Mechanistic insights into immunopathogenesis of murine cerebral malaria: Cues from "young" C57BL/6J and BALB/c mice.

Cerebral malaria (CM), a major cause of mortality in children <5 years, presents disparity in pathophysiological features and poor prognosis compared to adults. Adult C57BL/6J mice infected with Plasmodium berghei ANKA (PbA) are widely used to understand CM pathogenesis compared to relatively less prone BALB/c mice; however, age and immune status of the host also influence disease sequelae and cerebral manifestations. Murine models of CM known so far do not project complete disease spectrum of pediatric CM. The present study was designed to dissect and differentiate CM immunopathogenesis in "young" BALB/c and C57BL/6J mice infected with PbA, in search of a competent mouse model mimicking pediatric CM. Multipronged approach including the analysis of blood-brain barrier (BBB) permeability and parasite infiltration, histopathology, nitric oxide levels, and pro/anti-inflammatory (TNF-α, IFN-γ, IL-4, and IL-10) cytokine expression were compared in the cortices of both young BALB/c and C57BL/6J mice. The results illustrate severe course of infection and typical CM like histopathological alterations including monocytic plugging in PbA-infected "young" BALB/c compared to C57BL/6J mice. The decreased expression of tight junction proteins (ZO-1 and Claudin-3) and Evan's blue extravasation was also more evident in BALB/c mice indicating a more permeable BBB. The increased cortical expression of TNF-α, IFN-γ, IL-4, IL-10, iNOS, eNOS, nNOS, and associated activation of brain resident cells in cortices of BALB/c with progressive parasitaemia depicts the cumulative involvement of host immune responses and parasite accumulation in progression of CM. Thus, the incongruity of cytokine balance resulted in worsening of disease manifestation in "young" BALB/c similar to pediatric CM.

Non-small cell lung cancer tumour antigen, MUC-1 peptide-loaded non-aggregated poly (lactide-co-glycolide) nanoparticles augmented cellular uptake in mouse professional antigen-presenting cells: optimisation and characterisation.

Aim: MUC-1 lipopeptide vaccine exhibited immense potential in the treatment of non-small cell lung cancer (NSCLC) in both preclinical and clinical trials. However, it lacks triggering of mucosal immunity at the site of action. Therefore, in present investigation, MUC-1 peptide-loaded poly(lactide-co-glycolide) nanoparticles (MUC-1 peptide-PLGA-NPs) and MUC-1 peptide-loaded poly(lactide-co-glycolide) non-aggregated nanoparticles (MUC-1 peptide-PLGA-NA-NPs) using Central Composite Design (CCD) were customised.Methods and Results: The mean particle size of MUC-1 peptide PLGA-NPs was estimated to be 176.7 ± 32.7 nm, significantly (p < 0.05) higher than 100.3 ± 24.3 nm of MUC-1 peptide-PLGA-NA-NPs. Furthermore, integrity and stability of MUC-1 were maintained in MUC-1 peptide PLGA-NA-NPs. MUC-1 peptide-PLGA-NA-NPs exhibited augmented cellular uptake in mouse RAW264.7 macrophages preferably by clathrin-mediated endocytosis pathway as compared to phagocytosis followed by MUC-1-peptide PLGA-NPs owing to size ≤100 nm, and spherical shape.Conclusion: MUC-1 peptide-PLGA-NA-NPs may be a potential candidate to study antitumor potential in xenograft model of NSCLC through inhalation route of administration.

Metacaspases: Potential Drug Target Against Protozoan Parasites.

Among the numerous strategies/targets for controlling infectious diseases, parasites-derived proteases receive prime attention due to their essential contribution to parasite growth and development. Parasites produce a broad array of proteases, which are required for parasite entry/invasion, modification/degradation of host proteins for their nourishment, and activation of inflammation that ensures their survival to maintain infection. Presently, extensive research is focused on unique proteases termed as "metacaspases" (MCAs) in relation to their versatile functions in plants and non-metazoans. Such unique MCAs proteases could be considered as a potential drug target against parasites due to their absence in the human host. MCAs are cysteine proteases, having Cys-His catalytic dyad present in fungi, protozoa, and plants. Studies so far indicated that MCAs are broadly associated with apoptosis-like cell death, growth, and stress regulation in different protozoa. The present review comprises the important research outcomes from our group and published literature, showing the variable properties and function of MCAs for therapeutic purpose against infectious diseases.

