Structurally Dynamic Monocyte-Liposome Hybrid Vesicles as an Anticancer Drug Delivery Vehicle: A Crucial Correlation of Microscopic Elasticity and Ultrafast Dynamics.

A biomimetic cell-based carrier system based on monocyte membranes and liposomes has been designed to create a hybrid "Monocyte-LP" which inherits the surface antigens of the monocytes along with the drug encapsulation property of the liposome. Förster resonance energy transfer (FRET) and polarization gated anisotropy measurements show the stiffness of the vesicles obtained from monocyte membranes (Mons), phosphatidylcholine membranes (LP), and Monocyte-LP to follow an increasing order of Mons > Monocyte-LP > LP. The dynamics of interface bound water molecules plays a key role in the elasticity of the vesicles, which in turn imparts higher delivery efficacy to the hybrid Monocyte-LP for a model anticancer drug doxorubicin than the other two vesicles, indicating a critical balance between flexibility and rigidity for an efficient cellular uptake. The present work provides insight on the influence of elasticity of delivery vehicles for enhanced drug delivery.

microRNA-205 represses breast cancer metastasis by perturbing the rab coupling protein [RCP]-mediated integrin ß1 recycling on the membrane.

During cancer cell invasion, integrin undergoes constant endo/exocytic trafficking. It has been found that the recycling ability of integrin ß1 through Rab11-controlled long loop pathways is directly associated with cancer invasion. Previous studies showed that gain-of-function mutant p53 regulates the Rab-coupling protein [RCP]-mediated integrin ß1 recycling by inactivating tumor suppressor TAp63. So, we were interested to investigate the involvement of miR-205 in this process. In the current study first, we evaluated that the lower expression of miR-205 in MDA-MB-231 cell line is associated with high motility and invasiveness. Further investigation corroborated that miR-205 directly targets RCP resulting in attenuated RCP-mediated integrin ß1 recycling. Overexpression of TAp63 validates our in vitro findings. To appraise the anti-metastatic role of miR-205, we developed two in vivo experimental models- xenograft-chick embryo and xenograft-immunosuppressed BALB/c mice. Our in vivo results support the negative effect of miR-205 on metastasis. Therefore, these findings advocate the tumor suppressor activity of miR-205 in breast cancer cells and suggest that in the future development of miR-205-targeting RNAi therapeutics could be a smart alternative approach to prevent the metastatic fate of the disease.

A mechanistic insight into the potential anti-cancerous property of Nigella sativa on breast cancer through micro-RNA regulation: An in vitro & in vivo study.

Cancer continues to threat mortal alongside scientific community with burgeoning grasp. Most efforts directed to tame Cancer such as radiotherapy or chemotherapy, all came at a cost of severe side effects. The plant derived bioactive compounds on the other hand carries an inevitable advantage of being safer, bioavailable & less toxic compared to contemporary chemotherapeutics. Our strategic approach employed solvent extraction of Black Seed Oil (BSO) to highlight the orchestrated use of its oil soluble phytochemicals - Thymoquinone, Carvacrol & Trans-Anethole when used in cohort. These anti-cancer agents in unbelievably modest amounts present in BSO shows better potential to delineate migratory properties in breast cancer cells as compared to when treated individually. BSO was also observed to have apoptotic calibre when investigated in MDA-MB-231 and MCF-7 cell lines. We performed chemical characterization of the individual phytochemical as well as the oil in-whole to demonstrate the bioactive oil-soluble entities present in whole extract. BSO was observed to have significant anti-cancerous properties in cumulative proportion that is reportedly higher than the individual three components. Besides, this study also reports micro-RNA regulation on BSO administration, thereby playing a pivotal role in breast cancer alleviation. Thus, synergistic action of the integrants serves better combat force against breast cancer in the form of whole extract, hence aiming at a more lucrative paradigm while significantly regulating microRNAs associated with breast cancer migration and apoptosis.

Delivery of gefitinib in synergism with thymoquinone via transferrin-conjugated nanoparticle sensitizes gefitinib-resistant non-small cell lung carcinoma to control metastasis and stemness.

