Characterization and Exploration of Placket-Burman-Designed Porous Calcium Carbonate (Vaterite) Microparticles.

The objective of the research was to identify significant variables that impact the porosity-related properties of CaCO3 particles. The Placket-Burman design was employed to screen multiple variables, including pH, molar concentrations of calcium chloride and sodium carbonate, temperature, concentration of Gelucire 44/14, Cremophor RH40, Solutol HS15, Labrasol, mixing rate, reaction time, and order of addition. The response variables were surface area, pore radius, and pore volume. Influential methodologies such as XRD, FTIR, Raman spectroscopy, and TGA were utilized to validate the precipitate type. The BET surface area ranged from 1.5 to 16.14 m2/g, while the pore radius varied from 2.62 to 6.68 nm, and the pore volume exhibited a range of 2.43 to 37.97 cc/gm. Vaterite structures with spherical mesoporous characteristics were observed at high pH, whereas calcite formations occurred at low pH. The order of addition impacted the surface area but did not affect the pore volume. To maximize the surface area, a lower reaction time and molar concentrations of sodium carbonate were found to be advantageous. The pore radius was influenced by the pH, surfactants, and reaction conditions. The sediments were categorized based on the percentage of vaterite formation. The instrumental techniques effectively characterized the precipitates and provided a valuable complementary analysis.

Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry Estimation of Quercetin-Loaded Nanoemulsion in Rabbit Plasma: In Vivo-In Silico Pharmacokinetic Analysis Using GastroPlus.

In the present study, we developed and validated a rapid, specific, sensitive, and reproducible liquid chromatography-electrospray ionization tandem mass spectrometry method for quantifying quercetin (QT) in rabbit plasma using hydrochlorothiazide as the internal standard. Animals were orally administered with optimized QT-loaded nanoemulsion (QTNE) and QT suspension (QTS), equivalent to 30 mg/kg, to the test and control group, respectively. The blood samples were collected at pre-determined time points up to 48 h. The linearity range was from 5 to 5000 ng mL-1 with R 2 = 0.995. Further, we analyzed the various pharmacokinetic parameters and established the in vitro-in vivo correlation (IVIVC) of QTNE using GastroPlus software. The method was successfully developed and validated, and when applied for the determination of QT in rabbit plasma, it exhibited an increase in C max from 122.56 ng mL-1 (QTS) to 286.51 ng mL-1 (QTNE) (2.34-fold) and AUC0-48 from 976 ng h mL-1 (QTS) to 4249 ng h mL-1 (QTNE) (4.35-fold), indicating improved oral bioavailability QT when administered as QTNE. Statistical analysis revealed that the Loo-Riegelman method (two-compartmental method) best fitted the deconvolution approach (R 2 = 0.998, SEP = 4.537, MAE = 2.759, and AIC = 42.38) for establishing the IVIVC. In conclusion, the established bioanalytical method and IVIVC studies revealed that QTNE is a potential carrier for the effective delivery of QT with enhanced oral bioavailability.

Neuroprotective Role of Quercetin against Alpha-Synuclein-Associated Hallmarks in Parkinson's Disease.

Quercetin, a natural antioxidant, exhibits potential neuroprotective effects by efficiently downregulating α-synuclein protein aggregation and associated neurological hallmarks, responsible for the progression of Parkinson's Disease.

Therapeutic Potential of Quercetin-Loaded Nanoemulsion against Experimental Visceral Leishmaniasis: In Vitro/Ex Vivo Studies and Mechanistic Insights.

Visceral leishmaniasis (VL) is one of the most fatal and neglected tropical diseases caused by Leishmania donovani (L. donovani). The applications of currently available chemotherapy (amphotericin B, miltefosine, and others) in VL treatment have been limited due to their poor bioavailability, unfavorable toxicity profile, and prolonged parenteral dosing. Quercetin (QT), a potent natural antioxidant, is a prominent target when conducting investigations on alternative therapies against L. donovani infections. However, the therapeutic applications of QT have been restricted due to its low solubility and bioavailability. In the present study, we developed and evaluated the antileishmanial activity (ALA) of quercetin-loaded nanoemulsion (QTNE) against L. donovani clinical strains. In vitro anti-promastigote assay results demonstrated that QTNE (IC50 6.6 µM, 48 h) significantly inhibited the growth of parasites more efficiently than the pure QT suspension in a dose- and time-dependent manner. Results of the anti-amastigote assay revealed that the infected macrophages (%) of QTNE were significantly more than those of the pure QT suspension at all concentrations (6.6, 26.4, and 52.8 µM; p < 0.05, p < 0.01 compared to the control). Moreover, the results of in vitro and ex vivo studies assisted in determining the mechanistic insights associated with the ALA of QTNE. The overall findings suggested that QTNE exhibited potential ALA by enhancing the intracellular ROS and nitric oxide levels, inducing distortion of membrane integrity and phosphatidylserine release (AV/PI), rupturing the parasite DNA (late apoptosis/necrosis process), and upregulating the immunomodulatory effects (IFN-γ and IL-10 levels). Additionally, QTNE showed superior biocompatibility against all of the treated healthy cells (PBMCs, PECs, and BMCs) as compared to the control. In conclusion, QTNE acts as a potential antileishmanial agent targeting both promastigote and intracellular amastigote forms of L. donovani, which thus opens a new avenue for the use of QTNE in VL therapy.

