Experimental Study Of PTX/PFH Lipid Nanoparticles Conjugated With Escherichia Coli Expressing Gas Vesicles To Facilitate Hifu Therapy For Breast Cancer

BackgroundHigh intensity focused ultrasound(HIFU)as a new technique for the non-invasive local ablation of tumors,has been widely used in clinical applications.Nevertheless,tumors with irregular morphology and unclear boundaries may recur because of incomplete ablation of the cancer tissue.HIFU combined with chemotherapy drug-loaded nanoparticles can improve the therapeutic efficiency of residual tumor.However,these drug-loaded nanoparticles have certain limitations,such as poor tumor targeting and low accumulation at the tumor site,that restrict the therapeutic efficacy of HIFU.Therefore,how to increase the accumulation of drug-loaded nanoparticles in the tumor sites and improve the safety and efficacy of synergist to mediate HIFU ablation is an urgent problem to be solved.In this study,genetically modified Escherichia coli expressing gas vesicles(GVs-E.coli),which were gas-filled protein nanostructures,could specifically target into the tumor.GVs-E.coli was used as a vehicle by conjugating with PTX-CLs via electrostatic adsorption and subsequently attracting more PTX-CLs to the tumor site,and prolong its retention time in the target area,enhancing the therapeutic efficacy of HIFU and chemo-synergistic therapy.This novel combination strategy provides a new idea for tumor treatment,which is expected to improve the efficacy and safety of HIFU ablation.ObjectiveTo explore the efficacy and safety of PTX/PFH lipid nanoparticles(PTX and PFH co-loaded cationic liposomes,PTX-CLs)combined with GVs-E.coli for synergizing HIFU ablation therapy,which is expected to provide a new idea for clinical synergistic of HIFU to treat breast cancer.Methods1.GVs-E.coli was prepared by inducing gas vesicles(GVs)from E.coli BL21(AI)/PET28A-ARG1 using genetic engineering.Morphologic and structure characterization of GVs-E.coli was conducted using the transmission electron microscopy,the multi-angle particle size/zeta-potential analyzer was used to measure zeta-potential of GVs-E.coli.To observe the tumor-targeting of GVs-E.coli,20 Xenograft 4T1tumor-bearing mice were injected with 200μL GVs-E.coli(1×10~8CFU/m L)via tail vein.Mice were sacrificed at different time points after injection(days 1,3,7,and 14),and the major organs(heart,liver,spleen,lung,and kidney)and tumors were harvested and weighed to homogenate tissues.Paclitaxel and PFH coloaded cationic lipid nanoparticles(PTX-CLs)were prepared using film-ultrasonic method,the basic characterization of PTX-CLs(morphology,particle size,potential,drug encapsulation,loading efficiency)was performed,and the blood compatibility was detected by hemolysis test.To establish a link between PTX-CLs and GVs-E.coli in vitro,nanoparticles were mixed with GVs-E.coli in a 1:10 volume ratio.They were connected by electrostatic adsorption at room temperature for5-10 min.The connection effect was then observed using a confocal laser scanning microscope(CLSM)and the connection efficiency was determined using flow cytometry.2.To evaluate the cytotoxicity of PTX-CLs on breast cancer cells in vitro,the three groups were set as follows:(1)control group;(2)PTX-CLs with HIFU irradiation;(3)PTX-CLs without HIFU irradiation.The cytotoxicity of PTX-CLs on tumor cells was observed by flow cytometry apoptosis detection and CAM-PI fluorescence double staining.3.In vitro HIFU ablation therapy:Fresh isolated bovine liver tissue was sliced into 10 cm×6 cm×6 cm(L×W×H)and degassed,then placed in a special HIFU mold with a sound permeable film at the bottom.The four groups were set as follows:(1)PBS group;(2)GVs-E.coli group;(3)PTX-CLs group;(4)PTX-CLs+GVs-E.coli group,each group was irradiated with different HIFU energy(120 W,3 s;150 W,3 s;180 W,3 s)after injection of 100μL corresponding solution,and cavitation signals were detected simultaneously.The volume of coagulation necrosis and emergency efficiency factor(EEF)in the target area of bovine liver were measured and calculated after HIFU irradiation.4.To establish a connection between PTX-CLs and GVs-E.coli in vivo.The xenograft 4T1 tumor-bearing mice were randomly divided into two groups(5 per group):(1)PTX-CLs group;(2)PTX-CLs+GVs-E.coli group.The PTX-CLs distribution images in vivo were collected using fluorescence imaging in vivo at various time points:before injection,8 h,24 h,32 h,and 48 h after injection NPs.Tumors were immediately harvested to make frozen sections,and the distribution of PTX-CLs in the tumor at different time points was examined using CLSM.5.In vivo HIFU ablation therapy:The xenograft 4T1 tumor-bearing mice were randomly divided into four groups(5 per group):(1)PBS group;(2)PTX-CLs group;(3)GVs-E.coli group;(4)PTX-CLs+GVs-E.coli group.The tumor tissue was ablated using HIFU(120 W,3 s)and cavitation signal was detected simultaneously,the mice were sacrificed 24h after the HIFU ablation,and all tumors were harvested and incubated in TTC solution.Then,the volume of coagulative necrosis and EEF were calculated.H&E,TUNEL,and PCNA immunohistochemical analyses of tumor tissues were performed to observe the treatment effect of different groups.In addition,the four groups were set as follows(5 per group):(1)control group;(2)PTX-CLs group;(3)GVs-E.coli group;(4)PTX-CLs+GVs-E.coli group.The changes of tumor volumes and body weights of the mice were recorded within 14 days after HIFU treatment,and the inhibitory effect of synergizing HIFU therapy on breast cancer in different groups was evaluated.6.To establish the biosafety of PTX-CLs combined with