Design development and optimisation of multifunctional Doxorubicin-loaded Indocynanine Green proniosomal gel derived niosomes for tumour management.

This study presents the design, development, and optimization of multifunctional Doxorubicin (Dox)-loaded Indocyanine Green (ICG) proniosomal gel-derived niosomes, using Design of Experiments (23 factorial model). Herein, the multifunctional proniosomal gel was prepared using the coacervation phase separation technique, which on hydration forms niosomes. The effect of formulation variables on various responses including Zeta potential, Vesicle size, entrapment efficiency of Dox, entrapment efficiency of ICG, Invitro drug release at 72nd hour, and NIR hyperthermia temperature were studied using statistical models. On the basis of the high desirability factor, optimized formulation variables were identified and validated with the experimental results. Further, the chemical nature, vesicle morphology, surface charge, and vesicle size of optimized proniosomal gel-derived niosomes were evaluated. In addition, the effect of free ICG and bound ICG on NIR hyperthermia efficiency has been investigated to demonstrate the heating rate and stability of ICG in the aqueous environment and increased temperature conditions. The drug release and kinetic studies revealed a controlled biphasic release profile with complex mechanisms of drug transport for optimized proniosomal gel-derived niosomes. The potential cytotoxic effect of the optimised formulation was also demonstrated invitro using HeLa cell lines.

Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli.

Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2-6 disulfide bonds.