Optimization (central composite design) and validation of HPLC method for investigation of emtricitabine loaded poly(lactic-co-glycolic acid) nanoparticles: in vitro drug release and in vivo pharmacokinetic studies.

The objective of the current study is to develop nanoparticles (NPs) drug delivery system of emtricitabine solely using poly(lactic-co-glycolic acid) (PLGA) and evaluate its in vitro and in vivo release performance by systematically optimized HPLC method using Formulation by Design (FbD). NPs were evaluated for in vitro release and in vivo absorption study. The desired chromatographic separation was achieved on a Phenomenex C18 (250 mm × 4.6 mm I.D., 5 µm) column, under isocratic conditions using UV detection at 280 nm. The optimized mobile phase consisted of a mixture of 40 mM phosphate dihydrogen phosphate buffer (pH 6.8), methanol, and 2% acetonitrile in a ratio of (83 : 15 : 2, v/v/v) at a flow rate of 1 mL/min. The linear regression analysis for the calibration curves showed a good linear correlation over the concentration range 0.040-2.0 µg/mL, with retention time of 4.39 min. An average encapsulation efficiency of 74.34% was obtained for NPs. In vitro studies showed zero-order release and about 95% drug being released within 15 days in PBS (pH 7.4). In conclusion, the proposed optimized method was successfully applied for the determination of in vitro and in vivo release studies of emtricitabine NPs.

In vitro and in vivo performance of supersaturable self-nanoemulsifying system of trans-resveratrol.

To develop an optimized supersaturable self-nanoemulsifying drug delivery system (S-SNEDDS) in order to improve the oral bioavailability of trans-resveratrol (t-RVT), together with surmounting poor aqueous solubility, enterohepatic recirculation and controlling drug precipitation, by employing a precipitation inhibitor (PPI) that is, hydroxypropyl methylcellulose (HPMC). The long-term stability of a previously reported formulation optimized long chain triglycerides (OPT LCT-SNEDDS), consisting of Lauroglycol FCC and Transcutol P, indicated rapid precipitation of trans-resveratrol. Following incorporation of the selected PPI, the precipitates were differentiated using the X-ray diffraction (XRD) technique. An in vitro supersaturation test was carried out for the S-SNEDDS formulation. The S-SNEDDS formulation was appraised for pharmacokinetic and in situ perfusion studies. In vitro dilution of the S-SNEDDS formulation resulted in the formation of a nanoemulsion, followed by a slow precipitation of t-RVT in the S-SNEDDS formulation vis-à-vis SNEDDS formulated with OPT-LCT, where it undergoes rapid precipitation, yielding a low t-RVT concentration. The pharmacokinetic study indicated that the AUC(0-8h) of the S-SNEDDS formulation increased by nearly 1.33-fold in the presence of HPMC vis-à-vis the OPT LCT-SNEDDS, at a drug dose of 20 mg/kg. The in situ perfusion parameters, viz., fraction absorbed and effective permeability, demonstrated significant improvement in the rate and extent of absorption from the S-SNEDDS formulation. This case demonstrates that the supersaturatable approach is effective in delivering and in improving the oral bioavailability of t-RVT.

Atazanavir-loaded Eudragit RL 100 nanoparticles to improve oral bioavailability: optimization and in vitro/in vivo appraisal.

Atazanavir (ATV) is a HIV protease inhibitor. Due to its intense lipophilicity, the oral delivery of ATV encounters several problems such as poor aqueous solubility, pH-dependent dissolution, rapid first-pass metabolism in liver by CYP3A5, which result in low bioavailability. To overcome afore mentioned limitations, ATV-loaded Eudragit RL100 nanoparticles (ATV NPs) were prepared to enhance oral bioavailability. ATV NPs were prepared by nanoprecipitation method. The ATV NPs were systematically optimized (OPT) using 3(2) central composite design (CCD) and the OPT formulation located using overlay plot. The pharmacokinetic study of OPT formulation was investigated in male Wistar rats, and in-vitro/in-vivo correlation level was established. Intestinal permeability of OPT formulation was determined using in situ single pass perfusion (SPIP) technique. Transmission electron microscopy studies on OPT formulation demonstrated uniform shape and size of particles. Augmentation in the values of Ka (2.35-fold) and AUC0-24 (2.91-fold) indicated significant enhancement in the rate and extent of bioavailability by the OPT formulation compared to pure drug. Successful establishment of in vitro/in vivo correlation (IVIVC) Level A substantiated the judicious choice of the in vitro dissolution milieu for simulating the in vivo conditions. In situ SPIP studies ascribed the significant enhancement in absorptivity and permeability parameters of OPT formulation transport through the Peyer's patches. The studies, therefore, indicate the successful formulation development of NPs with distinctly improved bioavailability potential and can be used as drug carrier for sustained or prolonged drug release.

