Experimental Study On Visualization Of Nerve Density And Targeted Therapy For Prostate Cancer

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Experimental study on the targeting of NP41 to the autonomic nerve of prostate cancerObjective: To investigate the targeting of nerve peptide 41(NP41)to the autonomic nerve of orthotopic transplantation tumor model of prostate cancer(PCa)in Bal b/c Nude male mice,and to evaluate the feasibility of NP41 in the visualization of nerve density of PCa.Materials and methods: By building the orthotopic transplantation tumor model of PCa in Bal b/c Nude male mice,hematoxylin-eosin(H&E)staining and autonomic nerve markers(TH,VACh T,NF-H,NF-L)immunofluorescence staining to detect PCa autonomic nerve,western blot(WB)to detect expression levels of NP41-binding targets,laminins(Lams),in PCa tissues and healthy prostate tissues.Cy7-NP41 was synthesized and characterized by qualitative and quantitative analysis of fluorescence imaging(FLI),mass spectrometry,UV-vis spectrophotometer,excitation and emission spectra,serum stability and cytotoxicity.Through the orthotopic transplantation tumor model of PCa in mice,FLI was performed using IVIS Spectrum imaging system in vivo and in vitro,autonomic nerve markers(TH,VACh T,NF-H,NF-L)and Lams(Lam?4?Lam?2?Lam?2?Lam?1?Lam?1)colocalizes with NP41 binding,respectively.Results: The orthotopic transplantation tumor model of PCa in Bal b/c Nude male mice was successfully established.The distribution of PCa autonomic nerve were demonstrated by immunofluorescence staining of TH,VACHT,NF-H and NF-L.WB qualitative and quantitative results showed expression levels of Lam were up-regulated in PCa tissues in comparison with healthy prostate tissue.The successful synthesis of Cy7-NP41 was confirmed by FLI qualitative and quantitative analysis,mass spectrometry,UV-vis spectrophotometer,excitation and emission spectra,serum stability and cytotoxicity.In vivo,FLI revealed that Cy7-NP41 was superior to Cy7 in targeting autonomic nerve of orthotopic transplantation tumor of PCa in mice.In vitro,NP41 localized similarly in PCa tissue sections fluorescently stained not only for autonomic nerve markers(TH,VACh T,NF-H,NF-L)but also for Lams(Lam?4?Lam?2?Lam?2?Lam?1?Lam?1),suggesting that NP41 can target nerves in PCa with high specificity,revealing its potential in visualizing tumor innervation in PCa.Conclusion: The orthotopic transplantation tumor of PCa in mice was successfully constructed with rich distribution of autonomic nerve fibers.The expression level of Lam in PCa tissue was up-regulated.Through FLI,Cy7-NP41 and a variety of molecular biotechnologies revealed that NP41 is a remarkable nerve peptide and can enhance the specificity of visualization of nerve density of PCa.Part ? Magnetic resonance imaging and magnetic particle imaging of nerve density in prostate cancer mediated by PSN NPsObjective: To develop a molecular probe PSN NPs with magnetic resonance imaging(MRI)and magnetic particle imaging(MPI)signals,which can specifically target the autonomic nerve of prostate cancer(PCa).The orthotopic transplantation tumor model of PCa in mice was used to observe the imaging characteristics by MRI and MPI under the mediation of PSN NPs.Combined with histology to verify that PSN NPs can achieve high sensitivity and high specificity nerve density visualization of PCa.Materials and methods: On the basis of superparamagnetic iron oxide nanoparticles(SPIO NPs),PSN NPs were synthesized through coupling nerve peptide 41(NP41)and loading propranolol,and characterized by transmission electron microscope(TEM), malvern particle size analyzer,Zeta potential analyzer,UV-vis spectrophotometer,MRI,MPI,vibrating sample magnetometer(VSM)and high-performance liquid chromatography(HPLC).The orthotopic transplantation tumor model of PCa in mice was established.After the model was successfully constructed,the mice were randomly divided into two groups: PSN NPs with NP41(experimental group)and PS NPs without NP41(control group)were systemic administration,and MRI and MPI scans were performed at different time points.The imaging differences of MRI and MPI of orthotopic transplanted tumor of PCa in the two groups were qualitatively and quantitatively analyzed.Prussian blue staining was used to detect the distribution of PSN NPs and PS NPs in prostate tumor,liver,spleen and kidney of the two groups of mice.Results: The molecular probe PSN NPs with MRI and MPI signals coupled with NP41 and loaded propranolol was successfully synthesized through a series of characterization.PSN NPs has high monodispersity and homogeneity,high coupling rate of NP41 and loading content of propranolol.PSN NPs has superparamagnetism and excellent MRI and MPI signals.The MRI and MPI signal intensities of PCa orthotopic transplanted tumor in PSN NPs group(experimental group)were significantly higher than that in PS NPs group(control group)and there were statistically significant differences in tumor/normal tissue ratio(TNR)and MPI signal intensities between experimental group and control group.The aggregation of PSN NPs in PCa of the experimental group was significantly higher than that of the control group.There were different degrees of PSN NPs and PS NPs aggregation in the liver and spleen of the two groups of mice,but no PSN NPs and PS NPs aggregation in the kidney.Conclusion: A molecular probe,PSN NPs,which can specifically target the autonomic nerve of PCa was successfully developed.MRI and MPI mediated by PSN NPS can achieve highly sensitive and specific visualization of PCa nerve density in vivo.Part ? Application of PSN NPs combined with magnetic resonance imaging and magnetic particle imaging in evaluating nerve density of prostate cancerObjective: To investigate whether PSN NPs-mediated magnetic resonance imaging(MRI)and magnetic particle imaging(MPI)can distinguish between the high and low nerve density of prostate cancer(PCa),and to investigate the correlation of MRI and MPI signal intensities with nerve densities and Ki-67 indexes of PCa,respectively,so