p53 gain-of-function mutations increase Cdc7-dependent replication initiation.

Cancer-associated p53 missense mutants confer gain of function (GOF) and promote tumorigenesis by regulating crucial signaling pathways. However, the role of GOF mutant p53 in regulating DNA replication, a commonly altered pathway in cancer, is less explored. Here, we show that enhanced Cdc7-dependent replication initiation enables mutant p53 to confer oncogenic phenotypes. We demonstrate that mutant p53 cooperates with the oncogenic transcription factor Myb in vivo and transactivates Cdc7 in cancer cells. Moreover, mutant p53 cells exhibit enhanced levels of Dbf4, promoting the activity of Cdc7/Dbf4 complex. Chromatin enrichment of replication initiation factors and subsequent increase in origin firing confirm increased Cdc7-dependent replication initiation in mutant p53 cells. Further, knockdown of CDC7 significantly abrogates mutant p53-driven cancer phenotypes in vitro and in vivo Importantly, high CDC7 expression significantly correlates with p53 mutational status and predicts poor clinical outcome in lung adenocarcinoma patients. Collectively, this study highlights a novel functional interaction between mutant p53 and the DNA replication pathway in cancer cells. We propose that increased Cdc7-dependent replication initiation is a hallmark of p53 gain-of-function mutations.

Epoxyazadiradione suppresses breast tumor growth through mitochondrial depolarization and caspase-dependent apoptosis by targeting PI3K/Akt pathway.

BACKGROUND: Breast cancer is one of the most commonly diagnosed invasive cancers among women around the world. Among several subtypes, triple negative breast cancer (TNBC) is highly aggressive and chemoresistant. Treatment of TNBC patients has been challenging due to heterogeneity and devoid of well-defined molecular targets. Thus, identification of novel effective and selective agents against TNBC is essential. METHODS: We used epoxyazadiradione to assess the cell viability, mitochondrial potential, ROS level, cell migration, apoptosis and protein expression in cell culture models of TNBC MDA-MB-231 and ER+ MCF-7 breast cancer cells. The molecular mechanism was examined in two different type of breast cancer cells in response to epoxyazadiradione. We have also analyzed the effect of epoxyazadiradione on breast tumor growth using in vivo mice model. RESULTS: In this study, we for the first time investigated that out of 10 major limonoids isolated from Azadirachta indica, epoxyazadiradione exhibits most potent anti-cancer activity in both TNBC and ER+ breast cancer cells. Epoxyazadiradione induces apoptosis and inhibits PI3K/Akt-mediated mitochondrial potential, cell viability, migration and angiogenesis. It also inhibits the expression of pro-angiogenic and pro-metastatic genes such as Cox2, OPN, VEGF and MMP-9 in these cells. Furthermore, epoxyazadiradione attenuates PI3K/Akt-mediated AP-1 activation. Our in vivo data revealed that epoxyazadiradione suppresses breast tumor growth and angiogenesis in orthotopic NOD/SCID mice model. CONCLUSION: Our findings demonstrate that epoxyazadiradione inhibits PI3K/Akt-dependent mitochondrial depolarisation, induces apoptosis and attenuates cell migration, angiogenesis and breast tumor growth suggesting that this compound may act as a potent therapeutic agent for the management of breast cancer.

Therapeutic implications of cellular and molecular biology of cancer stem cells in melanoma.

Melanoma is a form of cancer that initiates in melanocytes. Melanoma has multiple phenotypically distinct subpopulation of cells, some of them have embryonic like plasticity which are involved in self-renewal, tumor initiation, metastasis and progression and provide reservoir of therapeutically resistant cells. Cancer stem cells (CSCs) can be identified and characterized based on various unique cell surface and intracellular markers. CSCs exhibit different molecular pattern with respect to non-CSCs. They maintain their stemness and chemoresistant features through specific signaling cascades. CSCs are weak in immunogenicity and act as immunosupressor in the host system. Melanoma treatment becomes difficult and survival is greatly reduced when the patient develop metastasis. Standard conventional oncology treatments such as chemotherapy, radiotherapy and surgical resection are only responsible for shrinking the bulk of the tumor mass and tumor tends to relapse. Thus, targeting CSCs and their microenvironment niche addresses the alternative of traditional cancer therapy. Combined use of CSCs targeted and traditional therapies may kill the bulk tumor and CSCs and offer a promising therapeutic strategy for the management of melanoma.

