A New Functionalized Nanoparticle HPPS-mAb For TfR Targeting Tumor Imaging And SiRNA Delivery

[Background and Objective]Conventional therapy of malignant tumor remains primarily surgery, radiotherapy and chemotherapy. Chemoradiotherapy is able to kill tumor cells and damage normal tissue terribly at the same time. This adverse effect goes against with patients’ prognosis and life quality. Targeted therapy is incapable of injuring healthy cells and has little side effect in comparison with traditional therapeutic method. Targeted nanomedicine is designed to improve the biodistribution of therapeutic drug and increase the drug accumulation in target site in systemic administration. One key point for that therapy is the selection of tumor specific antigen. Transferrin receptor (TfR) is a tumor relatively specific surface marker, and its high expression is closely related with tumor staging, grading and prognosis. TfR overexpression, accessible extracellular domain and receptor mediated endocytosis pathway makes TfR one of the hottest spot in tumor targeted therapy. Previous studies in our lab demonstrated TfR monoclonal antibody could recognize tumor cell with high efficiency and possess good synergistic antitumor effect when combined with chemo-drugs towards various different origins of tissue. However, its single treatment had relatively low antitumor effect. This research takes advantage of the characteristic that anti-TfR mAb can bind with TfR high expressing on tumor cells and establishes a TfR mAb functionalized nanoparticle HPPS for tumor imaging and drug delivery. It may shed new lights on the research of localization and tracing of therapeutics and tumor imaging and targeted therapeutic research. In this research, to achieve active targeting to tumor cell, our TfR antibody is designed to conjugate with a lipid-peptide nano-carrier mimicking HDL (HPPS) with long circulation time, hoping to solve problems typically existing in targeted research such as peripheral blood intercepting, nonspecific absorption and physiologic barriers. The nanoparticle in our research HPPS has controllable particle size with good biocompatibility and low immunogenicity. Small interfering RNA(siRNA) can exert antitumor effect by means of specifically knocking down a given gene. Nevertheless, the large molecular weight, polyanion, enzymatic degradation and hard delivery to cells of siRNA makes it hard to be used in vivo. According to the above, our research is designed to construct a new TfR nano-targeted molecular HPPS-mAb based on TfR targeting research and efficient conjugation of cholesterol modified siRNA to the lipid monolayer of HPPS. The nanoparticles are capable of targeting TfR high expressing tumor cell and delivering therapeutic siRNA to locate specifically in tumor site. So they can direct siRNA release to cytoplasm via receptor mediated endocytosis pathway and make a difference in tumor imaging and killing through siRNA and put the imaging feature of HPPS to good use. This research will explore new train of thought and new pathway to tumor-selective molecular imaging and therapeutic effect.[Methods]1. Targeting specificity of TfR mAb to TfR+ cell lines HepG2 and hTfR stably expressed CHO-hTfR cells.HepG2 cells and hTfR stably expressed CHO cells(CHO-hTfR)were used to detect TfR mAb binding capacity and specificity by immunofluorescent staining. CHOvec and CHO cells which have no hTfR expressed were used as control.2. Dynamic trafficking of TfR mAb in living cellsTrafficking of PE labeled TfR mAb was monitored immediately after their supplement into CHO-hTfR cell culture system using confocal scanning technique. At the same time, Ab-EGFP colocalization was confirmed in early endosome and late lysosome at different time points in fixed cells. The difference with classical Tf-TfR mediated endocytosis pathway was compared.3. The construction, purification and characterization of HPPS-mAbHPPS and-SH modifying mAb were coupled to generate TfR targeted HPPS-mAb nanoparticle which was purified and identified by FPLC. Particle size and zeta potential was obtained by nanosizer.4. In vitro identificationof TfR targeting of HPPS-mAbCHO-hTfR cells were incubated with HPPS-mAb(1μM D1R-BOA). HPPS-mAb and TfR colocalization within the cell was detected using confocal imaging.Multiple tumor cell lines were incubated with different concentrations of HPPS and HPPS-mAb respectively followed