Food-Grade Quercetin-Loaded Nanoemulsion Ameliorates Effects Associated with Parkinson's Disease and Cancer: Studies Employing a Transgenic C. elegans Model and Human Cancer Cell Lines.

A nanosized food-grade quercetin-loaded nanoemulsion (QNE) system comprising capmul MCM NF (oil) and cremophor RH 40 (surfactant) was developed using a high-speed homogenization technique. The developed QNE was studied for its significant neuroprotective (anti-Parkinsonism) and cytotoxicity (anticancer) effects against Caenorhabditis elegans (C. elegans) strains and human cancer cells, respectively. HR-TEM studies revealed that the QNE was spherical with a mean globule size of ~50 nm. Selected area electron diffraction (SAED) studies results demonstrated that QNE was amorphous. In vivo results show that QNE potentially reduced the α-Syn aggregation, increased mitochondrial and fat content, and improved the lifespan in transgenic C. elegans strain NL5901. QNE significantly downregulated the reactive oxygen species (ROS) levels in wild-type C. elegans strain N2. In vitro results of the MTT assay show that QNE significantly exhibited chemotherapeutic effects in all treated human cancer cells in an order of cytotoxicity: HeLa cells > A549 cells > MIA PaCa-2 cells, based on the IC50 values at 24 h. Conclusively, the QNE showed improved solubility, targetability, and neuroprotective effects against the PD-induced C. elegans model, and also cytotoxicity against human cancer cells and could be potentially used as an anti-Parkinson's or anticancer agent.

Studies on Core-Shell Nanocapsules of Felodipine: In Vitro-In Vivo Evaluations.

The present study aimed for in vitro-in vivo-in silico simulation studies of experimentally designed (32-factorial) Capmul PG-8-cored, Eudragit RSPO-Lutrol F 127 nanocapsules to ferry felodipine using GastroPlus™. The in silico parameter sensitivity analysis for pharmacokinetic parameters was initially assessed to justify the preparation of felodipine-loaded nanocapsules (FLNs) with enhanced solubility to overcome the bioavailability issues of felodipine. The overall integrated desirability ranged between 0.8187 and 0.9488 for three optimized FLNs when analyzed for mean particle size, zeta potential, encapsulation efficiency, and in vitro dissolution parameters. The morphological evaluation (SEM, TEM, and AFM) demonstrated spherical nanoparticles (200-300 nm). Validated LC-MS/MS analysis demonstrated enhanced relative bioavailability (13.37-fold) of optimized FLN as compared to suspension. The simulated regional absorption of the FLN presented significant absorption from the cecum (26.3%) and ascending colon (20.1%) with overall absorption of 67.4% from the GIT tract. Furthermore, in vitro-in vivo correlation demonstrated the Wagner-Nelson method as the preferred model as compared to mechanistic and numerical deconvolution on the basis of least mean absolute prediction error, least standard error of prediction, least mean absolute error, and maximum correlation coefficient (r 2 = 0.920). The study demonstrated enhanced oral absorption of felodipine-loaded nanocapsules, and GastroPlus™ was found to be an efficient simulation tool for in vitro-in vivo-in silico simulations.

Fabrication of lipidic nanocarriers of loratadine for facilitated intestinal permeation using multivariate design approach.