Cross-talk between endoplasmic reticulum (ER) stress and the MEK/ERK pathway potentiates apoptosis in human triple negative breast carcinoma cells: role of a dihydropyrimidone, nifetepimine.

Triple negative breast cancers (TNBC) are among the most aggressive and therapy-resistant breast tumors and currently possess almost no molecular targets for therapeutic options in this horizon. In the present study we discerned the molecular mechanisms of potential interaction between the endoplasmic reticulum (ER) stress response and the MEK/ERK pathway in inducing apoptosis in TNBC cells. Here we observed that induction of ER stress alone was not sufficient to trigger significant apoptosis but simultaneous inhibition of the MEK/ERK pathway enhanced ER stress-induced apoptosis via a caspase-dependent mechanism. Our study also demonstrated nifetepimine, a dihydropyrimidone derivative as a potent anti-cancer agent in TNBC cells. Nifetepimine down-regulated the MEK/ERK pathway in MDAMB-231 and MDAMB-468 cells and resulted in blockage of ER stress-mediated GRP78 up-regulation. Detailed mechanistic studies also revealed that nifetepimine by down-regulating pERK expression also declined the promoter binding activity of TFII-I to the GRP78 promoter and in turn regulated GRP78 transcription. Studies further extended to in vivo Swiss albino and SCID mice models also revalidated the anti-carcinogenic property of nifetepimine. Thus our findings cumulatively suggest that nifetepimine couples two distinct signaling pathways to induce the apoptotic death cascade in TNBC cells and raises the possibility for the use of nifetepimine as a potent anti-cancer agent with strong immune-restoring properties for therapeutic intervention for this group of cancer bearers.

Diosgenin Functionalized Iron Oxide Nanoparticles as Novel Nanomaterial Against Breast Cancer.

Iron oxide nanoparticles (IONPs) have gained immense importance recently as drug nanocarriers due to easy multifunctionalization, simultaneous targeting, imaging and cancer hyperthermia. Herein, we report a novel nanomedicine comprising of IONPs core functionalized with a potent anticancer bioactive principle, diosgenin from medicinal plant Dioscorea bulbifera via citric acid linker molecule. IONPs were synthesized by reverse co-precipitation and characterized using field emission scanning electron microscopy (FESEM), high resolution transmission electron microscopy (HRTEM) and dynamic light scattering (DLS). Diosgenin functionalization was confirmed using fourier transform infrared spectroscopy (FTIR) and biochemical methods. Synthesized IONPs, citrate linked IONPs (IONPs-CA), diosgenin functionalized IONPs (IONPs-D) along with free citric acid and diosgenin were checked for anticancer activity against MCF7 breast cancer cells by MTT assay, wound migration assay, confocal microscopy and protein expression by western blotting. Size of IONPs, IONPs-CA and IONPs-D gradually increased ranging from 12 to 21 nm as confirmed by FESEM and HRTEM. Signature peaks of diosgenin at 2914, 1166 and 1444 cm-1 IONPs-D, revealed in FTIR indicated the presence of functionalized diosgenin. IONPs-D exhibited 51.08 ± 0.37% antiproliferative activity against MCF7 cells, which was found to be superior to free citric acid (17.71 ± 0.58%) and diosgenin (33.31 ± 0.37%). Treatment with IONPs-D exhibited reduced wound migration upto 40.83 ± 2.91% compared to bare IONPs (89.03 ± 2.58%) and IONPs-CA (50.35 ± 0.48%). IONPs-D and diosgenin exhibited apoptosis induction, confirmed by Alexa Fluor 488 annexin V/PI double-stained cells indicating extensive cell membrane damage coupled with PI influx leading to nuclear staining in treated cells. IONPs-D mediated selective PARP cleavage strongly rationalized it as superior apoptotic inducers. Based on these findings, IONPs-D can be considered as first diosgenin functionalized novel magnetic nanomedicine with antiproliferative, migration inhibiting and apoptosis inducing properties against breast cancer.

Biomimetic bright optotheranostics for metastasis monitoring and multimodal image-guided breast cancer therapeutics.

