Synchronized Codelivery of Combination Chemotherapies Intratumorally Using a Lipidic Lyotropic Liquid Crystal System.

In this work, an injectable in situ depot-forming lipidic lyotropic liquid crystal (L3C) system is developed to codeliver a precisely synchronized combination of chemotherapeutics intratumorally. The developed L3C system is composed of amphiphilic lipids and surfactants, including monoolein, phosphatidylcholine, tocopherol acetate, and d-α-tocopherol polyethylene glycol 1000 succinate. Owing to its amphiphilic nature, the developed formulation can coaccommodate both hydrophobic and hydrophilic chemotherapeutic moieties simultaneously. The study presents a proof of concept by designing a combination chemotherapy regimen in vitro and demonstrating its in vivo translation using doxorubicin and paclitaxel as model hydrophilic and hydrophobic drug moieties, respectively. The synchronized combination of the two chemotherapeutics with maximum synergistic activity was identified, coloaded in the developed L3C system at predefined stoichiometric ratios, and evaluated for antitumor efficacy in the 4T1 breast tumor model in BALB/c mice. The drug-loaded L3C formulation is a low-viscosity injectable fluid with a lamellar phase that transforms into a hexagonal mesophase depot system upon intratumoral injection. The drug-loaded depot system locally provides sustained intratumoral delivery of the chemotherapeutics combination at their precisely synchronized ratio for over a period of one month. Results demonstrate that the exposure of the tumor to the precisely synchronized intratumoral chemotherapeutics combination via the developed L3C system resulted in significantly higher antitumor activity and reduced cardiotoxicity compared to the unsynchronized combination chemotherapy or the synchronized but uncoordinated drug delivery administered by a conventional intravenous route. These findings demonstrate the potential of the developed L3C system for achieving synchronized codelivery of the chemotherapeutics combination intratumorally and improving the efficacy of combination chemotherapy.

Simultaneous estimation of five biomarkers of neuroprotective herb Ashwagandha NMITLI-118R AF1 in rat plasma and brain using LC-ESI-MS/MS: Application to its pharmacokinetic and stability studies.

Withania Somnifera (WS) is a popular nutritional supplement in the USA, Europe, and Asia, known for its pharmacological effects on neurological disorders. However, the bioanalytical method development, validation, and pharmacokinetics of WS NMITLI-118R AF1 biomarkers Withanolide A (WLD A), Withanone (WNONE), Withanolide B (WLD B), Withaferin A (WF A), and 12 Deoxywithastramonolide (12 DEOXY) in rats have not been comprehensively explored. This study aimed to develop and validate a sensitive and selective LC-ESI-MS/MS method for these biomarkers in male Sprague Dawley rats plasma and brain matrix. Rats were divided into eight groups, each containing five rats. A plant extract of NMITLI-118R AF1 at 50 mg/kg was orally administered to the rats for in-vivo pharmacokinetic investigation. All the analytes had a linear calibration curve (r2 > 0.999), and intra-day and inter-day precision (%) were found in the range of 2.46 - 13.71% and accuracy were within the acceptable range (±15%). The biomarkers of NMITLI-118R AF1 were found stable in in-vitro plasma and simulated gastro-intestinal fluids. The observed (Cmax) and (Tmax) values for the biomarkers in the systemic circulation were WLD A (5.59 ± 0.34 ng/mL, Tmax 1.00 ± 0.00 h), WNONE (6.28 ± 0.41 ng/mL, Tmax 0.95 ± 0.11 h), WLD B (6.45 ± 2.87 ng/mL, Tmax 0.95 ± 0.11 h), WF A (6.50 ± 0.27 ng/mL, Tmax 1.00 ± 0.00 h), and 12 DEOXY (5.68 ± 0.39 ng/mL, Tmax 1.00 ± 0.00 h). In contrast to the old method, our approach exhibits a lower limit of quantification (LLOQ), shorter run time (less than10 min), and enables the detection of WF A and WNONE in fresh rat plasma by other quantitative analysis of mass spectrometry (m/z) [M]+. Shows high sample volumes for both, larger plasma volumes, costlier sample collection techniques dried blood spot (DBS), more expensive solid phase extraction techniques (SPE) and longer analysis time 14 min. Moreover, our method requires a smaller sample volume 10 µL, offers faster analysis time 4 min, and achieves a higher sensitivity 1 ng/mL. This is the first report of a comprehensive study on in-vitro and in-vivo pharmacokinetics of NMITLI-118R AF1 biomarkers, which may aid in further pre-clinical and clinical trial investigations.

