Selection Of Peptide Specifically Binding To Bladder Carcinoma By In Vivo Phage Display

Objective:To screen the peptide binding to human bladder carcinoma cells specifically by phage display technology in vivo. Methods:Nude mice were inoculated with bladder carcinoma cells BIU87 for establishing tumor-bearing mice model.The Ph.D.-C7 CTM Peptide Library was injected intravenously via tail vein. Then we screened Phage containing exogenous peptides binding to bladder transitional carcinoma cells specifically. The phage peptide homed to the tumor tissues was obtained after 3 rounds screening in vivo. The phage clones affinity to BIU87 were identified by immunohistochemistry and ELISA. The positive peptide was synthetized by chemical methods after sequencing the positive monoclonal phage DNA. Amino acid sequences of exogenous peptide phage were deduced and analysed. The synthetic peptides and control peptides were obtained commercially to our specifications, and FITC-conjugated peptides were also obtained. The cytotoxicity of peptide and FITC-conjugated peptide was detected with MTT. The specificity of homing peptide for tumor cell and pathology was identified by confocal laser scanning microscope and flow cytometry. Results:1. Tumor-bearing mice model with bladder carcinoma cells BIU87 was established successfully in nude mice. The phage titer and phage recovery were manipulated after every round of biopanning, which was 1.324×10-5 pfu/g,2.526×10-5 pfu/g,5.738×10-3 pfu/g respectively for three round. The phage specifically binding to the tumor was gradually increased, which was 4.334×102-fold at the third round.2. We randomly selected 30 monoclonal phage plaques after the third round for enzyme-linked immunosorbent assay with 96-well culture dishes. The results of ELISA showed that 24 monoclonal phages’ affinity was positive(P/N>2.0) and ten of them had a higher binding activity(P/N>5.0). Three scequences, CSSPIGRHC(8/10), CTMSNLKGC)(1/10) and CNNVLSQMC(1/10),were accquired at last. The test implied that the phage clone identified was not by chance with using the software Ex PASy /Prot Param and NCBI/BLAST. Therefore, the peptide CSSPIGRHC which was named NYZL1 was the most unique sequence for further testing.3. NYZL1 and FITC-NYZL1 were fused by way of solid-phase synthesis.Then we incubated them with BIU87 to observe cytotoxicity of peptides. The results show that peptides had no effect on proliferation and function of BIU87 at range of ~ 100μmol / L within 24 hours.4. The binding of FITC-NYZL1 and FITC-sv NYZL1 to BIU87 was observed via the confocal microscope. There was highly enriched fluorescent on the cytoplasm and the nucleus of BIU87 in group of FITC-NYZL1, suggesting that FITC-NYZL1 had affinity and specificity to BIU87.5. The binding rate of FITC-NYZL1 and FITC-sv NYZL1 to BIU87 was observed by flow cytometry. The difference was statistically significance(P <0.01).The value of fluorescence intensity in FITC-NYZL1 was 53.1925 ± 1.35, meanwhile that of FITC-sv NYZL1 was 15.17 ± 1.06 and the blank control was 6.67 ± 0.10. It implied that FITC-NYZL1 had affinity and specificity to BIU87.6. FITC-NYZL1 and FITC-sv NYZL1 were incubated with tissue paraffin from patients with bladder cancer and adjacent normal tissue. The results from fluorescence microscopy showed that, fluorescent of group FITC-NYZL1 had a higher enrichment than group FITV-sv NYZL1 in bladder tumor tissue sections. However, fluorescent display was weak in adjacent normal tissue sections.All of them indicated the specific and highly affinity of NYZL1 binding to bladder cancer tissues.Conclusions:We obtained the small molecular peptide NYZL1 binding to human bladder carcinoma specifically by means of phage display in vivo, which provide a theoretical basis for bladder carcinoma early diagnosis and targeted therapy.