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Multifunctional Immunocompatible NanoTheranostics to modulate tumor microenvironment and improve treatment monitoring: A double blow to pancreatic cancer

Periodic Reporting for period 1 - MINT (Multifunctional Immunocompatible NanoTheranostics to modulate tumor microenvironment and improve treatment monitoring: A double blow to pancreatic cancer)

Reporting period: 2019-09-01 to 2021-08-31

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal human malignancies having an overall 5-year survival rate less than 10%. Abundant fibrotic stroma and poor vascularization are the typical features of PDAC in humans limiting the delivery of chemotherapeutics to the tumor site. Herein, we have proposed to deliver VEGFR kinase inhibitor & HH pathway inhibitor simultaneously through a ZnO-based molecularly targeted and smart drug carrier to deplete tumor stroma, normalize tumor vascularity and show synergism to improve standard pancreatic cancer treatment by modulating tumor microenvironment.
Despite various nanomaterials were developed so far in particular against cancer, very little attention was paid to their potential immunogenicity, to their final destiny at the end of their functions, as well as to the off-target zero-delivery. Thus, there is still a huge disproportion between these nanotherapeutic treatments and the conventional ones proposed to patients (i.e. surgery, chemotherapy, radiotherapy, immunotherapy or their combinations). To address these important challenges and cover the gap between the present nanomedicine tools and the clinical requirements, a phospholipidic coating based on lipids derived from autologous extracellular vesicles (EVs) has been proposed, obtaining highly stable and non-immunogenic theranostic lipid coated ZnO nanocrystals (LZnO-NCs). Targeting is then accomplished by functional peptides, able to direct the drug-loaded nanoconstruct to the right site of action.
Successful delivery of these LZnO-NCs to target site can modulate tumor microenvironment and inhibit proliferation & migration of the tumor and stromal cells. This therapeutic outcome will have a significant impact on the standard treatment of PDAC and other stroma-rich cancers. Moreover, LZnO-NCs doped with transition elements is an increasingly attractive theranostic nanoparticle with the combined capabilities of contrast enhancement in magnetic resonance imaging (MRI) and as a drug carrier for therapeutics.
The main research objective of the present research project is to develop novel theranostic immunocompatible nanoparticles to target pancreatic tumor stroma as well as neoplastic cells, enable intracellular release of drugs and also provide contrast enhancement in MRI scanning for treatment monitoring. Therefore, it is articulated into two parts- i) Design of novel theranostic immunocompatible LZnO-NCs, ii) Evaluation of the therapeutic and imaging (theranostic) capabilities of the nanoconstruct (VEGFR inhibitor and Hh pathway inhibitor loaded doped ZnO-NCs coated with lipid bilayer) by performing various cellular functional assays with or without treatment of standard chemotherapy (Gemcitabine).
The coprecipitation method and the conventional sol-gel synthesis method has been used to synthesize and control the doping of ZnO:Gd and ZnO:Mn in NCs. Different characterization techniques including dynamic light scattering (DLS) and Z-Potential measurements, Transmission and Field Emission Scanning Electron Microscopies coupled to Energy Dispersive Spectroscopy, X-Ray Diffraction (XRD), X-Ray Photoelectron Spectroscopy (XPS), DC magnetometry have been used to analyze their structural, morphological, optical, and magnetic properties. I’m able to characterize the maintenance of the crystalline properties (wurtzitic structure) of ZnO in the doped structure and exclusion of the formation of undesired oxides made by doping elements (Barui S, Nanomaterials 2020).
Gemcitabine, the baseline drug of pancreatic cancer treatment, is successfully adsorbed on the surface of NCs. The freeze thaw technique has been exploited to prepare novel multifunctional lipid-coated nano-construct by coupling the Gemcitabine-loaded doped ZnO-NCs with lipidic bilayers derived from autologous EVs. I have also managed to incorporate targeting peptide ligand on the outer surface of the drug-loaded LZnO-NCs to deliver the drugs at the right site of action. The size and shape of nanoconstructs have been characterized by DLS and Nanoparticle Tracking Analysis (NTA). Energy Dispersive X-Ray for elemental analysis and DC magnetometry to determine MRI applicability of the nanoconstructs have also performed. Flow cytometric analysis of cell internalization in pancreatic cancer cells (e.g. AsPC-1, BxPC-3) shows potential advantage of introducing target peptide ligand in the nanoconstruct. Cellular cytotoxicity assay in pancreatic cancer cells dictates increased cell killing efficacy of Gemcitabine when delivered through the nanoconstruct than as free drug. Taken together, our designed theranostic EV-coated doped ZnO-NCs can have a significant impact on the standard treatment of pancreatic cancer (Poster at NanoMed 2021 Conference).
Thereafter, I have evaluated the synergistic effect of VEGFR inhibitor (Sorafenib) & Hh pathway inhibitor (Vismodegib) by performing in vitro cellular functional assays (WST-1 assay, Flow Cytometric analysis) by using human pancreatic cancer cells derived from metastasis site (AsPC-1) either untreated or pre-treated with the standard chemotherapy of pancreatic cancer i.e. Gemcitabine. I have determined that the synergistic cytotoxic effect of Sorafenib & Vismodegib is more promising in Gemcitabine pre-treated cells compared to native untreated cells. This therapeutic outcome of employing novel multifunctional lipid coated nanoconstruct having Gd-doped ZnO-NCs loaded with Sorafenib & Vismodegib can be expected to have a significant impact on PDAC therapy when treating with standard chemotherapy (Gemcitabine).
I have reported an easy and fast coprecipitation method exploiting oleic acid to synthesize spherical-shaped, small-sized doped ZnO-NCs. I have shown the improved colloidal stability of oleate-stabilized doped ZnO-NCs compared to the doped ZnO-NCs synthesized by conventional sol-gel synthesis method. This oleate-stabilized coprecipitation method can be subjected as a standard procedure to synthesize doped and also co-doped ZnO-NCs with any transition metal elements or rare earth elements. In the future, oleate-stabilized Gd/Mn-doped ZnO-NCs can be exploited as MRI contrast agents to increase the signal intensity on T1-weighted images or reduce the signal intensity on T2-weighted images.
Moreover, abundant fibrotic stroma and poor vascularization are the typical features of PDAC in humans limiting the delivery of standard chemotherapy to the tumor site. Thus, I aim to target signaling pathways correlating tumor microenvironment. I’m exploiting Hh pathway inhibitor (to deplete tumor stroma) and VEGFR kinase inhibitor (to normalize tumor vascularity) simultaneously through the multifunctional EV-coated doped ZnO-NCs to improve pancreatic cancer therapeutic index. The cytotoxicity assay results dictate that the therapeutic efficacy of drug molecule is going to enhance prominently when delivered through the designed lipid coated nanoconstruct than as free drug. I have evaluated improved synergistic effect of VEGFR inhibitor and Hh pathway inhibitor while treating with Gemcitabine. Therefore, successful delivery of the duel drug loaded nanoconstruct to the right site of action can modulate tumor microenvironment and inhibit proliferation and migration of the tumor and stromal cells. This therapeutic outcome can be expected to have a significant impact not only on PDAC treatment but also for other stroma-rich cancers.
Characterization of the synthesized doped ZnO-NCs
Multifunctionalized immunocompatible tumor targeted novel nanotheranostics in pancreatic cancer