Data on retinoic acid and reduced serum concentration induced differentiation of Neuro-2a neuroblastoma cells.

The present data describe the relative neuro-2a cellular differentiation induced by reducing serum concentration (0.1% FBS) in DMEM in the presence/absence of 20 µM retinoic acid (RA). Neurite outgrowth was observed within 24 h in DMEM supplemented with reduced serum and retinoic acid (GpIV). The CFSE based proliferation assay data signified cessation of neuro-2a cellular proliferation in GpIV. An increase in the number of cells arrested at G0/G1 phase was also evident in GpIV and DMEM supplemented with 0.1% FBS (GpIII). Moreover, GpIV cells had improved mRNA and protein expression of Rbfox3/NeuN and choline acetyltransferase (ChAT).

Antidepressant and anxiolytic like effects of Urtica dioica leaves in streptozotocin induced diabetic mice.

The present study was aimed to investigate the effect of Urtica dioica Linn. (UD) extract against chronic diabetes mediated anxiogenic and depressive like behavior in mice. Streptozotocin (STZ) (50 mg/kg, i.p.) for 5 consecutive days was used to induce diabetes followed by treatment with UD leaves extract (50 mg/kg, p.o.) and rosiglitazone (ROSI) (5 mg/kg, p.o.) for 8 weeks. STZ induced chronic diabetes significantly induced anxiety and depressive like behavior in mice. Chronic diabetes significantly downregulated BDNF (p < 0.001), TrKB (p < 0.001), Cyclin D1 (p < 0.001), Bcl2 (p < 0.05) and autophagy7 (p < 0.001), while upregulated iNOS (p < 0.05) mRNA expression in the hippocampus as compared to control mice. In addition, chronic diabetes significantly increased the expression of TNF-α in CA1 (p < 0.001), CA2 (p < 0.01), CA3 (p < 0.001) and DG (p < 0.001) regions of hippocampus as compared to control mice. Chronic diabetes mediated neuronal damage in the CA2, CA3 and DG regions of hippocampus. Chronic administration of UD leaves extract significantly reversed diabetes mediated anxiogenic and depressive like behavior in mice. Further, UD treatment significantly upregulated BDNF (p < 0.01), TrKB (p < 0.001), Cyclin D1 (p < 0.001), Bcl2 (p < 0.01), autophagy5 (p < 0.01) and autophagy7 (p < 0.001), while downregulated iNOS (p < 0.05) mRNA expression in the hippocampus of diabetic mice. Concomitantly, UD administration significantly decreased the expression of TNF-α in hippocampal CA1 (p < 0.001), CA2 (p < 0.01), CA3 (p < 0.001) and DG (p < 0.001) regions of diabetic mice. Diabetes mediated neuronal damage and DNA fragmentation in the hippocampus was substantially attenuated following UD treatment. UD leaves extract might prove to be effective for diabetes mediated anxiety and depressive like behavior.

Biochemical characterization of unusual cysteine protease of P. falciparum, metacaspase-2 (MCA-2).

