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With the rapid spread of diabetes in human society, the demand for insulin and its precursor (proinsulin) continues to rise. Therefore, the introduction of new methods for their production is essential. In the present study, human proinsulin, while ligated to αB-crystallin chaperone, was effectively expressed in the prokaryotic host system and then purified by the ion-exchange chromatography at high purity (97%). In the next step, human proinsulin with relatively high efficiency was released chemically from the hybrid protein (αB-pIns) and then purified using an appropriate gel filtration column. The SDS-PAGE and HPLC analyses confirmed the high purity, while mass spectroscopy assessment verified the exact molecular mass of the human proinsulin. Using a well-established protocol, the protein was folded in a one-step folding process with a yield of about 70%. The assessment of the secondary structures of the human proinsulin by Raman and FTIR spectroscopy suggested that this protein is rich in α-helix. Also, the conformation of disulfide bonds in the folded proinsulin was confirmed by Raman spectroscopy. The recombinant human proinsulin also demonstrated hypoglycemic activity and mitogenic action (induction of cell proliferation). The method proposed in this work for the production of human proinsulin is easy to run and does not depend on expensive and complex equipment. Thus, it can be used in the industrial production of human proinsulin.

Among the various treatments, GLP-1 receptor agonists (incretin mimics) such as liraglutide and exenatide have been well received in treating type 2 diabetes mellitus (T2DM) and obesity. In this study, an exenatide analogue, in which methionine at position 14 substituted with leucine, was ligated to human αB-crystallin (αB-Cry) and then expressed in the bacterial host cells. In the next step, the exenatide analogue was effectively released from the hybrid protein (αB-Ex) and subsequently purified using gel filtration chromatography. The HPLC and electrospray ionization mass spectrometry (ESI-MS) analyses respectively suggested a high purity (more than 97%) and an accurate molecular mass for the exenatide analogue (4168.22 Da and 835.01, z = 5). Also, the molecular mass of the αB-Ex hybrid protein based on the MALDI-TOF analysis was 24,702.162 Da. The secondary structure assessment by the three spectroscopic methods revealed that exenatide analogue and αB-Ex hybrid protein have an α-helix and a β-sheet rich structure, respectively. Also, according to the results of the DLS analysis, the αB-Ex hybrid protein indicated a high tendency to form large oligomeric structures. The NMR assessment suggested that the hybrid protein exists in its folding state. Both exenatide analogue and the αB-Ex hybrid protein revealed a crucial ability to reduce the blood sugar levels in healthy and diabetic mice. They were also capable of inducing insulin secretion to the bloodstream. Overall, our study introduces the αB-Ex hybrid protein as a novel incretin mimic, exerting its biological activity for a longer period of time. It might also be considered a potential drug candidate in the treatment of T2DM.

Nanotechnology has brought a great revolution in cancer detection, screening, and imaging through its various novel innovative nanodelivery systems. Tracking of cancerous site and cells via in vivo imaging using excellent nano tools or modifying the nano tools in a different way is the greatest achievement toward advancement in nanoparticle-based cancer therapy. Anticancer drugs used in chemotherapy cause severe adverse effect due to its high-dose requirement; however, the advent of nanotechnology and drug delivery at nanoplatform has lessen this to an extent. The nanotechnology-based drug delivery systems due to their specificity, selectivity, and sensitivity target the site of interest in a programmed manner without harming the healthy tissues. In addition, quantum dots, gold nanoparticles, carbon nanotubes, etc., are advanced nanodevices that have emerged out as promising tool in cancer imaging and diagnosis. The present chapter summarizes the advanced progress and application in nanotechnology for cancer diagnosis and imaging.

In recent years pH-sensitive interpenetrating polymeric network (IPN) has emerged out as an excellent drug delivery vehicle in controlling the release of numerous therapeutics. Application of ‘smart’, stimuli sensed polymers and the topology of polymeric network of IPN in combination have made this possible. These intelligent delivery systems through mechanism of swelling, extent of cross-linking, concentration of polymeric ratio and change in pH of the environment or media release the entrapped drug at specific time and site in a programmed way. Due to variation in pH in different parts of the body in normal or pathological condition, preparation of such pH sensed IPN could be useful. The literature evidenced development of large numbers of intelligent IPNs with excellent and efficient results. This chapter highlights a brief overview on hydrogel, its translation to smart IPN and its application in delivery of several drugs belonging to the different classes or category.

