Identification Of A Peptide Capable Of Targeting Human Osteosarcoma Cells And Its Preliminary Application

Osteosarcoma(OS)is the most frequent malignant musculoskeletal tumor occurring primarily in childhood and adolescence.The treatment regime for osteosarcoma has advanced from amputation to the combination of complex limb-sparing surgeries and intensive multi-agent chemotherapy,while about 30%of OS patients would not able to survive over five years.In fact,in the past 30 years,the survival rate has not been improved[2-5]Sarcoma therapy is currently limited by the obstacle of achieving efficient drug delivery into target cells,and ligands specific to the surface receptors of cancer cell have been intensively searched in cancer research.The emerging phage display technology is a powerful tool in this field and may impact clinical issues including functional diagnosis and targeted therapy.ObjectiveIn our present study,we aimed to develop a targeted therapy for treating OS by employing phage display technique(Fig.1).Specifically,we first identified a peptide that can target OS cancer cells.Then we conjugated the targeting peptides to the surface of gold nanocages(GNCs),a model cancer nanomedicine,to develop targeted cancer nanotherapeutics.Methods1.To identify a ligand that can guide inorganic nanoparticle drugs to human osteosarcoma cells for targeted therapy,we employed the Ph.D.TM-12 Phage Displayed Peptide Library to search for a peptide that can not only target the surface of a human osteosarcoma cells(U2OS)but also selectively become internalized in the target cells,by a subtractive screening(called biopanning)in vitro.2.The selected phages were titrated on LB/IPTG/X-Gal plates through blue-white plaque forming assay for phage clone identification and the number of blue colonies on the plates was counted.During the four rounds of in vitro panning,blue plaques were randomly picked in round 2,3 and 4 and their sequences were analyzed by the ABI Automatic DNA Analyzer.3.The individual phage clone instead of the phage-displayed library was added to an empty culture flask and hFOBl.19 cells sequentially,the remaining steps were similar to the steps of Phage Display Biopanning described above.4.The binding ability and specificity to U2OS cells of the selected phages were measured by phage capture ELISA assay and immunohistochemical staining.The control cells were hFOB1.19,MG-63 and MCF-7.5.The selected peptides(translated from the selected phages sequence),and random peptide(termed null peptide:HGVAELVKSSTA)were synthesized with fluorescein isothiocyanate(FITC)labeling and purified.6.The targeting capability of the selected peptide toward U2OS cells was studied by flow cytometry through FITC label.In brief,U2OS,hFOBl.19,MG-63 and MCF-7 cells were seeded into 12-well plates,5 ?M and 10 ?M FITC-labeled peptides,were added into the target U2OS cells and incubated,respectively.The cells were detached from the culture plates and analyzed on a Guava EasyCyte 5HT flow cytometer(Millipore,USA).7.The competitive inhibition effect of the selected peptide with its corresponding phage counterpart was performed by Flow Cytometry and blue-white plaque forming assay,respectively.8.To investigate the photothermal effect induced by NIR laser irradiation due to the association of the peptide-conjugated GNCs with the target cells,each well was exposed to a NIR coherent diode laser light(808 nm),and the elevation of the solution temperature was monitored by a Fluorescence-Based Fiber Optical Monitoring system.Afterwards,cell proliferation was quantified by a colorimetric assay based on the mitochondrial oxidation of CCK-8 and cell viability was detected by the Live/Dead staining assay following the manufacturer' s protocol.Results1.The results of blue-white plaque forming assay confirmed that the amount of cell-surface-bound phages recovered from U2OS cells increased about 144-fold(from 1.0×106 pfu to 1.45×108 pfu)after enrichment,and the corresponding value for the cell-internalized phages was 15.53-fold(from 8.95×104 pfu to 1.39 × 106 pfu),as shown in Fig.2A,B.2.81,123 and 200 blue plaques were randomly picked and sequenced in round 2,3 and 4,respectively,giving rise to a total of 404 blue plaques being sequenced.We gave a sequential name to each of the 404 plaque clones and the corresponding exogenous peptide sequence from OS-1 to OS-404.3.12 phage clones screening from biopanning procedure(Repetition frequency ? 2)were amplified for the enrichment analysis of individual phage colony through the blue-white plaque forming assay.The results of the enrichment analysis of individual phage colony indicating the specific binding ability of each phage clone derived from DNA sequencing to osteosarcoma U2OS cells.4.As shown in Fig.3A,cell-bound Phage OS-7 had higher affinity against U2OS cells than other selected phages(p<0.01),showing 6.5-fold than Wild-type phages,respectively.As expected,similar to the wild-type phages,none of the selected phages exhibited binding ability to the surface of or being internalized in the human breast cells(MCF-7)and human osteoblasts(hFOB1.19)(p>0.05).Thus,we chose Phage OS-7 and the corresponding peptide as the U2OS-targeted peptide candidate for the following studies5.Immunohistochemical staining was performed to observe the specific binding of Phage OS-7 to human osteosarcoma tissue sections(Fig.4),and the cells in tumor tissue sections were stained dark brown distinctly.However,both the osteosarcoma tissue sections interacted with wild-type phage clone and the para-carcinoma tissue sections interacted with OS-7 clone showed negative staining.6.Fig.5A showed that targeting peptide group(FITC-OS-7)displayed higher targeting ability(the curve shifted towards right)compared with the non-targeting control peptide group(FITC-Null peptide)and blank control group at the concentration of both 5 ?M and 10 ?.These data indicate the highest binding capability to U2OS cells of Peptide OS-7(APWTEAYWWHLP)as well as no binding evidene exhibited in blank control group.7.Competitive Inhibition StudiesT confirm Phage OS-7 and the corresponding synthetic peptide competed for the same binding site.8.The results Photothermal Therapy of osteosarcoma cells with NIR laser irradiation demonstrated the feasibility of Peptide OS-7-GNCs as a OS-targeting photothermal therapy agent.ConclusionOur study provide a clear example of the screening and identification of human osteosarcoma cells U2OS-specific binding peptides by phage display technology and the use of such peptides in the development of tumor targeting nanodrugs.The sequence of the peptide that we screened and identified by this method was:APWTEAYWWHLP.The peptides has a high affinity and specific binding properties for human osteosarcoma cells U2OS.In addition,the targeting peptides may be coupled to gold nanocomposites so that they can target tumor cells.Under the irradiation of near infrared laser,the photothermal properties of gold nanocages and the targeting ability of target peptides were synergistic to achieve selective inhibition of U2OS cell proliferation.Our work shows that the use of phage library can identify short cell targeting/internalizing peptides,which seems to be a promising ligand for the applications in targeted treatment of human osteosarcoma.