| background:With the continuous development of medical imaging,the emergence of injection developers in various development methods has greatly assisted in enhancing the imaging effect in vivo and more accurately determining the extent and nature of lesions.Among them,ultrasound combined with microbubble/nanobubble contrast agent can not only be used as a clearer way of ultrasound development,but because of its precise targeting and visibility,it is convenient to load and promote the ability of cells to ingest drugs or genes.UTMD has become one of the most promising options for targeted diagnostic treatmentChemotherapy,as a major treatment for malignant tumors,can significantly improve the survival rate of cancer patients.However,many chemotherapy drugs have significant side effects:cisplatin,which has irreversible nephrotoxicity,myelosuppression,neurotoxicity;carboplatin has myelosuppressive;and doxorubicin has significant cardiotoxicity.In order to reduce systemic side effects,the dose of these chemotherapeutic drugs is limited,so many of these drugs have limited anticancer therapeutic effects.Local delivery of a chemotherapeutic drug can be achieved by increasing its therapeutic dose at the local or target site of the tumor,while correspondingly reducing the concentration of the drug in blood circulation and other tissues.In this way,the side effects of the chemotherapeutic drugs are correspondingly reduced,and the content and efficacy of the tumor are increased locally.Therefore,targeted therapy can help promote the efficacy of the drug while reducing its systemic side effectsThere are many types of targeted delivery systems in current research,including nanoparticles,micro micelles,liposomes,etc.The routes of administration are also divided into oral administration,intravenous administration,nasal administration,colon administration and the like.The targeting system of ultrasound combined with microbubble destruction(UTMD)has its unique therapeutic advantages,such as more accurate positioning,visibility and real-time control relative to other development methods,and targeted delivery can promote the entry of anticancer drugs into tumor cells.Internal to strengthen drug efficacy,ultrasound-targeted nano/microbubble destruction has been widely used as an effective drug delivery system due to its advantages of visibility,non-invasiveness,and high targeting efficiency.Compared with conventional ultrasound contrast agent microbubbles,the nano-sized microbubbles or droplets can pass through the tumor neocapsule wall more easily,and thus can be more efficiently delivered to the target site tumor tissue.The biosafety of ultrasound contrast agents is one of the most important metrics for true clinical applications.Ultrasonic nano contrast agents usually consist of two parts:the outer shell and the inner core.Due to the biological toxicity of some chemical high molecular substances,the addition of materials as a developer may pose a potential threat to the health of the body.Therefore,it is important to find a safer material and to produce a contrast agent that can achieve the same development effect and targeted load capacity.Chitosan is known as a natural polysaccharide with good biodegradability,biocompatibility,low immunogenicity,antibacterial activity and practicability,and is one of the most abundant biological materials on earth.In addition,studies have shown that in the presence of IFN-y,water-soluble chitosan oligomers can activate macrophages to kill cancer cells.Therefore,chitosan itself has direct and indirect anti-tumor effects,making it more suitable as a carrier for anti cancer drugs.Because chitosan has been widely used in medical gels,scaffolds,excipients,and in vivo nanocarriers due to its high biocompatibility and biosafety,studies have shown that it can be used for the fabrication of ultrasonic nanobubbles and the carrying of genes.Based on the above studies,we used chitosan and other biocompatible surfactants and inert gases to make chitosan nanobubbles that can be used for contrast-enhanced ultrasound,and to support the anticancer drug model doxorubicin.We hope to have high biosafety,ultrasound contrast capabilities,and the ability to target delivery under ultrasound guidance.objecitve:The purpose of this study was to produce a highly biocompatible chitosan contrast agent and to detect its particle size,biocompatibility,drug loading