Research On The Application Of ZIF-8-based Integrated Multifunctional Nanoparticles For Prostate Cancer Diagnosis And Treatment

PurposeIn recent years,the incidence of tumors has been increasing year by year,and prostate cancer has become the second most common disease in the world for male malignant tumors.In China,it ranks sixth in the incidence of male malignant tumors.At present,the exact pathogenesis of prostate cancer is still unclear.In clinic,screening and anti-tumor treatment are the main focus for prostate cancer.Chemotherapy,as one of the main methods for the treatment of malignant tumors,also plays an extremely important role in the treatment of prostate cancer.However,while traditional chemotherapy drugs inhibit tumor cell proliferation or kill tumor cells,they often have serious adverse effects on healthy tissues,and even the immune system.In addition,the commonly used tumor diagnostic techniques,such as magnetic resonance imaging,pathological slices,and other technologies have limited application value in the early detection and diagnosis of tumors.Therefore,there is an urgent need to develop new tumor early diagnosis technologies and treatment methods to improve the early screening and the efficiency of anti-tumor treatment for prostate cancer.With the continuous development of biomedical technology and nanomaterial science,various types of stimulus-responsive drug delivery nanoparticles have been developed to achieve the controlled release and specific response release of chemotherapeutic drugs at targets.However,chemotherapeutic drugs alone are not enough to completely kill cancer cells,so there is a great need to develop multifunctional nano-platforms for more effective tumor treatments.In recent years,the combination of photothermal therapy and chemotherapy is considered to be a promising strategy to improve treatment efficiency and reduce side effects.In addition,in addition to being a drug delivery system,the rational design of nanoparticles can also make them a good imaging contrast agent.In this study,the metal-organic framework ZIF-8 was first used to load the chemotherapeutic drug DOX,and then the surface was functionalized.The nanoparticles can achieve pH-responsive drug release and photothermal therapy,which can be used as a contrast agent for magnetic resonance and photoacoustic imaging.Prostate cancer cell line PC-3 in vitro experiments and PC-3 tumor-bearing mice in vivo experiments confirmed that the nanoparticle-mediated chemotherapy-photothermal synergistic treatment can effectively kill tumor cells and inhibit tumor tissue proliferation.It can also perform high-quality imaging of tumor tissues.In addition,the materials used in this study are all organic materials,which greatly improves the biocompatibility of nanoparticles.This study provides a theoretical basis and strong evidence support for the application of the multifunctional nano drug delivery system in the diagnosis and treatment of prostate cancer.Methods1.Synthesis and characterization of ZIF-8/DMPP nanoparticlesFirst,ZIF-8 nanoparticles were prepared by stirring at room temperature,and then DOX-incorporated ZIF-8 nanoparticles,which was named ZIF-8/DOX,were synthesized by in-situ one-pot method.Then ZIF-8/DOX was modified with poly dopamine(PDA)shell,onto which Mn ions were subsequently chelated and PEG was grafted to obtain the final product ZIF-8/DMPP.The morphology of ZIF-8/DMPP nanoparticles was observed by transmission electron microscopy(TEM).High-angle annular dark-field scanning transmission electron microscopy(HAADF-STEM)imaging and energy-dispersive X-ray spectroscopy(EDX)elemental mapping were performed for element mapping analysis.The crystal structure of ZIF-8/DMPP nanoparticles was recorded by X-ray diffraction(XRD),the size distribution was assessed by dynamic light scattering(DLS),and the Zeta potential measurement was conducted on a Nano ZS apparatus,and its spectroscopic characterization was analyzed by ultraviolet-visible spectroscopy.2.The study on pH/near infrared dual response and NMR/photoacoustic dual-mode imaging capabilities of ZIF-8/DMPP nanoparticlesTo evaluate its in vitro photothermal performance,the aqueous dispersion of ZIF-8/DMPP nanoparticles with various concentrations was illuminated by an 808 nm near-infrared(NIR)laser for 5 min and the solution temperature was monitored and recorded.The prostate cancer cells PC-3 were subcutaneously injected into the back of nude mice to establish the xenografted tumor model,into which the suspension of ZIF-8/DMPP nanoparticles was injected into the tumor site in situ,and then the tumor site was subjected to 808 nm near-infrared laser irradiation to assess the in