Earlier studies on Plasmodium apoptosis revealed the presence of proteases with caspases like- activity, which are known as "metacaspases". Although this family of cysteine proteases is structurally similar to caspases with Cys-His dyad but their evolutionary significance and functional relevance remains largely unknown. These proteases are considered to be an important target against malaria due to their absence in humans. In this report, we have biochemically characterized metacaspase-2 (PfMCA-2) of P.falciparum. Enzymatic assay showed that PfMCA-2 efficiently cleaved arginine/lysine specific peptide, but not caspase-specific substrate. Consistently, PfMCA-2 activity was sensitive to effector caspases inhibitor, Z-FA-FMK, and mildly inhibited by aprotinin and E-64. However, general caspase inhibitors such as Z-VAD-FMK and Z-DEVD-FMK had no effect on PfMCA-2 activity. Z-FA-FMK inhibits parasite growth with an IC50 value of 2.7 µM along with the notable morphological changes. PfMCA-2 specifically expressed in schizonts and gametocyte stages and there was a notable depletion of PfMCA-2 expression in Z-FA-FMK treated schizonts and gametocytes stages of parasite. Notably, PfMCA-2 cleaves a phylogenetically conserved protein, TSN (Tudor staphylococcal nuclease) and the proteolysis of PfTSN did not occur after treatment with the Z-FA-FMK. The production of large amount of reactive oxygen species in presence of Z-FA-FMK caused oxidative stress which in turn leads to loss of cell viability. The oxidative stress further generates positive feedback for the occurrence of cell death in term of phosphatidylserine externalization and DNA fragmentation in vitro.

Noscapinoids bearing silver nanocrystals augmented drug delivery, cytotoxicity, apoptosis and cellular uptake in B16F1, mouse melanoma skin cancer cells.

PURPOSE: Noscapine (Nos) and reduced brominated analogue of noscapine (Red-Br-Nos) prevent cellular proliferation and induce apoptosis in cancer cells either alone or in combination with other chemotherapeutic drugs. However, owing to poor physicochemical properties, Nos and Red-Br-Nos have demonstrated their anticancer activity at higher and multiple doses. Therefore, in present investigation, silver nanocrystals of noscapinoids (Nos-Ag2+ nanocrystals and Red-Br-Nos-Ag2+ nanocrystals) were customized to augment drug delivery, cytotoxicity, apoptosis and cellular uptake in B16F1 mouse melanoma cancer cells. METHODS AND RESULTS: Nos-Ag2+ nanocrystals and Red-Br-Nos-Ag2+ nanocrystals were prepared separately by precipitation method. The mean particle size of Nos-Ag2+ nanocrystals was measured to be 25.33±3.52nm, insignificantly (P>0.05) different from 27.43±4.51nm of Red-Br-Nos-Ag2+ nanocrystals. Furthermore, zeta-potential of Nos-Ag2+ nanocrystals was determined to be -25.3±3.11mV significantly (P<0.05) different from -15.2±3.33mV of Red-Br-Nos-Ag2+ nanocrystals. The shape of tailored nanocrystals was slightly spherical and or irregular in shape. The architecture of Nos-Ag2+ nanocrystals and Red-Br-Nos-Ag2+ nanocrystals was crystalline in nature. FT-IR spectroscopy evinced the successful interaction of Ag2+ nanocrystals with Nos and Red-Br-Nos, respectively. The superior therapeutic efficacy of tailored nanocrystals was measured in terms of enhanced cytotoxicity, apoptosis and cellular uptake. The Nos-Ag2+ nanocrystals and Red-Br-Nos-Ag2+ nanocrystals exhibited an IC50 of 16.6µM and 6.5µM, significantly (P<0.05) lower than 38.5µM of Nos and 10.3µM of Red-Br-Nos, respectively. Finally, cellular morphological alterations in B16F1 cells upon internalization of Nos-Ag2+ nanocrystals and Red-Br-Nos-Ag2+ nanocrystals provided the evidences for accumulation within membrane-bound cytoplasmic vacuoles and in enlarged lysosomes and thus triggered mitochondria mediated apoptosis via caspase activation. CONCLUSION: Preliminary investigations substantiated that Nos-Ag2+ nanocrystals and Red-Br-Nos-Ag2+ nanocrystals must be further explored and utilized for the delivery of noscapinoids to melanoma cancer cells.

Preclinical Evaluation of DMA, a Bisbenzimidazole, as Radioprotector: Toxicity, Pharmacokinetics, and Biodistribution Studies in Balb/c Mice.