Nanoparticle formulations blending optical imaging contrast agents and therapeutics have been a cornerstone of preclinical theranostic applications. However, nanoparticle-based theranostics clinical translation faces challenges on reproducibility, brightness, photostability, biocompatibility, and selective tumor targeting and penetration. In this study, we integrate multimodal imaging and therapeutics within cancer cell-derived nanovesicles, leading to biomimetic bright optotheranostics for monitoring cancer metastasis. Upon NIR light irradiation, the engineered optotheranostics enables deep visualization and precise localization of metastatic lung, liver, and solid breast tumors along with solid tumor ablation. Metastatic cell-derived nanovesicles (∼80 ± 5 nm) are engineered to encapsulate imaging (emissive organic dye and gold nanoparticles) and therapeutic agents (anticancer drug doxorubicin and photothermally active organic indocyanine green dye). Systemic administration of biomimetic bright optotheranostic nanoparticles shows escape from mononuclear phagocytic clearance with (i) rapid tumor accumulation (3 h) and retention (up to 168 h), (ii) real-time monitoring of metastatic lung, liver, and solid breast tumors and (iii) 3-fold image-guided solid tumor reduction. These findings are supported by an improvement of X-ray, fluorescence, and photoacoustic signals while demonstrating a tumor reduction (201 mm3) in comparison with single therapies that includes chemotherapy (134 mm3), photodynamic therapy (72 mm3), and photothermal therapy (88mm3). The proposed innovative platform opens new avenues to improve cancer diagnosis and treatment outcomes by allowing the monitorization of cancer metastasis, allowing the precise cancer imaging, and delivering synergistic therapeutic agents at the solid tumor site.

Poly(d,l-lactide-co-glycolide) Surface-Anchored Biotin-Loaded Irinotecan Nanoparticles for Active Targeting of Colon Cancer.

A poly(d,l-lactide-co-glycolide) (PLGA) copolymer was synthesized using the ring-opening polymerization of d,l-lactide and glycolide monomers in the presence of zinc proline complex in bulk through the green route and was well characterized using attenuated total reflectance-Fourier transform infrared, 1H and 13C nuclear magnetic resonance, gel permeation chromatography, differential scanning calorimetry, X-ray diffraction, matrix-assisted laser desorption/ionization time-of-flight, etc. Furthermore, PLGA-conjugated biotin (PLGA-B) was synthesized using the synthesized PLGA and was employed to fabricate nanoparticles for irinotecan (Ir) delivery. These nanoparticles (PLGA-NP-Ir and PLGA-B-NP-Ir) were tested for physicochemical and biological characteristics. PLGA-B-NP-Ir exhibited a stronger cellular uptake and anticancer activity as compared to PLGA-NP-Ir in CT-26 cancer cells (log p < 0.05). The accumulation and retention of fluorescence-labeled nanoparticles were observed to be better in CT-26-inoculated solid tumors in Balb/c mice. The PLGA-B-NP-Ir-treated group inhibited tumor growth significantly more (log p < 0.001) than the untreated control, PLGA-NP-Ir, and Ir-treated groups. Furthermore, no body weight loss, hematological, and blood biochemical tests demonstrated the nanocarriers' nontoxic nature. This work presents the use of safe PLGA and the demonstration of a proof-of-concept of biotin surface attached PLGA nanoparticle-mediated active targeted Ir administration to combat colon cancer. To treat colon cancer, PLGA-B-NP-Ir performed better due to specific active tumor targeting and greater cellular uptake due to biotin.

Sponging of five tumour suppressor miRNAs by lncRNA-KCNQ1OT1 activates BMPR1A/BMPR1B-ACVR2A/ACVR2B signalling and promotes chemoresistance in hepatocellular carcinoma.