as to provide imaging basis for targeting PCa neural microenvironment.Materials and methods: The experimental animals were orthotopic transplantation tumor models of PCa with high-and low-nerve-density in mice.Orthotopic PCa models with high-nerve-density(PBS group and sham-operated group)were constructed by phosphate buffered saline(PBS)or sham operation(sham-operated),and by 6-hydroxydopamine(6OHDA)or surgically severed afferent hypogastric nerves(HGNx)to build a low-nerve-density of orthotopic PCa models(6OHDA group and surgical HGNx group).Bioluminescence imaging(BLI)and histological methods(immunofluorescence staining of autonomic nerve markers of PCa and Ki-67 immunohistochemical staining)were used to verify the successful construction of the models.After the establishment of orthotopic transplantation tumor models of PCa with high-and low-nerve-density in mice,MRI and MPI scans were performed by tail vein injection of PSN NPs(6mg/kg)to observe the enrichment of PSN NPs in PCa regions of different groups.The differences of MRI and MPI signal intensities between high-and low-nerve-density group of PCa were compared by unpaired t-test.P<0.05 was considered statistically significant.Spearman rank correlation test was used to analyze the correlation of MRI and MPI signal intensities with nerve densities(TH,VACHT,NF-H and NF-L)and Ki-67 indexes of orthotopic transplantation tumor models of PCa with high-and low-nerve-density in mice.Further using FLI to verify PSN NPs is helpful to distinguishing between the high and low nerve density of PCa.Results: The orthotopic transplantation tumor models of PCa with high-and low-nerve-density in mice were successfully generated by drug and surgical intervention,which realistically simulate the neural microenvironment of PCa.Qualitatively and quantitatively analyzed MRI and MPI images after systemic administration of PSN NPs in mice with high-and low-nerv-density PCa orthotopic transplantation tumor models.The results showed that there were statistically significant differences in TNR and MPI signal intensities between the high-nerve-density group and the low-nerve-density group of PCa.By analyzing the correlation of MRI and MPI signal intensities with nerve densities and Ki-67 indexes of orthotopic PCa in different groups,it was found that MRI signal intensities were negatively correlated with nerve densities and Ki-67 indexes,while the MPI signal intensities were positively correlated with nerve densities and Ki-67 indexes.These results show that MRI and MPI signal intensities were significantly correlated with the nerve densities and Ki-67 indexes of PCa.Conclusion: PSN NPs-mediated MRI and MPI contribute to evaluate the high and low nerve density and invasiveness of PCa in vivo,and have potential application value in the clinical diagnosis and treatment of PCa.Part ? PSN NPs-mediated targeted therapy for prostate cancerObjective: To evaluate the efficacy of PSN NPs,loaded with the ?-blocker propranolol,mediated targeted therapy for prostate cancer(PCa).And to improve the effectiveness and application range of PSN NPs,and provide a new strategy and new method for the treatment of PCa.Materials and methods: The orthotopic transplantation tumor models of PCa in mice were established.After 10 days,the mice were randomly divided into PBS group,SN NPs group,P group and PSN NPs group.The mice in different groups were treated with PBS,SN NPs,P and PSN NPs twice a week,for 3 weeks.The progress and metastasis of orthotopic transplantation tumor models of PCa in mice of the PBS group,SN NPs group,propranolol group and PSN NPs group were evaluated by BLI,body weight and survival time observation,and the detection values of each group were compared by statistical analysis.The ex vivo specimen,H&E staining and tumor cell apoptosis of tumor tissues and metastatic lymph nodes were measured and evaluated in the PBS group,SN NPs group,propranolol group and PSN NPs group on day 45 of treatment.The nerve densities,laminin(Lam)densities and Ki-67 indexes of orthotopic transplantation tumor models of PCa in mice in PBS group,SN NPS group,propranolol group and PSN NPS group were detected by immunofluorescence staining.The systemic toxicity and side effects of PSN NPs on mice in acute,subchronic and chronic stages were evaluated by detecting the biochemical function indexes of normal organs,histological morphology,nerve function and the expression of nerve markers in normal organs of mice.Statistically different significance between groups was determined by nonparametric one-way analysis with Kruskal-Wallis.The Kaplan-Meier method was used to illustrate the survival curves and the log-rank test was used to determine the statistical significance of difference between survival curves.P < 0.05 was considered statistically significant.Results: The progress of orthotopic transplantation tumor models of PCa in mice in PBS group,SN NPs group,propranolol group and PSN NPs group was evaluated by qualitative and quantitative analysis of BLI,body weight,survival curve,ex vivo specimen and H&E staining of tumor tissue and metastatic lymph nodes,and apoptosis analysis of tumor cells.It was confirmed that PSN NPs had the most significant inhibitory effect on the progression of PCa.Compared with the control group,PSN NPs increased the survival rate of mice with orthotopic PCa to 83.3%,and the nerve densities,Lam densities and Ki-67 indexes of PCa tissue decreased by more than twofold times.After PSN NPs treatment,the important blood biochemical indexes and histomorphology of heart,liver and kidney in mice were not abnormal in acute,subchronic or chronic stages,and nerve function and the expression of nerve markers in normal tissues were not affected.Conclusion: PSN NPs can targete delivery of propranolol to the tumor neural microenvironment and target therapy for PCa,and inhibit the progress of PCa.PSN NPs not only have no toxic effects on normal tissues,but also had no side effect toward the nerve function and expression of nerve markers in normal tissues.