Preparation Of Hypoxia-responsive Nanomaterials For Enhanced Radiosensitization Of Cervical Cancer

Background:Cervical cancer（CC）is the most common malignant tumor of the female reproductive tract.According to the National Cancer Report in 2022,the number of cervical cancer patients remains as high as 110,000,and a trend of younger ages is becoming more and more prominent,posing a serious threat to the physical and mental health and life safety of women.Radiation Therapy（RT）is an effective means of adjuvant therapy for early cervical cancer（with high-risk factors）and controlling tumor progression when radical tumor surgery cannot be performed in the late stage,which is currently an irreplaceable treatment strategy in clinical practice.However,the rapid proliferation of cancer cells leads to a hypoxic state inside the tumor,which becomes a major cause of radiotherapy failure in solid tumors^[1].Studies have shown that the level of nitroreductase（NTR）is positively correlated with the degree of tumor hypoxia,making it a new target for tumor detection^[2].Therefore,the construction of an NTR-responsive radiotherapy sensitization nanoplatform has become an important research topic.Photoacoustic imaging（PAI）is an emerging medical imaging modality that combines the excellent resolution of optical imaging with the deep penetration of acoustic imaging,and has promising applications for early detection,accurate diagnosis and precise therapeutic evaluation of cancer^[3].Currently,PAI in the second near-infrared（NIR-Ⅱ,1000-2000 nm）window has deeper tissue penetration,higher optical resolution and lower background than visible（400-700 nm）and first near-infrared（NIR-I,700-1000 nm）windows,which have attracted increasing research interest in biomedical imaging applications^[4].Small-sized nanoparticles（NPs）can be easily removed after reaching the tumor site,thus affecting the effectiveness of diagnostic imaging and therapy.Therefore,the development of multifunctional nanoprobes with long-term retention and enrichment for NIR-Ⅱ PA imaging and enhanced radiotherapy has become particularly important.Objectives:The aim of this study is to construct a diagnostic and therapeutic nanoprobe that integrates a depleted oxygen-responsive nanoprobe to form larger aggregates at the tumor site and thus increase its enrichment and retention,enabling in vivo precision imaging-guided radiation therapy at the tumor site with NIR-Ⅱ PA.Methods:1.First,prepare the hypoxia response type with core shell structure AuNNP@PAA/NIC NPs nanoprobes.Through Transmission electron microscope（TEM）,Dynamic light scattering（DLS）and ultraviolet spectrum AuNNP@PAA/The morphology,structure,particle size distribution and UV absorption of NIC NPs were characterized and their responsiveness to NTR was verified.2.We used CCK-8,live/dead staining,and apoptosis flow cytometry to determine the in vitro cytotoxicity of AuNNP@PAA/NIC NPs nanomaterials.Further evaluation of DNA damage after X-ray irradiation was carried out usingγ-H2AX immunofluorescence.Reactive oxygen species（ROS）production was detected using DCFH-DA after radiation therapy.Finally,we co-cultured the AuNNP@PAA/NIC NPs nanoprobes with cells under different oxygen concentration conditions and observed the intracellular aggregation.3.By constructing He La nude mouse subcutaneous transplantation tumor models with different tumor volumes（20 mm³ and 60 mm³）to simulate hypoxic and normoxic tumor models,NIR-Ⅱ PA imaging was performed and the in vivo hypoxic response ability of AuNNP@PAA/NIC NPs nanomaterials was evaluated.After determining the optimal enrichment time of the tumor in vivo,the anti-tumor therapeutic effect and biocompatibility of AuNNP@PAA/NIC NPs were further evaluated.Results:1.The AuNNP@PAA/NIC NPs nanomaterials with core-shell structure were successfully synthesized,and the results of UV-vis-NIR,TEM,and DLS indicated that the probe had good NTR responsiveness,and its loudness was concentration and time dependent on NTR.The good linear relationship between different concentrations of NTR and the post-response photoacoustic intensity of nanomaterials（y=0.036x+0.158,R²=0.994）can be further used for the quantitative determination of NTR.2.The probe had a good biocompatibility with the survival rate of 90.37%and93.33%in He La and LO2 cells at a concentration of up to 200μg/m L.X-ray tumor cell toxicity test results showed that under hypoxia and normoxia conditions,the survival rate of He La cells in AuNNP@PAA/NIC NPs+X-ray（4 Gy）group were（45.2%and 32.2%）significantly lower than those of alone X-ray group（62.1%and 42.3%）,indicating that AuNNP@PAA/NIC NPs has radiation sensitization effect on hypoxic and normoxic tumors.Flow cytometry and dead cell staining results were consistent with the above results.In addition,DCFH-DA fluorescence detection and DNA damage assay showed that after endogenous NTR response in tumor cells,AuNNP@PAA/NIC NPs induced the production of ROS in tumor cells,promoting tumor cell apoptosis and necrosis.3.The NIR-Ⅱ PA_1200nm photoacoustic signal intensity peaked at 24 hours（photoacoustic intensity increased from 0.5 to 1.92,about 3.84-fold）after 6 hours of tail vein injection in mice,indicating that AuNNP@PAA/NIC NPs self-assembled and aggregated in oxygen-depleted tumors to achieve NIR-Ⅱ photoacoustic imaging.In the He La tumor mouse model,the AuNNP@PAA/NIC NPs+X-ray group showed significant inhibition of tumor growth compared to the single X-ray group,indicating that AuNNP@PAA/NIC NPs had a significant radiosensitizing effect on cervical cancer mice.The mice body weight did not change significantly and no obvious damage was seen in the main organ tissue pathological examination,indicating that the AuNNP@PAA/NIC NPs nanoprobe has good biocompatibility.Conclusion:1.We successfully synthesized a multifunctional gold-based nano-material（AuNNP@PAA/NIC NPs）with tumor hypoxia-responsive core-shell structure,constructed a reproducible nanoparticle preparation process and provided a new idea for the early diagnosis and treatment of cancer.2.The small-sized gold-based nanoparticles synthesized in this study have deep tumor penetration ability,and can undergo self-assembly in the hypoxic tumor microenvironment,prolonging their high enrichment and long retention in tumor tissues.This is of great significance for improving the radio-sensitizing effect of live tumor and reducing damage to normal tissues.3.The oxygen-deficient NTR-responsive aggregation of gold-based nanomaterials（AuNNP@PAA/NIC NPs）exhibits excellent radiosensitization effect under the guidance of NIR-Ⅱ photoacoustic imaging for cervical cancer,providing a new strategy for promoting the integration of tumor diagnosis and treatment based on nanoparticle platforms and clinical translation.