capacity,development ability,and ultrasound-targeted drug-mediated drug entry into cells to exert anticancer effects.At the same time,the application of fluorocarbon substances with different object morphology as the core of the ultrasound contrast agent,its particle size,carrying charge,developing ability and biosafety in the body were evaluated.We hope to establish an ultrasound contrast agent with good biocompatibility,imaging ability,load and targeted delivery of anticancer drugs,which will lay a foundation for future application in clinical ultrasound imaging.Method:Part I:Biocompatible Chitosan Nanobubble Ultrasound-Mediated Targeted Doxorubicin(1)Making chitosan nanobubbles:The oscillating method was used to make nanobubbles using chitosan,lecithin,palmitic acid mixed solution as the material,and pre-adding the doxorubicin to be loaded,filling the perfluoropropane inert gas,and replacing the solution.The mechanical oscillator is oscillated to form nanobubbles,and a small amount of stabilizer is added to prevent bubble fusion.The ultrafiltration tube is centrifuged for use.(2)The average particle size,size distribution and charge amount of the chitosan nanobubbles were measured by dynamic light scattering method.The stability of chitosan nanobubbles was measured by measuring the change of particle size and observed an inverted microscope by dynamic light scattering method for a period of time.(3)The drug-loading capacity of the anticancer drug doxorubicin loaded with chitosan nanobubbles was determined by uv spectrophotometer.The release rule of doxorubicin loaded with nanobubbles under ultrasonication was examined in vitro by dialysis method,and the difference in drug release between nanobubbles and non-ultrasound was compared.(4)An in vitro ultrasound imaging device was established to evaluate the ultrasound enhanced imaging capability of the prepared chitosan nanobubbles using a clinical ultrasound imaging system.(5)The biosafety of blank chitosan nanobubbles was evaluated in vitro using the cck8 assay and assessed by their cytotoxicity assay in mcf-7 breast cancer cells.(6)We determined the ability of doxorubicin chitosan nanobubbles,doxorubicin chitosan nanobubbles plus ultrasound,and free doxorubicin to mediate the uptake of doxorubicin in breast cancer cells in vitro by flow cytometry along with their ability to induce apoptosis in breast cancer cells.(7)All experiments were performed in triplicate and the data were expressed as mean ± standard deviation.Statistical analysis was performed using SPSS version 18.0.A p value<0.05 was considered statistically significant.Part ?:Preparation and characterization of liquid fluorocarbon and perfluoropropane chitosan nanoparticles(1)The blank perfluoropropane-chitosan nanobubbles and perfluorohexane-chitosan nanodroplets were also prepared by the oscillating method.The perfluoropropane gas was replaced by the addition of liquid perfluorohexane during the production of the nanoparticles.(2)The average particle size of perfluoropropane-chitosan nanobubbles PP nanobubbles and perfluorohexane-chitosan Nanodroplet PE nanodroplets were measured and compared by dynamic light scattering method,size distribution and the amount of charge carried.(3)The stability of the PP nanobubbles and PE nanodroplets in the PBS solution and blood at 4 0 C and room temperature was observed by an inverted microscope.(4)The ultrasound imaging effect and stability of PE nanobubbles and PE nanodroplets was compared using a clinical ultrasound scanner.(5)All experiments were performed in triplicate and the data was expressed as an average.Statistical analysis was performed using SPSS version 18.0.A p value<0.05 was considered statistically significant.Part ?:Evaluation of the safety of PP nanobubbles and PE nanodrops(1)The corresponding concentration of PP nanobubbles and PE nanodrop solution was configured.A random sample of 55 healthy Kunming mice were selected and randomly divided into 9 groups according to the mode of administration and dosage(Table 1).Oral gavage was according to the total dose of 2100mg/kg,the dosage volume of 0.8ml was used per each mouse;the administration time was within the same day(8:00a.m,4:00 pm);the total dose was 240 mg/kg,administered a volume of 0.5 ml/each mouse was intraperitoneally injected;a total dose of 80 mg/kg,a dosing volume of 0.5 ml for each intravenous injection from the tail vein of the mouse.30 mice were used as controls.