Visualized Nanoparticles Guided By Phtotacoustic Imaging For Multi-strategy Synergistic Reversal Of Multidrug Resistance In Breast Cancer

PART ONE:PREPARATION,CHARACTERIZATION AND STABILITY DETECTION OF VISUALIZED ANTI-DRUG RESISTANCE NANOPARTICLESObjective:1.To prepare a peptide functionalized(t Ly P-1)polythylenimine(PEI)-modified Poly(lactic-co-glycolic acid)(PLGA)nanoparticle loadded with paclitaxel(PTX)and Porphyrin copper(Cu TCPP).The size,zeta potential,UV spectrum and other basic characterization of anti-drug resistant nanoparticles were detected.2.The drug loading performance,drug release capacity and biosafety of multi-strategy synergistic based anti-drug resistance nanoparticles(TPP@PTX-Cu TCPP)were evaluated.Method:tLy P-1-PEI was synthesized by maleimide-thiol method.PLGA nanoparticles loaded with PTX and Cu TCPP were prepared by ultrasonic double emulsification method.Finally,t Ly P-1-PEI was covalently linked with carboxylated PLGA nanoparticles by carbon diimide method.Fourier Transform Infrared spectroscopy(FTIR)and nuclear magnetic resonance spectrum(NMR)and time of flight mass spectrometer(TOF-MS)were used to detect the synthesis of t Ly P-1-PEI.The morphology of nanoparticles was detected by transmission electron microscopy(TEM)and scanning electron microscopy(SEM),and the loading performance and capacity of Cu TCPP was determined by ultraviolet spectrophotometer(UV).High performance liquid chromatography(HPLC)was used to detect PTX loading performance and loading capacity.HPLC was used to detect the release of PTX from TPP@PTX-Cu TCPP in different PH environments,and CCK-8 method was used to detect the biosafety of TPP@Cu TCPP without PTX.Result:tLy P-1-PEI was successfully synthesized by maleimide method.NMR results showed that t Ly P-1-PEI had a peak value close to PEI at 2.6～3.0ppm.FTIR results showed that t Ly P-1-PEI-PLGA and PEI-PLGA nanoparticles showed characteristic peaks representing the-CH group of PLGA near 3002/cm and 2948/cm,and characteristic peaks representing the-CO-NH-and-NH2 groups near 1649/cm and 3426/cm.However,only the-CH group representing PLGA appeared near 3002/cm and 2948/cm in PLGA empty nanoparticles.TOF-MS results showed that the molecular weight of PLGA nanoparticles increased from 14955.22 to 20722.68 Da after covalently bonded with t Ly P-1-PEI.These results indicated that the amino group in t Ly P-1-PEI formed an amide bond with the carboxyl group of PLGA nanoparticles.Transmission electron microscopy and scanning electron microscopy showed that the nanoparticles were spherical,evenly distributed and uniform in size.The particle size of the nanoparticles measured by Malvern analyzer was 354.8 nm,PDI: 0.14.Meanwhile,the particle size of the nanoparticles did not change significantly after re-suspension in deionized water,1640 medium containing fetal bovine serum and phosphate buffer salt solution(PBS).The zeta potenial of unmodified PLGA nanoparticles loaded with Cu TCPP and PTX(P@PTX-Cu TCPP),PEI-modified PLGA nanoparticles loaded with Cu TCPP and PTX(PP@PTX-Cu TCPP)and t Ly P-1 functionalized PEI-modified PLGA nanoparticles loaded with Cu TCPP and PTX(TPP@PTX-Cu TCPP)were-13.6 m V,3.92 m V and 8.42 m V,respectively.Ultraviolet spectrophotometer showed no obvious characteristic peak was observed in TPP@PTX,a characteristic peak at 412 nm for Cu TCPP,and a characteristic peak at 415 nm for TPP@PTX-Cu TCPP,which had a slight red shift than the original solution of Cu TCPP.According to the calculation of Cu TCPP standard curve,the encapsulation rate and drug loading of the resistant nanoparticles to Cu TCPP were 27.1% and 2.25% respectively.The encapsulation rate and drug loading of PTX were 70.1% and 5.84%respectively.HPLC analysis showed that TPP@PTX-Cu TCPP had the ability to release PTX slowly,and the release rate was higher in weakly acidic environment(PH = 6.3)than in neutral environment(PH = 7.4).CCK-8 method showed that the survival rate of MCF-7/Taxol,human umbilical vein vascular endothelial cells were all greater than 80% under different concentrations of PTX-free nano-carrier TPP@Cu TCPP after 24 h of co-incubation.Conclusion:Peptide-functionalized PEI modified PLGA nanoparticles loaded with PTX and Cu TCPP(TPP@PTX-Cu TCPP)were successfully prepared.The nanoparticles were spherical and uniformly distributed,and had good stability in different solution,with good loading capacity of PTX and Cu TCPP.TPP@PTX-Cu TCPP nanoparticles had good drug release ability in weak acid environment,and PLGA as a drug delivery carrier had good biocompatibility.PART TWO:EVALUATION OF AGAINST TUMOR MULTIDRUG RESISTANCE EFFICACY AND PHOTOACOUSTIC IMAGING PERFORMANCE OF