Selection Of Cervical Cancer Specific Binding Peptides And Study On Targeted Radiosensitization Of AuNPs-PEG-(CSP-GD)

Cervical cancer is a common malignant tumor in the female.At present,Radiotherapy is a one of main method of the clinical treatment About 80%of patients with cervical cancer have been treated with radiotherapy,however,30%-50%of those with advanced cancer appeared radiotherapy resistance.Therefore,it is very important to improve the sensitivity of radiotherapy for patients with advanced cervical cancer.The targeted radiosensitizer markedly enhances radiosensitization of cancer cells.Its application is an effective way for improved efficacy of Radiotherapy.But,now there were not yet suited sensitizer using to clinicThe development of nanotechnology provides a lot of theoretical basis and technical support for the research and development of new targeted radiosensitizer.Gold nanoparticles(AuNPs,GNPs)possess a number of unique properties that make them ideal candidates as radiosensitizers on the basis of their strong photoelectric absorption coefficient,surface plasmon resonance,enhanced permeability and retention effect(EPR)with tumor tissues,ease of synthesis and modification,controllable for particle size and morphology,and good biocompatibility.In this project,AuNPs have been modified with cervical cancer specific binding peptides(CSPs)selected by in vivo phage display technology for achieving its active targeting to cervical cancer,enhancing its tumor radiosensitization efficacy,and reducing side effects.It will provide new methods and effective exploration for targeted radiosensitization therapy of cervical cancerThe experimental study was carried out in three parts to develop a new targeted radiosensitizer for cervical cancer,and to investigate its targeting and radiosensitizing effect on cervical cancerPart Ⅰ Selection of cervical cancer specific binding peptidesObjective To select CSPs with specific target binding ability for cervical cancer by in vivo phage display technologyMethods:Peptide library screening in vivo was performed on human cervical cancer xenografts with Ph.D.TM-12 and C7C phage display peptide libraries.The phage library was injected into the tail vein of the tumor-bearing nude mice.After screening 3 or 4 rounds,the phages in tumor tissues and control tissues(heart,liver,kidney and brain tissues)were recovered.The enrichment of the phage in the tumors was detected by phage recovery rate and immunohistochemical staining.After the third or fourth round,individual phage clones were selected.Phage monoclonal affinity to cervical cancer cells was determined by phage ELISA.High affinity phage monoclonal DNA extracted by the QIAprep Spin M13 Kit were sequenced.The amino acid sequences of the displayed peptides were deduced from the DNA sequences of the phages.The basic physicochemical properties and homology of polypeptides were analyzed Selected CSPs phages amplified individually were administered into the blood circulation of tumor-bearing mice via tail veins injection to validate the in vivo tumor-targeting ability.Immunofluorescent cytochemical staining was carried out to confirm the targeting ability of CSPs to cervical cancer cells.Tissue microarrays were used to confirm the affinity of CSPs to human cervical cancer tissuesResults:After screening 3 or 4 rounds on human cervical cancer xenografts(SiHa,C-33A,ME-180)with Ph.D.TM-12 and C7C phage display peptide libraries,the phages were significantly enriched in the tumor tissues,and the recovery rate was significantly increased(P<0.05 vs.Round 1),while there was no such change in the corresponding control tissues(heart,liver,kidney and brain),which was also confirmed by immunohistochemical staining.The results of cell ELISA showed that each group of phage monoclones had good specific binding ability to cervical cancer cells.A total of 162 individual phage clones[SiHa(12p)48,SiHa(7p):10;C-33A(12p):40,C-33A(7p):35);ME-180(12p):17,ME-180(7p):12]of the plated bacterial culture from the final round selection,including those phage clones used for cell-based ELISA,were sequenced.The amino acid sequences of the displayed peptides were deduced from the DNA sequences of the phages.The five predominant peptide sequences(GDALFSVPLEVY,TLHQPPSSANWI,FTPGGNTYAGQP,SIDDQR DVAEFA,and KQNLAEG)were identified and sequentially named as CSP-GD,CSP-TL,CSP-FT,CSP-SI,and CSP-KQ,respectively,for further experiments.To validate the in vivo specific targeting of the CSP phages,we injected the CSP phages(CSP-GD,CSP-TL,CSP-FT,CSP-SI,and CSP-KQ phages)and insertless phage as a negative control into the circulation of the whole body through the tail vein of the tumor-bearing mice for 15 min.The results showed that CSP-GD,CSP-FT,CSP-SI and CSP-KQ phages were significantly enriched in tumor tissues,and the recovery rate was significantly increased compared with the control tissues(liver and kidney)(P<0.05),while CSP-TL phage was not significantly enriched in tumor tissues,and its recovery rate was not significantly increased compared with control tissues(P>0.05).These indicate that the four CSP phages(CSP-GD,CSP-FT,CSP-SI,and CSP-KQ phages)can specifically target the cervical cancer xenograft in vivo.The tumor-targeting ability of CSPs was evaluated in human cervical cancer cells in vitro through immunofluorescent cytochemical staining.The results confirmed that CSP-GD,CSP-FT,CSP-SI,and CSP-KQ possess specific targeting ability to cervical cancer cells,and the binding sites are mainly located on cell membrane and cytoplasm.Tissue immunofluorescence staining showed that CSP-GD,CSP-FT,CSP-SI and CSP-KQ had certain antigen specificity to human cervical cancer tissues,especially CSP-GD and CSP-KQConclusions:Four CSPs(12-peptide CSP-GD,CSP-FT and CSP-SI,7-peptide CSP-KQ)were screened;CSPs have specific targeting to human cervical cancer cells and antigen specificity to human cervical cancer tissues,especially CSP-GD and CSP-KQPart Ⅱ Synthesis and characterization of AuNPs-PEG-(CSP-GD)as a gold nanoparticle targeting carrier systemObjective:To develop a gold nanoparticle targeting carrier system,AuNPs-PEG-(CSP-GD),for targeted radiotherapy of cervical cancerMethods:In a particular situation,AuNPs were synthesized by reducing gold chloride in the presence of sodium citrate.And then modified with polyethylene glycol(PEG)and CSP-GD,AuNPs-PEG and AuNPs-PEG-(CSP-GD)were synthesized successively.The preparation conditions of AuNPs-PEG-(CSP-GD)were optimized.The samples were characterized by transmission electron microscopy(TEM),Malvern laser particle size analyzer,X-ray photoelectron spectrometer(XPS),ultraviolet-visible spectrometer(UV-Vis),fluorescence spectrophotometer,etc.The concentrations of gold in gold nanoparticles solution were measured by inductively coupled plasma mass spectrometer(ICP-MS)and the concentrations of CSP-GD were detected by Micro BCA methodResults:AuNPs with uniform size and good monodispersity were successfully synthesized by reducing gold chloride in the presence of sodium citrate,with particle size of 14.48 ± 1.17 nm.The optimized conditions for the preparation of AuNPs-PEG-(CSP-GD)were as follows the mass of gold element in the reaction was 0.1 mg,the pH value of the reaction system adjusted by MES solution was 6.5,the reaction temperature was 16℃,the reaction duration was 8 h,and the reaction mass ratio of AuNPs-PEG to CSP-GD was 1:20.The AuNPs-PEG-(CSP-GD)synthesized under the optimized conditions had good monodispersity and stability with a particle size of 65.91±1.44 nm.The concentration of gold in gold nanoparticles solution was 9.966±0.013 g/mL measured by ICP-MS,and the CSP-GD amount coupling in AuNPs-PEG-(CSP-GD)solution was 3.22±0.11g/mL indirectly detected by Micro BCA method.The results of XPS,UV-Vis and fluorescence spectroscopy showed that CSP-GD was successfully modified to the surface of AuNPs-PEGConclusions:Gold nanoparticles targeting carrier system AuNPs-PEG-(CSP-GD)was successfully synthesizedPart Ⅲ Targeting and radiosensitization of AuNPs-PEG-(CSP-GD)Objective:To verify the targeting of AuNPs-PEG-(CSP-GD)to cervical cancer cells in vitro and in vivo,and to further explore its radiosensitizing effect on cervical cancer cells in vitroMethods:The in vivo and in vitro targeting ability of AuNPs-PEG-(CSP-GD)to cervical cancer cells was analyzed by cell immunofluorescence,flow cytometry and small animal in vivo imaging CCK-8 assay was used to detect the proliferative toxicity of CSP-GD and AuNPs-PEG-(CSP-GD)on cervical cancer cells SiHa and C-33A.Under irradiation,cell clonogenic assay and flow cytometry were used to detect the radiosensitizing effect of AuNPs-PEG-(CSP-GD)on cervical cancer cells.Results:The results of cellular immunofluorescence showed that the uptake of AuNPs-PEG-(CSP-GD)by SiHa or C-33A cells co-incubated with coumarin 6-labeled gold nanoparticles(Au final concentration of 10 g/mL)for 2,6 and 12 h was significantly higher than that of AuNPs-PEG and AuNPs(P<0.05).The results of flow cytometry were consistent with this.In vivo imaging results of small animals showed that AuNPs-PEG-(CSP-GD)was significantly enriched in tumor tissues(P<0.05),indicating that AuNPs-PEG-(CSP-GD)had good targeting ability to cervical cancer tissues in vivo.The CCK-8 assay showed that CSP-GD did not significantly inhibit the growth of Siha and C-33A cells(P>0.05),and Low concentration of AuNPs,AuNPs-PEG and AuNPs-PEG-(CSP-GD)had no significant toxic effect on SiHa and C-33A cells(P>0.05).The results of cell clone formation showed that when the concentration of AuNPs-PEG was 5 μg/ml and 10 μg/ml,the SERD0 was 1.05 and 1.10 respectively;when the concentration of AuNPs-PEG-(CSP-GD)was 5 μg/ml and 10 μg/ml,the SERD0 was 1.49 and 1.57 respectively,and the sensitization effect of AuNPs-PEG-(CSP-GD)was significantly better than that of AuNPs-PEG(P<0.05).The results of flow cytometry showed that the combination of gold nanoparticles and radiation had the strongest ability to induce apoptosis of SiHa cells,and the apoptosis rate of AuNPs PEG-(CSP-GD)+radiation group(30.43±3.32%)was significantly higher than that of AuNPs-PEG+radiation group(21.24±3.95%)(P<0.05)Conclusions:AuNPs-PEG-(CSP-GD)has good targeting in vivo and in vitro on human cervical cancer cells,and radiosensitizing effect in vitro.