Experimental Study On Mutifunctional Targeting Lipid Nanobubbles For Ultrasound Targeted Diagnosis And Therapy Of Multiple Tumors

Malignant tumors have become one of the main threats to human life and health.By 2025,an estimated 19.3 million new cases will be diagnosed annually.Chemotherapy is currently one of the most common methods for cancer treatment,especially for tumors with metastatic tendency or having undergone metastasis.At present,chemotherapy is mostly achieved by means of systemic administration of drugs,but the chemotherapeutic agents used in clinics may not be delivered effectively and specifically at tumor tissues.This not only reduces the effectiveness of antitumor therapy but also has detrimental effects in normal tissues and causes cardiac,liver,and kidney toxicity.To improve the therapeutic effect of chemotherapeutic agents and minimize their impact on normal tissues,targeted delivery systems may be developed to enhance their intended release in tumor tissues.This has become one of the main research directions of cancer therapy in recent years.It is confirmed that lipid carriers can be used as targeted drug delivery system with some unique advantages:(1)lipids are the main component of cell membranes,feature great biocompatibility and safety in vivo and do not manifest toxicity or immunogenicity.(2)Lipids can have different configurations and carry hydrophobic chemotherapeutic agents via noncovalent bonds to improve their aqueous solubility.(3)In the host,the chemotherapeutic drugs in lipid carriers can be released in a targeted fashion upon specific environmental metrics(pH,temperature,light,and ultrasound);the lipid surface can also be subject to polyethylene glycol(PEG)modification to avoid phagocytic ingestion,thereby prolonging the circulation time of the drugs in the body and enhancing their stability and biological activity.(4)Lipids are easy to modify and can be attached to specific ligands to form targeted carriers,thereby facilitating targeted delivery of chemotherapeutic drugs.(5)Lipids can also encapsulate gases to form ultrasound contrast agents and in turn greatly augment ultrasound imaging quality.Lipid ultrasound contrast agents have been widely used in the diagnosis of thrombus,tumors,inflammation,and other conditions.These materials allow ultrasound-assisted,targeted release of the drug or gene that they carry and in turn improve the therapeutic outcome.At present,ultrasound contrast agents are mostly at the micron scale,and thus,they cannot penetrate the endothelial space of tumor vessels to allow ultrasound molecular imaging or focused delivery of drugs or genes.Ultrasound contrast agents with a nanoscale particle size and strong permeability can pass through tumor blood vessels and accumulate in tumor extravascular tissues.Related studies have confirmed that lipid nanobubbles(NBs)represent a common nanoscale ultrasound contrast agent with strong permeability and high stability that can not only enhance extravascular ultrasound imaging of tumors but also allow ultrasoundassisted focused delivery of drugs or genes to tumor parenchymal cells.Moreover,specific antibodies or ligands that target tumor tissue can be conjugated to the surface of drug-or gene-loaded NBs,thereby improving the aggregation ability of NBs in tumor extravascular tissue and in turn improving the outcome of extracellular ultrasound molecular imaging and targeted therapy.CXCR4 is a G protein-coupled receptor that is highly expressed in various tumor cells and is closely related to tumor prognosis.CXCR4 interacts with its cognate ligand CXCL12 for constituting a CXCL12/CXCR4 axis to encourage tumor cell proliferation and invasion,but blocking the axis can inhibit tumor growth and metastasis.Therefore,CXCR4 can simultaneously serve as a specific target for tumor-targeted diagnosis and therapy.Thus far,several CXCR4-targeted drugs have been developed,among which AMD070,a small-molecule antagonist of the receptor,has entered the second phase of clinical trials.This compound boasts several advantages,including low molecular weight,strong permeation ability,and high aqueous solubility.A large body of experimental evidence has demonstrated that AMD070 not only specifically binds to CXCR4-positive cells but also effectively inhibits tumor cell proliferation and metastasis.PTX is a microtubule stabilizer and is widely used as a chemotherapy agent against breast cancer,cervical cancer,and other tumors in the clinic.However,its antitumor therapeutic performance is reduced by certain issues,such as strong side effects,poor aqueous solubility,and inadequate specificity.In light of this,herein,we conjugated AMD070,a small-molecule CXCR4 antagonist,to paclitaxel-carrying NBs,thereby generating a NB(PTX-AMD070 NBs)with dual therapeutic effects(Chemistry and biological targeting).The material was then subject to in vivo and in vitro studies in which we examined its binding