Oridonin-loaded And GPC1-targeted Multimodal Nanoparticles For Simultaneous Diagnosis And Treatment Of Pancreatic Cancer

Objective and Significance:Pancreatic cancer is a digestive system malignancy with high morbidity and mortality.The average surv ival time of untreated patients is 3 months and the patients after surgery are 10 to 20 months,with a 5-year survival is 5%-7%.Due to the lack of specific symptoms and effective biomarkers,early diagnosis of pancreatic cancer patients is difficult.Less than 20%of patients can be diagnosed at early stage and given surgery,while most patients are diagnosed with advanced or metastatic disease and cannot be treated surgically.Although existing imaging technologies can provide morphological information about pancreatic tissues,single-modality imaging does not have sufficient sensitivity and specificity for the diagnosis of pancreatic cancer.A major challenge in treatment is that pancreatic cancer is not sensitive to traditional chemotherapy.Therefore,early diagnosis methods and effective treatment methods are urgently needed.Molecular imaging offers an approach to combine of multimodal imaging and targeted therapy,which provides hope for early diagnosis and effective treatment of pancreatic cancer.Gold nanocages(AuNCs)are widely used in cancer theranostics due to their inner hollow space,good biocompatibility,and easy modification.By loading a variety of functional materials such as imaging agents,drugs,targeting molecules,AuNCs not only can extend the half-life of the contrast agent,improve the imaging mode,improve the biocompatibility,but also reduce the toxic side effects of the therapeutic drugs,and achieve the integration of diagnosis and treatment of pancreatic cancer.This study constructed the AuNCs as a carrier,with pH/hyaluronidase as the control valve,carrying oridonin(ORI),targeting glypican-1(GPC1)multimodal theranostic nanoparticles GPC1-Gd-ORI@HAuNCs-Cy7(ORI-GPC1-NPs).Through in vivo and in vitro experiments,the ability of near-infrared fluorescence(NIRF)and magnetic resonance(MR)imaging,and anti-tumor effects were verified.Methods:ORI was added at room temperature and stirred to load the ORI inside the AuNCs.A solution of hyaluronic acid-coated ORI@AuNCs(ORI@HAuNCs)was obtained by Au-S bond bonding.GPC1 antibody,Gd,Cy7 was coupled to the nanoparticles by esterification reaction,and finally the multimodal nanoparticles ORI-GPCI-NPs were synthesized.Meanwhile,non-targeted Gd-ORI@HAuNCs-Cy7 NPs(ORI-NPs)were synthesized as control.The morphological structure,average hydrodynamic size,Zeta potential,contained elements,functional groups,stability,drug loading and release,targeting ability,and in vitro multimodal imaging performance of ORI-GPC1-NPs were evaluated by performance testing.The effects of ORI-GPC1-NPs on proliferation,apoptosis,cycle distribution,and invasion of pancreatic cancer cells were analyzed by MTT,flow cytometry,Western Blot and transwell experiments.Healthy nude mice were selected and injected with high,medium and low doses of ORI-GPC1-NPs through the tail vein for 21 days.The body weight of the mice was monitored,and the effects of the hematological system,liver function,and renal function on the mice were analyzed by routine biochemical tests of blood,and the doses administered in vivo were determined.The orthotopic pancreatic tumor model of nude mice was constructed,and in vivo NIRF and MR imaging were performed by injecting with ORI-GPC1-NPs through a lateral tail vein at specific time points(before injection,12 h,24 h,and 48 h post-injection)to evaluate the ability of multirmodal imaging in vivo.After imaging,the mice were sacrificed and tumor tissues and main organs were harvested.The in vivo biological distribution of ORI-GPC1-NPs was analyzed by ex vivo NIRF imaging and inductively coupled plasma mass spectrometry(ICP-MS).Tumor-bearing mice were randomly divided into negative control group,gemcitabine(GEM)group,ORI group,ORI-NPs group,and ORI-GPC1-NP group.Different drugs were injected through the tail vein for 14 days.The body weight and tumor volume of the tumor-bearing mice were monitored,and time-weight curves and time-tumor volume curves were plotted.On the last day,the tumor and organ samples were harvested from euthanized mice and were subjected to hematoxylin and eosin(H&E)staining and immunohistochemistry(IHC)assay,the antitumor effect of ORI-GPC1-NPs was further verified from the in vivo level.Results:1.The performance test results showed that the target nanoparticles ORI-GPC1-NPs were successfully synthesized.ORI-GPC1-NPs had a nearly spherical shape,excellent dispersity,good stability,and NIRF/MR multimodal imaging performance in vitro.ORI-GPC1-NPs held an adequate amount of ORI content and a pH/enzyme sensitive controlled release.2.Real-time PCR and flow cytometry analysis showed that high expression of GPC1 in pancreatic cancer cell lines PANC1,BXPC-3,and SW1990,and low expression of GPC1 in human embryonic kidney 293T cells.3.Transmission electron microscopy showed that ORI-GPC1-NP samples were found in PANC-1 and BXPC-3 cells(high expression of GPC1),while ORI-GPC1-NP samples could not be found in 293 T cells(low expression of GPC1)at higher magnification.4.In vitro cytology results showed that ORI-GPC1-NPs can inhibit the proliferation of pancreatic cancer cells,promote cell apoptosis,arrest cell cycle,and inhibit cell migration.5.In vivo toxicity assays showed that compared with the negative control group,there were no significant changes in blood routine indicators,liver and kidney function biomarkers in high,medium,and low dose ORI-GPC1-NPs group,P>0.05.6.In vivo imaging results showed NIRP/MR signals at the tumor site of the ORI-NP group reached peak intensity at 12 hours post-injection.These signals gradually diminished as time passed,and nearly disappeared after 48 hours.For the ORI-GPC1-NPs group,NIRF/MR signals were also detected in the tumor 12 hours after injection.These signals gradually increased and reached peak intensity at 24 hours post-injection,significantly higher than the control group,P<0.05.The tumor NIRF/MR intensity remained steady even 48 hours after injection,significantly higher than that the control group,P<0.05.7.The ex vivo NIRF and ICP-MS results showed that ORI-GPC1-NPs mainly accumulatcd in the liver,tumor and kidney.At 12 hours post-injection,ORI-NPs and ORI-GPC1-NPs were consistently distributed in tissues.At 24 and 48 hours post-injection,the accumulation of ORI-GPC1-NP in tumor tissues was significantly higher than that of ORI-NPs.8.The body weight of mice did not significantly change over the course of the treatment period.After treatment,the tumor volume and weight in GPC 1-ORI-NPs group were lighter than those in the other control groups.HE results showed that the structure of tumor tissue in the ORI-GPC1-NPs group suffered more obvious damage than the other groups;this damage included irregular widening intercellular spaces.No obvious major organ damage was observed in liver and kidney.IHC results showed that ORI-GPC1-NPs can reduce the expression of Bcl-2 and enhance the expression of Caspase-3 in tumor tissues.Conclusions:1.ORI-GPC1-NPs could specifically bind to pancreatic cancer cells with high expression of GPC1,prolonging the blood circulation time of ORI-GPC1-NPs,and reducing the influence on normal tissue cells.2.GPC1-GEM-NPs were biocompatible and did not cause any obvious adverse effect in vivo.3.ORI-GPC1-NPs were mainly metabolized by liver and kidney.4.ORI-GPC1-NPs were ideal NIRF/MR multimodal imaging probes,and had significant anti-tumor effect,which could be used for early diagnosis and targeted treatment of pancreatic cancer.