Pulmonary route of administration is considered as the best alternative to deliver drugs and is suitable for both systemic and local applications. Incorporation of therapeutics into polymeric microparticle offers additional advantages for delivery of drugs to the lungs. Microparticle drug delivery system exhibits high payload efficiency, suitability for both hydrophilic and hydrophobic drugs, and good ability to control the drug release, thus emerging out as an attractive vehicle. Fabricating the simple polymeric microparticle into different multiparticulate forms at microlevel such as porous microparticle or stimuli-sensitive hydrogel microparticles has shown excellent results against pulmonary diseases. A number of microparticle delivery system carrying therapeutics for lung diseases have been reported. The present chapter is focused on the role of different types of microparticles in the treatment of pulmonary diseases.

Considering alarming projections in the prevalence of periodontitis, following study was undertaken to develop chitosan-vanillin crosslinked microspheres loaded in-situ gel (MLIG) implants containing ornidazole and doxycycline hyclate for the treatment of pocket infections. Firstly, microspheres were formulated and optimized using response surface methodology for particle size 80%, in-vitro drug release (T80%) >7 days and acceptable mucoadhesion. Further, MLIG were optimized for gelation temperature of 34–37 °C and viscosity

The research aims the development and optimization of capecitabine loaded interpenetrating polymeric network by ionotropic gelation method using polymers locust bean gum and sodium alginate by QbD approach. FMEA was performed to recognize the risks influencing CQAs. BBD was applied to study the effect of factors (polymer ratio, amount of cross-linker and curing time) on responses (particle size, % drug entrapment and % drug release). Polynomial equations and 3-D graphs were plotted to relate between factors and responses. The results of the optimized batch viz. particle size (457.92 ± 1.6 μm), % drug entrapment (74.11 ± 3.1%) and % drug release (90.23 ± 2.1%) were close to the predicted values generated by Minitab® 17. Characterization techniques SEM, EDX, FTIR, DSC and XRD were also performed for the optimized batch. To study the water transport inside IPN microbeads, swelling study was done. In vitro drug release of optimized batch showed controlled drug release for 12 h. Pharmacokinetic study carried out following oral administration in Albino Wistar rats exhibited that optimized microbeads had better PK parameters than free drug. In vitro cytotoxicity against HT-29 cells revealed significant reduction of the cell growth when treated with optimized formulation indicating IPN microbeads as effective dosage form for treating colon cancer.

Aim To design, develop, optimize and evaluate sustained-release dasatinib-loaded gold nanoparticles (DSB-GNPs) to treat chronic myeloid leukemia (CML) by using quality by design. Materials & methods In this study, we performed risk assessment, optimization, in vitro characterizations, stability study, drug release studies, cytotoxicity study and in vivo pharmacokinetic evaluation. Results DSB-GNPs of desired size, entrapment, smooth, spherical, stable and sustained drug release for 48 h were achieved. DSB-GNPs exhibited significantly more percentage growth inhibition and enhanced systemic bioavailability compared with pure DSB. Conclusion The in vitro and in vivo evaluation exhibited that the DSB-GNPs have a potential cytotoxic effect, systemic bioavailability and sustained release making them a promising system of DSB delivery in the treatment of chronic myeloid leukemia.