Radiotherapy, a therapeutic modality of cancer treatment, nonselectively damages normal tissues as well as tumor tissues. The search is ongoing for therapeutic agents that selectively reduce radiation-induced normal tissue injury without reducing tumoricidal effect, thereby increasing the therapeutic ratio of radiation therapy. Our laboratory established 5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxyphenyl)-5'benzimidazolyl] benzimidazole (DMA) as noncytotoxic radioprotector in mammalian cells. DMA showed an excellent radioprotection in mice at single nontoxic oral dose by a dose-reduction factor of 1.28. An oxygen radical absorbing capacity assay confirmed its free-radical quenching ability. Single bolus dose and 28-days of repeated administration of DMA in mice for toxicity studies determined an LD50 of >2000 mg/kg body weight (bw) and 225 mg/kg bw, respectively, suggesting DMA is safe. Histopathology, biochemical parameters, and relative organ weight analysis revealed insignificant changes in the DMA-treated animals. The pharmacokinetic study of DMA at oral and intravenous doses showed its C(max) = 1 hour, bioavailability of 8.84%, elimination half-life of 4 hours, and an enterohepatic recirculation. Biodistribution study in mice with Ehrlich ascites tumors showed that (99m)Tc-DMA achieved its highest concentration in 1 hour and was retained up to 4 hours in the lungs, liver, kidneys, and spleen, and in a low concentration in the tumor, a solicited property of any radioprotector to protect normal cells over cancerous cells. We observed that the single-dose treatment of tumor-bearing mice with DMA 2 hours before 8 Gy total body irradiation showed an impressive rescue of radiation-induced morbidity in terms of weight loss and mortality without a change in tumor response.

Improved cisplatin delivery in cervical cancer cells by utilizing folate-grafted non-aggregated gelatin nanoparticles.

PURPOSE: Cisplatin is highly effective in the treatment of cervical cancer. However, in therapeutic doses, cisplatin induces several adverse effects due to undesirable tissue distribution. Therefore, it is worth targeting cisplatin in cervical cancer cells by implicating non-aggregated ligand-modified nanotherapeutics. METHODS AND RESULTS: Here, we report the preparation of non-aggregated folic acid-conjugated gelatin nanoparticles of cisplatin (Cis-GNs-FA) by two-step desolvation method with mean particle size of 210.6±9.6nm and 140.5±10.9nm for Cis-GNs to improve the drug delivery in cervical cancer, HeLa cells. FTIR and DSC spectra confirmed the presence and stability of cisplatin in gelatin matrix. Furthermore, amorphization of cisplatin in nanoparticles was ascertained by PXRD. Drug release followed a first-order release kinetic at both pH âˆ¼ 5.6 (cervical cancer pH) and pH âˆ¼ 7.4. In addition, a significant (P<0.05) decrease in IC50 value (8.3µM) and enhanced apoptosis were observed in HeLa cells treated with Cis-GNs-FA as compared to Cis-GNs (15.1µM) and cisplatin solution (40.2µM). In contrast, A549 lung cancer cells did not discriminate between Cis-GNs-FA and Cis-GNs due to the absence of folate receptors-α (FR-α). Consistently, higher cellular uptake, 80.54±7.60% was promoted by Cis-GNs-FA significantly (two-way ANOVA, P<0.05) greater than 51.68±9.78%, by Cis-GNs. This was also illustrated by CLSM images, which indicated that Cis-GNs-FA preferably accumulated in the cytoplasm of HeLa cells nearby nucleus by following receptor-mediated endocytosis pathway as compared to Cis-GNs. CONCLUSION: Therefore, Cis-GNs-FA warrants further in-depth in vitro and in vivo investigations to scale up the technology for clinical translation.

Enhanced noscapine delivery using estrogen-receptor-targeted nanoparticles for breast cancer therapy.