In this investigation, multivariate design approach was employed to develop self-nanoemulsifying drug delivery system (SNEDDS) of loratadine and to exploit its potential for intestinal permeability. Drug solubility was determined in various vehicles and existence of self-nanoemulsifying region was evaluated by phase diagram studies. The influence of formulation variables X1 (Capmul MCM C8) and X2 (Solutol HS15) on SNEDDS was assessed for mean globule sizes in different media (Y1-Y3), emulsification time (Y4) and drug-release parameters (Y5-Y6), to improve quality attributes of SNEDDS. Significant models were generated, statistically analyzed by analysis of variance and validated using the residual and leverage plots. The interaction, contour and response plots explicitly demonstrated the influence of one factor on the other and displayed trend of factor-effect on responses. The pH-independent optimized formulation was obtained with appreciable global desirability (0.9266). The strenuous act of determining emulsification time is innovatively replaced by the use of oil-soluble dye to produce visibly distinct globules that otherwise may be deceiving. TEM images displayed non-aggregated state of spherical globules (size < 25 nm) and also revealed the structural transitions occurring during emulsification. Optimized formulation exhibited non-Newtonian flow justified by the model-fit and also presented the stability to dilution effects and thermodynamic stress testing. The ex vivo permeation study using confocal laser scanning microscopy indicate strong potential of rhodamine 123-loaded loratadine-SNEDDS to inhibit P-gp efflux and facilitate intestinal permeation. To conclude, the effectiveness of design yields a stable optimized SNEDDS with enhanced permeation potential, which is expected to improve oral bioavailability of loratadine.

LC-ESI-MS/MS analysis of quercetin in rat plasma after oral administration of biodegradable nanoparticles.

A simple, rapid and sensitive LC-MS/MS method was developed and validated for the determination of free quercetin in rat plasma, using fisetin as internal standard. The detection was performed by negative ion electrospray ionization under selected reaction monitoring. Chromatographic separation (isocratic elution) was carried out using acetonitrile-10 m m ammonium formate (80:20, v/v) with 0.1% v/v formic acid. The lower limit of quantification (4.928 ng/mL) provided high sensitivity for the detection of quercetin in rat plasma. The linearity range was from 5 to 2000 ng/mL. Intra- and inter-day variability (RSD) of quercetin extraction from rat plasma was <4.19 and 1.37% with accuracies of 98.77 and 99.67%. The method developed was successfully applied for estimating free quercetin in rat plasma, after oral administration of quercetin-loaded biodegradable nanoparticles (QLN) and quercetin suspension. QLN (C(max), 1277.34 ± 216.67 ng/mL; AUC, 17,458.25 ± 3152.95 ng hr/mL) showed a 5.38-fold increase in relative bioavailability as compared with quercetin suspension (C(max), 369.2 ± 108.07 ng/mL; AUC, 3276.92 ± 396.67 ng hr/mL).

Evidence for bactericidal activities of lipidic nanoemulsions against Pseudomonas aeruginosa.

Pseudomonas aeruginosa has been implicated in a broad range of infections and shown to acquire rapid resistance to anti-microbial agents. In the present, study we have used particular amalgamation of specific lipids that hold innate antibacterial activities, which can be transformed into cationized and non-cationized nanoemulsions. The anti-Pseudomonas activities were then elucidated by transmission/scanning electron microscopy, and atomic force microscopy. The microscopic studies revealed the cell lysis due to the formation of blebs, exudation of essential cellular contents and loss of characteristics contour of the cells. The microscopic studies were then corroborated by zone of inhibition, cytoplasmic release studies, time dependent killing and MIC determination. Conclusively, it can be inferred that the delivery issues of antibiotics could be reassessed by using certain excipients that possess inherent antibacterial properties. This will not only avoid unnecessary introduction of inactive excipients in the body, but will also reduce the dose of antibiotics because of synergistic effects of excipients and drug acting together.

New insights into the diagnosis and chemotheraphy for visceral leishmaniasis.

Visceral leishmaniasis (VL) is a severe form of leishmaniasis - a disease caused by protozoan parasites and transmitted by the bite of certain species of sand fly. In VL, parasites migrate to the vital organs and bone marrow, destroying white and red blood cells. VL has been called the parasitic version of HIV/AIDs (Human immunodeficiency virus infection / acquired immunodeficiency syndrome), as it attacks the immune system. The most common form of the disease is cutaneous leishmaniasis, which causes skin lesions and often leaves the infected individual permanently disfigured. Even though the disease is treatable, current treatments are difficult to administer, too expensive, or toxic for extensive use in developing nations. Furthermore, resistance to treatment is an increasing problem, particularly in India. 90% of people with VL die if the infection is left untreated, and death can come within 2 years, significantly faster than AIDS. The search for new drugs continues, with new chemical and natural compounds. Many potential drug targets have been identified in biochemical and molecular studies, and some have been validated. Attempts to exploit these targets are in progress. This article reviews the epidemiology, clinical features, control Strategies, diagnosis and current treatment about visceral leishmaniasis.