The present research deals with the optimization and preparation of aluminium ion cross-linked buoyant interpenetrating polymeric network (IPN) microbeads (MBs) of locust bean gum (LBG) and sodium alginate (NaAlg) carrying Capecitabine (CAP). The formulation was prepared by ionotropic gelation method. The optimization was done by Response Surface Methodology based Box Behnken Design (BBD) involving a significant changes or effect on responses, particle size, drug entrapment and buoyancy on varying the concentration of polymeric blend (LBG:NaAlg), cross-linker (Aluminum chloride; AlCl3) and pore former (sodium bicarbonate; NaHCO3). Finally, the best optimized batch of buoyant IPN MBs of CAP obtained through BBD was examined through different evaluations that exhibited particle size of 402.12 ± 1.9 μm, drug entrapment 82.45 ± 2.5% and % buoyancy 85.03 ± 1.1% with total floating time of greater than 10 h. The formed IPN MBs showed good buoyancy in gastric pH and better mucoadhesion in intestinal pH. The results of pharmacokinetic study displayed improved systemic circulation, bioavailability and extended plasma half-life of encapsulated CAP within polymeric shell than free CAP. To ensure the gastroretention of formed buoyant IPN MBs, the gamma scintigraphy study performed revealed the gastroretention of MBs for more than 6 h suggesting the designed and developed optimized batch is effective for gastroretention with improved bioavailability.

A combination of biopolymers sodium alginate and locust bean gum has been used to prepare an interpenetrating polymeric network of an anticancer drug Capecitabine by ionotropic gelation method. For the optimization 32 levels, a full factorial design was employed to examine the influence of independent factors, i.e. polymer ratio and cross-linker concentration on responses particle size and drug entrapment. The obtained optimized formulation was examined for solid-state characterization, swelling study, in vitro drug release, SRB study, oral toxicity study, in vivo pharmacokinetic and in vivo antitumor study. The results of all the studies performed were found suitable in extending the release of a short elimination half-life drug with improved bioavailability and suggesting it to be safe and effective for oral drug delivery in treating colon cancer.

The purpose of this research study was to develop, optimize, and characterize dasatinib loaded polyethylene glycol (PEG) stabilized chitosan capped gold nanoparticles (DSB-PEG-Ch-GNPs).Gold (III) chloride hydrate was reduced with chitosan and the resulting nanoparticles were coated with thiol-terminated PEG and loaded with dasatinib (DSB). Plackett-Burman design (PBD) followed by Box-Behnken experimental design (BBD) were employed to optimize the process parameters. Polynomial equations, contour, and 3D response surface plots were generated to relate the factors and responses. The optimized DSB-PEG-Ch-GNPs were characterized by FTIR, XRD, HR-SEM, EDX, TEM, SAED, AFM, DLS, and ZP.The results of the optimized DSB-PEG-Ch-GNPs showed particle size (PS) of 24.39 ± 1.82 nm, apparent drug content (ADC) of 72.06 ± 0.86%, and zeta potential (ZP) of -13.91 ± 1.21 mV. The responses observed and the predicted values of the optimized process were found to be close. The shape and surface morphology studies showed that the resulting DSB-PEG-Ch-GNPs were spherical and smooth. The stability and in vitro drug release studies confirmed that the optimized formulation was stable at different conditions of storage and exhibited a sustained drug release of the drug of up to 76% in 48 h and followed Korsmeyer-Peppas release kinetic model.A process for preparing gold nanoparticles using chitosan, anchoring PEG to the particle surface, and entrapping dasatinib in the chitosan-PEG surface corona was optimized.

The objective of the study was to develop interpenetrating polymeric network (IPN) of capecitabine (CAP) using natural polymers locust bean gum (LBG) and sodium alginate (NaAlg).The IPN microbeads were optimized by Box-Behnken Design (BBD) to provide anticipated particle size with good drug entrapment efficiency. The comparative dissolution profile of IPN microbeads of CAP with the marketed preparation proved an excellent sustained drug delivery vehicle.Ionotropic gelation method utilizing metal ion calcium (CaThe particle size and % drug entrapment of the optimized batch was 494.37 ± 1.4 µm and 81.39 ± 2.9%, respectively, closer to the value predicted by Minitab 17 software. The in vitro drug release study showed sustained release of 92% for 12 h and followed anomalous drug release pattern. The derived pharmacokinetic parameters of optimized batch showed improved results than pure CAP.Thus, the formed IPN microbeads of CAP proved to be an effective extended drug delivery vehicle for the water soluble antineoplastic drug.