Noscapine (Nos), an orally available plant-derived antitussive alkaloid, is in phase II clinical trials for cancer chemotherapy. It has extensively been shown to inhibit tumor growth in nude mice bearing human xenografts of hematopoietic, breast, lung, ovarian, brain, and prostate origin. However, high tumor-suppressive Nos dosages encumber the development of oral controlled-release formulations because of a short biological half-life (<2 h), poor absorption, low aqueous solubility, and extensive first-pass metabolism. Here, we present the design, fabrication, optimization, characterization, and biological evaluation of estrone-conjugated noscapine-loaded gelatin nanoparticles (Nos-ES-GN) for targeting estrogen-receptor-positive breast cancer MCF-7 cells. Gelatin nanoparticles (GN) were a uniformly compact size, stable at physiological pH, and showed a drug entrapment efficiency of 66.1±5.9 and 65.2±5.6% for Nos-GN and Nos-ES-GN, respectively. The secondary structure of gelatin nanocoacervates was predicted using circular dichroism and in-silico molecular modeling. Our data suggest that ethanol-fabricated GN retained the α-helical content of gelatin, whereas acetone favored the formation of random coils. The conjugation of estrone to Nos-GN did not affect the release rate of the drug, and both formulations followed first-order release kinetics with an initial burst, followed by a slow release. The IC50 value of Nos-ES-GN was 21.2 µmol/l, which was ∼50% lower than the free drug (43.3 µmol/l), suggesting targeted drug delivery. Our cell uptake study carried out in an estrogen-receptor-positive (MCF-7) and negative (MDA-MB-231) cancer cell lines showed greater accumulation of Nos-ES-GN in MCF-7 cells instead of MDA-MB-231 cells. Our data indicated that estrone-conjugated nanoparticles may potentially be used for targeting breast cancer cells.

Sustained-release protamine sulphate-impregnated microspheres may reduce the frequent administration of recombinant interferon α-2b in ovarian cancer: in-vitro characterization.

Parenteral administration of recombinant interferon-α-2b (rINF-α-2b) at a dose of 50×10 IU once a week for 8 weeks is recommended for ovarian cancer. However, short half-life, small therapeutic index and proteolytic degradation cause fluctuations in plasma level and pose barriers in the development of a clinically viable dosage form. Therefore, in the present investigation, fluorescein isothiocynate-tagged rINF-α-2b was loaded into stearic acid (*rINF-α-2b-SMs), pectin (*rINF-α-2b-PMs) and gelatin (*rINF-α-2b-GMs) microspheres. Parameters such as particle size, ζ potential, encapsulation efficiency and in-vitro release were studied to follow the optimization process. The formulation, *rINF-α-2b-GMs of particle size 8.3±2.1 µm with an encapsulation efficiency of 76.0±7.4%, offered 97.4% of *rINF-α-2b release at 288 h. Thus, negatively charged extended-release formulation *rINF-α-2b-GMs was then tethered with a gradient concentration (5-20 mg/ml) of a cationic arginine-rich protein stabilizer, protamine sulphate (Pt). The nanoformulation, *rINF-α-2b-Pt-GMs-15 superimposed with 15 mg/ml of Pt, released 95.0% of *rINF-α-2b at 336 h and was designated as the optimized formulation. The optimized formulation also conserved the primary and secondary structure of *rINF-α-2b as analysed by gel electrophoresis and circular dichroism. Moreover, in-vitro cytotoxicity analysis of SKOV3 cells of the optimized nanoformulation reported significantly (one-way analysis of variance test, P<0.05) lower IC50 (414.3 IU/ml) compared with *rINF-α-2b-GMs (514.3 IU/ml) and pure rINF-α-2b (628.6 IU/ml) at 72 h by offering a prolonged cytotoxic effect. Therefore, *rINF-α-2b-Pt-GMs-15, a promising nanomedicine, warrants further in-depth in-vivo study to scale up the technology for clinical translation.

Sterically stabilized gelatin microassemblies of noscapine enhance cytotoxicity, apoptosis and drug delivery in lung cancer cells.