by conducting flow cytometry to detect the contrast fluorescence intensity of DIR-BOA to characterize the nanoparticle uptake by cell.The same amount of no hTfR expressing cells CHO and stable expressing hTfR cells CHO-hTfR were mixed. The mixture was incubated with the same concentration of HPPS and HPPS-mAb respectively followed by detecting uptake of HPPS-mAbin CHO-hTfR cells.5. Identification of HPPS-Isotype and inhibition evaluation of nanoparticle uptake under HDL treatmentHPPS-Isotype was constructed as an isotype control to HPPS-mAb. Comparation was performed among the three nanoparticles in cellular uptake. Meanwhile, HDL was used to block the surface SR-BI in HepG2 cells. The uptake inhibition was detected using flow cytometric analysis.6. Accumulation of HPPS-mAb in U87 bearing nude miceU87 bearing nude mice model was established to be injected with HPPS or HPPS-mAb containing 10nmol DIR-BOA through tail vein.3h,12h,24h,48h,72hafter injection, the D1R-BOA fluorescence distribution was detected using whole body imaging system.7. Accumulation of HPPS-mAb in double grafted CHOvec/CHO-hTfR mice model.CHO/CHO-hTfR double bearing nude mice model was established. The left flank was injected with CHO cells and the right was CHO/hTfR bearing. The nanoparticles were injected into mice through tail veins. To compare the time dependent accumulation effect between the two different xenografts, whole body imaging technology was also used to determine the nanoparticle accumulation between two sides at different time point. Tumors were isolated and prepared for single cell suspension with tumor cell uptake detection.8. Survivin mRNA silencing induced by HPPS-mAb/siRNA in HepG2 cellsDifferent concentration of chol-si-survivin and chol-NC was mixed and incubated with HPPS-mAb. The mixture was self-assembling HPPS-mAb/siRNA. HepG2 was treated with HPPS-mAb/siRNA for 48h using different concentration of siRNA followed by detection of survivin mRNA by RT-PCR.9. Survivin protein silencing induced by HPPS-mAb/siRNA in HepG2 cells and cell apoptosis detectionHepG2 was treated with different concentration of HPPS-mAb/siRNA for 48h followed by detection of survivin protein expression by Western Blot. Cell apoptosis was detected in HepG2 cells by Annexin V/PI staining using flow cytometry.[Results]1. TfR mAb binds specifically with TfR+ cells99.3% hTfR stably expressed CHO-hTfR cells emit the PE fluorescence which was used to label TfR mAb. This figure showed no difference with that of HepG2 cells (98.03%). It was suggested that CHO-hTfR cells could stably express human TfR in high intensity and this cell line could be used as a model to study receptor mediated endocytosis. CHO cells and CHOvec cells showed lower than 5% binding with PE-TfR mAb. PE was colocalized with TfR-EGFP. This feature had no significant difference comparing with TfR natural ligand Tf. mAb had no binding with CHOvec cells suggesting CHO and CHOvec cell may serve as negative control in research.2. Endocytosis dynamics and cellular localization of TfRmAb in CHO-hTfR cellsConfocal laser microscopy of living CHO-hTfR real-time imaging showed that TfR-EGFP mediated endocytosis of PE-mAb. PE fluorescence was evenly distributed on the cell surface, then diffused to become’grainy’,thereafter, a large part of the grains migrated to the cytoplasm even toward a perinuclear area. At 20min, a small portion of vesicle structure lost EGFP. Furthermore, the cellular localization showed that in the early stage yellow merged fluorescence was colocalized with endosome marker EEA-1. And at 30min, TfR mAb was colocalized with lysosome marker LAMP-1.The above results suggested that mAb triggered TfR mediated internalization was generally consistent with natural TfR, but owned its characteristics.3. The construction, purification and characterization of targeted nanoparticle HPPS-mAbEngineering HPPS was prepared by Brritton chance center for biomedical photonics of Wuhan national laboratory for optoelectronics. TfR mAb was modified by -SH and conjugate to the surface of engineering HPPS. Through FPLC purification, the successful conjugation was collected. The FPLC purification profiles showed that the retention time of HPPS-mAb shifted earlier than TfR mAb apparently and the peak time was distinctly different with HPPS, which suggested that mAb had been coupled with HPPS. The size of HPPS-mAb was about 29nm, which was a little bigger