This research was motivated due to substantial requirement of improved treatment for breast cancer which accounts for over 0.52 million deaths annually worldwide. Utilizing nanoparticles carrying active medicaments as targeted delivery carrier is emerging as a promising approach. For a drug to be clinically effective, it needs to be suitably protected in the biological fluid till it is delivered to the targeted site. Keeping above in mind, we prepared and optimized polymeric nanoparticles by polyhydroxybutyrate-co-hydroxyvalerate (PHBV) a biodegradable polymer utilizing modified emulsification solvent evaporation method. The optimized formulation had particle size of 349±3.51nm with entrapment efficiency of 69±1.28%. Nanoparticle formation and its surface morphology were confirmed by various electron microscopes. The in vitro and pharmacokinetic studies demonstrated a sustained release of drug in a non-biological system and into rat's bloodstream respectively. Also, the in vitro cytotoxicity and in vivo toxicological evaluation at the therapeutic dose demonstrated the safety and antitumor efficacy of the formulation. Due to formulation characteristic properties, it was found to be effective in enveloping and chaperoning the drug to the suitable site of action. The PHBV-TPGS combination causes the drug to be released in controlled and sustained modes, thereby reducing drug dose and toxicity.

Long-circulating nanoparticles (NPs) are promising drug delivery vehicles which target solid tumors via enhanced permeation and retention effect. Plackett-Burman (PBD) and Box-Behnken (BBD) designs were adopted to study the effects of factors viz. polymer concentration, surfactant concentration, homogenizer speed, homogenization time and ultrasonication time on responses. A graphical and numerical optimization technique was used to obtain predicted value of the response. The drug entrapment efficiency was approximately 39±0.85%. The particle size of the nanoparticles was found to be 260±2.85nm, while the zeta potential was -18±2.12mV, indicating more stable particles. SEM, TEM, and AFM were used for characterization of surface morphology and the physicochemical characters of NPs. A pharmacokinetic evaluation carried out intravenous administration in healthy Charles Foster rats displayed enhanced systemic bioavailability and plasma drug concentration. The in vivo-in silico assessment by GastroPlus™ showed good prediction accuracy and presented best-fit model. Nanoparticles were also studied for stability testing and were found to be stable concerning their drug content and physical characters. In vitro cytotoxicity was assessed using MCF-7 for percentage inhibition of human breast cancer cell line. Anticancer studies of optimized NPs showed a significant increase in efficacy as observed by relative tumor volume up to 30 days.

The investigation was aimed for designing pH-sensitive, polymeric surface modified Guar Gum Nanoparticles (GgNP) with Folic acid (FA) loaded with curcumin (CU), a natural anti-cancer agent, for the treatment of colon cancer with the objective to enhance the bioavailability of CU, concurrently reducing the required dose through selective targeting to colon. The CU loaded FA functionalized GgNP (CU-FA-GgNP) has been prepared by emulsion cross linking method. These surface modified nanoparticles were compared with unmodified CU loaded GgNP (CUGgNP). Various process parameters were optimized and the optimized formulation was evaluated for particle size distribution, % drug entrapment and in vitro drug release in simulated gastrointestinal fluids (SGIF). IR spectroscopy confirms the successful modification of CU-FAGgNP and TEM confirms the surface morphology. Anti-cancer potential of the formulation was demonstrated by MTT assay in Caco-2 cells line. The average size of CU-FA-GgNP was found to be 245 nm in diameter having polydispersity index (PDI) 0.165 indicating mono-disperse particles. The zeta potential of the particles was found to be - 38.1 mV. The CU-FA-GgNP protects the release of CU in upper gastrointestinal tract while maximum release of CU occurred in simulated colonic fluids of pH 6.8. The nanoparticles (NP) were found to be non-toxic at the dose equivalent to 2000 mg/kg of body weight of CU in the acute-toxicity study. Sub-acutetoxicity study proved the safety of the formulation for prolonged administration at the commonly used therapeutic dose of 100 mg/kg of body weight of CU. These studies provide evidences that CU-FA-GgNP holds promise to address colorectal cancer over-expressing folate receptors and they were found to be safe for oral administration for a short as well as a prolonged duration. Keyword: Nanoparticles, Curcumin, Guar gum, Caco-2 cell lines, Folic acid, Toxicity, cancer.