Ratiometric codelivery of Paclitaxel and Baicalein loaded nanoemulsion for enhancement of breast cancer treatment.

The most prevalent clinical option for treating cancer is combination chemotherapy. In combination therapy, assessment and optimization for obtaining a synergistic ratio could be obtained by various preclinical setups. Currently, in vitro optimization is used to get synergistic cytotoxicity while constructing combinations. Herein, we co-encapsulated Paclitaxel (PTX) and Baicalein (BCLN) with TPP-TPGS1000 containing nanoemulsion (TPP-TPGS1000-PTX-BCLN-NE) for breast cancer treatment. The assessment of cytotoxicity of PTX and BCLN at different molar weight ratios provided an optimized synergistic ratio (1:5). Quality by Design (QbD) approach was later applied for the optimization as well as characterization of nanoformulation for its droplet size, zeta potential and drug content. TPP-TPGS1000-PTX-BCLN-NE significantly enhanced cellular ROS, cell cycle arrest, and depolarization of mitochondrial membrane potential in the 4T1 breast cancer cell line compared to other treatments. In the syngeneic 4T1 BALB/c tumor model, TPP-TPGS1000-PTX-BCLN-NE outperformed other nanoformulation treatments. The pharmacokinetic, biodistribution and live imaging studies pivoted TPP-TPGS1000-PTX-BCLN-NE enhanced bioavailability and PTX accumulation at tumor site. Later, histology studies confirmed nanoemulsion non-toxicity, expressing new opportunities and potential to treat breast cancer. These results suggested that current nanoformulation can be a potential therapeutic approach to effectively address breast cancer therapy.

Enhanced apoptosis and mitochondrial cell death by paclitaxel-loaded TPP-TPGS1000-functionalized nanoemulsion.

Background: The present research was designed to develop a nanoemulsion (NE) of triphenylphosphine-D-α-tocopheryl-polyethylene glycol succinate (TPP-TPGS1000) and paclitaxel (PTX) to effectively deliver PTX to improve breast cancer therapy. Materials & methods: A quality-by-design approach was applied for optimization and in vitro and in vivo characterization were performed. Results: The TPP-TPGS1000-PTX-NE enhanced cellular uptake, mitochondrial membrane depolarization and G2M cell cycle arrest compared with free-PTX treatment. In addition, pharmacokinetics, biodistribution and in vivo live imaging studies in tumor-bearing mice showed that TPP-TPGS1000-PTX-NE had superior performance compared with free-PTX treatment. Histological and survival investigations ascertained the nontoxicity of the nanoformulation, suggesting new opportunities and potential to treat breast cancer. Conclusion: TPP-TPGS1000-PTX-NE improved the efficacy of breast cancer treatment by enhancing its effectiveness and decreasing drug toxicity.

Biocompatible fluorescent carbon quantum dots prepared from beetroot extract for in vivo live imaging in C. elegans and BALB/c mice.

Luminescent carbon quantum dots (CQDs) prepared from aqueous beetroot extract were developed as unique fluorescent nanomaterials for in vivo live animal imaging applications. Blue (B) and green (G) emitting environmentally benign CQDs (particle size of 5 nm and 8 nm, respectively) exhibited bright fluorescence in aqueous medium and were found to be biocompatible, photostable and non-toxic in animal models. The in vivo imaging and toxicity evaluation of both CQDs were performed for the first time in the Caenorhabditis elegans (C. elegans) model, which revealed consistent fluorescence in the gut tissues of the worms without exerting any sign of toxic effects on the nematodes. The in vivo bio-distribution of G-CQDs given by tail vein injection in live BALB/c mice showed optical signals in the lower abdominal regions, mainly in the intestine, and cleared from the body through faeces. The tremendous potential shown by these eco-friendly CQDs in the C. elegans and mice models advocates new hopes for greener CQD nanomaterials as diagnostic tools in the biomedical field.