Diverse mechanisms have been established to understand the chemoresistance of hepatocellular carcinoma (HCC), but the contribution of non-coding RNAs is not surveyed well. Here, we aimed to explore the lncRNA-miRNA axis in Hepatitis C and B virus (HCV and HBV) infected HCC to investigate the molecular mechanism of chemoresistance and to identify a potential therapeutic target for HCC. The small RNA transcriptome analysis followed by qRT-PCR validation with the liver tissues of both HCV and HBV infected HCC patients revealed that miR-424-5p, miR-136-3p, miR-139-5p, miR-223-3p, and miR-375-3p were the most downregulated miRNAs in HCC compared to normal (log2 fold change ≤-1.5, Padj ≤ 0.05). In silico pathway analysis with the validated targets of each miRNA revealed that the signalling pathway regulating pluripotency of stem cells is commonly targeted by these five miRNAs. Subsequent validation by 3'UTR-luciferase assay and western blot analysis unveiled that these five miRNAs impeded either same or diverse genes, but all linked to BMP signalling pathway such as BMPR1A/BMPR1B by miR-139-5p, miR-136-3p, and miR-375-3p, and ACVR2A/ACVR2B by miR-424-5p and miR-223-3p. Furthermore, restoration of each miRNA in Huh7/SNU449 cells inhibited phosphorylation of downstream SMAD1/5 and ERK1/2, and attenuated Epithelial-mesenchymal transition, stemness, spheroid formation, chemoresistance, invasion and migration of cells. To investigate the mechanism of suppression of these miRNAs, "DIANA" tool was employed and lncRNA-KCNQ1OT1 was retrieved as interacting partner of all the five miRNAs. In vitro RNA pull-down assay revealed that lncRNA-KCNQ1OT1 physically interacted and sequestered these five miRNAs in the cytoplasm. Hence, KCNQ1OT1 was suppressed in Huh7/SNU449 cells using CRISPR technology and observed regression of oncogenic properties with enhanced chemosensitivity and reduced metastasis in cancer cells. Shrinkage of tumour size and volume in NOD-SCID mice injected with KCNQ1OT1-sgRNA cells further strengthened our observations. Thus, lncRNA-KCNQ1OT1 is the main regulator, which reduces the level of beneficiary miRNAs in the tumour milieu and modulates BMP signalling pathway to promote chemoresistance in HCC, suggesting lncRNA-KCNQ1OT1 might have robust potential to be a therapeutic target in HCC.

Hybrid Inorganic Complexes as Cancer Therapeutic Agents: In-vitro Validation.

A series of novel mixed transition metal-Magnesium tartarate complexes of general formulation [MMg(C4H4O6)2 .xH2O] (where M = Mn, Fe, Co, Ni, Cu and Zn) is prepared with bidentate tartarate ligand. The synthesized complexes (C1 to C6) are characterized by various analytical techniques such as Elemental analysis, Thermo gravimetric analysis, FT-IR Spectroscopy, X-ray Diffraction, Magnetic susceptibility study etc. All complexes exhibit the composition MMgL2 where M = Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II) and L = bidentate tartarate ligand. Analytical data reveals all complexes possesses 1:1 (metal: ligand) ratio. FT-IR spectral study shows that bidentate tartarate ligand coordinate with metal ion in a bidentate manner through two oxygen atoms. Thermo gravimetric analysis of all complexes shows that degradation curves of complexes agrees with recommended formulae of the complexes. X-ray diffraction technique suggests that all complexes (C1 to C6) are polycrystalline in nature. All newly synthesized metal tartarate complexes and ligand were screened in vitro for their anticancer activity against human breast cancer (MDA-MB-231) cell line. The bioassays of all these complexes showed C3 (Co) and C5 (Cu) Mg-tartarate complexes contains maximum antiproliferative activity at 200 µg/ml concentration on MDA-MB-231 cells as compared to other complexes. MDA-MB-231 cells treated with C3 (Co) and C5 (Cu) Mg-tartarate complexes also showed inhibition in cell migration.

Bioinspired and biomimetic cancer-cell-derived membrane nanovesicles for preclinical tumor-targeted nanotheranostics.

Bioinspired cell-membrane-camouflaged nanohybrids have been proposed to enhance tumor targeting by harnessing their immune escape and self-recognition abilities. In this study, we introduce cancer-cell-derived membrane nanovesicles (CCMVs) integrated with gold nanorods (AuVNRs) in addition to therapeutic and imaging cargos such as doxorubicin and indocyanine green. This approach enhances targeted tumor imaging and enables synergistic chemo-phototherapeutics for solid tumors. CCMVs demonstrate significant tumor penetration and retention, serving as nanotheranostics with accessible surface biomarkers, biomimicking properties, and homologous targeting abilities. By evading uptake by the mononuclear phagocytic system, CCMVs can diffuse into the deep tumor core, leading to precise tumor reduction while preserving the surrounding healthy tissues. Notably, intravenous administration of these theranostic agents ensures biocompatibility, as evidenced by a survival period of approximately two months (up to 63 days) without any observed side effects. Our findings underscore the diagnostic and therapeutic potential of this biomimetic nanotheranostics platform.