Correlation of Beta Angle with Antero-Posterior Dysplasia Indicators and FMA: An Institution Based Cephalometric Study.

INTRODUCTION: Beta angle utilizes three skeletal landmarks - point A, point B, and point C (the apparent axis of the condyle). It is formed between A-B line and point A perpendicular to C-B line. Further this angle indicates the severity and the type of skeletal dysplasia in the sagittal dimension and it changes with the growth pattern of the patient. Hence, it is important to study the dependence of beta angle on the growth pattern. AIM: The present study was designed to evaluate the correlation of Beta angle with point A-Nasion-point B (ANB) angle, points A and B to palatal plane (App-Bpp), Wit's appraisal and Maxillary-Mandibular plane angle Bisector (MMB) and Frankfort-Mandibular plane Angle (FMA) in Skeletal Class I, Class II and Class III malocclusion groups. MATERIALS AND METHODS: Pre-treatment lateral head cephalo-grams of 120 subjects in age group of 15-25 years were obtained. Three skeletal Class I, Class II and Class III malocclusion groups (40 each) were assorted on the basis of ANB, MMB, App-Bpp, Wit's appraisal and FMA. Analysis of variance (ANOVA) and mean differences were calculated to compare the study groups. Bivariate correlations among different parameters of these groups were obtained. RESULTS: Normal values of beta angle in skeletal Class I group, skeletal Class II group and skeletal Class III group was 31.33±3.25, 25.28±4.28 and 40.93±4.55 respectively. Overall beta angle showed a strong correlation with all parameters of anterio-posterior dysplasia indicators except FMA. CONCLUSION: Beta angle shows weak correlation with FMA and is not affected by growth pattern/jaw rotation. The normal values are in same range irrespective of the differences in craniofacial morphology.

In-vitro/in-vivo characterization of trans-resveratrol-loaded nanoparticulate drug delivery system for oral administration.

OBJECTIVES: The current studies entail successful formulation of systematically optimized (OPT) nanoparticulate drug delivery system to increase the oral bioavailability using Eudragit RL 100 of trans-resveratrol (t-RVT), and evaluate their in-vitro/in-vivo performance. METHODS: t-RVT-loaded Eudragit RL 100 nanoparticles (t-RVT NPs) were prepared by nanoprecipitation method. The nanoparticles (NPs) were systematically optimized using 3(2) central composite design and the OPT formulation located using overlay plot. The pharmacokinetic and in-vivo biodistribution of t-RVT NPs were investigated in rats, and various levels of in-vitro/in-vivo correlation (IVIVC) were established. KEY FINDINGS: The OPT formulation (mean particle size: 180 nm) indicated marked improvement in drug release profile vis-à-vis pure drug and marketed formulation (MKT). Augmentation in the values of Ka (5.64-fold) and AUC0-24 (7.25-fold) indicated significant enhancement in the rate and extent of bioavailability by the optimized trans-resveratrol-loaded Eudragit RL 100 nanoparticles (OPT-t-RVT NPs) compared with pure drug. Level A of IVIVC was successfully established. OPT-t-RVT NPs showed 4.11-fold rose in the values of t-RVT concentrations in liver. In-situ single pass intestinal perfusion studies construed remarkable enhancement in the absorptivity and permeability parameters of OPT-t-RVT NPs. CONCLUSIONS: The results, therefore, insight into the role of solubility enhancement and trounce enterohepatic recirculation for improving the oral bioavailability of t-RVT.

Optimized PLGA nanoparticle platform for orally dosed trans-resveratrol with enhanced bioavailability potential.