Synergistic enhancement of parasiticidal activity of amphotericin B using copaiba oil in nanoemulsified carrier for oral delivery: an approach for non-toxic chemotherapy.

BACKGROUND AND PURPOSE: The aim of this study was to devise a nanoemulsified carrier system (CopNEC) to improve the oral delivery of amphotericin B (AmB) by increasing its oral bioavailability and synergistically enhance its antileishmanial activity with copaiba oil (Cop). EXPERIMENTAL APPROACH: The AmB encapsulated NEC (CopNEC-AmB) comprised of Cop, d-α-tocopheryl polyethylene glycol 1000 succinate and phosphatidylcholine was prepared by high-pressure homogenization method. Stability study of CopNEC-AmB was carried out in simulated gastric fluid and simulated intestinal fluid. The CopNEC-AmB and plain AmB were compared as regards their in vitro antileishmanial activity, pharmacokinetics, organ distribution and toxicity. KEY RESULTS: The optimal CopNEC-AmB had a small globule size, low polydispersity index, high ζ potential and encapsulation efficiency. The high resolution transmission electron microscopy illustrated spherical particle geometry with homogeny in their sizes. The optimal CopNEC-AmB was found to be stable in gastrointestinal fluids showing insignificant changes in globule size and encapsulation efficiency. The AUC0-48 value of CopNEC-AmB in rats was significantly improved showing 7.2-fold higher oral bioavailability than free drug. The in vitro antileishmanial activity of CopNEC-AmB was significantly higher than that of the free drug as Cop synergistically enhanced the antileishmanial effect of AmB by causing drastic changes in the morphology of Leishmania parasite and rupturing its plasma membrane. The CopNEC-AmB showed significantly less haemolytic toxicity and cytotoxicity and did not change the histopathology of kidney tissues as compared with AmB alone. CONCLUSIONS AND IMPLICATIONS: This prototype CopNEC formulation showed improved bioavailability and had a non-toxic synergistic effect on the antileishmanial activity of AmB.

Resveratrol as an adjunct therapy in cyclophosphamide-treated MCF-7 cells and breast tumor explants.

Cyclophosphamide (CPA) has efficacy as a breast cancer therapy. However, toxicity to CPA limits its clinical applications. Hence there is a need to develop compounds that may be combined with it to improve the efficacy and overcome toxicity. We showed previously that Resveratrol (RES), a chemopreventive agent, increased the growth inhibitory effect of CPA-treated MCF-7 cells. Here we have explored the molecular basis of 5 mM CPA and 50 µM RES as a combination on cell-cycle progression, apoptosis and oxidative stress in MCF-7 breast cancer cells. Efficacy of the combination was also evaluated in a serum-free tumor explant culture model. The combination elicited enhanced anti-proliferative action coupled with differential expression of cell-cycle, apoptosis and stress factors. Furthermore, co-treatment superiority in histologically validated ER positive breast cancer explants suggests that this combination may be a worthy future clinical anti-neoplastic regimen.

RNAi-mediated silencing of nuclear factor erythroid-2-related factor 2 gene expression in non-small cell lung cancer inhibits tumor growth and increases efficacy of chemotherapy.

Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non-small cell lung cancer. In this study, we show that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by up-regulation of glutathione, thioredoxin, and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth, and results in increased sensitivity to chemotherapeutic drug-induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance.

Simultaneous application of t-test and fold change criteria to identify acetaminophen and carbon tetrachloride affected genes in mice liver.

t-Test and fold change criteria were employed simultaneously following acute exposure to acetaminophen and carbon tetrachloride to provide high statistical confidence in the identification of affected genes in mice livers. Furthermore, gene expression data was also investigated with either t-test or fold change criteria alone. Gene expression studies were also accompanied by liver histological and serum biochemical studies for toxicity evaluation. We identified a large number of affected genes using both filtering criteria (p<0.01 and twofold) simultaneously following both the hepatotoxicants. In some cases gene expression studies provided the earliest evidence of tissue response in the absence of traditional markers at histological and biochemical level. We conclude that simultaneous application of t-test and fold change criteria helps to identify important genes with greater statistical confidence than the use of either of them alone, however, this approach results in the reduction of identified probes. Thus, data analysis at different statistical stringencies is needed to know exact outcome of any toxicological event.