In Vivo Multi-scale Photoacoustic Imaging Guided Photothermal Therapy Of Cervical Cancer Based On Customized Laser System And Targeted Nanoparticles

Background and objectiveCervical cancer is the most commom malignant tumor in female reproductive system,which is also one of the main causes of women’s death.Cervical cancer is closely related to the social development and human papillomavirus(HPV)infection.Existing treatment strategies not only include traditional treatment methods such as surgical resection,radiotherapy and chemotherapy,but also emerging treatments including gene therapy,biological immunotherapy etc have been gradually applied in clinical practice,which have brought good news to cervical cancer for women.However,these treatments also have their respective limitations,such as recurrence caused by incomplete treatment,various complications,severe drug side effects,and cost-effectiveness.Therefore,It is our common expectation to find an efficient and accurate treatment method for cervical cancer to improve the life quality and survival rate of patients.In this paper,a conjugated polymer PIIGDTS nanoparticle that is targeted to HeLa cells with excellent photoacoustic effect and strong photothermal conversion efficiency was designed for in vitro cell experiments and in vivo animal experiments by utilizing customized laser systems,in order to evaluate the effect of photoacoustic imaging(PAI)-guided-photothermal therapy(PTT)for cervical cancer.This research is expected to provide a new vision of efficient and accurate treatment against cervical cancer.Methods(1)The conjugated polymer PIIGDTS(PD)nanoparticles with strong near infrared region(NIR)absorption was prepared by one-step nanoprecipitation method.Folic acid(FA)that targeted to HeLa cells was modified in surface of PD nanoparticles and named PD-FA nanoparticles.The morphology of the PD-FA nanoparticles was observed by transmission electron microscope(TEM),and the particle size of PD-FA nanoparticles was measured by Malvern Nano-ZS particle sizer.The absorption spectra of the PD-FA nanoparticles were recorded by UV-spectrophotometer.The photoacoustic performances of the PD-FA nanoparticles including the photoacoustic spectrum,photoacoustic stability and concentration-dependence of photoacoustic signal were measured by our self-established photoacoustic system.The in vitro photothermal effects of the PD-FA nanoparticles including photothermal stability and photothermal conversion efficiency were evaluated by the infrared thermal imagery and customized near infrared laser system.(2)Subsequently,in vitro cell experiment was carried out.The cellular toxicity of PD-FA nanoparticles on normal cells(Cos-7)was detected by MTT colorimetric method firstly.Then,in order to explore the cellular uptake of nanoparticles in HeLa cells,PD-FA nanoparticles and PD nanoparticles were labeled with fluorescent dye CU-6.The cellular uptake of two nanoparticles to HeLa cells at different time points was observed by confocal laser scanning microscope(CLSM).On this basis,the best time point for HeLa cells to internalize nanoparticles was determined.The HeLa cells were irradiated with laser at this best time point,the survival rate of HeLa cells after laser irradiation with nanoparticles were detected by the living/dead cell staining method and MTT colorimetric method to evaluate the photothermal killing effect.(3)The subcutaneous tumor model was established to assess the in vivo photoacoustic performance and photothermal therapy effect of the PD-FA nanoparticles.A tumor-mimicking medical coupling gel phantom with different concentrations of PD-FA nanoparticles was detected by our photoacoustic system to verify the photoacoustic performance of nanoparticles in mice.Further,the photoacoustic images were obtained by our photoacoustic system at different time points after intravenous injection of PD nanoparticles or PD-FA nanoparticles to monitor the aggregation of two nanoparticles in the tumor region.To evaluate the antitumor effect of nanoparticles in HeLa tumor-bearing nude mouse,mice were randomly divided into 5 groups and were treated as follows: PD-FA nanoparticles +laser,PD nanoparticles + laser,PD-FA nanoparticles,laser,control group.Tumor volume and mouse weight were monitored every three days.The last,all mice were sacrificed,the Hematoxylin and Eosin(HE)analysis of major organs including the heart,liver,spleen,lung,and kidneys after treatments were conducted.Results(1)PD-FA nanoparticles showed a regular spherical structure in the TEM images and the hydrodynamic diameter of the PD-FA nanoparticles was about 50 nm,and the absorption spectra of the PD-FA nanoparticles showed that PD-FA nanoparticles displayed a broad absorption spectrum with a peak at 845 nm.The excellent photoacounstic performance in vitro was confirmed by our photoacounstic system,the PD-FA nanoparticles exhibited photoacoustic effect even at a low concentration,and the intensity of the photoacoustic signal could present a good linear correlation with the concentration of PD-FA nanoparticles.The photoacoustic stability was verified by the unchanged photoacoustic signal under continuous laser irradiation for 20 min.In vitro photothermal experiments demonstrated that the photothermal effect of PD-FA nanoparticles is concentration-,power-and time-dependent.Moreover,as-prepared PD-FA nanoparticles possessed excellent photostability and admirable photothermal conversion efficiency up to 62.6%.(2)There is no significant cytotoxicity was noticed when the PD-FA nanoparticles was co-incubated with normal cell line Cos-7.The results of cellular uptake showed that the PD-FA nanoparticles have good selectivity toward HeLa cells,and the amount of internalized PD-FA nanoparticles achieved a high level after 8hours incubations.HeLa cells photothermal killing experiment showed that PD-FA nanoparticles have strong photothermal killing effect after laser irradiation in HeLa cells,and the cell mortality rate was higher than 80%.The cell mortality was significantly reduced when HeLa cells was incubated with PD nanoparticles.This phenomenon verified the targeting ability and photothermal killing effect of PD-FA nanoparticles to HeLa cells.(3)The photoacoustic images of tumor tissue in tumor-bearing nude mouse showed that the PD-FA nanoparticles reached the tumor tissue after 2 h intravenous injection and gradually accumulated up to 24-h post injection.After that they were excreted from the mice.In contrast,the signal was relatively weak after injection of the PD nanoparticle.This phenomenon indicated that the PD-FA nanoparticles possessed the ability to target HeLa cells and accumulate in the tumor site.In vivo photothermal therapy showed that the Hela tumor can be completely eliminated within 18 days after injection of PD-FA nanoparticles under laser iradiation,with no toxicity and side effects.ConclusionMulti-dimensional PAI-guided PTT by aid of multi-functional PD-FA nanoparticles is effective,which is expected to broaden the field of diagnosis and treatment of cervical cancer.