VISUALIZED ANTI-RESISTANCE NANOPARTICLES WITH MULTIPLE STRATEGIES IN VITROSECTION 1: EVALUATION OF SYNERGISTIC EFFECT OF LYSOSOME ESCAPE,GLUTATHIONE CONSUMPTION AND TUMOR HOMING-PENETRATION STRATEGY FOR REVERSAL OF DRUG RESISTANCE IN VITROObjective:To evaluate the synergistic effect of lysosome escape,glutathione depletion and tumor homing-penetrating strategy on reversing multidrug resistance in vitro.Method:Confocal laser fluorescence microscopy and bio-transmission electron microscopy were used to observe the effect of lysosome escape triggered by PEI modified nanoparticles(PP@PTX)and non-PEI modified nanoparticles(P@PTX).Glutathione detection kit and X-ray photoelectron spectrometer(XPS)were used to detect glutathione consumption from nanoparticles loaded Cu TCPP in vitro and in vivo.Three-dimensional tumor spheres of drug-resistant cells and subcutaneous transplanted tumor models of drug-resistant breast cancer(MCF-7/Taxol)were established. Confocal laser fluorescence microscopy and small animal fluorescence imaging were used to evaluate the in vitro and in vivo targeting and membrane penetration effects of anti-resistant nanoparticles after tumor homing-penetrating peptides functionalization.CCK-8 assay,apoptosis staining by flow cytometry and calcein-AM/propyl iodide(CAM/PI)double staining were used to evaluate the inhibitory effect of MCF-7/Taxol cells by different anti-resistance strategies and multi-strategy synergistic anti-resistance nanoparticles.Result:Confocal laser fluorescence microscope observed that the co-localization of red fluorescence representing nanoparticles and green fluorescence representing lysosomes in the PP@PTX group was lower than that in the P@PTX group.Image J software showed that the Person’s correlation coefficient(PCC)between nanoparticles and lysosomes in the PP@PTX group was 0.511,while that in the P@PTX group was 0.871,showing a statistical difference(P < 0.05).At the same time,PP@PTX,P@PTX and FITC-PTX were co-incubated with MCF-7/Taxol cells for 24 hours,and then the solution was replaced by serum-free cell culture medium,which was observed again.The PP@PTX group still retained a large number of fluorescence of nanoparticles,while the P@PTX and FITC-PTX groups had less fluorescence.Bio-transmission electron microscopy showed that the lysosome in MCF-7/Taxol cells was oval with intact lysosome membrane before co-incubation with PP@PTX.When cells co-incubated with PP@PTX for 6 h,the lysosome membrane in MCF-7/Taxol cells lost integrity,and some lysosomes swelled and burst.Glutathione assay kit detected glutathione solution and MCF-7/Taxol cells co-incubated with Cu TCPP and PLGA nanoparticles loaded with Cu TCPP(PLGA-Cu TCPP),and found that glutathione content in both solution and cells decreased.XPS detection showed that the characteristic satellite peak of 2-valent copper ion in Cu TCPP solution decreased,and the 2-valent copper ion content decreased from 96.4% to 30.7%.Confocal laser fluorescence microscopy was used to evaluate the targeting and membrane penetrating effect of t Ly P-1-functionalized anti-resistant nanoparticles(TPP@PTX-Cu TCPP)and non-t Ly P-1-functionalized nanoparticles(PP@PTX-Cu TCPP)on MCF-7/Taxol single cell and MCF-7/Taxol three-dimensional tumor cell spheres.It was found that TPP@PTX-Cu TCPP nanoparticles could accumulate in large quantities around MCF-7/Taxol cells after incubating with them for 2 hours.The penetration depth of TPP@PTX-Cu TCPP nanoparticles in MCF-7/Taxol three-dimensional tumor sphere model was 21.3 μ m,while that of PP@PTX-Cu TCPP was only 7.55 μ m.In MCF-7/Taxol subcutaneous transplanted tumor model,the fluorescence signal of TPP@PTX-Cu TCPP in tumor tissue was stronger than that of PP@PTX-Cu TCPP,and it peaked at 6 h.Immunofluorescence sections of tumor tissue showed that TPP@PTX-Cu TCPP nanoparticles could penetrate the vascular barrier of tumor and enter deeper regions of tumor tissue.CCK-8,apoptosis staining by flow cytometry and CAM/PI double staining showed that TPP@PTX-Cu TCPP group had the lowest survival rate(P < 0.05)and the combination index(CI)of anti-resistance nanoparticels containing lysosome escape,glutathione consumption and tumor homing-penetrating strategies was 0.201,which exhibided a strong synergistic effect on inhibiting MCF-7/Taxol cells..SECTION TWO: EVALUATION OF PHOTOACOUSTIC IMAGING PERFORMANCE OF VISUALIZED ANTI-RESISTANCE NANOPARTICLES FOR BREAST CANCER IN VITRO AND IN VIVOObjective:To observe the ability of TPP@PTX-Cu TCPP as photoacoustic contrast agent in vitro and in vivo imaging of breast cancer.Method:The gel model was established,and the photoacoustic signal intensity of TPP@PTX-Cu TCPP with