able to reduce the blood sugar level (BSL) for a prolonged period of time. Further, the stability study data showed the formulations were found stable at both temperature and having the shelf life of 488 d. CONCLUSIONS: This research has shown that SIT-NPs based on experimental design offers a new and better approach to delivering SIT, thus encouraging further development of this formulation.

In vitro and preclinical assessment of factorial design based nanoethosomes transgel formulation of an opioid analgesic.

CONTEXT: Tramadol is a centrally acting analgesic and requires frequent dosing. Hence, judicious selection of retarding formulations is necessary. Transdermal ethosomal gel delivery has been recognized as an alternative route to oral delivery. OBJECTIVE: The objective was to develop statistically optimized ethosomal systems for enhanced transdermal activity of tramadol vis-à-vis traditional liposomes. MATERIALS AND METHODS: Box-Behnken design was employed for optimization of nanoethosomes using phospholipon 90G (A), ethanol (B), and sonication time (C) as independent variables while dependent variables were the vesicle size (Y1), entrapment efficiency (Y2), and flux (Y3). It was prepared by rotary evaporation method and characterized for various parameters including entrapment efficiency, size and transflux. Preclinical assessments were conducted on Wistar rats to measure the performance of developed formulations. RESULTS: The optimized formulation provided mean vesicles size, reasonable entrapment efficiency and enhanced flux when compared with liposome (control). In-vivo absorption study showed a significant increase in bioavailability (7.51 times) compared with oral tramadol. The average primary irritancy index was found to be 1.4, indicating it to be non-irritant and safe for use. DISCUSSION AND CONCLUSION: The results also demonstrated that encapsulated tramadol increases its biological activity due to the superior skin penetration potential. The preclinical study indicates a significant (P < 0.05) extended analgesic effect compared to oral solution using the hot plate test method. The overall results suggest that developed formulation is an efficient carrier for transdermal delivery of tramadol.