Screening And Identification Of The Peptides Specifically Binding To Human Non-small Cell Lung Cancer NCI-H1299 Cells

Objective:Using in vivo phage display technology to screen small-molecular peptide that bind to human non-small cell lung cancer NCI-H1299 cells and to identify their binding specificity and targeting in vivo and in vitro.Methods:1.Human non-small cell lung cancer NCI-H1299 cells were inoculated into nude mice to prepare tumor-bearing nude mice models,using Ph.D.-C7 CTM Peptide Library to screen tumor-bearing nude mice for 3 rounds.Immunohistochemistry was used to identify the distribution of phages in tumor tissues and normal control tissues.Enzyme-linked immunosorbent assay(ELISA)was used to detect the binding affinity of phage clones to NCI-H1299 cells and identify positive monoclonal phages.2.The positive monoclonal phages DNA were extracted and sequenced to obtain the amino acid sequence of the exogenous poly-peptide.The sequence with the highest repetition rate was synthesized into a targeting peptide NSP1,and fluorescein(FITC)labeling was performed to synthesize a fluorescent probe FITC-NSP1.The binding specificity of the synthesized targeting peptide to NCI-H1299 cells was identified by the competitive inhibition experiment of peptide and phage.3.CCK8 method was used to detect the effect of fluorescent probe FITC-NSP1 on the proliferation and activity of NCI-H1299 cells;the cell scratch test and Transwell test were used to detect the effect of fluorescent probe FITC-NSP1 on the migration and invasion of NCI-H1299 cells.4.Through cell immunofluorescence experiments,observe the binding of FITC-NSP1 and cells under an inverted fluorescent microscope to identify the specificity of FITC-NSP1 to NCI-H1299 cells;flow cytometry detects the FITC-NSP1 binding specific for NCI-H1299 cells.5.Detect the targeting of fluorescent probes FITC-NSP1 to tumor tissues in vivo by small animal in vivo imaging experiments,and identify the optimal concentration of fluorescent probes and the best imaging time.Separate tumor tissues and normal tissues for in vitro experiments to further identify the targeting of fluorescent probes.Results:1.After three rounds of screening of the phage peptide library in tumor-bearing nude mice,phage clones that specifically bind to NCI-H1299 cells were enriched.The phage titer results showed that the phages enrichment rate was 341.3 times compared with the first round,and the enrichment effect was obvious.2.Immunohistochemical staining showed that with the increase of screening times,the phages binding to tumor tissues continued to increase.After the third round of screening,the phages binding to tumor tissues were significantly higher than normal tissues.3.ELISA results showed that 20 of the 30 phage clones picked at random were positive phage clones(affinity>2.5).4.The sequencing results of positive phage clones showed that the sequence CTNESIGTC had the highest repetition rate,and there was no similarity with the known amino acid sequence in the database.This sequence was synthesized targeting peptide and named NSP1,and FITC labeling was carried out at the same time,the targeting fluorescent probe FITC-NSP1 and the random control fluorescent probe FITC-sv NSP1 were synthesized.5.The competitive inhibition experiment between peptide and phage showed that the targeting peptide NSP1 can compete with the corresponding positive phage clone to bind to NCI-H1299 cells.6.The results of CCK8 showed that FITC-NSP1 has no significant effect on the proliferation of NCI-H1299;the results of cell scratch test and Transwell test showed that FITC-NSP1 will not affect the migration and invasion of NCI-H1299 cells in each detection period.7.Cellular immunofluorescence results showed that the fluorescence intensity of FITC-NSP1 combined with NCI-H1299 cells was 103.553±8.412,while the fluorescence intensity of FITC-sv NSP1 combined with NCI-H1299 cells was9.653±0.880.The difference between the two was statistically significant(P<0.01).And the FITC-NSP1 was not combined with other cancer cells and normal cells(P<0.01),indicating that FITC-NSP1 is specific to NCI-H1299 cells.8.The results of flow cytometry showed that the positive percentage of FITC-NSP1 labeled NCI-H1299 cells was 70.54±2.15,which was significantly higher than other cells(P<0.01).The percentage of FITC-sv NSP1 labeled NCI-H1299 cells was 7.22±0.36,and the difference between with the FITC-NSP1 was statistically significant(P<0.01).9.The results of in vivo imaging of small animals showed that FITC-NSP1 gradually accumulate in the tumor site in vivo,which is targeted,and the fluorescence intensity of tumor tissue reaches the maximum at 4h.The results of in vitro experiments showed that FITC-NSP1 will not be enriched in other normal tissues.Conclusion:In this study,we used in vivo phage display technology to screen the small peptide NSP1 that specifically binds to NCI-H1299 cells,and identified the binding specificity and targeting of NSP1 optical molecular probes to lung cancer cells in vivo and in vitro,which lays a foundation for the early diagnosis and targeted therapy of non-small cell lung cancer.