Smart Fluorescent Nanoparticle For MicroRNA Delivery And Tumor Imaging And Therapy

Although significant progress had been made in diagnostic techniques and treatment,cancer is the major disease that threats human life seriously and is also the leading cause of deaths worldwide.In recent years,theranostic nanoparticles which combined imaging and therapy in a single platform for image-guided cancer therapy,have attracted more and more attention in nanomedicine and cancer precision medicine.Nanomaterials have special physcial and chemical properties,which could be obtained by various synthesis and assembly methods.The combination of imaging probes with therapeutic drugs/genes in nanoparticles not only could mediate effective delivery,but also allows us to monitor the accumulation and distribution of drugs/genes in an animal model to facilitate the evaluation of treatment effects and optimize treatment dose for improving therapeutic efficacy,which provide novel methods and strategies in cancer theranostics.Traditional near-infrared?NIRF?organic fluorophores have been receiving increasing attention in biomedical applications,which offers significant advantages in living cells and tissue imaging due to its low autofluorescence and deeper tissue penetration abilities in the NIR region?700-900 nm?.However,the confinement of NIR fluorescence showed a notorious phenomenon with weakened or self-quenched fluorescence intensity due to?–?stacking and other nonradiative pathways,named as aggregated-caused quenching?ACQ?.In addition,Most NIR dyes have some limitions for in vivo applications,including short half-life in blood,quick clearance from the body and lack of the targeting ability.Recent years,the utilization of fluorescent nanoparticles?FNPs?,which consist of organic fluorophores embedded into a nanoparticle,seems to be a promising concept for in vitro/vivo imaging,multimodal imaging,photodynamic/phototheramal therapy and imaging-guided cancer therapy.Rectification of miRNA abnormality by miRNA replacement has emerged as one of novel strategies for alternative miRNA-based tumor therapy.However,transportation of miRNA to the tumor sites and cells is still dramatically hindered by multistage biological barriers.Therefore,non-viral nanoparticles-mediate effective miRNA delivery and modulation is of great significance in miRNA-based gene therapy.In this thesis,we constructed a series of designed nanostructures to develop NIR fluorescence“OFF-ON”smart nanoparticles for miRNA delivery and accurate tumor imaging and miRNA-based therapy.The detailed work is described below:1.Two-step assembling of Cy-5 molecules and miR-203-co-loaded nanohybrids:Nanodiamond clusters?NDs?were first functionalized for protamine sulfate immobilization?PNDs?on their surfaces via a noncovalent self-assembling approach and simultaneous encapsulation of NIR emitting fluorescence dye cyanine 5?Cy-5??F-PNDs?.Tumor suppressor miRNA-203?miR-203?was then adsorbed onto the surface of F-PNDs to form miR-203/F-PNDs via electrostatic interactions.The size,morphology,photophysical stability and optical properties of miR-203/F-PNDs were analyzed.Our results indicated that Cy-5 molecule can be loaded into F-PNDs with high efficacy and F-PNDs had better colloidal stability compared with bare ND clusters.After loading into miR-203/F-PNDs,no fluorescence of Cy-5 could be observed due to the ACQ effect.Trypsin could accelerate the release of Cy-5 and miR-203 from miR-203/F-PNDs,thus leading to the recovery of Cy-5 fluorescence.2.Application of the near-infrared“OFF-ON”fluorescent nanohybrids for synchronous tumor imaging and miRNA modulation-based therapy:We found that the NIR fluorescence of miR-203/F-PNDs could be activated to the“ON”state in intracellular environment while remaining in the“OFF”state in extracellular or blood environment.In vitro studies indicated that miR-203/F-PNDs could effectively inhibit proliferation and migration of Ec-109 cells.Western-blot and qRT-PCR results demonstrated that miR-203/F-PNDs could lead to effieient reduction in the downstream genes and proteins.Furthermore,in vivo live imaging experiments showed that miR-203/F-PNDs could be predominantly accumulated in tumor tissues and image the tumor sites 24 h postintravenous injection.In addition,intravenous and intratumoral injection of miR-203/F-PNDs could efficiently inhibit tumor growth through down-regulation of the expressions of oncogenes Ran and?p63.Our study indicated that miRNA/F-PNDs could serve as a promising theranostic platform for synchronous tumor imaging and miRNA-based modulation therapy against cancer.3.Construction and characterization of glutathione?GSH?-responsive light-up inter-polyelectrolyte nanocomplexes?CMINs?:Glutathione?GSH?-responsive peptide-polysaccharide-inter-polyelectrolyte nanocomplexes were established through self-assembly of carboxymethyl dextran?CMD?with disulfide-bridged?“S-S”?oligoarginine peptide?S-Arg₄?,in which microRNA-34a?miR-34a?and indocyanine green?ICG?were simultaneously embedded and the nanocomplexes were subsequently stabilized by intermolecular cross-linking.ICG was used as the NIRF probe which tends to aggregate while entrapped within the nanocomplexes and exhibits no fluorescence?“OFF”?due to self-quenching effect.The disulfide bond in S-Arg₄ could be cleaved by intracellular GSH,which led to the dissociation of nanocomplexes and triggered the simultaneous release of miR-34a and ICG.The NIRF of ICG was concomitantly activated through dequenching of the aggregated ICG.Thus,we could monitor miR-34a release by assessing the“OFF-ON”NIRF.4.In situ monitoring of miR-34a replacement efficacy and accurate imaging-guided cancer therapy through CMINs:In vitro experiments,we performed confocal microscopy and flow cytometry analysis to investigate the miR-34a delivery ability by CMINs.We could monitor miR-34a release by the activated ICG fluorescence.CMINs-mediated miR-34a delivery and replacement could inhibit cancer cell proliferation and migration,and induce apoptpsis.CMD could lead to enhanced blood circulation of CMINs and thus accumulate in tumor site due to the enhanced permeability and retention?EPR?effect,which could image the tumor site effectively.In addition,we could monitor miR-34a release and replacement efficacy in vivo conditions by activated ICG NIRF.Furthermore,replacement of miR-34a with CMINs resulted in efficient tumor-suppressive effects in vivo.The data demonstrated that the established nanocomplexes have a great potential to be used as a light-up theranostic platform with excellent biocompatibility for realtime monitoring of miR-34a replacement efficacy and accurate imaging-guided therapy strategy against tumor.