vivo photothermal imaging ability.To evaluate the degradation performance of ZIF-8/DMPP in response to pH,ZIF-8/DMPP nanoparticles were dispersed in a medium of pH=7.4 and pH=5.5,respectively.Then the morphological changes of nanoparticles in the two solutions was observed by TEM at predetermined time points,and the concentrations of Zn2+and Mn2+ were detected by ICP-MS.To assess the in vitro drug release,the DOX release of ZIF-8/DMPP nanoparticles solutions in pH 7.4 and pH 5.5 medium was measured by ultraviolet-visible light spectroscopy.For photothermal-triggered drug release,the solutions were irradiated by an 808 nm NIR laser for 10 min,and the medium was also extracted and subjected to UV-vis spectroscopy.Furthermore,the DOX release experiment was also conducted under the same condition in the absence of NIR laser irradiation.To assess the in vitro magnetic resonance(MR)imaging ability,ZIF-8/DMPP dispersion with different concentrations,in which the Mn2+ concentrations were 0.05 mol/L,0.1 mol/L,0.2 mol/L,0.4 mol/L and 0.8 mol/L,was subjected to an NMR analyzing and imaging system to measure their relaxation times,the transverse relaxivity(r1)of the nanoparticles is calculated,and then the T1-weighted MR imaging of each dispersion was collected.For in vivo MR imaging ability assessment,ZIF-8/DMPP nanoparticles suspension(2 mg/mL,0.1 mL)was injected into the tumor area of prostate cancer subcutaneously transplanted tumor-bearing mice,and the T1-weighted MR imaging of tumor site was collected before injection and 30 minutes after injection,respectively.To perform in vivo photoacoustic(PA)imaging,the ZIF/DMPP suspension was also injected into the tumor(0.1 mL,2 mg/mL)and the PA images of the tumor were acquired on Vevo LAZR photoacoustic imaging system.3.Evaluation of the anti-tumor effect of ZIF-8/DMPP nanoparticlesTo explore the cellular uptake of ZIF-8/DMPP nanoparticles,PC-3 cells were incubated with free DOX,ZIF-8/DMPP nanoparticles loaded with the same amount of DOX,and FITC-labeled ZIF-8/MPP nanoparticles without DOX,and then irradiated with or without an 808 nm near-infrared laser.Then cells were collected and stained with DAPI,following by collecting images to observe the uptake of nanoparticles using Confocal laser scanning microscope(CLSM).The flow cytometry was also conducted to detect the fluorescence intensity of each group of cells.To evaluate the cytotoxicity of the nanoparticles,PC-3 cells and HUVEC cells were incubated with different concentrations of ZIF-8/MPP,following by CCK-8 to determine their cell viability.To assess the therapeutic efficacy on PC-3 cells in vitro,the cells were co-incubated with free DOX,ZIF-8/DOX,ZIF-8/MPP and ZIF-8/DMPP,respectively,and irradiated with or without an 808 nm near-infrared laser.Then cells in each treatment group were collected for CCK-8 and Calcein AM and PI stain to detect cell viability and cell survival.To evaluate the anti-tumor effect of nanoparticles in vivo,ZIF-8/DMPP and ZIF-8/MPP dispersion were injected into tumor-forming sites of tumor model mice,respectively,and subjected to near-infrared laser irradiation.And then the tumor sizes and mice body weights were recorded every day.After 14 days,the tumor tissue was isolated for histological analysis.To evaluate the histological toxicity of drug-free nanoparticles,the main organs were isolated for histological analysis after intravenous injection of ZIF-8/MPP into mice at different doses(10 and 20 mg/kg).Results1.ZIF-8/DMPP nanoparticles loaded with chemotherapeutic drug DOX were successfully synthesized,which are regular spheres with good crystal structure and Zn,C,O,N,and Mn are uniformly distributed in the nanoparticles.The average hydrodynamic diameter and average Zeta potential of ZIF-8/DMPP is about 285 nm and-(22.4±0.9)mV,respectively.The high negative surface charge of ZIF-8/DMPP makes them have good dispersibility and colloidal stability in PBS,cell culture fluid and water.2.After being irradiated by 808 nm near-infrared laser(2 W/cm2),the temperature of ZIF-8/DMPP nanoparticle dispersion(200 μ g/mL)quickly increased and maintained at 45-50℃,and its photothermal efficacy increased corresponding with the concentration of nanoparticles.The evaluation of the photothermal effect in vivo found that the temperature of tumor site injected with ZIF-8/DMPP increased quickly and maintained at about 47.5℃ after 7 minutes.These results indicating that the nanoparticles possess good photothermal efficacy in vitro and in vivo.TEM results showed that the morphological characteristics of ZIF-8/DMPP nanoparticles dispersed in a neutral matrix(pH=7.4)are stable,and the release of Zn2+and Mn2+is extremely low.While the