different concentrations in the gel model was analyzed by photoacoustic imaging machine.MCF-7/Taxol subcutaneous transplanted tumor model was established to observe the photoacoustic imaging of breast cancer in vivo with and without targeted anti-resistance nanoparticles.Result:In vitro photoacoustic results showed that TPP@PTX-Cu TCPP nanoparticles had an obvious absorption peak at 695 nm in the near infrared region,and the photoacoustic signal of the nanoparticles increased linearly with the increase of Cu TCPP concentration.In vivo breast cancer imaging results showed that obvious photoacoustic signal appeared in tumor region of tumor-bearing mice after injection of TPP@PTX-Cu TCPP via tail vein,and reached a peak at 6 h,while the photoacoustic signal was weak in tumor region of PP@PTX-Cu TCPP group.Conclusion:Multi-strategy synergistic anti-resistance nanoparticles with the ability of lysosome escape,glutathione consumption and tumor homing-penetrating has an obvious synergistic effect on inhibiting the growth of MCF-7/Taxol cells in vitro.TPP@PTX-Cu TCPP nanoparticles show good photoacoustic imaging performance in vivo and in vitro,demonstrating their application prospects as photoacoustic contrast agents.PART THREE:EFFICACY OF ANTI-RESISTANCE NANOPARTICLES WITH MULTIPLE STRATEGIES IN VIVO AGAINST TUMOR MULTIDRUG RESISTANCEObjective:To evaluate the biosafety of TPP@PTX-CuTCPP nanoparticles in mice and the results of chemotherapy in nude mice with drug-resistant breast cancer MCF-7/TaxolMethod:The 200μL TPP@PTX-Cu TCPP nanoparticle solution(PLGA concentration 5 mg/ m L)was injected into Kunming mice via tail vein,and then the same dose and concentration of TPP@PTX-Cu TCPP nanoparticles were injected into the mice 14,21,25 and 27 days later.On day 27,mice were injected with saline(control).On day 28,the mice were sacrificed and blood samples were collected through orbital vein to detect the physiological indexes of liver,kidney and heart function and blood routine.The main organs(including heart,liver,spleen,lung and kidney)of each group were removed and H&E staining was performed to observe the morphological changes of each organ section.MCF-7/Taxol subcutaneous transplanted tumor model was established,and the tumor-bearing nude mice were randomly divided into:(1)control group;(2)free PTX group;(3)P@PTX group;(4)P@PTX-Cu TCPP group;(5)PP@PTX group;(6)TPP@PTX group;(7)PP@PTX-Cu TCPP group and(8)TPP@PTX-Cu TCPP group.The reagents were injected into tumor-bearing mice through tail vein every 4 days and the body weight and tumor volume of tumor-bearing nude mice were measured every 4 days.On day 16,the mice were sacrificed and the main organs and tumor tissues of one mouse in each group were randomly removed.The tumor tissues were stained with H&E,TUNEL and PCNA,and the main organs were stained with H&E.Result:After the injection of TPP@PTX-Cu TCPP nanoparticles,there were no significant differences in liver,kidney,heart function,blood routine and other indexes of kunming rats at different time nodes compared with the control group(P >0.05),no obvious abnormality was observed in H&E staining of main organs in each group.There was no difference in body weight of tumor-bearing nude mice in each group after treatment,and no obvious pathological damage was observed in H&E staining of major organs.The relative tumor volume in the control group,free PTX group and P@PTX group increased 7.21 times,5.50 times and 4.97 times,respectively.The tumor growth rate of tumor-bearing mice in the nanoparticles group containing one or multiple anti-resistance strategies was slower than that in the control group.The TPP@PTX-Cu TCPP containing lysosome escape,glutathione consumption and tumor homing-penetrating strategies could not only inhibit tumor growth,but also reduce tumor volume(tumor relative volume was about 0.988,tumor inhibition rate was 91.4%).H&E staining of tumor tissue showed that free PTX group and P@PTX group had less damage to tumor cell morphology.With the increase of anti-drug resistance strategies,some tumor cells showed nuclear pyknosis,fragmentation and dissolution,among which the TPP@PTX-Cu TCPP group was the most obvious.In addition,PCNA and TUNEL staining results showed that the proliferation index of TPP@PTX-Cu TCPP group was decreased and apoptosis index was increased compared with other groups.Conclusion:TPP@PTX-Cu TCPP has good short-term and long-term biocompatibility and has broad clinical application prospects.TPP@PTX-Cu TCPP has the best inhibitory effect on MCF-7/Taxol tumors.Multi-strategy synergistic anti-resistant nanoparticles can reverse the sensitivity of drug-resistant breast cancer to chemotherapy.