Noscapine, recently identified as anticancer due to its microtubule-modulating properties. It is presently in Phase I/II clinical trials. The therapeutic efficacy of noscapine has been established in several xenograft models. Its pharmacokinetic limitations such as low bioavailability and high ED50 impede development of clinically relevant treatment regimens. Here we present design, synthesis, in vitro and in vivo characterization of sterically stabilized gelatin microassemblies of noscapine (SSGMS) for targeting human non-small cell lung cancer A549 cells. The average size of the sterically stabilized gelatin microassemblies of noscapine, SSGMS was 10.0±5.1 µm in comparison to noscapine-loaded gelatin microassemblies, GMS that was 8.3±5.5 µm. The noscapine entrapment efficiency of SSGMS and GMS was 23.99±4.5% and 24.23±2.6%, respectively. Prepared microassemblies were spherical in shape and did not show any drug and polymer interaction as examined by FTIR, DSC and PXRD. In vitro release data indicated that SSGMS and GMS follow first-order release kinetics and exhibited an initial burst followed by slow release of the drug. In vitro cytotoxicity evaluated using A549 cells showed a low IC50 value of SSGMS (15.5 µM) compared to GMS (30.1 µM) and free noscapine (47.2 µM). The SSGMS can facilitate a sustained therapeutic effect in terms of prolonged release of noscapine as evident by caspase-3 activity in A549 cells. Concomitantly, pharmacokinetic and biodistribution analysis showed that SSGMS increased the plasma half-life of noscapine by ~9.57-fold with an accumulation of ~48% drug in the lungs. Our data provides evidence for the potential usefulness of SSGMS for noscapine delivery in lung cancer.

Poly (ethylene)-glycol conjugated solid lipid nanoparticles of noscapine improve biological half-life, brain delivery and efficacy in glioblastoma cells.

Noscapine crosses blood-brain-barrier and inhibits proliferation of glioblastoma cells. However, short plasma half-life and rapid elimination necessitate the administration of multiple injections for successive chemotherapy. Noscapine bearing solid lipid nanoparticles, Nos-SLN and poly (ethylene)-glycol conjugated solid lipid nanoparticles of noscapine, Nos-PEG-SLN of 61.3 ± 9.3-nm and 80.5 ± 8.9-nm containing 80.4 ± 3.2% and 83.6 ± 1.2% of Nos, were constructed. First order kinetic and Higuchi equation were followed to release the Nos at intracellular pH~4.5. Further, a decrease in IC50 (Nos; 40.5 µM>Nos-SLN; 27.2 µM>20.8 µM) and enhanced subG1 population were observed in U87cells. Plasma half-life was enhanced up to ~11-fold and ~5-fold by Nos-PEG-SLN and Nos-SLN which significantly (P<0.05) deposits 400.7 µg/g and 313.1 µg/g of Nos in comparison to 233.2 µg/g by drug solution. This is first report demonstrating a workable approach to regulate the administration of multiple injections of Nos, warranting further in vivo tumor regression study for superior management of brain cancer. FROM THE CLINICAL EDITOR: This report describes a possible approach to regulate the administration of multiple injections of Noscapine using solid lipid nanoparticles. The data warrant further in vivo tumor regression studies for optimal management of glioblastoma, a generally very poorly treatable brain cancer.

Immunoproteomic identification of biotransformed self-proteins from the livers of female Balb/c mice following chronic ethanol administration.

Chronic alcohol consumption culminates in alcoholic hepatitis which is characterized by ballooning degeneration of hepatocytes and perivenous inflammation. The aldehydes produced by ethanol oxidation and lipid peroxidation form adducts with the hepatic proteins rendering them immunogenic and initiating an autoimmune response. The present study was designed to identify these immunoreactive hepatic proteins in ethanol-treated Balb/c mice. Liver cytosolic, mitochondrial, and microsomal proteins from the ethanol-treated and control female Balb/c mice were size fractionated on SDS-PAGE and immunoblotted with the sera from the individual animal. The immunoreactive proteins were identified using antimouse IgG antibody and characterized by MALDI-TOF. It is the first report demonstrating that 15 hepatic proteins show immunoreactivity following alcohol administration. The identified autoreactive proteins ranged in function from metabolism to cytoskeletal support. Remarkably, three key enzymes of ethanol metabolism, namely alcohol dehydrogenase, aldehyde dehydrogenase I and III as well as important antioxidant enzyme glutathione S-transferase were found to be autoreactive upon ethanol treatment. We conclude that ethanol treatment induces biotransformation of host proteins from almost every compartment of the cell, especially the enzymes involved in the detoxification of ethanolic insult being the major target for biotransformation. Hence, we propose that these proteins can be the potential candidates for the biomarker studies.

