Asymmetric fabrication and in vivo evaluation of the wound healing potency of electrospun biomimetic nanofibrous scaffolds based on collagen crosslinked modified-chitosan and graphene oxide quantum dot nanocomposites.

Asymmetric scaffolds were developed through electrospinning by utilizing biocompatible materials for effective wound healing applications. First of all, the chitosan surface was modified with decanoyl chloride and crosslinked with collagen to synthesize collagen crosslinked modified-chitosan (CG-cross-CS-g-Dc). Then, the asymmetric scaffolds were fabricated through electrospinning, where the top layer was a monoaxial nanofiber of the PCL/graphene oxide quantum dot (GOQD) nanocomposite and the bottom layer was a coaxial nanofiber having PCL in the core and the CG-cross-CS-g-Dc/GOQD nanocomposite in the shell layer. The formation of monoaxial (∼130 ± 50 nm) and coaxial (∼320 ± 40 nm) nanofibers was confirmed by transmission electron microscopy (TEM). The presence of GOQDs contributed to antioxidant and antimicrobial efficacy. These scaffolds showed substantial antibacterial activity against the common wound pathogens Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The scaffolds exhibited excellent cytocompatibility (MTT assay) and anti-inflammatory behaviour as analysed via the cytokine assay and biochemical analysis. The in vivo wound healing potential of the nanofibrous scaffolds was assessed with full-thickness excisional wounds in a rat model. The scaffolds accelerated the re-epithelialization as well as the collagen deposition, thereby facilitating the wound healing process in a very short span of time (10 days). Both in vitro and in vivo analyses thus provide a compelling argument for the use of these scaffolds as therapeutic biomaterials and their suitability for application in rapid wound regeneration and repair.

Hyaluronic acid engrafted metformin loaded graphene oxide nanoparticle as CD44 targeted anti-cancer therapy for triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with limited treatment modalities. It is associated with high propensity of cancer recurrence. METHODS: UV Spectroscopy, FTIR, DLS, Zeta potential, TEM and SEM were employed to characterize nanoparticles. MTT assay, Wound healing assay, SEM, Immunocytochemistry analysis, Western blot, RT-PCR, mammosphere formation assay were employed to study apoptosis, cell migration and stemness. Tumor regression was studied in chick embryo xenograft and BALB/c mice model. RESULTS: Hylaluronic acid engrafted metformin loaded graphene oxide (HA-GO-Met) nanoparticles exhibited an anti-cancer efficacy at much lower dosage as compared to metformin alone. HA-GO-Met nanoparticles induced apoptosis and inhibited cell migration of TNBC cells by targeting miR-10b/PTEN axis via NFkB-p65. Upregulation of PTEN affected pAKT(473) expression that induced apoptosis. Cell migration was inhibited by reduction of pFAK/integrinß1 expressions. Treatment inhibited epithelial mesenchymal transition (EMT) and reduced stemness as evident from the increase in E-cadherin expression, inhibition of mammosphere formation and low expression levels of stemness markers including nanog, oct4 and sox2 as compared to control. Moreover, tumor regression was studied in chick embryo xenograft and BALB/c mice model. HA-GO-Met nanoparticle treatment reduced tumor load and nullified toxicity in peripheral organs imparted by tumor. CONCLUSIONS: HA-GO-Met nanoparticles exhibited an enormous anti-cancer efficacy in TNBC in vitro and in vivo. GENERAL SIGNIFICANCE: HA-GO-Met nanoparticles induced apoptosis and attenuated cell migration in TNBC. It nullified overall toxicity imparted by tumor load. It inhibited EMT and reduced stemness and thereby addressed the issue of cancer recurrence.

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.

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.

Nanotailored hyaluronic acid modified methylcellulose as an injectable scaffold with enhanced physico-rheological and biological aspects.

The collaborative endeavor in tissue engineering is to fabricate a bio-mimetic extracellular matrix to assist tissue regeneration. Thus, a novel injectable tissue scaffold was fabricated by exploring nanotailored hyaluronic acid (nHA) and methylcellulose (MC) (nHAMC) along with pristine HA based MC scaffold (HAMC). nHA with particle size ∼22 ±â€¯5.3 nm were obtained and nHAMC displayed a honeycomb-like 3D microporous architecture. Nano-HA bestowed better gel strength, physico-rheological and biological properties than HA. It creditably reduced the high content of salt to reduce the gelation temperature of MC. The properties ameliorated with increased in-corporation of nano-HA. The addition of salt showed more prominent effect on gelation temperature of nHAMC than in HAMC; and salting-out effect was dependent on nHA/HA content. Biocompatible nHAMC assisted adequate cell adherence and proliferation with more extended protrusions with better migration rate than control. Thus, biomodulatory effect of nanotailored glycosaminoglycan could be asserted to design an efficient thermo-responsive scaffold.