(2)The appetite and activity status of each group of mice after administration were observed for 14 consecutive days.(3)After 14 days of feeding,blood was collected from each mouse's eye,and the differences in biochemical parameters between PP nanobubble and PE nanodrop group and control group were detected and compared,including urea nitrogen,alanine aminotransferase,glutamic oxaloacetic aminotransferase,total protein,albumin,triglyceride,cholesterol,blood sugar.(4)Each group of experimental mice was sacrificed after 14 days of feeding.Four mice were randomly selected from each group for anatomy.The liver,spleen,kidney,heart,and lung were taken to observe the color and shape of the organs,and the pathological sections of the organs were taken.HE stained to observe the cells have no edema,deformation,necrosis and so on.(5)All experiments were performed in triplicate and the data was expressed as an average.Statistical analysis was performed using SPSS version 18.0.A p value<0.05 was considered statistically significant.Result:Part I:Biocompatible Chitosan Nanobubble Ultrasound-Mediated Targeted Doxorubicin(1)The average diameter of the doxorubicin-loaded chitosan nanobubbles was 641 nm,PI:0.256,and the average zeta potential of the nanobubbles was+67.12±2.1 mV.There was no significant change in the particle size of the doxorubicin-loaded chitosan nanobubbles at 4? for 48 h(p<0.05).After storage at room temperature,the average particle size of the doxorubicin-loaded chitosan nanobubbles in PBS and human serum were slightly increased.(2)The final loading of doxorubicin-loaded chitosan nanobubbles was 64.12 mg/g,which corresponds to a drug encapsulation efficiency of 54.18%.The detection of chitosan nanobubbles in the ultrasound group and the non-ultrasound group release drug process are shown in Fig.2.Within 24 hours,the release rate and release of doxorubicin in the ultrasound group were seen to be significantly greater than those in the non-ultrasound group.(3)The doxorubicin ultrasound contrast agent has good enhanced ultrasound imaging ability,and the sound attenuation is slow within 15 minutes of detecting development,indicating that the ultrasonic enhanced signal of chitosan nanobubbles is sufficient for stable contrast imaging for a certain period of time.(4)The blank chitosan nanobubbles have high biosafety.When used in combination with ultrasound,the survival rate of MCF-7 cells in a 10%nanobubble suspension was 99.53%at an ultrasound intensity of 0.5 W/cm2 and 30 seconds of irradiation.Under the same ultrasound,the survival rate of MCF-7 cells in 20%chitosan nanobubbles was also greater than 90%.(5)The average fluorescence intensity of mcf-7 cells in the doxorubicin nanobubble group was much lower than that of the cells in the free doxorubicin group.This indicates that under non-ultrasonic conditions,chitosan nanobubbles reduce the uptake of doxorubicin by cells,which can protect cells in non-ultrasound irradiation sites from doxorubicin uptake and side effects during circulation in vivo.Under ultrasound irradiation,doxorubicin uptake in nanobubble mcf-7 cells increased significantly,while drug uptake in mcf-7 cells in the free doxorubicin group increased only slightly,indicating that ultrasound-assisted DOX-NBs could deliver more adriamycin drugs to mcf-7 cells for action.(6)Cytotoxicity assay and flow cytometry were used to measure the viability of each group of mcf-7 cells.The results showed that the activity of mcf-7 cells loaded with doxorubicin nanobubble group was higher than that of free doxorubicin group in non-ultrasound state.Correspondingly,the viability of the doxorubicin-loaded mcf-7 cells under ultrasound irradiation was significantly lower than that of the free doxorubicin group.The trend of the results of the apoptosis experiment was consistent with the cell viability experiment.The percentage of apoptosis in MCF-7 cells in the free doxorubicin group was 4.4±0.9%,and there was no significant change in the proportion of apoptosis in the 30 seconds and 60 seconds after sonication.Under the same ultrasound,the percentage of apoptotic cells in the doxorubicin nanobubble group was significantly increased(4.4 ± 0.9%vs.45.7 ± 1.1%,p<0.01).In addition,the percentage of apoptotic cells in the nanobubble group was lower than that in the free doxorubicin-treated group(3.2 ± 0.9%vs.4.4 ± 0.9%).Part ?:Preparation and characterization of liquid fluorocarbon and perfluoropropane chitosan contrast agent(1)The average particle diameter of PE nanodroplets is 641 nm,and PI:0.219;the average particle diameter of PP nanobubbles is 762 nm,and PI:0.328,which is significantly larger than the average particle diameter of nanodroplets.The zeta potential of the PE nanodrops is +17.89±3.47mV.The zeta potential of PP nanobubbles was +18.21±4.91mV.There was no significant difference shown between the two.