Galactosylated gelatin nanovectors of doxorubicin inhibit cell proliferation and induce apoptosis in hepatocarcinoma cells.

We have synthesized and characterized doxorubicin (DOX)-loaded galactosylated gelatin nanovectors using in vitro and in vivo for targeting liver cells including hepatocarcinoma cells. Galactosylated and nongalactosylated gelatin nanovectors (GL-GN-DOX and GN-DOX) were spherical in shape and had mean sizes of about 95.1 and 88.3 nm, respectively. In-vitro release of DOX from nanovectors followed first-order kinetics and was pH dependent. Galactosylated formulation released 95.2% of DOX compared with 86.6% by nongalactosylated formulation at pH 5.8. However, the release rate was suppressed at pH 7.4. Further, we showed that GL-GN-DOX had greater growth inhibitory effect on HepG2 in terms of low inhibitory concentration (IC(50); 0.35 µg/ml) compared with GN-DOX (0.75 µg/ml) and induced caspase-3-mediated apoptosis in HepG2 cells. This might be due to efficient internalization of galactosylated nanovectors by HepG2 cells compared with unmodified formulation. Pharmacokinetic and biodistribution analyses show that galactosylated formulation deposits 24.5 µg/g of DOX in targeted tissue (liver) in comparison with heart (0.3 µg/g) at a single dose of 10 mg/kg. These results suggest that DOX-loaded galactosylated gelatin nanovectors warrant future in-depth antitumor study to scale-up technology and may be used for the management of hepatocarcinoma.

Long-circulating poly(ethylene glycol)-grafted gelatin nanoparticles customized for intracellular delivery of noscapine: preparation, in-vitro characterization, structure elucidation, pharmacokinetics, and cytotoxicity analyses.

Noscapine, the tubulin-binding anticancer agent, when administered orally, requires high ED(50) (300-600 mg/kg), whereas intravenous administration (10 mg/kg) results in rapid elimination of the drug with a half-life of 0.39 h. Hence, the development of long-circulating injectable nanoparticles can be an interesting option for designing a viable formulation of noscapine for anticancer activity. Noscapine-enveloped gelatin nanoparticles and poly(ethylene glycol)-grafted gelatin nanoparticles were constructed and characterized. Data indicate that smooth and spherical shaped nanoparticles of 127 ± 15 nm were engineered with maximum entrapment efficiency of 65.32 ± 3.81%. Circular dichroism confirms that nanocoacervates retained the α-helical content of gelatin in ethanol whereas acetone favored the formation of a random coil. Moreover, the Fourier transform infrared and powder X-ray diffraction pattern prevents any significant change in the noscapine-loaded gelatin nanoparticles in comparison with individual components. In-vitro release kinetic data suggest a first-order release of noscapine (85.1%) from gelatin nanoparticles with a release rate constant of 7.611×10(-3). It is to be noted that there is a 1.43-fold increase in the area under the curve up to the last sampling point for the noscapine-loaded poly(ethylene glycol)-grafted gelatin nanoparticles over the noscapine-loaded gelatin nanoparticles and a 13.09-fold increase over noscapine. Cytotoxicity analysis of the MCF-7 cell line indicated that the IC(50) value of the noscapine-loaded poly(ethylene glycol)-grafted gelatin nanoparticles was equivalent to 20.8 µmol/l, which was significantly (P<0.05) lower than the IC(50) value of the noscapine-loaded gelatin nanoparticles (26.3 µmol/l) and noscapine (40.5 µmol/l).Noscapine-loaded poly(ethylene glycol)-grafted gelatin nanoparticles can be developed as a promising therapeutic agent for the management of breast cancer.