Folic-Acid-Adorned PEGylated Graphene Oxide Interferes with the Cell Migration of Triple Negative Breast Cancer Cell Line, MDAMB-231 by Targeting miR-21/PTEN Axis through NFκB.

Triple negative breast cancer (TNBC), characterized by its aggressive and highly metastatic nature, is difficult to cure by the currently available therapies. In our investigation, folic-acid-adorned PEGylated graphene oxide (FA-PEG-GO) was synthesized by modifying graphene oxide (GO) with folic acid-PEG conjugate (FA-PEG-NH2) by EDC/NHS coupling reaction. FA-PEG-GO exhibited an exceptional potential to attenuate cell migration of TNBC cell line ,MDAMB-231 as compared to GO because of the adorned folic acid moiety, which rendered better targeting. FA-PEG-GO inhibited cell migration by actin depolymerization and perturbing lamellipodia formation. The immunocytochemistry and western blot data unraveled the fact that FA-PEG-GO inhibited cell migration by targeting miR-21 by restricting the nuclear translocation of NFκB. The downregulation of miR-21 resulted in the elevation of PTEN expression which sequentially downregulated pFAK resulting in inhibition of cell migration. Moreover, upregulation of PTEN in FA-PEG-GO treated cells led to the decrease in expression of the downstream regulators including pAkt(Ser473) and pERK1/2, which contributed to the retardation of cell migration. Interestingly, the overexpression of NFκB-p65 by the transfection of NFκB-p65 expression plasmid in TNBC cells reversed the inhibitory effect of FA-PEG-GO on the nuclear translocation of NFκB-p65 which stabilized miR-21 expression and successively downregulated PTEN expression in FA-PEG-GO treated cells. Furthermore, miR-21 overexpression by transfection of miR-21 mimic in turn downregulated PTEN expression and sequentially restored the expression of pFAK even upon FA-PEG-GO treatment. miR-21 overexpression also compensated the inhibitory effect of FA-PEG-GO on pAkt(Ser473) and pERK1/2 which was evident from their significant expression in FA-PEG-GO-treated cells. The studies on chick embryo model ratified the ex ovo antimigratory efficacy of FA-PEG-GO. Altogether, our study unveiled the enormous potential of FA-PEG-GO to attenuate migration of TNBC cell line, MDAMB-231 by targeting the miR-21/PTEN axis through NFκB and thereby providing insights on cancer treatment.

Tailoring the Efficacy of Multifunctional Biopolymeric Graphene Oxide Quantum Dot-Based Nanomaterial as Nanocargo in Cancer Therapeutic Application.

Nanotechnology has acquired an immense recognition in cancer theranostics. Considerable progress has been made in the development of targeted drug delivery system for potent delivery of anticancer drugs to tumor-specific sites. Recently, multifunctional nanomaterials have been explored and used as nanovehicles to carry drug molecules with enhanced therapeutic efficacy. In this present work, graphene oxide quantum dot (GOQD) was conjugated with folic acid functionalized chitosan (FA-CH) to develop a nanocargo (FA-CH-GOQD) for drug delivery in cancer therapy. The synthesized nanomaterials were characterized using Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, scanning electron microscopy, transmission electron microscopy, and dynamic light scattering. Photoluminescence spectroscopy was also employed to characterize the formation of GOQD. To validate the efficacy of FA-CH-GOQD as nanocarriers, doxorubicin (DOX) drug was chosen for encapsulation. The in vitro release pattern of DOX was examined in various pH ranges. The drug release rate in a tumor cell microenvironment at pH 5.5 was found higher than that under a physiological range of pH 6.5 and 7.4. An MTT assay was performed to understand the cytotoxic behavior of GOQD and FA-CH-GOQD/DOX. Cytomorphological micrographs of the A549 cell exhibited the various morphological arrangements subject to apoptosis of the cell. Cellular uptake studies manifested that FA-CH-GOQD could specifically transport DOX within a cancerous cell. Further anticancer efficacy of this nanomaterial was corroborated in a breast cancer cell line and demonstrated through 4',6-diamidino-2-phenylindole dihydrochloride staining micrographs.