Theranostic Nanoplatform With Sequential Sdt And Adv Effects In Response To Well-programmed LIFU Irradiation For Cervical Cancer

Background Globally,cervical cancer remains one of the most common cancers in women and the fourth most common cancer after breast,colorectal and lung cancers.The current treatment strategy for cervical cancer is local therapy through surgery and radiotherapy.As the disease progresses,multidisciplinary therapy and chemotherapy can be considered.However,traditional treatment methods have various drawbacks,such as serious complications after surgery in some patients;low radiosensitivity for advanced cervical cancer,which cannot achieve satisfactory therapeutic effects;limited treatment methods for recurrent cervical cancer and poor prognosis.In addition,some young cervical cancer patients have the need to preserve fertility,while some elderly patients cannot tolerate traditional radiotherapy,chemotherapy and surgery.Therefore,there is an urgent need for a minimally invasive treatment scheme that can effectively kill cervical cancer cells.Object To prepare an integrated diagnosis and treatment nanoprobe(termed IGP@P)carrying IR780,perfluorohexane(PFH)and gadolinium diethylene-trianmine pentaacetic acid(Gd-DTPA)to achieve sequential killing of cervical cancer cells under low-intensity focused ultrasound(LIFU)irradiation,using photoacoustic(PA)and magnetic resonance(MR)molecular imaging to achieve target-specific diagnosis and treatment effect evaluation of cervical cancer.Methods1.Preparation and characterization of the integrated nanoprobe IGP@P for diagnosis and treatment:the integrated nanoprobe for diagnosis and treatment of cervical cancer He La cells was prepared by phacoemulsification.Dynamic light scattering(DLS)was used to detect the particle size and zeta potential of nanoparticles.Scanning electron microscopy(SEM)was used to characterize the three-dimensional structure of IGP@P nanoparticles.Confocal laser scanning microscopy(CLSM)was used to characterize Di I-labeled IGP@P nanoparticles.High-resolution transmission electron microscopy(HRTEM)was used to examine the morphology and structure of IGP@P nanoparticles with electron-dense gadolinium(Gd)and IP@P nanoparticles without Gd,energy dispersive spectroscopy(EDS)was used to observe the distribution of various elements including iodine(I),Gd and fluorine(F)in the nanoparticles.Inductively coupled plasma optical emission spectrometer(ICP-OES)was used to measure the encapsulation efficiency of Gd-DTPA in each group of Gd-containing nanoparticles.The encapsulation efficiency of IR780 in IG@P and IGP@P nanoparticles was determined using a ultraviolet-visible-near-infrared(UV-Vis-NIR)spectrophotometer.The PA and MR imaging capabilities of IGP@P nanoparticles in vitro were evaluated.2.Targeting and therapeutic effect evaluation of integrated nanoprobes for diagnosis and treatment of He La cells under LIFU irradiation in vitro:the targeted uptake of IGP@P nanoparticles by He La cells was evaluated by CLSM,and the targeting affinity of IGP@P nanoparticles to mitochondria was further verified by their co-localization with mitochondria.The reactive oxygen species(ROS)generation of IGP@P nanoparticles after LIFU irradiation for different time(2.5 W/cm~2;2 min,4 min and 6 min)was measured,and the characteristics of acoustic droplet vaporization(ADV)effect of nanoparticles were observed by optical microscope at corresponding time points.The production of ROS in He La cells,the phase transition of NPs,and the apoptosis/necrosis rate of He La cells before and after LIFU irradiation were evaluated to analyze the therapeutic effect and treatment mechanism at the cellular level in vitro.The safety of nanoparticles on cells in vitro was determined by CCK-8 assay.3.In vitro and in vivo experiments of LIFU irradiation to promote the penetration of integration of diagnostic and therapeutic nanoprobes into cervical cancer:at the in vitro level,the ability of LIFU irradiation to promote the penetration of IGP@P nanoparticles into 3D multicellular tumor spheroids(MCTSs)was verified.At the in vivo level,the targeting ability of IGP@P nanoparticles to the tumor of nude mice was evaluated by PA and MR imaging,and the ability of the first penetration-enhancing LIFU irradiation to promote the penetration of IGP@P nanoparticles into the tumor was verified by MR T1-weighted image(T1WI).4.In vivo experiments of sonodynamic therapy(SDT)and ADV effects after re-therapeutic LIFU irradiation on sequential synergistic treatment for cervical cancer:MR T2-weighted image(T2WI)was used to evaluate the T2 signal changes of tumor in nude mice after re-therapeutic LIFU irradiation,and the in vivo treatment mechanism was analyzed in comparison with pathological sections.Tumor-bearing mice were treated with intermittent IGP@P+LIFU irradiation and observed for16 days to evaluate the long-term efficacy of the treatment.After the treatment,the hematological indexes of nude mice were detected,and the heart,liver,spleen,lung and kidney of nude mice were collected for pathological section analysis to evaluate the in vivo safety of IGP@P nanoparticles in the treatment of cervical cancer.Results1.Using poly(lactic-co-glycolic acid)(PLGA)as a carrier,IR780was loaded on the shell of nanoparticles,Gd-DTPA and PFH were encapsulated into the core of nanoparticles,and IGP@P nanoparticles were successfully prepared by phacoemulsification.The nanoparticles were spherical,uniform in particle size,and have good dispersibility.The average particle size of the IGP@P