(2)Under microscopic observation,there was no significant change in the particle size of PP nanobubbles and PE nanodroplets placed at 4 0 C after 48 hours.After 6 hours at room temperature,the particle size of PP nanobubbles in PBS and serum increased slightly,and the particle size of PE nanodrops did not change significantly.(3)The same concentration of PP nanobubbles and PE nanodroplets showed good ultrasound-enhanced imaging under the clinical ultrasonic diagnostic apparatus,and the imaging effect was not significantly different.After 20 minutes of ultrasonic irradiation at the same frequency and depth,the acoustic attenuation rate of PP nanobubbles on the display screen was slightly larger than that of PE nanodroplets.Part ?:Evaluation of the safety of pp nanobubbles and pe nanodrops(1)General:The mice in each group were generally in good condition after administration,and no deaths occurred.On the day of intravenous administration,14 mice showed transient instability,loss of appetite,and increased water intake.Three experimental mice developed lethargy and returned to normal the next day.The other groups and control mice showed no abnormalities in activity and diet.(2)The main biochemical indicators of each group of mice,including urea nitrogen,alanine aminotransferase,glutamic oxaloacetic aminotransferase,total protein,albumin,triglyceride,cholesterol,blood sugar.There was no significant difference in uric acid(UA),total bilirubin(TBIL)and total bile acid(TBA)between the groups and the control group(p<0.05).(3)Pathological observation of the main organs of the mice after administration.There was no significant difference in the color and morphology of the gross specimens,and no significant abnormalities were observed.There was no significant difference in the organ coefficients between the groups.There was no significant difference in cell morphology and tissue arrangement between the heart,lung,liver,spleen,and kidney sections,and there was no inflammatory cell infiltration.Analysis conclusion:In this study,we developed a biocompatible chitosan nano-ultrasound contrast agent,and observed for the first time its ability to load and deliver anticancer drugs targeting cells.For the first time,we compared the performance and stability of fluorocarbon-chitosan nano-contrast agent with that of liquid fluorocarbon-chitosan nano-contrast agent.The safety of the object was evaluated.The results showed that PP nanobubbles had good drug loading ability and enhanced drug release ability under ultrasound.PP nanobubbles have very low cytotoxicity and high biocompatibility under ultrasound,and have a good enhancement effect and a long duration of development.In vitro experiments showed that PP nanobubbles could significantly improve the cell uptake and anti-cancer efficacy of drugs.The average particle size of PE nanodroplets was slightly smaller than that of PP nanobubbles,and there was no significant difference in charge between them.The stability of PE nanodroplets was higher in vitro,and there was no significant difference between the ultrasonic development effect of PE nanodroplets and PP nanobubbles.Animal experiments in vivo confirmed that neither PE nanodroplets nor P P nanobubbles caused blood biochemical indexes and pathological changes of organs in mice.Both ultrasound developers had high biological safety.Studies have shown that the application of chitosan nano-contrast agent in contrast-enhanced ultrasound is a safe and effective way for both imaging diagnosis and targeted anticancer therapy.The disadvantage is that the development effect and anti-tumor effect of chitosan nano-contrast agent in vivo need to be confirmed by research.In conclusion,biocompatible chitosan contrast agents are suitable for ultrasound-targeted drug/gene delivery.As one of the promising strategies for non-invasive targeted therapy,they deserve further study. |
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