nanoparticles dispersed in an acidic matrix(pH=5.5)displayed obvious framework disintegration at 0.5 h,only small fragments were observed after 6 h incubation,and the average release of Zn2+and Mn2+reached 57.1%and 12.5%,respectively.These results revealed that ZIF-8/DMPP can achieve pH-responsive degradation.The DOX release of ZIF-8/DMPP dispersion with pH=7.4 was detected at 5%after 24 hours incubation,and NIR laser irradiation did not promote the release of DOX.While the DOX release of ZIF-8/DMPP dispersion with pH=5.5 was at about 30%after 24 hours incubation,and the amount of DOX released increases significantly after NIR laser irradiation.These results indicated that the drug release of ZIF-8/DMPP nanoparticles is pH-dependent and can be accelerated by NIR laser.The relaxation performance test found that the r1 relaxation rate of ZIF-8/DMPP nanoparticles was 6.03 mM-1s-1,and its in vitro MR imaging ability increased corresponding with Mn2+ concentration.In addition,the MRI brightness of tumor region was significantly enhanced after in situ injection with ZIF-8/DMPP,indicating that the nanoparticles can be applied as a good contrast agent for in vivo MR imaging.Furthermore,the PA signals in the tumor region are clearly stronger after injection of ZIF-8/DMPP,demonstrating the PA imaging ability of the prepared nanocomposites.3.The results of CLSM showed that red fluorescence was visible in the cytoplasm of PC-3 cells after incubation with ZIF-8/DMPP,and the fluorescence intensity increased after NIR laser irradiation.The flow cytometry analysis also revealed that the DOX fluorescence peak of the NIR irradiated group was distinctly right-shifted,indicating that ZIF-8/DMPP can be taken up by PC-3 cells and the photothermal effect can enhance the uptake effect.CCK-8 analysis showed that all concentrations of ZIF-8/MPP did not affect the viability of PC-3 and HUVEC cells,indicating that the cytotoxicity of the nanoparticle was low.The cell viability of PC-3 cells without drug treatment remained the same after irradiation with different intensities of NIR lasers.The cell viability of PC-3 in the ZIF-8/DOX treatment group and the free DOX group did not decrease significantly,and no significant change was observed after the NIR laser irradiation,suggesting that neither photothermal nor chemotherapy alone had a better killing effect on PC-3 cells.By comparing blank control,the cell viability in ZIF-8/DMPP group and ZIF-8/MPP combined with NIR laser irradiation group,was found to be significantly higher than that in ZIF-8/DMPP combined with NIR laser groups.Furthermore,the higher the concentration of ZIF-DMPP nanoparticles,the stronger the killing effect on PC-3 cells.After in situ injection 14 days with ZIF-8/DMPP into tumor site and NIR laser irradiation,the tumor volume was reduced to 0.17 times of that before treatment,while the tumor volume of mice in ZIF-8/DMPP group and ZIF-8/MPP combined with NIR laser group was about 7 times and 4 times that before treatment,respectively.Histopathological evaluation found that the chemotherapy-photothermal treatment group had a large area of cell fragments and necrosis in the tumor tissue,while the tumor tissue of the other groups maintained retained relatively complete cell morphology.The main organs of the mice after intravenous injection of ZIF-8/MPP showed no abnormal histological morphology,indicating that it has good histocompatibility.ConclusionsZIF-8/DOX synthesized by one-pot approach achieves high loading of chemotherapeutic drug DOX.The modification of PDA not only renders the resultant nanoconstruct with good photothermal effect but also facilitates its further surface functionalization with Mn ions and PEG.The ZIF-8/DMPP exhibits desirable degradation behavior and sensitive pH-dependent drug release.Meanwhile,the data demonstrate that the photothermal effect of ZIF-8/DMPP could also accelerate the drug release rate,thereby making ZIF-8/DMPP a smart pH/NIR dual-responsive drug delivery system.More importantly,it is also affirmed that the photothermal effect could significantly enhance the cellular uptake of ZIF-8/DMPP.As a consequence,the intracellular drug concentration was improved and the therapeutic efficiency was augmented due to the synergistic effect.In addition,both the T1-weighted MR imaging and PA imaging capacities are demonstrated in vivo.The in vitro and in vivo antitumor experiment also suggests the excellent therapeutic efficiency of ZIF-8/DMPP under laser irradiation on prostate cancer cells and tumor-bearing mice.Taken together,the presented data manifest that the developed nanoconstruct holds great potential for smart drug delivery and prostate cancer theranostics.