nanoparticles was 174.5±3.10 nm,and the zeta potential was-5.5±3.31 m V.HRTEM images show the existence of electron dense Gd in IGP@P NPs,EDS shows that I element is uniformly distributed on the nanoparticle shell in a ring shape,and Gd and F elements are uniformly distributed in the core of the nanoparticle.The encapsulation efficiency of Gd-DTPA in IGP@P NPs was 57.60±5.42%,and that of IR780 was 67.39±4.07%.The PA and MR imaging capabilities of the nanoparticles were evaluated,showing that the PA intensity increased with the increase of the concentration of IGP@P nanoparticles and was linearly correlated;meanwhile,the IGP@P nanoparticles had good MR positive contrast enhancement ability,the signal intensity(SI)of T1 is positively correlated with the concentration of IGP@P nanoparticles.2.CLSM showed that IGP@P nanoparticles not only have high affinity for He La cells and can be endocytosed by He La cells in large quantities,but also can selectively localize to mitochondria in cells due to the assistance of IR780.After irradiating IGP@P nanoparticles with LIFU in vitro,it was shown that under the experimental conditions,IR780 in IGP@P nanoparticles could be induced to generate a large amount of ROS after 2 and 4 min of irradiation,and when the irradiation time was prolonged to 6 min,the generation of ROS was significantly reduced.The phase transition of PFH in IGP@P nanoparticles became obvious with the prolongation of irradiation time,and extensive and sufficient phase transition could be observed after 6 min of LIFU irradiation.Cell experiments showed that in the IGP@P+LIFU group,the anti-tumor effect mainly relied on ROS in the first half of LIFU irradiation,and the second half mainly relies on ADV effect,resulting in cascaded sequential therapy on tumor cells.The CCK-8 assay showed that IGP@P nanoparticles had reliable cellular safety.3.After LIFU irradiation,CLSM showed that a large number of Di I-labeled IGP@P nanoparticles penetrated into the tumor and were uniformly distributed throughout the 3D MCTSs,confirming that in vitro LIFU irradiation could help IGP@P nanoparticles penetrate inside the tumor.In vivo PA imaging showed that the PA intensity in the tumor area gradually increased with time,and was the strongest 12 h after the injection of IGP@P nanoparticles.In vivo MR imaging showed that the T1 signal in the tumor area gradually increased 3 h and 6 h after the injection of IGP@P nanoparticles.6 h after IGP@P nanoparticle injection,we applied the first penetration-enhancing LIFU irradiation at the tumor site,6 h after irradiation,T1WI showed that the T1 signal at the tumor site was further enhanced compared with the group that did not apply the first penetration-enhancing LIFU irradiation,indicating that a large number of IGP@P nanoparticles accumulated at the tumor.4.12 h after of injection of IGP@P nanoparticles into nude mice,re-therapeutic LIFU irradiation was applied,and 6 h after irradiation,MR T2WI showed a heterogeneous decrease in T2 signal in the tumor area.Pathological sections showed that there were scattered bleeding spots in the interstitium of the tumor in the IGP@P+LIFU group,and the structure of the nucleus and cell membrane was blurred.Both pathological sections and MR showed that the tumor had coagulative necrosis after re-therapeutic LIFU irradiation.In the observation of tumor-bearing nude mice for a period of 16 days after injection of IGP@P nanoparticles and re-therapeutic LIFU irradiation,it was shown that the tumor volume in the IGP@P+LIFU group gradually decreased and the body weight of nude mice did not change significantly.After treatment,no significant abnormality was found in hematological tests and pathological analysis of nude mice,indicating the safety of this method in vivo.Conclusion In this experiment,we designed and fabricated IGP@P nanoparticles carrying IR780 and PFH,which have high affinity for He La cells and can selectively achieve mitochondrial localization in cells due to the assistance of IR780.Under LIFU irradiation,the ROS generated by IR780 and the ADV effect induced by the phase transition of PFH can achieve synergistic minimally invasive treatment of cervical cancer.LIFU irradiation played two roles of promoting penetration and synergistic therapy in this experiment.We used first penetration-enhancing LIFU irradiation and validated its ability to promote nanoparticles penetration into tumors in 3D MCTSs and nude mice.Then under re-therapeutic LIFU irradiation,the IR780 carried in the nanoparticles can generate ROS through sonodynamic therapy(SDT).The ROS generated in the first half of LIFU irradiation was not enough to exert a strong antitumor effect,so in the second half of the re-therapeutic LIFU irradiation,the physical ADV effect generated after the phase transition of the PFH carried in the nanoparticles was observed in vitro and in vivo,thus achieved sequential antitumor effects in vitro and in vivo.Linking ROS+ADV effects can induce coagulative necrosis of cells and have comprehensive,long-term effects on tumor tissue.We applied well-programmed LIFU irradiation to promote nanoparticle penetration into deep tumor tissues and simultaneously achieve antitumor effects.In addition,due to the nanoparticle-loaded IR780 and Gd-DTPA,we used photoacoustic(PA)and magnetic resonance(MR)imaging to monitor and evaluate the targeting and therapeutic effects of these nanoparticles on tumor tissue,providing a novel target for cervical cancer.An integrated nano-platform for minimally invasive diagnosis and treatment of cervical cancer.