Formulation by design of felodipine loaded liquid and solid self nanoemulsifying drug delivery systems using Box-Behnken design.

OBJECTIVE: To design and develop liquid and solid self-nanoemulsifying drug delivery systems (SNEDDS and S-SNEDDS) of felodipine (FLD) using Box-Behnken design (BBD). METHODS: Solubility study was carried out in various vehicles. Ternary phase diagram was constructed to delineate the boundaries of the nanoemulsion domain. The content of formulation variables, X1 (Acconon E), X2 (Cremophor EL) and X3 (Lutrol E300) were optimized by assessment of 15 formulations (as per BBD) for mean globule sizes in Millipore water (Y1), 0.1 N HCl (Y2), phosphate buffer (pH 6.4) (Y3); emulsification time (Y4) and T85% (Y5). The responses (Y1-Y5) were evaluated statistically by analysis of variance and response surface plots to obtain optimum points. The optimized formulations were solidified by adsorption to solid carrier technique using Aerosil 200 (AER). RESULTS AND DISCUSSION: Transmission electron microscopy images confirmed the spherical shape of globules with the size range concordant with the globule size analysis by dynamic light scattering technique (<60 nm). The surface morphology of S-SNEDDS (before release) by scanning electron microscopy and atomic force microscopy indicated that SNEDDS are adsorbed uniformly on the surface of AER. The dried residue of S-SNEDDS (after release) revealed the presence of nanometric pores vacated by the previously adsorbed SNEDDS onto AER. Differential scanning calorimetry and X-ray powder diffraction studies illustrated the change of FLD from crystalline to amorphous state. CONCLUSION: This study indicates that owing to nanosize, SNEDDS and S-SNEDDS of FLD have potential to enhance its absorption and may serve an efficient oral delivery.

Development of doxorubicin loaded novel core shell structured nanocapsules for the intervention of visceral leishmaniasis.

The aim of this study was to develop novel nanoemuslion core loaded nanocapsules (NCs) with high payload of doxorubicin (DOX) and to assess its efficacy against Leishmania donovani. The low energy emulsification method was used to obtained nanoemulsion core as template, followed by stepwise addition of additional layer components protamine sulphate and sodium alginate. Zeta potential studies revealed that there was reversal in charge after each layering. NCs were characterized on the basis of size (340 nm) and entrapment efficiency (>80%). The drug release behaviour was studied by in vitro method. The NCs loaded with DOX (NCs-DOX) is completely internalized into macrophages showing improved efficacy (IC50 of formulation is almost ≤ 1.9-fold as compared to plain drug, p < 0.05) against intracellular amastigotes.

Partial biodistribution and pharmacokinetics of isoniazid and rifabutin following pulmonary delivery of inhalable microparticles to rhesus macaques.

Dry powder inhalations (DPI) of microparticles containing isoniazid (INH) and rifabutin (RFB) are under preclinical development for use in pulmonary tuberculosis. Microparticles containing 0.25, 2.5, or 25 mg of each drug were administered daily for 90 days to rhesus macaques (n = 4/group). Single inhalations or intravenous (i.v.) doses were administered to separate groups. Drugs in serum, alveolar macrophages, and organ homogenates were assayed by high-performance liquid chromatography (HPLC). The RFB/INH in the lungs (101.10 ± 12.90/101.07 ± 8.09 µg/g of tissue) was twice that of the liver concentrations (60.22 ± 04.97/52.08 ± 4.62 µg/g) and four times that of the kidneys (22.89 ± 05.22/30.25 ± 3.71 µg/g). Pharmacokinetic parameters indicated the operation of flip-flop kinetics. Thus, the elimination half-life (t(1/2)) of RFB and INH was calculated as 8.01 ± 0.5 and 2.49 ± 0.23 h, respectively, upon intravenous (iv) administration, and as 13.8 ± 0.8 and 10.43 ± 0.77 h following a single inhalation; or 13.36 ± 3.51 and 10.13 ± 3.01 at a presumed steady state (day 60 of dosing). Targeted and sustained drug delivery to nonhuman primate lungs and alveolar macrophages was demonstrated. Flip-flop serum pharmacokinetics was observed, and nonlinearity in some pharmacokinetic parameters at logarithmic dose increments was indicated. The results suggest that human patients would benefit through improvement in biodistribution following DPI.

