Investigating The Potential Of Ultrasound In Combination With Multifunctional Nanocomplexes In Molecular Targeting Therapy Of NSCLC

Part Ⅰ Experimental study on the effect of the gene therapy targeted inhibition of M2-like TAMs polarization on NSCLC invasive metastasisPurpose: This study was designed to investigate the crucial role of the JAK/STAT6 signaling pathway in the polarization of M2-like tumor-associated macrophages(TAMs)and its consequential impact on the invasive metastatic ability of non-small cell lung cancer(NSCLC)by employing a JAK/STAT6 signaling pathway blockade in TAMs to provide a possible effective target for molecularly targeted therapy of NSCLC.Methods: The STAT6 gene in the JAK/STAT6 signaling pathway of macrophages was suppressed using commercial transfection reagents with STAT6 siRNA.The transfection efficiency was assessed via fluorescence microscopy,and the silencing efficiency of the STAT6 gene and the inhibitory effect on M2-like TAMs were measured using q RT-PCR.Furthermore,the effect of blocking the JAK/STAT6 signaling pathway in TAMs on the metastatic and invasive capabilities of NSCLC was evaluated using scratch and Transwell assays.Results: M2-like TAMs can facilitate the metastasis and invasion of NSCLC.However,utilizing siRNA technology to silence the STAT6 gene can effectively impede M2-like TAMs polarization,subsequently reducing NSCLC metastasis and invasion.Conclusion: The JAK/STAT6 signaling pathway plays an important role in the polarization of TAMs,which is closely linked to the development of NSCLC and therefore represents a promising target for NSCLC molecularly targeted therapy.Part Ⅱ Multifunctional ultrasound nanocarriers for targeted inhibition of M2-like TAMs polarization to enhance photothermal therapy in NSCLC Purpose: The utilization of commercial transfection reagents for effective gene transfection and silencing in immune cells poses significant challenges.Furthermore,the inflammatory response triggered by residual tumor tissue following tumor photothermal therapy(PTT)may lead to a massive enrichment of M2-like TAMs,increasing the risk of tumor recurrence and metastasis.To overcome these obstacles,the development of multifunctional gold-based nanocomplexes,capable of efficient gene therapy to inhibit M2-like TAMs polarization and possessing photothermal properties,represents a promising new strategy for immunotherapy-enhanced PTT of NSCLC that can be visualized via ultrasound.Methods: Multifunctional ultrasound nanocomplexes(PGMP-siRNA)were synthesized using a double emulsion method,citric acid reduction method,amide reaction method,and positive and negative charge attraction method.The physical properties of the PGMP-siRNA nanocomplexes were evaluated through various methods including transmission electron microscopy(TEM),scanning electron microscopy(SEM),particle size potential analyzer,agarose gel electrophoresis,UV-visible spectrometer,laser,and photothermal imager.Contrast-enhanced ultrasound(CEUS)imaging was employed to assess the in vitro and in vivo capabilities of the PGMP-siRNA nanocomplexes.In vitro experiments were conducted to evaluate the biosafety of the nanocomplexes using the CCK-8 assay,and the gene transfection rate of macrophages was determined by laser confocal and flow cytometry.Additionally,the effect of STAT6 gene silencing in macrophages was analyzed using q RT-PCR and Western Blot assays.The PGMP-siRNA nanocomplex’s functionality was tested through scratch and Transwell assays,and the ability of photothermal action to kill NSCLC cells was measured using the CCK-8 assay,Calcein-AM/PI staining assay,and flow apoptosis assay.In vivo feasibility of synergistic therapy with the nanocomposite was evaluated using photothermal imaging assay,biosafety assay,tumor suppression assay,and immunohistochemical staining assay.Results: The PGMP-siRNA nanocomplex had good stability,resulting in a stable particle size of approximately 258.7 ± 26.96 nm and a zeta potential of around-14.9 ± 4.5 m V.Gel electrophoresis demonstrated that the nanocomplex effectively transported siRNA with high UV absorption and 808 nm photothermal conversion.The nanocomplex exhibited excellent CEUS capability in both in vitro and in vivo tumor models.In vitro experiments revealed that the PGMPsiRNA nanocomplex achieved a gene transfection efficiency of 90% in macrophages and inhibited87% of M2-like TAMs polarization,which was 40% higher than commercial transfection reagents.Furthermore,the nanocomplex was