Epidermal growth factor receptor (EGFR) normally over-expresses in non-small cell lung cancer (NSCLC) cells. Its mutations act as oncogenic drivers in the cellular signal transduction pathway, and induce the downstream activation of numerous key cellular events involved in cellular proliferation and survival. EGFR tyrosine kinase inhibitors (EGFR-TK inhibitors), such as gefitinib and erlotinib, have been used for a long time in the treatment of NSCLC. However, they fail to overcome the EGFR-TK mutation due to the acquisition of drug resistance. It is strongly believed that the epithelial-to-mesenchymal transition (EMT) is a key player for acquired resistance and consequent limitation of the clinical efficiency of EGFR-TKIs. Therefore, a new strategy needs to be developed to overcome the resistance in NSCLC. In this current study, we have disclosed for the first time the efficiency of transferrin-modified PLGA-thymoquinone-nanoparticles in combination with gefitinib (NP-dual-1, NP-dual-2 and NP-dual-3) towards gefitinib-resistant A549 cells. The gefitinib-resistant A549 cells (A549/GR) showed 12.3-fold more resistance to gefitinib in comparison to non-resistant A549 cells. The phenotypic alteration resembling spindle-cell shape and increased pseudopodia integuments featured the EMT phenomena in A549/GR cells. EMT in A549/GR was later coupled with the loss of Ecad and expansion of Ncad, along with upregulated vimentin expression, as compared to the control A549 cells. Moreover, the invasive nature and migration potential are more amplified in A549/GR cells. Pre-incubation of A549 cells with TGFß1 also initiated EMT, leading to drug resistance. Conversely, treatment of A549 or A549/GR cells with NP-dual-3 effectively retrieved the sensitivity to gefitinib, restricted the EMT phenomenon, and impaired the TGFß1-induced EMT. On unveiling the underlying mechanism of therapeutic action, we found that STAT3 and miR-21 were individually overexpressed in the A549/GR cells by transfection, and followed by treatment with NP-dual-3. Simultaneously, NP-dual-3 fragmented HIF1-α induced EMT in A549/GR cells and reduced the CSCs markers, viz., Oct-4, Sox-2, Nanog, and Aldh1. These data are self-sufficient to suggest that NP-dual-3 re-sensitizes the drug-resistant A549/GR cells to gefitinib, possibly by retrieving MET phenomena via modulation of STAT3/mir-21/Akt/PTEN/HIF1-α axis. Thus, TQ nanoparticles combined with TKI gefitinib may provide an effective platform to treat NSCLC.

Delivery of novel coumarin-dihydropyrimidinone conjugates through mixed polymeric nanoparticles to potentiate therapeutic efficacy against triple-negative breast cancer.

To date, most of the accessible therapeutic options are virtually non-responsive towards triple-negative breast cancer (TNBC) due to its highly aggressive and metastatic nature. Interestingly, chemotherapy reacts soundly in many TNBC cases compared to other types of breast cancer. However, the side effects of many chemotherapeutic agents are still under cross-examination, and thus prohibit their extensive uses. In this present study, we have developed a series of coumarin-dihydropyrimidinone conjugates (CDHPs) and subsequently their poly(lactic-co-glycolic acid) (PLGA)-PEG4000 mixed copolymer nanoparticles as excellent chemotherapeutic nanomedicine to control TNBC. Among all the synthesized CDHPs, CDHP-4 (prepared by the combination of EDCO with 3,4-difluorobenzaldehyde) showed excellent therapeutic effect on a wide variety of cancer cell lines, including TNBC. Besides, it can control the metastasis and stemness property of TNBC. Furthermore, the nano-encapsulation of CDHP-4 in a mixed polymer nanoparticle system (CDHP-4@PP-NPs) and simultaneous delivery showed much improved therapeutic efficacy at a much lower dose, and almost negligible side effects in normal healthy cells or organs. The effectiveness of the present therapeutic agent was observed both in intravenous and oral mode of administration in in vivo experiments. Moreover, on elucidating the molecular mechanism, we found that CDHP-4@PP-NPs could exhibit apoptotic, anti-migratory, as well as anti-stemness activity against TNBC cell lines through the downregulation of miR-138. We validated our findings in MDA-MB-231 xenograft chick embryos, as well as in 4T1-induced mammary tumor-bearing BALB/c mice models, and studied the bio-distribution of CDHP-4@PP-NPs on the basis of the photoluminescence property of nanoparticles. Our recent study, hence for the first time, unravels the synthesis of CDHP-4@PP-NPs and the molecular mechanism behind the anti-migration, anti-stemness and anti-tumor efficacy of the nanoparticles against the TNBC cells through the miR-138/p65/TUSC2 axis.

