Development And Validation Of A Targeted Nanotheranostic Platform For Triple Negative Breast Cancer

Background:Triple negative breast cancer(TNBC)is negative for estrogen receptor(ER),progesterone receptor(PR)and without human epidermal growth factor receptor 2(HER2)overexpression.It has the worst prognosis among all subtypes of breast cancer,with high invasiveness,early metastasis and diagnosis at a later stage.The mainstay of the systemic therapy for TNBC remains to be cytotoxic chemotherapy due to its insensitivity to endocrine therapy and lack of available targeted therapy.However,the efficacy of chemotherapy is limited.Therefore,it is urge to identify its specific target and develop new strategy for the diagnosis and therapy,so as to improve the prognosis of TNBC.Although efforts have been devoted to develop targeted therapy for TNBC and recently obtained preliminary results,no cell surface protein has been identified as the specific target of TNBC due to the huge inherent heterogeneity of TNBC.The rise and rapid development of nanomedicine has brought new strategies for the traditional mode of diagnosis and therapy of tumor.Various multifunctional hybrid nanoparticles with at least two functions(such as targeting,controlled release,imaging and therapy etc.)have been designed to boost medicine due to the progress in nanomaterials science and technology.Among them,theranostic nanoparticles which can provide simultaneous imaging and therapy of tumor have caught the most attention from researchers.However,no theranostic platform has been successfully put into clinical trial until now.Purpose:Therefore,the purpose of this study is to identify the specific membrane protein as the target of TNBC,and then develop a biocompatible,effective targeted nanotheranostic platform for TNBC for the image-guided precision therapy of TNBC to improve its prognosis.Methods:(1)Confirmation the target membrane protein of TNBC: Real-time quantitative PCR and Western-Blot were conducted to analysis the m RNA and protein level of intercellular adhesion molecule 1(ICAM1).Immunofluorescence was conducted to locate the ICAM1 protein.(2)Synthesis and characterization of TNBC-targeted nanotheranostic platform: To ensure the biosafety and utility of the platform,the biocompatible poly ethylene glycolpoly ε-caprolactone(PEG-PCL)polymer was used to formed the carriers,Gd-based contrast used for magnetic resonance imaging(MRI),and cytotoxic chemotherapy drug doxorubicin(DOX)as the therapeutic drug.In general,Gd and DOX were encapsulated in the hydrophilic core of vesicles which were based on the PEG-PCL polymer and modified with anti-ICAM1 antibody on the surface,yielding the target product AntiGd-Dox@PEG/PCL multifunctional hybrid nanoparticles.The characteristics of these nanoparticles were analyzed with dynamic light scattering(DLS),and the morphology was analyzed with transmission electron microscope(TEM).The in vitro T1 contrastenhanced ability of these nanoparticles was evaluated with MRI T1 WI image.(3)In vitro and in vivo evaluation the efficacy and biosafety of the nanotheranostic platform:1)targeting ability: MCF7 and MDA-MB-231 cells were incubated with Anti-GdDox@PEG/PCL-FITC nanoparticles respectively for different time,and the cell uptake of nanoparticles was observed with fluorescence microscope.Flow cytometer was used to quantified the uptake difference.2)biocompatibility: MCF7 and MDA-MB-231 cells were incubated with blank PEG/PCL vesicles of different concentrations,and the cell viability was evaluated with MTT.3)cytotoxicity: MCF7 and MDA-MB-231 cells were incubated with Anti-GdDox@PEG/PCL nanoparticles of different concentrations.The cell viability was evaluated with MTT and the half inhibiting concentrations(IC50s)of nanoparticles were calculated and compared.4)biodistribution: TNBC tumor-bearing models were built by subcutaneous injection of MDA-MB-231 