Glibenclamide-loaded self-nanoemulsifying drug delivery system: development and characterization.

OBJECTIVE: To develop and characterize self-nanoemulsifying drug delivery system (SNEDDS) of the poorly water-soluble drug, glibenclamide (GBD). METHODS: Solubility of GBD was determined in various vehicles. Phase diagrams were constructed to identify efficient self-emulsification region using oils, surfactants, and cosurfactants in aqueous environment. Formulations were assessed for drug content, spectroscopic clarity, emulsification time, contact angle, zeta potential, particle size, and dissolution studies. On the basis of similarity and dissimilarity of particle size distribution, formulations were further characterized using principal component analysis and agglomerative hierarchy cluster analysis. RESULTS: Among the formulations prepared and evaluated, optimized formulation showed mean particle size between 15.65 and 32.70 nm after 24 hour postdilution in various media. Dilution volume had no significant effect on particle size. Transmission electron microscopy of these formulations confirmed the spherical shape of globules with no signs of coalescence of globules and precipitation of drug. The relevance of difference in t(50%) and percent dissolution efficiency were evaluated statistically by two-way ANOVA. Infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction studies indicated compatibility between drug, oil, and surfactants. CONCLUSIONS: The results of this study indicate that the self-nanoemulsifying drug delivery system of GBD, owing to nanosize, has potential to enhance its absorption and without interaction or incompatibility between the ingredients.

Development and characterization of a lovastatin-loaded self-microemulsifying drug delivery system.

The objective of the work was to develop, optimize and evaluate a self-microemulsifying drug delivery system of the poorly water soluble drug, lovastatin. Solubility of lovastatin was determined in various vehicles. Ternary phase diagrams were constructed to identify the efficient self-emulsification region using oils, surfactants, and co-surfactants in aqueous environment. Optimized formulations were assessed for drug content, spectroscopic clarity, emulsification time, contact angle, zeta potential, particle size and dissolution studies. Zeta potential was measured in absence and presence of oleylamine, a positive charge inducer. On the basis of similarity and dissimilarity of particle size distribution, formulations were characterized using principal component analysis and agglomerative hierarchy cluster analysis, the multivariate statistical analysis. Transmission electron microscopy of selected formulations (F5-F7) confirmed the spherical shape of globules with no signs of coalescence of globules and precipitation of drug, even after 24 h post dilution in distilled water. The relevance of differences in t(50%) and percentage dissolution efficiency was evaluated statistically by two-way ANOVA. Infrared spectroscopy, differential scanning calorimetric and x-ray diffraction studies indicated no incompatibility between drug, oil and surfactants. The results of this study indicate that the self-microemulsifying drug delivery system of lovastatin, owing to nanosize, has potential to enhance its absorption and without interaction or incompatibility between the ingredients.

[Design and development of hydroxypropyl methycellulose (HPMC) based polymeric films of methotrexate: physicochemical and pharmacokinetic evaluations].

The present investigation was aimed to evaluate the possibility of using different concentrations and polymeric grades of hydroxypropyl methylcellulose (K4M, K15M and K100M) for transdermal delivery of methotrexate, an immunosuppressant drug for rheumatoid arthritis. The matrix films were evaluated for their physicochemical characterization followed by in vitro and in vivo evaluation. Selected formulations were subjected for their in vivo studies on healthy rabbits following balanced incomplete block design. The relevance of difference in the in vitro dissolution rate profile and pharmacokinetic parameters (C(max), t(max), AUC((s)), t(1/2), K(el), and MRT) were evaluated statistically. The thickness and weight of the patch increased with the increase in polymeric grade and content. Fourier transform infrared spectroscopy and differential scanning calorimetry results confirm that there is no interaction between drug and polymer used. X-ray diffraction study reveals an amorphous state of drug in the matrix films. The in vitro drug release followed Higuchi kinetics (r=0.972-997; p<0.001) as its coefficient of correlation values predominates over zero order and first order release kinetics. In vitro dissolution profiles and pharmacokinetic parameters showed a significant difference between test products (p<0.01), but not within test products. A quantitatively good correlation was found between per cent of drug absorbed from the transdermal patches and AUC((s)). A significant in vitro/in vivo correlation was observed when per cent drug released was correlated with serum drug concentration. Out of the various formulations made, the selected formulations are better in their in vitro dissolution and pharmacokinetic characteristics and thus hold potential for transdermal delivery.