Ultrasound-Mediated Cavitation Enhances Drug Delivery And Antitumor Immune Response

Part 1 Ultrasound-Mediated Cavitation Enhances EGFR-Targeting PLGA-PEG Nano-Micelle Delivery for Triple-Negative Breast Cancer TreatmentBackground:Triple-negative breast cancer（TNBC）has a poor prognosis and is prone to recurrence and metastasis.Currently,there is a lack of drugs for effective targeted therapy.Epidermal growth factor receptor（EGFR）overexpression is highly correlated with poor prognosis;therefore,it may serve as a potential target for enhancing the delivery of chemotherapeutic drugs to the tumor site in patients with TNBC,thereby increasing the antitumor effect.In addition to active targeting strategies,ultrasound-mediated cavitation（UMC）effects have been reported to increase the permeability of vascular and tumor physiological barriers to enhance intratumoral drug enrichment.DOX has potent antitumor activity,but its toxicity and side effects significantly limit its clinical application.In this study,we used EGFR-targeted functionalnanomicelles（PLGA-PEG/DOX@anti-EGFR）andthe penetration-enhancing effect of UMC to enhance drug delivery in TNBC.Moreover,we evaluated the efficacy of the combination of functional nanomicelles and UMC in treating TNBC.Methods:1.Construction and performance characterization of functional nanomicelles（PLGA-PEG/DOX@anti-EGFR）.Functional nanomicelles were constructed based on hydrophilic and hydrophobic self-assembly.Characterization was using a particle size analyzer,measurement of the zeta potential,transmission electron microscopy,and UV-vis spectrophotometry to detect the cumulative drug release of DOX under different pH conditions.2.Uptake of functional nanomicelles by human TNBC MDA-MB-468 cells;evaluation of the combined therapeutic efficacy of functional nanomicelles and UMC using the CCK-8 assay and live-dead cell staining;use of endothelial cells and Transwell models to evaluate the effects of UMC in vitro.3.Human TNBC MDA-MB-468 tumor-bearing nude mice were used as research objects and the enhancement effect of ultrasonic cavitation penetration was evaluated using different ultrasonic powers.Moreover,the therapeutic effect of the combination of functional nanomicelles and UMC was evaluated in vivo.The biosafety and pharmacokinetics of functional nanomicelles in healthy nude mice were also evaluated.Results:Functional nanomicelles（PLGA-PEG/DOX@anti-EGFR）with high DOX loading were successfully constructed,and their EGFR active targeting could effectively enhance drug delivery at tumor sites,thereby enhancing the antitumor effect of chemotherapeutic drugs while reducing side effects to nontargeted sites.At the same time,Sono Vue^TM as a cavitation nucleus could significantly enhance blood perfusion at the tumor site under the action of ultrasound（0.5 W/cm² output power）,further improving drug enrichment at the tumor site and effectively targeting TNBC cells at lower drug concentrations.The combination of functional nanomicelles and UMC could significantly prolong the survival of mice.Moreover,the combination treatment approach demonstrated good safety,highlighting its potential for use in a clinical setting.Conclusions:PLGA-PEG@DOX/anti-EGFR functional nanomicelles combined with UMC were found to be effective in the treatment of TNBC.This combination treatment demonstrated good safety and highlighted its use in clinical applications.Part 2 Ultrasound-mediated cavitation improves immune adjuvant liposome delivery and enhances antitumor immune response for pancreatic ductal adenocarcinomaBackground: Pancreatic ductal adenocarcinoma（PDAC）is the fourth leading cause of tumor-related deaths worldwide.PDAC is characterized by a dense,rich extracellular matrix and high interstitial fluid pressure,which can compress local blood vessels and lead to hypoperfusion,thereby limiting treatment efficacy.Toll-like receptors（TLRs）can stimulate the immune system and improve the effect of immunotherapy;however,their clinical application is limited due to hydrophobicity.pH-responsive liposomes can actively release the drug in the acidic tumor microenvironment of the pancreas and improve drug accumulation at tumor sites.Ultrasound-mediated cavitation（UMC）significantly increases