able to inhibit the invasion and metastasis abilities of NSCLC and induce more than 90% NSCLC cell death via PTT.In vivo experiments demonstrated that the PGMP-siRNA nanocomplex possessed efficient photothermal conversion,leading to inhibition of tumor cell proliferation and reduction of M2-like TAM polarization,which in turn potentiated the photothermal treatment ability of NSCLC.Conclusion: The use of PGMP-siRNA nanocomplexes provides a promising approach for targeted delivery of STAT6 siRNA to macrophages,effectively inhibiting the polarization of M2-type TAM induced by PTT.This intervention strategy can significantly prevent the invasion and metastasis of cancer cells,which in turn potentiates the photothermal treatment ability of NSCLC,offering a novel avenue for the clinical application of ultrasound-assisted visualization and treatment of NSCLC.Part Ⅲ Multifunctional nanoparticles targeted to overcome the EGFR-TKI resistance of NSCLC via low-temperature photothermal therapy combined with sonodynamic therapyPurpose: Drawing on the clinical use of EGFR-TKI as a first-line molecular targeting agent for the treatment of advanced NSCLC,the development of acquired drug resistance has been observed,and the underlying mechanisms are not fully understood.To address this challenge,we sought to construct multifunctional gold-based nanocomplexes with lung cancer targeting properties,investigate the potential mechanisms underlying EGFR-TKI resistance in NSCLC,and ultimately provide a novel nanoplatform for combating EGFR-TKI resistance in NSCLC.Methods: A series of functionally targeted multifunctional ultrasound nanocomplexes(cRGDGIPG)were synthesized based on the gold nanoparticle framework structure established in Part II.These nanocomplexes were designed to carry the EGFR-TKI inhibitor(gefitinib)and IR780,and were modified with a cRGD targeting peptide via covalent linkage.The physical properties of cRGD-GIPG nanocomplexes were comprehensively evaluated using a range of techniques,including TEM,SEM,particle size potential analyzer,UV-Vis spectrometer,ESR detector,fluorescence imager,laser,and photothermal imager.In vitro experiments were conducted to evaluate the functions of the nanocomplexes,including a CCK-8 assay to assess biosafety,laser confocal and flow cytometry for tumor targeting,DCFH-DA for intracellular ROS production,and mitochondrial membrane potential kit for changes in mitochondrial membrane potential.The efficacy of low-temperature PTT combined with sonodynamic therapy(SDT)in the treatment of EGFR-TKI resistance in NSCLC was assessed through CCK-8,Calcein-AM/PI staining assay,and flow apoptosis assay.A WB assay was conducted to investigate whether the mechanism of the cRGD-GIPG nanocomplexes for EGFR-TKI resistance treatment was associated with the TGF-β/PDLIM5/SMAD pathway and mitochondrial apoptosis pathway.The targeting ability of the nanocomplexes in vivo was evaluated using fluorescence imaging experiments,and the feasibility of their application in vivo was further verified through photothermal imaging experiments,biosafety experiments,and tumor suppression experiments.Results: The cRGD-GIPG nanocomplexes exhibited a particle size of approximately 370 nm and a zeta potential of-13.7±4.5 m V,displaying excellent stability,photothermal conversion,and ROS generation upon exposure to ultrasound.Laser irradiation triggered the targeted release of gefitinib,with a release rate of around 76%.In vivo experiments demonstrated the nanocomplex’s strong active targeting to PC-9GR cells.The combination of low-temperature PTT and SDT effectively increased intracellular temperature and ROS production,suppressed the activation of the TGF-β/PDLIM5/SMAD resistance pathway,and induced the mitochondrial apoptosis pathway,resulting in the treatment of EGFR-TKI resistance.Notably,low-temperature PTT and SDT demonstrated potent tumor suppression against EGFR-TKI-resistant lung cancer cells while maintaining excellent tumor targeting and biosafety profiles,as supported by the in vivo data.Conclusion: The cRGD-GIPG nanocomplex synergistically deactivated the drug-resistant TGF-β/PDLIM5/SMAD signaling pathway via low-temperature PTT and SDT,activated the mitochondrial apoptotic pathway,and effectively sensitized EGFR-TKI therapy to eradicate drugresistant tumor cells.The nanotherapeutic system developed in this study presents a novel approach to effectively overcome EGFR-TKI resistance and significantly enhance the antitumor efficacy of NSCLC treatment.