The Experimnetal Study Of LIP4 Loaded HMME And AQ4N Targeting Lipid Nanoparticles On Bi-mode Imaging And Combined With Sdt Cascade Therapy Against Mammary Carcinoma

PARTⅠ THE PREPARATION AND CHARACTERIZATION AND BI-MODE IMANGING IN VITRO OF LIP4 AND EXPERIMENTAL STUDY ON SDT POSSIBILITYObjectives To prepare a lipid nanoparticle lip4 with integrated diagnosis and therapy function.The nanoparticle has dual-modality imaging and cascade therapy for breast cancer,and has the function of actively targeting cancer cells.The properties of lip4 were characterized,and ultrasound and photoacoustic imaging were observed,and the amount of singlet oxygen produced by lip4 was further examined.Methods Lip4 was prepared by conventional film dispersion-mechanical oscillation method.Lip4 was quantitatively analyzed by measuring its particle size and surface potential;the morphology of lip4 was observed by transmission electron microscopy,and its particle size and overall particle size distribution were observed;UV spectrophotometer was used to detect the drug encapsulation efficiency and drug loading of HMME and AQ4 N in lip4.meanwhile,to further proved that the lip4 contains the above drugs;LIFU irradiation was applied to observe the lip4 phase transition ability;using ultrasound and small animal photoacoustic imager to observe ultrasound and photoacoustic imaging in vitro;using SOSG probe to detect Singlet oxygen production of LIP4 combined with LIFU irradiation.Results LIP4 was successfully prepared.The appearance was red opaque emulsion.LIP4 was negatively charged.Its surface Zeta-potential was-35.9m V,the particle size was about 183.9nm,PDI was 0.116.Under TEM,LIP4 was spherical with a shell-core structure,relatively uniformity particle size,about 200nm;LIP4 loadings of HMME and AQ4 N were 9.05% and 1.7% respectively.Under the light microscope,the degree of phase tansition of lip4 increased with the increase of time after LIFU irradiation.The increasing of sound intensity and time,the clearer of the imaging of contrast-enhanced ultrasound observed.In vitro,photoacoustic imaging observations show that more thicker of LIP4 concentration,more stronger of the photoacoustic signal(PAavr),quantitatively analyze the relationship between the two paramers,they display relation of linear correlation,correlation coefficient r=0.992,P<0.01;SOSG probe test results proved that LIP4 combined with LIFU,sufficient singlet oxygen can be produced to act on tumor cells,while both sigle LIFU or single lip4 cannot produce singletoxygen.Conclusions The lip4 lipid nanoparticles were successfully prepared.The nanoparticles can be phase-transition under the action of LIFU,and have the ability of ultrasound imaging.At the same time,photoacoustic imaging can be performed under the action of LIFU and sound sensitive agent.At the same time,a large amount of singlet oxygen is generated.The therapeutic aftermath of SDT developped a very favorable microenvironment for the conversion of prodrug AQ4 N to the cytotoxic drug aq4.PART II THE EXPERIMENTAL STUDY OF LIP4 COMBINED WITH LIFU ON TARGETING AND KILLING 4T1 CELLSObjective To explore the targeting and cytotoxicity of LIP4 nanoparticles on mammary cancer cells at the cell level,and further explore the mechanism of cytotoxicity.Methods(1)Normal cells(HUVEC cells)and 4T1 cells in logarithmic growth phase were inoculated into CLSM-specific culture dishes,and incubated with LIP4 nanoemulsion for 1 h,2 h,3 h.CLSM was used to observe the binding state of LIP4 and 4T1 cells,HUVEC cells;(2)different concentrations of LIP6(containing AQ4 N 0,2.5,5,10,20 μM respectively)in hypoxia and normoxia were incubated with4T1 cells in the logarithmic growth phase for 24 h.The cell viability of each group was determined by CCK-8 method,and the cytotoxicity of LIP6 encapsulating AQ4 N was observed.(3)Different concentrations of LIP4(conctaining HMME 0,2.5,5,10,20 μg/m L respctvely)were divided into LIFU group and no LIFU Group,co-incubated with 4T1 cells in logarithmic growth phase,CNK-8 method was exployed to determine the cell livability of eachgroup,and the cytotoxic effect of LIP4 nanoparticles combined with LIFU sonodynamic therapy was observed.(4)LIP4+LIFU,LIP4,and PBS 3 groups,they were inoculated into a laser confocal culture dish with 4T1 cells,and each group were incubated in a cell incubator for 24 hours after different treatments.The DCFH-DA method was wielded to observed reactive oxygen species produced incells by CLSM.Results(1)There was no red fluorescence in the HUVEC cell group within 3 h.LIP4 and the adherent HUVEC cells did not bind at all.While he 4T1 cell group always showed red fluorescence.The results display that LIP4 possess the character of actively targeting to4T1 cells,at the same time no targeting to HUVEC cells at all.And it can be observed that the two combine together and be swallowed into intracelluar by 4T1 cells.It was apparent that the two were combined and phagocytosed into the cells;(2)There was no statistical difference in thecell liviablity of 4T1 cells between the PBS group under hypoxic conditions and any groups under normoxic conditions,P>0.05;Under hypoxic conditions,the toxic effects on 4T1 cells were enhanced with LIP6 concentrations increased,and there exsit a statistically significant difference between the groups,P < 0.001.Especially in the 2.5μg/ml and 5μg/ml groups,the cell livability decreased at an exponentially velocity.Between each two groups,there was a dramatically significant statistical difference P < 0.001.The drug effect is emerging prominently at AQ4 N concentration up to5μg/ml;while the AQ4 N concentration of 