Polyethylene Glycol-Mediated Fusion of Extracellular Vesicles with Cationic Liposomes for the Design of Hybrid Delivery Systems.

To realize a customizable biogenic delivery platform, herein we propose combining cell-derived extracellular vesicles (EVs) derived from breast cancer cell line MCF-7 with synthetic cationic liposomes using a fusogenic agent, polyethylene glycol (PEG). We performed a fluorescence resonance energy transfer (FRET)-based lipid-mixing assay with varying PEG 1000 concentrations (0%, 15%, and 30%) correlated with flow cytometry-based analysis and supported by dimensional analysis by dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) to validate our fusion strategy. Our data revealed that these hybrid vesicles at a particular concentration of PEG (∼15%) improved the cellular delivery efficiency of a model siRNA molecule to the EV parental breast cancer cells, MCF-7, by factors of 2 and 4 compared to the loaded liposome and EV precursors, respectively. The critical rigidity/pliability balance of the hybrid systems fused by PEG seems to be playing a pivotal role in improving their delivery capability. This approach can provide clinically viable delivery solutions using EVs.

Combinatorial therapy of Thymoquinone and Emodin synergistically enhances apoptosis, attenuates cell migration and reduces stemness efficiently in breast cancer.

BACKGROUND: Breast cancer intimidates the contemporary medical advances, attempting to revolutionize cancer therapeutics. While patients suffering an advanced breast cancer are dependent on mono drugs, yet the build out of resistance leading to treatment fails has become inevitable. METHODS: Cell viability Assay with MTT revealed the "IC50" concentrations of the drugs in both cancer as well as PBMC. Cell cycle arrest, flow cytometric ROS analysis & apoptosis evaluation pointed out the efficacy of the dual drug. Wound Healing, Transwell Migration & Immunocytochemistry indicated anti-migratory potential of TQ-Emo while expression patterns of Cl-Cas3, p53, Bax, Bcl2 & the stemness markers further vouched the potential of the combinatorial drug. Furthermore, validation of tumor inhibitory effect was earned by an ex-ovo xenograft model. RESULTS: Dual dosage enhanced apoptosis through ROS generation, anti- migratory effect by targeting FAK &Integrins, displaying effective stemness control by assessing regulatory proteins- Oct4, Sox2, Nanog, ALDH1/2. Ex-ovo xenograft model validated tumor regression. Our study thereby deals with devastating effects of cancer drug resistance while trying to abate enhanced migratory potential & stemness, utilizing the synergism of the combinable therapy. CONCLUSION: TQ/Emo inhibited breast cancer proliferation synergistically while enhancing cytotoxicity, inducing apoptosis on MCF-7 cells while curbing migration & stemness. GENERAL SIGNIFICANCE: Employment of the combinatorial phytochemicals, Thymoquinone & Emodin attempted to achieve deliverables like reduced cellular toxicity, drug resistance, anti-migratory potency & stemness. Besides, decreased p-FAK expression or regression in Mammosphere & tumor size in ex-ovo xenograft model is indicative of the better anti-tumorigenic potential of the dual formulation.

Delivery of dual miRNA through CD44-targeted mesoporous silica nanoparticles for enhanced and effective triple-negative breast cancer therapy.

The development of new therapeutic strategies to target triple-negative breast cancer (TNBC) is in much demand to overcome the roadblocks associated with the existing treatment procedures. In this regard, therapies targeting the CD44 receptor have drawn attention for more than a decade. MicroRNAs (miRNAs) modulate post-transcriptional gene regulation and thus, the correction of specific miRNA alterations using miRNA mimics or antagomiRs is an emerging strategy to normalize the genetic regulation in the tumor microenvironment. It has been acknowledged that miR-34a is downregulated and miR-10b is upregulated in TNBC, which promotes tumorigenesis and metastatic dissemination. However, there are a few barriers related to miRNA delivery. Herein, we have introduced tailored mesoporous silica nanoparticles (MSNs) for the co-delivery of miR-34a-mimic and antisense-miR-10b. MSN was functionalized with a cationic basic side chain and then loaded with the dual combination to overexpress miR-34a and downregulate miR-10b simultaneously. Finally, the loaded MSNs were coated with an hyaluronic acid-appended PEG-PLGA polymer for specific targeting. The cellular uptake, release profile, and subsequent effect in TNBC cells were evaluated. In vitro and in vivo studies demonstrated high specificity in TNBC tumor targeting, leading to efficient tumor growth inhibition as well as the retardation of metastasis, which affirmed the clinical application potential of the system.