Identification of a physiologic vasculogenic fibroblast state to achieve tissue repair.

Tissue injury to skin diminishes miR-200b in dermal fibroblasts. Fibroblasts are widely reported to directly reprogram into endothelial-like cells and we hypothesized that miR-200b inhibition may cause such changes. We transfected human dermal fibroblasts with anti-miR-200b oligonucleotide, then using single cell RNA sequencing, identified emergence of a vasculogenic subset with a distinct fibroblast transcriptome and demonstrated blood vessel forming function in vivo. Anti-miR-200b delivery to murine injury sites likewise enhanced tissue perfusion, wound closure, and vasculogenic fibroblast contribution to perfused vessels in a FLI1 dependent manner. Vasculogenic fibroblast subset emergence was blunted in delayed healing wounds of diabetic animals but, topical tissue nanotransfection of a single anti-miR-200b oligonucleotide was sufficient to restore FLI1 expression, vasculogenic fibroblast emergence, tissue perfusion, and wound healing. Augmenting a physiologic tissue injury adaptive response mechanism that produces a vasculogenic fibroblast state change opens new avenues for therapeutic tissue vascularization of ischemic wounds.

Antiproliferative and apoptotic potential of methotrexate lipid nanoparticles in a murine breast cancer model.

Aim: To evaluate the efficacy of novel methotrexate-loaded nanoparticles (MTX-NPs) in vitro and in vivo in the treatment of breast cancer. Materials & methods: MTX-NPs were tested for cellular uptake, cell viability, cell cycle, cellular wound migration and changes in tumor volume using characterized NPs. Results: The solid lipid NPs (SLNPs) showed strong cellular uptake, increased apoptosis, controlled cytotoxicity at lower IC50 of methotrexate and a sizable reduction in tumor burden. Conclusion: MTX-NP oral formulation can be a promising candidate in breast cancer treatment with improved cellular uptake and in vivo efficacy.

Oncostatin M Improves Cutaneous Wound Re-Epithelialization and Is Deficient under Diabetic Conditions.

Impaired re-epithelialization characterized by hyperkeratotic nonmigratory wound epithelium is a hallmark of nonhealing diabetic wounds. In chronic wounds, the copious release of oncostatin M (OSM) from wound macrophages is evident. OSM is a potent keratinocyte (KC) activator. This work sought to understand the signal transduction pathway responsible for wound re-epithelialization, the primary mechanism underlying wound closure. Daily topical treatment of full-thickness excisional wounds of C57BL/6 mice with recombinant murine OSM improved wound re-epithelialization and accelerated wound closure by bolstering KC proliferation and migration. OSM activated the Jak-signal transducer and activator of transcription pathway as manifested by signal transducer and activator of transcription 3 phosphorylation. Such signal transduction in the human KC induced TP63, the master regulator of KC function. Elevated TP63 induced ITGB1, a known effector of KC migration. In diabetic wounds, OSM was more abundant than the level in nondiabetic wounds. However, in diabetic wounds, OSM activity was compromised by glycation. Aminoguanidine, a deglycation agent, rescued the compromised KC migration caused by glycated OSM. Finally, topical application of recombinant OSM improved KC migration and accelerated wound closure in db/db mice. This work recognizes that despite its abundance at the wound site, OSM is inactivated by glycation, and topical delivery of exogenous OSM is likely to be productive in accelerating diabetic wound closure.

MiRNA-146a/AKT/ß-Catenin Activation Regulates Cancer Stem Cell Phenotype in Oral Squamous Cell Carcinoma by Targeting CD24.