to breast cancer and cervical cancer cells,its ability to enhance ultrasound imaging,its targeted release of paclitaxel,and the process and mechanism underlying AMD070-based tumor inhibition.This study provides a means to integrate ultrasound molecular imaging and targeted therapy by conjugating an ultrasound contrast agent with a drug carrier,which can be used for treatment of multiple tumor types.Objective1.The goal of this study was to generate nanobubbles(PTX-AMD070 NBs)for targeted delivery of paclitaxel(PTX),a chemotherapy drug,and AMD070,a smallmolecule CXCR4 antagonist,and to examine their performance in ultrasound molecular imaging of breast cancer and cervical cancer.This provides a safe,efficient and penetrating targeted contrast agent for tumor-targeted ultrasound molecular imaging.2.The therapeutic effect and mechanism on breast cancer and cervical cancer when PTX-AMD070 NBs were applied in conjunction with UTND technology.This study provides a detailed experimental foundation for targeted drug-loaded nanobubbles to achieve integrated diagnosis and treatment of multiple tumors.Methods1.Experimental study on PTX-AMD070 NBs for tumor ultrasound molecular imaging(1)PTX-AMD070 NBs were prepared via an amide reaction and mechanical mixing.The particle size,zeta potential,encapsulation rate,drug loading efficiency,and in vitro agarose model ultrasound intensity were examined.(2)Laser confocal microscopy and flow cytometry were used in vitro to analyze the targeted binding ability of PTX-AMD070 NBs to CXCR4 human breast cancer MCF-7 cells and CXCR4 human cervical cancer C33 a cells.(3)The ultrasound-enhanced imaging features between vivo xenografts treated with PTX-AMD070 NBs and those treated with PTX NBs were compared.(4)Fluorescence immunohistochemistry(IHC)was employed to investigate the distribution and penetration ability of PTX-AMD070 NBs in xenograft tissues.2.Experimental study of PTX-AMD070 NBs combined with UTND in the therapeutic of xenografts in nude mice(1)CCK-8 assays and flow cytometry were performed to detect the effect of PTXAMD070 NBs combined with UTND on proliferation inhibition and apoptosis induction in tumor cells.(2)The therapeutic outcome of PTX-AMD070 NBs combined with UTND against xenograft tumors was assessed by measuring the weight of nude mice,tumor volume,and tumor traits and by performing HE staining and TUNEL staining.Results1.Experimental study on PTX-AMD070 NBs for tumor ultrasound molecular imaging(1)PTX-AMD070 NBs showed a particle size of 494.3±61.2 nm,a zeta potential of-22.4±1.75 MV,an encapsulation rate of 53.73±7.87%,and a drug loading efficiency of 4.48±0.66%.The in agarose ultrasound image quality of PTX-AMD070 NBs decreased in response to a reduction in concentration.(2)PTX-AMD070 NBs displayed significantly higher targeted binding to MCF-7 cells and C33 a cells than that of PTX NBs(P<0.05).(3)In vivo experiments revealed that PTX-AMD070 NBs aggregated specifically at xenograft sites in the nude mice,where the nanoparticles significantly improved the image quality.(4)DiI-labeled PTX-AMD070 NBs can enter the extravascular tissue through the interstitial space of the xenograft tumor tissues through MCF-7,C33 a and MDA-MB-468.The distribution of PTX-AMD070 NBs was not observed in the interstitial space of the thigh muscle.2.Experimental study of self-made lipid NBs combined with UTND in the therapeutic of xenografts in nude mice(1)PTX-AMD070 NBs that combined with UTND manifested a more pronounced effect in inhibiting cell proliferation and promoting apoptosis than other treatments(Control?AMD070?PTX?AMD070+PTX?PTX-AMD070 NBs).(2)In comparison with other treatment groups(Control ? AMD070 ? PTX ?AMD070+PTX?PTX-AMD070 NBs),the combined PTX-AMD070 NBs and UTND treatment group exhibited the smallest tumor volume and weight and the highest degree of apoptosis and necrosis.Conclusions1.The targeted drug-loaded NBs(PTX-AMD070 NBs)were synthesized in this study with the characteristics of small particle size,uniform dispersion and stable property,which laid a foundation for ultrasound imaging by using tumor EPR effect to enter the tumor extravascular tissue space.2.PTX-AMD070 NBs were shown to bind specifically to CXCR4-positive MCF-7 cells and C33 a cells in vitro,and them specifically enhance ultrasound imaging capability of CXCR4-positive MCF-7 and C33 a xenografts in vivo.This study provides a new ultrasound contrast agent for targeted diagnosis of tumor.3.PTX-AMD070 NBs combined with UTND could significantly enhance the apoptotic rate of tumor cells in vitro,and have the potential to inhibit tumor growth of tumor xenograft experiments in vivo.This result is closely related to the molecular targeting of AMD070 and the cavitation effect of ultrasound irradiation.Meanwhile,this technology provides new research ideas and detailed experimental basis for realizing the integration of tumor-targeted diagnosis and therapy.