drug delivery at the tumor site,and the resulting tumor fragments can act as tumor-associated antigens（TAAs）to increase T cell infiltration in the tumor microenvironment by activating dendritic cells（DCs）.In this study,pH-responsive liposomes loaded with the TLR agonist R837 were fabricated and combined with UMC to achieve high-efficiency immune adjuvant delivery to the tumor site,thereby enhancing the antitumor immune response in the pancreatic cancer microenvironment and inhibiting its recurrence and metastasis.Methods: 1.Preparation and characterization of pH-responsive liposomes（PEOz-Lip@ R837）.pH-responsive liposomes were synthesized using the standard reverse evaporation method and characterized based on particle size analysis,zeta potential,transmission electron microscopy findings,and UV-vis spectrophotometry.The dialysis-diffusion method was used to evaluate R837 release at different pH conditions.The accumulated drug release was analyzed to determine the pH responsiveness of the liposomes.2.Bone marrow dendritic cells were prepared by extracting bone marrow cells from healthy C57BL/6 mice.The Transwell model was used to verify the activation of DCs by UMC-responsive immune adjuvant delivery in vitro.Real-time quantitative PCR was used to detect immunogenicity cytokine levels.Murine pancreatic cancer cells panc02 were used in this study and treatment efficacy using UMC-responsive immune adjuvant delivery was evaluated.3.Panc02 pancreatic cancer C57BL/6 mice were used as the research object to study the effect of UMC on the distribution of pH-responsive liposomes in tumors and major organs,to evaluate the penetration-enhancement effect of UMC,and to guide UMC-responsive immune adjuvant therapy.The dosing window of the drug delivery combination therapy,changes in interstitial fluid pressure in tumors,and the vascular morphology after UMC intervention were determined.The impact mechanism of tumor permeability was evaluated.Effects of the pancreatic cancer microenvironment on antitumor immune responses were also evaluated.The aim of this study was to evaluate the efficacy and immunological mechanism;the survival rate of mice with primary tumors and distant tumors subjected to UMC-responsive immune adjuvant delivery;and to study the long-term immune memory function of the combination therapy and evaluate tumor recurrence.Results: pH-responsive liposomes（PEOz-Lip@R837）were successfully prepared using a high R837 loading rate.When administered intravenously,the pH-responsive liposome PEOz-Lip@R837 released the active drug in the tumor acidic microenvironment,enabling the delivery of hydrophobic immune adjuvants to the tumor site.In combination with enhanced permeability due to UMC,PEOz-Lip@R837 administration led to the achievement of effective drug concentration at the tumor site and induced a synergistic antitumor immune response through DC activation.UMC has been confirmed to downregulate multiple immune checkpoint molecules including Foxp3,CD274,CTLA4,and some costimulatory molecules,and to participate in the release of R837 in the tumor immunosuppressive microenvironment.Our results showed that UMC-mediated pH-responsive immune adjuvant delivery could effectively inhibit the progression of primary tumors,protect from tumor rechallenge,and lead to effective immune memory function.The antitumor effect was mainly related to the increase in antitumor immune response through DC activation,increased cytokine secretion,and enhanced proportion of CD45+ lymphocytes and CD8+ T cells.Additionally,cavitated core Sono VueTM and liposomes are FDA-approved drugs;thus,UMC-mediated pH-responsive immune adjuvant delivery therapy shows potential for use in a clinical setting.Conclusion: pH-responsive liposomes（PEOz-Lip@R837）have tumor microenvironment–responsive drug-release properties.In combination with UMC,there is a further improvement in the delivery of immune adjuvants to tumor sites and an increase in the antitumor immune response in the pancreatic cancer microenvironment.pH-responsive liposomes combined with UMC have a synergistic effect on the immunosuppressive microenvironment in PDAC,thereby enhancing the anti-tumor immune response and inhibiting the recurrence and metastasis of PDCA.