CD44highCD24low population has been previously reported as cancer stem cells (CSCs) in Oral Squamous Cell Carcinoma (OSCC). Increasing evidence suggests potential involvement of microRNA (miRNA) network in modulation of CSC properties. MiRNAs have thus emerged as crucial players in tumor development and maintenance. However, their role in maintenance of OSCC stem cells remains unclear. Here we report an elevated expression of miR-146a in the CD44highCD24low population within OSCC cells and primary HNSCC tumors. Moreover, over-expression of miR-146a results in enhanced stemness phenotype by augmenting the CD44highCD24low population. We demonstrate that miR-146a stabilizes ß-catenin with concomitant loss of E-cadherin and CD24. Interestingly, CD24 is identified as a novel functional target of miR-146a and ectopic expression of CD24 abrogates miR-146a driven potential CSC phenotype. Mechanistic analysis reveals that higher CD24 levels inhibit AKT phosphorylation leading to ß-catenin degradation. Using stably expressing miR-146a/CD24 OSCC cell lines, we also validate that the miR-146a/CD24/AKT loop significantly alters tumorigenic ability in vivo. Furthermore, we confirmed that ß-catenin trans-activates miR-146a, thereby forming a positive feedback loop contributing to stem cell maintenance. Collectively, our study demonstrates that miR-146a regulates CSCs in OSCC through CD24-AKT-ß-catenin axis.

Ultrahigh Penetration and Retention of Graphene Quantum Dot Mesoporous Silica Nanohybrids for Image Guided Tumor Regression.

So far, near-infrared (NIR) light responsive nanostructures have been well-defined in cancer nanomedicine. However, poor penetration and retention in tumors are the limiting factors. Here, we report the ultrahigh penetration and retention of carbanosilica (graphene quantum dots, GQDs embedded mesoporous silica) in solid tumors. After NIR light exposure, quick (0.5 h) emission from the tumor area is observed that is further retained up to a week (tested up to 10 days) with a single dose administration of nanohybrids. Emissive and photothermally active GQDs and porous silica shell (about 31% drug loading) make carbanosilica a promising nanotheranostic agent exhibiting 68.75% tumor shrinking compared to without NIR light exposure (34.48%). Generated heat (∼52 °C) alters the permeability of tumor enhancing the accumulation of nanotheranostics into the tumor environment. Successive tumor imaging ensures the prolonged follow-up of image guided tumor regression due to synergistic therapeutic effect of nanohybrids.

Urolithin A augments angiogenic pathways in skeletal muscle by bolstering NAD+ and SIRT1.

Urolithin A (UA) is a natural compound that is known to improve muscle function. In this work we sought to evaluate the effect of UA on muscle angiogenesis and identify the underlying molecular mechanisms. C57BL/6 mice were administered with UA (10 mg/body weight) for 12-16 weeks. ATP levels and NAD+ levels were measured using in vivo 31P NMR and HPLC, respectively. UA significantly increased ATP and NAD+ levels in mice skeletal muscle. Unbiased transcriptomics analysis followed by Ingenuity Pathway Analysis (IPA) revealed upregulation of angiogenic pathways upon UA supplementation in murine muscle. The expression of the differentially regulated genes were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC). Angiogenic markers such as VEGFA and CDH5 which were blunted in skeletal muscles of 28 week old mice were found to be upregulated upon UA supplementation. Such augmentation of skeletal muscle vascularization was found to be bolstered via Silent information regulator 1 (SIRT1) and peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1α) pathway. Inhibition of SIRT1 by selisistat EX527 blunted UA-induced angiogenic markers in C2C12 cells. Thus this work provides maiden evidence demonstrating that UA supplementation bolsters skeletal muscle ATP and NAD+ levels causing upregulated angiogenic pathways via a SIRT1-PGC-1α pathway.

RGD functionalized chitosan nanoparticle mediated targeted delivery of raloxifene selectively suppresses angiogenesis and tumor growth in breast cancer.