BACKGROUND: Trans-resveratrol (t-RVT) is a potent antioxidant. The drug suffers enterohepatic recirculation and extensive first-pass metabolism by CYP3A4 in liver, resulting in very low bioavailability (almost zero). OBJECTIVE: The current studies entail a novel formulation approach to develop systematically optimized (OPT) nanoparticles (NPs) to enhance the oral bioavailability potential using poly (dl-lactide-co-glycolide) (PLGA) of t-RVT and overcome enterohepatic recirculation. METHODS: T-RVT-loaded PLGA NPs were prepared by nanoprecipitation method. Delineating the NP regions, the amounts of polymer and emulsifier were selected as the critical factors for systematically formulating the OPT NPs employing 3(2) central composite design. The pharmacokinetics, in vivo biodistribution and in situ single-pass intestinal perfusion (SPIP) studies of OPT formulation were investigated in male Wistar rats. RESULTS: Augmentation in the values of Ka (7.17-fold) and AUC0 - ∞ (10.6-fold) indicated significant enhancement in the rate and extent of bioavailability by the OPT formulation compared to pure drug and marketed product. OPT formulation showed a 2.78-fold rise in the values of t-RVT concentrations in liver. In situ SPIP studies ascribed the significant enhancement in absorptivity and permeability parameters of OPT NPs to transport through the Peyer's patches. Successful establishment of in vitro/in vivo correlation substantiated the judicious choice of the in vitro dissolution milieu for simulating the in vivo conditions. CONCLUSION: The studies, therefore, could provide another useful tool for successful development of t-RVT NPs and an in vivo approach to designate nanoparticulate system of t-RVT with distinctly improved bioavailability and to overcome enterohepatic recirculation.

Pharmacokinetics and in vivo biodistribution of optimized PLGA nanoparticulate drug delivery system for controlled release of emtricitabine.

The objective of this study was to develop systematically optimized (OPT) nanoparticles (NPs) providing a controlled release using PLGA of emtricitabine (FTC) employing Formulation by Design (FbD), and evaluate their in vitro and in vivo performance. FTC generates severe adverse effects with risks of toxicity. Thus, NPs were prepared to reduce these drawbacks in this study. The NPs were prepared by water-in-oil-in-water (w/o/w) emulsion method, followed by high-pressure homogenization. The FTC NPs were systematically OPT using 3(2) central composite design and the OPT formulation located using overlay plot. The pharmacokinetics and in vivo biodistribution of OPT-FTC NPs were investigated in male Wistar rats via the oral administration. Transmission electron microscopy studies on OPT-FTC NPs demonstrated uniform shape and size of particles. In vitro release was sustained up to 15 days in PBS pH 7.4. Augmentation in the values of Cmax (1.63 fold) and AUC0-∞ (5.39 fold) indicated significant enhancement in the rate and extent of bioavailability by the OPT-FTC NPs compared to pure drug. OPT-FTC NPs showed 2.325 fold increase in the values of FTC concentrations in liver. The OPT-FTC NPs was found to be quite stable during 6 months of study period. Hence, the developed OPT-FTC NPs can be used as drug carrier for sustained/prolonged drug release and/or to reduce toxic effects.

Taste Masked Orally Disintegrating Pellets of Antihistaminic and Mucolytic Drug: Formulation, Characterization, and In Vivo Studies in Human.

The main aim of the present study was to evaluate the potential of orally disintegrating pellets (ODPs) as an approach for taste masking of bitter drugs, namely, Ambroxol hydrochloride (A-HCl) and Cetirizine dihydrochloride (C-DHCl). Pellets were prepared by extrusion/spheronization with Eudragit EPO, kyron T-134, Kyron T-314, mannitol, sorbitol, MCC (Avicel PH-101), sucralose, chocolate flavor, and 5% xanthum gum. The prepared pellets were characterized for percentage yield, drug content, particle size, in vitro drug release, and in vivo evaluation on humans for taste, mouth feel, and in vivo disintegration time. The results revealed that the average size of pellets was influenced greatly by the percentage of binder and extrusion speed. The optimized ODPs disintegrated in less than 20 s and showed more than 98% of drugs in ODPs dissolved within 15 min. Taste perception study was carried out on human volunteers to evaluate the taste masking ability of ODPs for taste, mouth feel, and in vivo disintegration time. Crystalline state evaluation of drugs in the optimized ODPs was conducted for X-ray powder diffraction. In conclusion, the study confirmed that ODPs can be utilized as an alternative approach for effective taste masking and rapid disintegration in the oral cavity.

Relative rigidity of cell-substrate effects on hepatic and hepatocellular carcinoma cell migration.