Transferrin-decorated thymoquinone-loaded PEG-PLGA nanoparticles exhibit anticarcinogenic effect in non-small cell lung carcinoma via the modulation of miR-34a and miR-16.

Non-small cell lung carcinoma (NSCLC) is a highly lethal type of cancer with limited therapeutic avenues available to date. In the present study, we formulated PEGylated PLGA thymoquinone nanoparticles (TQ-Np) for improved TQ delivery to NSCLC cells. Transferrin (TF), a biodegradable, non-immunogenic and non-toxic protein, is well known to bind to TFR (transferrin receptor) over-expressed in non-small cell lung carcinoma A549 cells. Thus, the further decoration of the PEGylated PLGA thymoquinone nanoparticles with transferrin (TF-TQ-Np) enhanced the internalization of the nanoparticles within the A549 cells and the activity of TQ. We established TF-TQ-Np as a potent anti-tumorigenic agent through the involvement of p53 and the ROS feedback loop in regulating the microRNA (miRNA) circuitry to control apoptosis and migration of NSCLC cells. TF-TQ-Np-mediated p53 up-regulation favored the potential simultaneous activation of miR-34a and miR-16 targeting Bcl2 to induce apoptosis in the A549 cells. Additionally, TF-TQ-Np also restricted the migration through actin de-polymerization via activation of the p53/miR-34a axis. Further studies in chick CAM xenograft models confirmed the anti-cancer activity of TF-TQ-Np by controlling the p53/miR-34a/miR-16 axis. Furthermore, in vivo experiments conducted in a xenograft model in immunosuppressed Balb/c mice also proved the efficacy of the nanoparticles as an antitumor agent against NSCLC. Thus, our findings cumulatively suggest that the transferrin-adorned TQ-Np successfully coupled two distinct miRNA pathways to potentiate the apoptotic death cascade in the very lethal NSCLC cells and also restricts the migration of these cells without imparting any significant toxicity, which occurs in the widely used chemotherapeutic combinations. Thereby, our findings rekindle new hopes for the development of improved targeted therapeutic options with specified molecular objectives for combating the deadly NSCLC.

Methylglyoxal at metronomic doses sensitizes breast cancer cells to doxorubicin and cisplatin causing synergistic induction of programmed cell death and inhibition of stemness.

Potent anticancer activity coupled with absence of toxicity at therapeutic dose established the glycolytic metabolite, methylglyoxal, as a promising candidate against malignant neoplasia. In this preclinical study we illustrate the applicability of methylglyoxal in formulating an optimally designed combination regimen with chemotherapeutic drugs against breast cancer. Results demonstrated a synergistic augmentation in doxorubicin and cisplatin mediated cytotoxicity in human breast cancer cell lines MDA MB 231 & MCF 7 with methylglyoxal co-treatment at metronomic concentrations. The cell death due to combination treatment was significantly prevented by N-Acetylcysteine and the synergistic effects were attenuated in presence of inhibitors for apoptosis and necroptosis, in MDA MB 231 and MCF 7 cells, respectively. Additionally, acridine orange staining and immunoblotting with LC3B antibody indicated the suppression of doxorubicin induced autophagy flux with methylglyoxal co-treatment. This report documents for the first time the preferential targeting of breast cancer stem cells by methylglyoxal. Combination treatment with doxorubicin or cisplatin hindered mammosphere forming efficiency and inclusively eliminated both cancer stem as well as non-stem cancer cells. The synergistic effect was validated in Ehrlich mammary carcinoma cell induced murine ascites model and the combination advantage in vivo was achieved without any additional deleterious effect to liver and kidney. Our present study evidences the implications of methylglyoxal inclusion in adjuvant multimodal chemotherapeutics against breast cancer and offers noteworthy insights into the possible outcome.