than unconjugated HPPS.4. Uptake of HPPS-mAb through TfR mediated endocytosis by TfR+ cells and mAb conjugation accelerating the uptake.Confocal imaging showed that DIR-BOA labeled HPPS was colocalized with TfR-EGFP in CHO-hTfR cells, indicating that the uptake of HPPS-mAb was mediated by TfR. HepG2 cells treated with different concentrations of HPPS and HPPS-mAb showed a higher uptake of HPPS-mAb. When acting on other cell lines, HPPS-mAb showed various targeting facilitation, ranging from 2-5 folds.5. Targeting identification of HPPS-mAb in mixed cellsCHO-hTfR cells and the same amount of CHO cells were mixed, then cultured with HPPS-mAb and HPPS respectively. The result showed that HPPS-mAb can be taken up by TfR+ CHO-hTfR cells preferentially. And the amount of HPPS-mAb uptake was parallel with TfR expression level.6.Function identification of HPPS-Isotype and HDL inhibition effectThe isotype construction of HPPS-mAb benefited the HPPS-mAb in vivo targeting imaging research. HPPS-Isotype had no evident distinction with HPPS in cellular uptake but far less than HPPS-mAb uptake. When human HDL was used to block SR-BI on HepG2 cell surface, the uptake was partialy reduced of HPPS-mAb and almost totally suppressed of HPPS, promting a dual targeting mechanism of TfR and SR-BI once more.7. In vivo HPPS-mAb imaging in U87 bearing miceThe long circulation characteristic of nanoparticle was examined via in vivo imaging. Results showed that the nanoparticle accumulation in tumor site became stronger over time, which peaked at 48h. The whole body fluorescence distribution of HPPS was more widespread than that of HPPS-mAb, suggesting that HPPS-mAb benefited the accumulation on the tumor site and reduction of other organs.8. HPPS-mAb targeting imaging in double grafted mice modelMoreover, a double grafted transplantation mouse model was prepared for in vivo targeting identification. The result showed that before 24h, HPPS-mAb gathered preferentially in the CHO-hTfR bearing side and HPPS-Isotype accumulation showed no difference in bilateral xenografts.9. Survivin mRNA expression in HepG2 cells treated by HPPS-mAb/siRNAHepG2 cells were treated with HPPS-mAb/siRNA for 48h using different concentration of siRNA. RT-PCR results showed at 400nM of chol-si-survivin, survivin mRNA expression was suppressed apparently. The silence efficiency could reach 62%. A lower suppression effect was induced by HPPS-mAb/siRNA which was below 50%.10. Survivin protein expression and apoptosis effects induced by HPPS-mAb/siRNA in HepG2HepG2 cells were treated 48h using different concentration of HPPS-mAb/siRNA. WB showed the survivin protein expression viation. At 400nM of chol-si-survivin, the silence effect was apparent which was 64.8% reduction compared to blank group. The early apoptosis rate was as much as 19.7% detected by flow cytometry.[Conclusions]Based on TfR antibody targeting research, this project has been designed to construct a new nanoparticle HPPS-mAb and investigated its TfR targeted tumor imaging in vitro and in vivo and siRNA delivery. The research found that ①TfR mediated mAb endotytosis showed that the pathway of TfR-mAb compound was not completely consistent with Tf-TfR cycle and was also different with the TfR degradation triggered by TfR antibodies performing Fe deprivation. Our TfR mAb had its own charccteristics as targeting ligand used in nanocarrier delivery which could accumulate high concentration of drugs in target site.②HPPS-mAb can be taken up efficiently by TfR+ tumor cells in vitro. The mAb conjugation facilitated the cellular HPPS uptake evidently. And HDL was able to inhibit the HPPS uptake but partially to the HPPS-mAb, pointing out that the TfR mAb functionalized HPPS-mAb nanoparticle possesses a dual targeting mechanism to tumor cells. Whole body imaging showed that within 24h, HPPS-mAb had a preferred accumulation in CHO-hTfR bearing xenograft, nevertheless, this targeting effect was not obvious at later stages, prompting for the possibility of tumor early stage diagnosis application. We have constructed HPPS-mAb/siRNA nanoparticle successfully and confirmed that HPPS-mAb could serve as a TfR oriented nanocarrier tosilence survivin mRNA and protein expression in survivin overexpression cells and induce tumor cell apoptosis. And then establish a solid foundation for TfR mediated mAb functionalized HPPS-mAb tumor molecular imaging and anti-tumor research.