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

Funding: This work was supported by Department of Biotechnology, Government of India. J.C. acknowledges the European Research Council Starting Grant (ERC-StG-2019-848325). We thank the staff of animal house, NCCS, Pune for supporting us during animal studies. We also thank Mr. Sumit for the discussion and Dr. Mukesh K. Kumawat for providing GQDs.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.publishersversionpublishe

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.publishersversionpublishe

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

Functional characterization of stromal osteopontin in melanoma progression and metastasis.

BACKGROUND: Recent studies demonstrated that not only tumor derived- but stroma derived factors play crucial role in cancer development. Osteopontin (OPN) is a secreted non-collagenous, sialic acid rich, chemokine-like phosphoglycoprotein that facilitates cell-matrix interactions and promotes tumor progression. Elevated level of OPN has been shown in melanoma patient and predicted as a prognostic marker. Recent reports have indicated that stroma-derived OPN are involved in regulating stem cell microenvironment and pre-neoplastic cell growth. However, the function of stroma derived OPN in regulation of side population (SP) enrichment leading to melanoma growth, angiogenesis and metastasis is not well studied and yet to be the focus of intense investigation. METHODOLOGY/PRINCIPAL FINDINGS: In this study, using melanoma model, in wild type and OPN knockout mice, we have demonstrated that absence of host OPN effectively curbs melanoma growth, angiogenesis and metastasis. Melanoma cells isolated from tumor of OPN wild type (OPN(+/+)) mice exhibited more tumorigenic feature as compared to the parental cell line or cells isolated from the tumors of OPN KO (OPN(-/-)) mice. Furthermore, host OPN induces VEGF, ABCG2 and ERK1/2 expression and activation in B16-WT cells. We report for the first time that stroma derived OPN regulates SP phenotype in murine melanoma cells. Moreover, loss in and gain of function studies demonstrated that stroma-derived OPN regulates SP phenotype specifically through ERK2 activation. CONCLUSIONS: This study establish at least in part, the molecular mechanism underlying the role of host OPN in melanoma growth and angiogenesis, and better understanding of host OPN-tumor interaction may assist the advancement of novel therapeutic strategy for the management of malignant melanoma

Host OPN selectively enriches stem-like cancer cells in B16F10 cells.

<p>(<b>A-D</b>) Cells (B16F10, B16-WT, B16-KO) were stained with Hoechst 33342 dyes in the absence or presence of reserpine and analyzed using flow cytometer for SP population. Lung adenocarcinoma (A549) cells used as positive control for SP phenotype. The average size of the SP was around 8–9% in B16F10 and B16-KO cells whereas 21% in B16-WT cells. All data are representation of three independent experiments exhibiting similar results.</p

ERK2 but not ERK1 regulates SP phenotype in B16F10 cells in response to stromal OPN.

<p>(<b>A</b>) Western blot analysis of ABCG2 expression in the lysates of B16F10, B16-KO and B16-WT cells treated with either wortmannin or U0126. (<b>B</b>) <i>Panels I and II</i>, B16F10 cells, stably transfected with either ERK1-dn or ERK2-dn were treated with conditioned media of B16-WT cells, stained with Hoechst and SP phenotype was analyzed using flow cytometer. (<b>C</b>) <i>Panels I and II</i>, B16F10 cells stably transfected with either ERK1-wt or ERK2-wt were treated with CM collected from B16-WT cells, stained with Hoechst and SP phenotype was analyzed. <i>Inset:</i> control setup for SP analysis treated with reserpine for respective treatment group. (<b>D</b>) Analysis of ABCG2 expression from lysates of B16F10 cells stably transfected with ERK1-dn, ERK2-dn, ERK1-wt or ERK2-wt in presence of CM of B16-WT. Actin was used as loading control. All the figures are representative of three independent experiments exhibiting similar results.</p

Stromal OPN regulates SP phenotype in B16F10 through ERK pathway.

<p>(<b><i>A</i></b>) B16-WT cells were stained with Hoechst and SP analysis was performed with flow cytometer. (<b><i>B</i></b>) B16-WT cells were treated with wortmannin for 24 h, stained with Hoechst and analyzed for SP phenotype. (<b><i>C</i></b>) B16-WT cells were treated with U0126 and SP analysis was performed. (<b><i>D</i></b>) Flow cytometeric analysis of SP phenotype in B16F10 cells. (<b><i>E</i></b>) B16F10 cells were treated with conditioned media of B16-WT cells for 24 h and SP analysis was performed. (<b><i>F</i></b>) B16F10 cells were pre-treated with conditioned media of B16-WT cells and then treated with wortmannin for 24 h and SP analysis were performed. (<b><i>G</i></b>) B16F10 cells were pre-treated with conditioned media of B16-WT cells and then treated with U0126 for 24 h and SP analysis was performed. <b><i>Inset:</i></b> control setup for SP analysis treated with reserpine for respective panels. All the panels are representative of three independent repeats exhibiting similar results.</p

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 αβ 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

Sorting and characterization of SP and non-SP from B16F10 cells.

<p>(<b>A</b>) Sorted SP and non-SP cells were treated with mitoxantrone (0–50 nM). In addition, SP cells were treated with or without reserpine (5 µM) and growth inhibition analysis was performed by MTT assay. Results are mean ± SEM (^$, P = 0.001; ^$$, P = 0.3; ^#, P = 0.006; ^##, P = 0.002; *, P<0.001). The data shown is the representation of three independent experiments showing similar results. (<b>B</b>) Differentiation assay was performed with <i>in vitro</i> cultured SP cells. The data are the representation of three independent experiments showing similar results. (<b>C</b>) <i>In vitro</i> tumorigenicity of SP and NSP cells were checked by Matrigel-based colony formation assay. Colonies formed were photographed at 10× magnification. The data shown is representation of three separate experiments showing similar results. (<b>D</b>) (<i>panel I</i>), Sorted SP cells (1×10³) were injected into the OPN^+/+ and OPN^−/− and kept for 5 weeks. Mice were sacrificed and tumors were collected and photographed. Mean tumor volume was calculated, analyzed statistically and represented graphically. Bar represents mean±SEM; *, P = 0.007. <i>Panel II</i>, Mean tumor weight was calculated, analyzed statistically and represented in the form of bar graph. Bar represents mean±SD; *P = 0.007. (<b>E</b>) Sorted SP and non-SP cells were injected intravenously into NOD/SCID mice (n = 9) and metastasis to lungs and liver was analyzed using IVIS system. <i>Lower panel</i>: Mice were sacrificed. Lung and liver were dissected out and metastases were analyzed using IVIS.</p