GVs-E.coli in vivo.12 healthy BALB/c mice were randomly divided into four groups:(1)control group;(2)3 d group;(3)7 d group;(4)14 d group.All mice in the control group were raised normally without any treatment,the mice in the other groups were injected with GVs-E.coli(1×10~8CFU/m L)via tail vein,three days later,the mice were administrated a second round of injection containing PTX-CLs(6 mg/m L)via tail vein.Blood was drawn from mice on 3 d,7 d,and 14 d after the second injection for blood routine and blood biochemistry tests.Results1.E.coli BL21(AI)successfully formed gas vesicles(GVs)after induction,according to TEM,E.coli BL21(AI)had hollow,cylindrical,or spindle-like GVs.The zeta potential of GVs-E.coli was-35.20±0.79m V,the homogenate tissues result showed that the number of GVs-E.coli in the tumor tissue peaked at day 3,conversely,the mount of GVs-E.coli in the heart,liver,spleen,lung,and kidney tissue gradually decreased after injection and became undetectable on day 14.The PTX-CLs prepared by film-ultrasonic method showed uniform in shape and had good dispersibility under optical microscope.The SEM and TEM images showed that PTX-CLs had a spherical shape and smooth surface.The mean zeta potential was 35.37±2.18 m V,and an average particle size was278.87±1.30 nm,the encapsulation and loading efficiency of PTX in PTX-CLs were 45%and 3.5wt%.The hemolysis rate of nanoparticles was below 5%,which demonstrated that PTX-CLs have good blood compatibility.CLSM showed that PTX-CLs could effectively bind to GVs-E.coli by electrostatic adsorption,with fluorescence fusion of the two appearing in yellow,the connection rate of the two in vitro was99.66%measured by flow cytometry.2.The flow cytometry apoptosis results showed that the apoptosis rate of breast cancer cells was 3.62%±0.48%in the control group,8.45%±1.75%in the group without HIFU irradiation,and 73.93%±5.66%in the HIFU irradiated group,compared to the first two groups,the difference was statistically significant(***P<0.001).The CAM-PI fluorescence double staining results showed that fewer breast cancer cells died in the PTX-CLs group without HIFU irradiation,whereas the majority of the breast cancer cells died in the PTX-CLs group with HIFU irradiation,which was consistent with the flow cytometry apoptosis results.3.A grey-white coagulative necrotic region appeared in the targeted area of the bovine liver after HIFU irradiation.With the increase of HIFU irradiation energy,the coagulation necrosis volume increased,and EEF decreased.Under the same HIFU irradiation energy,coagulative necrosis volume of each group was as follows:PTX-CLs+GVs-E.coli group>PTX-CLs group>GVs-E.coli group>PBS group,The EEF of each group was inversely proportional to the coagulative necrosis volume.PTX-CLs+GVs-E.coli group had the highest ablation efficiency,compared to the other groups,the difference was statistically significant(***P<0.001).Cavitation signal detection results showed that the RMS value was positively proportional to the irradiation energy of HIFU,and cavitation effect in PTX-CLs+GVs-E.coli group was the most significant.4.In vivo fluorescence imaging and the frozen section of the tumor results showed that in the PTX-CLs+GVs-E.coli group,fluorescence at the tumor site gradually increased over time and reached a maximum level32?h after the second injection,with strong fluorescence still visible at 48 h.However,in the PTX-CLs group,the fluorescence intensity at the tumor site reached a peak at 24 h and then gradually decreased.By comparing the fluorescence intensity at the tumor site between the two groups at each time point,the fluorescence in the PTX-CLs+GVs-E.coli group was stronger.these results validated that GVs-E.coli which specially target the tumor site could attract more nanoparticles to the tumor site and cause aggregation and retention effect.5.The efficacy of PTX-CLs combined with GVs-E.coli for synergizing HIFU tumor ablation in breast cancer treatment,after HIFU irradiation,PTX-CLs+GVs-E.coli group had the largest coagulation necrosis volume(103.96±0.44 mm~3)and the smallest EEF value(2.42±0.01 J/mm~3),compared to other groups,the ablation efficiency was the highest,and the difference was statistically significant(***P<0.001).The most significant increase in RMS was detected in the PTX-CLs+GVs-E.coli group with the most significant cavitation effect.The results of H&E,TUNEL,and PCNA staining showed that coagulation necrosis of tumor target area in PTX-CLs+GVs-E.coli group was the most serious,proliferation of tumor cells around target area was reduced,and apoptosis was the most significant.Tumor suppressive experiment in mice further verified that GVs-E.coli combined with PTX-CLs for synergizing HIFU ablation had good anti-tumor effect.6.The biosafety of PTX-CLs combined with GVs-E.coli in vivo:In the experimental group,some blood indexes had slight fluctuations on day 3 post injection,but gradually returned to normal on day 7.On the14th day,there was no significant difference between the blood biochemical and blood routine indicators compared to the control group.The weight of the mice among the groups did not differ significantly during the experiment,demonstrating that the treatment was biosafe.ConclusionsIn this study,we successfully constructed PTX-CLs conjugated with GVs-E.coli which can selectively colonize the tumor site to facilitate HIFU ablation for breast cancer,enhancing the therapeutic efficacy of HIFU and chemo-synergistic therapy.This novel combination strategy can improve the efficacy and safety of HIFU ablation,and provide a new idea for clinical HIFU treatment of breast cancer.