Modification of mouse A2M B (620-792) and A2M N (168-230) by malondialdehyde and acetaldehyde attenuates the proteinase and TGF-ß1 binding ability of A2MB.

Acetaldehyde and malondialdehyde react covalently with cellular proteins forming protein-malondialdehyde-acetaldehyde adducts thus modulating their biochemical functions. Alpha-2 macroglobulin, an acute phase protein produced by liver binds to cytokines, growth factors and neutralizes proteinases. In this study we examined the formation of MAA adducts of N-terminal and bait region of mouse A2M and their effect on modulating its proteinase and TGF-ß1 binding activities. Adduct formation abrogated the binding of bait region with TGF-ß1, trypsin, and elastase. TGF-ß1 induced NO production was also suppressed. Acetaldehyde and MDA adduction of A2M may have physiological consequences in alcoholic patients.

Inclusion complexes of noscapine in beta-cyclodextrin offer better solubility and improved pharmacokinetics.

PURPOSE: Noscapine (NOS) is a unique class of tubulin-binding anticancer agents. Their potential usefulness as anticancer drugs is however limited by the poor bioavailability, thus necessitating administration of a higher dose regime in the range of 300-600 mg/kg for tumor growth inhibition. To augment bioavailability, we prepared an inclusion complex of NOS in beta-cyclodextrin (beta-CD) and evaluated its physico-chemical characteristics. METHOD AND RESULTS: Our phase-solubility analysis shows a 1:1-complexation (Kc approximately 0.454 mM(-1)) of NOS with beta-CD that offers better dissolution properties. We confirmed complex formation in solid state by differential scanning calorimetry, powder X-ray diffractometry, Fourier-transform infrared spectroscopy, 1H nuclear magnetic resonance spectroscopy, rotating frame Overhauser enhancement spectroscopy and by molecular modeling methods. Based upon theoretical calculations in gas phase, we propose O-CH2-O- in orientation of NOS in the beta-CD cavity. The thermal behavior data also provides complementary evidences of complex formation. The pharmacokinetic studies showed a 1.87-fold increase in bioavailability of NOS upon complexation in the beta-CD inclusion complex state as compared to free NOS. Furthermore, the complex retains the anticancer attributes of NOS. CONCLUSION: Our studies propose for the first time a stable NOS-beta-CD inclusion complex as an effective approach to enhance the solubility and bioavailability of NOS for anticancer therapy.

Calcium channel antagonist (nifedipine) attenuates Plasmodium berghei-specific T cell immune responses in Balb/C mice.

Nifedipine and verapamil (Martin et al. Science 1987;235:899-901) are a class of calcium channel blockers involved in the reversal of chloroquine (CQ) drug resistance in CQ-sensitive Plasmodium spp. Nifedipine alters calcium-dependent functions of macrophages and neutrophils during Plasmodium berghei malaria. However, knowledge of nifedipine-induced immunomodulation of T cell functions during P. berghei malaria is still limited. We investigated the effect of nifedipine on the immune status of splenic T cells during P. berghei malaria. The intracellular calcium levels were determined in the FURA-2A/M loaded T cells by spectrofluorometry. Splenic T cell proliferation, phosphatidylserine (PS) externalization, Fas expression and Bcl2/Bax expression were determined by flow cytometry. We report a significant increase in mean percent parasitemia in nifedipine-treated and P. berghei-infected mice. Although nifedipine treatment alone did not affect the resting state free calcium levels in splenic T cells, the rise in intracellular calcium levels of T cells following P. berghei infection was significantly less in nifedipine-treated mice compared to untreated groups at various parasitemia levels. Antigen-specific splenic T cell proliferation and apoptosis was ablated in nifedipine-treated and untreated groups at various parasitemia levels. The study unequivocally reflects the suppression of P. berghei-specific T cell immune responses by nifedipine.