Polyacrylamide gels with different stiffness and glass were employed as substrates to investigate how substrate stiffness affects the cellular stiffness of adherent hepatocellular carcinoma (HCCLM3) and hepatic (L02) cells. The interaction of how cell-substrate stiffness influences cell migration was also explored. An atom force microscope measured the stiffness of HCCLM3 and L02 cells on different substrates. Further, F-actin assembly was analyzed using immunofluorescence and Western blot. Finally, cell-surface expression of integrin ß1 was quantified by flow cytometry. The results show that, while both HCCLM3 and L02 cells adjusted their cell stiffness to comply with the stiffness of the substrate they were adhered to, their tuning capabilities were different. HCCLM3 cell stiffness complied when substrate stiffness was between 1.1 and 33.7 kPa, whereas the analogous stiffness for L02 cells occurred at a higher substrate stiffness, 3.6 kPa up to glass. These ranges correlated with F-actin filament assembly and integrin ß1 expression. In a migration assay, HCCLM3 cells migrated faster on a relatively soft substrate, while L02 cells migrated faster on substrates that were relatively rigid. These findings indicate that different tuning capabilities of HCCLM3 and L02 cells may influence cell migration velocity on substrates with different stiffness by regulating cy- toskeleton remodeling and integrin ß1 expression.

Novel multi-biotin grafted poly(lactic acid) and its self-assembling nanoparticles capable of binding to streptavidin.

Targeted drug delivery requires novel biodegradable, specific binding systems with longer circulation time. The aim of this study was to prepare biotinylated poly(lactic acid) (PLA) nanoparticles (NPs) which can meet regular requirements as well conjugate more biotins in the polymer to provide better binding with streptavidin. A biotin-graft-PLA was synthesized based on previously published biodegradable poly(ethylene glycol) (PEG)-graft-PLA, with one polymer molecule containing three PEG molecules. Newly synthesized biotin-graft-PLA had three biotins per polymer molecule, higher than the previous biotinylated PLA (≤1 biotin per polymer molecule). A PEG with a much lower molecular weight (MW ~1900) than the previous biotinylated PLA (PEG MW ≥ 3800), and thus more biocompatible, was used which supplied good nonspecific protein-resistant property compatible to PEG-graft-PLA, suggesting its possible longer stay in the bloodstream. Biotin-graft-PLA specifically bound to streptavidin and self-assembled into NPs, during which naproxen, a model small molecule (MW 230 Da) and hydrophobic drug, was encapsulated (encapsulation efficiency 51.88%). The naproxen-loaded NPs with particle size and zeta potential of 175 nm and -27.35 mV realized controlled release within 170 hours, comparable to previous studies. The biotin-graft-PLA NPs adhered approximately two-fold more on streptavidin film and on biotin film via a streptavidin arm both in static and dynamic conditions compared with PEG-graft-PLA NPs, the proven nonspecific protein-resistant NPs. The specific binding of biotin-graft-PLA NPs with streptavidin and with biotin using streptavidin arm, as well as its entrapment and controlled release for naproxen, suggest potential applications in targeted drug delivery.

Novel PEG-graft-PLA nanoparticles with the potential for encapsulation and controlled release of hydrophobic and hydrophilic medications in aqueous medium.

This study concerns the encapsulation and controlled release of both hydrophobic and hydrophilic medications with one polymer, which are delivered together as a combined therapy to treat diseased tissue. To test our hypothesis that the novel PEG-graft-PLA (PEG, polyethylene glycol; PLA, polylactic acid) can deliver both the hydrophobic and hydrophilic medications on account of its amphiphility, charge, and graft structure, PEG-graft-PLA (molecular weight of PEG = 1900) with very low critical micelle concentration was synthesized. One hydrophilic (insulin) and one hydrophobic (naproxen) model medication were loaded in separately during its self-assembly in aqueous solution. The resulting nanoparticles (NPs) were narrowly distributed and spherical, with average particle size around 200 nm, zeta potential >-10 mV, and encapsulation efficiency >50%. The NPs realized controlled release of insulin and naproxen for over 24 and 160 hours, respectively. Specifically, the bioactivity of the insulin released from the NPs was maintained. Owing to encapsulation, both for hydrophobic and hydrophilic medicines, and NPs obtained with similar size and zeta potential, as well as maintenance of bioactivity of loaded protein, we expect the applications of PEG-graft-PLA NPs in combination therapy.