10μg/ml,the 20μg/ml group show statistically vital significant(P<0.01),and the group of20μg/ml AQ4 N concentration performed the most cytotoxicity effects,the cell livability was only 0.20±0.02.The toxic effect on cells was 80%;(3)There was no statistical difference in the 4T1 cells livability in different concentrations of LIP4 cells under no LIFU condition,P>0.05,neither LIFU+PBS group.There were significant differences in cell livability between the LIFU group and the LIP4 nanoparticle emulsion group,P<0.01;the 20 μg/ml group had the lowest cell survival rate of0.24±0.01;(4)PBS and LIP4 alone groups,no green fluorescence appeared,but the LIP4+LIFU group showed obvious and volume green fluorescence.Conclusions(1)Lip4 lipid nanoparticles have obvious targeting effect on 4T cells,and there is no targeted binding ability to normal cells;(2)LIFU and LIP4 are the two essential factors for SDT treatment;SDT of HMME combined LIFU,amplified AQ4 N drug efficacy leading to a synergistic,cascaded amplification therapy for mammary cancer;(3)LIP4 combined with LIFU characterize producing a large amount of singlet oxygen,thereby exerting the effect of SDT which could provides a favourable microenvironment for the reduction of AQ4 N to AQ4,thereby exerting synergistic antitumor effects.PART III THE EXPERIMENTAL STUDY OF LIP4 LOADED HMME AND AQ4 N LIPID NANOPARTICLES COMBINED WITH LOW-INTENSITY FOCUSED ULTRASOUND ON PRECISION THERANSOTICS AGAINST MAMMARY CARCINOMAAND TOXICITY ON VITAL ORGANS IN VIVOObjectives To observe the bi-modal imaging effect of LIP4 nanoparticles in vivo;to conduct in vivo experiments with lip4 combined with LIFU on mammary cancer of nude mice,and to observe whether LIP4 have the ability of targeting accumulation,targeted unloading and cascade amplification therapy can be achieved;further observation t o x i c effect of LIP4 on the vi t a l or g a n s.Methods(1)The tumor-bearing mouse model was developped successfully by subcutaneous injection of 4t1 cell suspension.ultrasound and photoacoustic imaging in vivo were performed;(2)40 tumor-bearing mice were randomly divided into 8 groups: pbs,pbs+lifu,lip4,lip4+lifu,lip5,lip5+lifu,lip6,Lip6+lifu,5 in each group,number was identified;each group was given the corresponding treatment to observe the therapeutic effect,and the corresponding parameters included: nude mouse body mass,tumor volume,tumor tissue h&e staining,tumor tissue immunohistochemistry to test cell proliferation And apoptotic conditions;(3)12 nude mice randomly was divided into three groups: experimental group and control group.200 μl LIP4 nanoparticles or PBS were injected into the tail vein,and the mice were sacrificed at 1d,7d,and 14 d after injection.Blood was taken for routine blood test,blood biochemistry,and main organs were taken.Heart,liver,spleen,lung and kidney were observed by H&E staining.Results(1)In vivo contrast-enhanced ultrasonography PBS group showed no obvious eacho signal at three time points;in the LIP4 group,significant echo signals in the tumor was observed in 3 minutes after LIFU irradiation and echogenicity was significantly faded in the tumor tissue in 4 hours after LIFU intervention.The quantitative analysis results of DFY diagnostic apparatus showed that the ultrasound gray value was the highest at LIFU3 min,which was significantly different from other time groups,P<0.0001;in vivo photoacoustic imaging showed only LIP4 group nanoparticle photoacoustic imaging,The peak time of PA imaging is at 4h after intravenous injection LIP4,and then the degree of imaging becoming weaken.Further analysis of PA avr show that the PA avr values of LIP4 nanoparticles fluctuated regularly,peaked at 4h,and then gradually decreased;while PA avr values in LIP3 and PBS groups did not change significantly with time,the curve tends to be a straigh line.(2)In the nanoparticle group with LIFU intervention,tumor volume and body mass were generally significantly different,P < 0.0001.The tumor volume was significantly different between the LIP4+LIFU,LIP5+LIFU,and LIP6+LIFU groups,P<0.0001.Moreover,it is worth noting that the LIP4+LIFU group the tumor growth was the slowest,which was 246.40±20.58 on the14 th day,moreover,the weight of the nude mice in this group has an increasing trend;the nanoparticle group(LIP4+LIFU,LIP5+LIFU,LIP6+LIFU)and the nanoparticle group(LIP4,LIP5,LIP6)without LIFU intervention correspond to each other,there was a particularly significant statistical difference,P<0.0001;tumor tissue sections and immunohistochemistry also confirmed the above statistical results(3)compared with the control group,liver and kidney function changes and blood routine on the 1st,7th,14 th day There was no statistical difference,P>0.05;H&E staining of heart,liver,spleen,lung and kidney showed that all organs had normal morphological structure,cell membrane intact,without cell edema,shrinkage,nuclear chromatin edge fragmentation,vacuole structure,regular nuclear morphology.Conclusions(1)Lip4 lipid nanoparticles have obvious targeting effect on 4T1 cells,and can be used for contrast enhanced ultrasonography and photoacoustic imaging,which can be used as molecular imaging agents for early diagnosis of mammary cancer;(2)LIFU and LIP4 are the two essential factors for SDT treatment;the LIP4 nanosystem combined with SDT treatment of HMME and LIFU has a targeted unloading feature for mammary cancer,and has a synergistic,cascade-amplified anticancer effect;Lip4 does not have any effect on other organs and blood components in the body,liver and kidney function,etc.It is an ideal molecular developer,and is integrated into diagnosis and treatment.