Acidic pH is a crucial intrinsic property of the microenvironment of most solid tumors. Hence, the use of pH sensitive tumor targeting nanoparticles is an attractive approach to enhance the therapeutic efficacy of anti-cancer agents in solid tumors. Chitosan nanoparticles (CHNPs) have been widely explored in the area of cancer drug delivery; nevertheless their true potential as a pH responsive targeted drug delivery vehicle in cancer therapy has not been deciphered yet as most of the research is limited to pH dependent stability and drug release. In the present study, we investigate the direct effect of pH in synergy with RGD peptide based targeting on the therapeutic efficacy of chitosan nanoparticles (RGD-CHNPs) in breast cancer. Furthermore, for the first time we performed a comprehensive study showing the anti-tumor, anti-migratory and anti-angiogenic effect of raloxifene (Rlx) loaded CHNPs in breast cancer. We prepared stable formulations of raloxifene encapsulated CHNPs and RGD-CHNPs by the nontoxic ionic gelation method. pH dependent studies revealed that NPs possess higher stability and zeta potential along with enhanced cellular uptake at acidic pH (as present in solid tumors) compared to physiological pH. Furthermore, RGD conjugation enhanced the in vitro cellular uptake of CHNPs in αvß3 integrin expressing breast cancer cells and induced higher cellular apoptosis in breast cancer cells which was further augmented by lower pH. Moreover, Rlx-RGD-CHNPs significantly inhibited breast cancer cell migration and angiogenesis. In vivo studies showed that Cy5.5 conjugated RGD-CHNPs can distinctly visualize tumors and Rlx-RGD-CHNPs significantly inhibit breast tumor growth without causing any toxic effect to normal tissue as confirmed by hematology and blood biochemical studies. Therefore, RGD-CHNPs could potentially enhance the therapeutic efficacy of chemotherapeutic drugs due to the synergistic effect of pH responsiveness and tumor specific targeting in breast cancer.

Liposomal nanotheranostics for multimode targeted in vivo bioimaging and near-infrared light mediated cancer therapy.

Developing a nanotheranostic agent with better image resolution and high accumulation into solid tumor microenvironment is a challenging task. Herein, we established a light mediated phototriggered strategy for enhanced tumor accumulation of nanohybrids. A multifunctional liposome based nanotheranostics loaded with gold nanoparticles (AuNPs) and emissive graphene quantum dots (GQDs) were engineered named as NFGL. Further, doxorubicin hydrochloride was encapsulated in NFGL to exhibit phototriggered chemotherapy and functionalized with folic acid targeting ligands. Encapsulated agents showed imaging bimodality for in vivo tumor diagnosis due to their high contrast and emissive nature. Targeted NFGL nanohybrids demonstrated near infrared light (NIR, 750 nm) mediated tumor reduction because of generated heat and Reactive Oxygen Species (ROS). Moreover, NFGL nanohybrids exhibited remarkable ROS scavenging ability as compared to GQDs loaded liposomes validated by antitumor study. Hence, this approach and engineered system could open new direction for targeted imaging and cancer therapy.

Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing.

Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exoκ-GFP) using tissue nanotransfection (TNT), and they were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low-base-pair RNA and was selectively engulfed by wound macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFPin vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to the murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to the TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, the TLNPκ/si-hnRNPA2B1 treated group showed a significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to the TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP is recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties postinjury.

In Vivo Wound Healing Performance of Halloysite Clay and Gentamicin-Incorporated Cellulose Ether-PVA Electrospun Nanofiber Mats.

Wound healing is a dynamic and complex process that requires a suitable environment to enhance the rapid healing process. In this context, fabrications of nanofibrous materials with antibiotic and antibacterial properties are becoming extremely important. In this present work, we report on the fabrication and characterization of electro-spun cellulose ether-PVA nanofiber mats loaded with halloysite clay (HNT) and gentamicin sulfate (GS) for faster wound healing applications. The morphology of nanofiber mats was examined by SEM and TEM. The average diameter of the nanofiber mats were in the range of 325 ± 30 nm. The physicochemical characterizations were done by FT-IR and XRD, which reveal the presence of HNT and GS into the nanofibers. The incorporation of halloysite gave good mechanical strength to the nanofiber mats. Swelling studies indicated the hydrophilicity of the mats. In vitro studies revealed that HNTs are nontoxic to L929 fibroblast cells and also promote cell growth and proliferation. The antibacterial property of HNT was also studied. The slow release of GS from the nanofiber mats was observed for a period of 18 days. The in vivo wound healing studies on the wistar rats for 21 days revealed the wound healing faster within 2 weeks by the incorporation of HNT and GS into the nanofiber mats and hence these nanofiber mats show great potential in acute and chronic wound healing applications.