cells into the flank of nude mice.Anti-Gd-Dox@PEG/PCLcy5.5 nanoparticles were intravenously injected into tumor-bearing mice.The biodistribution of cy5.5 fluorescence in tumor-bearing mice as well as ex vivo tumors and organs were observed with in vivo imaging of small animal(IVIS).5)contrast-enhanced(CE)MRI: Anti-Gd-Dox@PEG/PCL,Gd-Dox@PEG/PCL nanoparticles and Gd were intravenously injected into tumor-bearing mice,respectively.T1 WI were acquired before and after intravenous injection of any drugs.The contrastnoise ratios(CNRs)of tumors on T1 WI were calculated and compared among different groups.6)In vivo therapeutic efficacy and safety: tumor-bearing mice were intravenously treated with DOX,Gd-Dox@PEG/PCL,and Anti-Gd-Dox@PEG/PCL,respectively every 3 days for 2 weeks.Tumor volume and body weight were monitored and compared among different groups.The whole blood was collected and analyzed for routine blood parameters and serum biochemical parameters.Tumor apoptosis and proliferation were assessed using TUNEL and Ki-67 staining,respectively.Results:(1)ICAM1 was expressed significantly higher in MDA-MB-231 cells than in other types of breast cancer cells and normal breast epithelium in both m RNA and protein levels.ICAM1 was expressed mainly on cell surface,indicating ICAM1 could be the specific surface target of TNBC.(2)The target products Anti-Gd-Dox@PEG/PCL nanoparticles were obtained by modification with anti-ICAM1 antibodies on the surface of vesicles which encapsulated Gd and DOX within their hydrophilic inner core.The hydrodynamic size of these nanoparticles was 279 ± 7.6 nm,ζ-potential-24.7 ± 0.9 m V and PDI 0.155 ± 0.02.TEM revealed the irregular shape these nanoparticles.T1 WI images of Anti-GdDox@PEG/PCL solutions became observably brighter as the concentration of Anti-GdDox@PEG /PCL was increased,suggesting its excellent T1 contrast ability.The drug loading efficiency(DLE)and drug loading content(DLC)for DOX were 13.23% and1.75%,and for Gd 28.80% and 67.54%,respectively.(3)MDA-MB-231 cells absorbed more Anti-Gd-Dox@PEG/PCL-FITC nanoparticles than Gd-Dox@PEG/PCL-FITC nanoparticles after 3 h treatment,and the difference of absorption increased as the incubation time was extended to 6 h.The analysis of flow cytometry displayed that the percentage of FITC positive MDA-MB-231 cells was 3.98% ± 1.21% in Anti-Gd-Dox@PEG/PCL-FITC treated group and 0.95%± 0.18% in Gd-Dox@PEG/PCL-FITC treated group after 3 h incubation(p = 0.005),and the percentage increased to 21.57% ± 2.96% in Anti-Gd-Dox@PEG/PCL-FITC treated group but was almost unchanged in Gd-Dox@PEG/PCL-FITC treated group(1.64% ± 0.25%)(p < 0.001 in comparison with Anti-Gd-Dox@PEG/PCL-FITC treated group)as the incubation time was extended to 6 h.In contrast,the absorption of Anti-Gd-Dox@PEG/PCL-FITC nanoparticles or Gd-Dox@PEG/PCL-FITC nanoparticles was barely observed in MCF7 cells even though the incubation time was extended to 6 h.These results indicated that the Anti-Gd-Dox@PEG/PCL nanoparticles could specifically target to TNBC via binding to ICAM1(4)Blank PEG/PCL nanoparticles had little cytotoxicity to MDA-MB-231 and MCF7 cells even at concentration up to 500 μg/m L,indicating good biocompatibility of the carriers.Anti-Gd-Dox@PEG/PCL nanoparticles were more effective in inhibiting the proliferation of MDA-MB-231 cells than Gd-Dox@PEG/PCL nanoparticles or DOX,with its half inhibitory concentration(IC50)(0.29 ± 0.06 μg/m L)significantly lower than that of Gd-Dox@PEG/PCL nanoparticles(0.66 ± 0.02 μg/m L,p < 0.0001)or DOX(0.57 ± 0.03μg/m L,p < 0.001).For MCF7 cells,comparable cytotoxicity to MCF7 cells was observed for Anti-Gd-Dox@PEG/PCL nanoparticles,Gd-Dox@PEG/PCL nanoparticles and DOX,with their IC50 s to be 0.67 ± 0.09 μg/m L,0.91 ± 0.19 μg/m L and 0.59 ± 0.14 μg/m L(p = 0.078),respectively.Furthermore,the IC50 of Anti-Gd-Dox@PEG/PCL nanoparticles for MDA-MB-231 cells was significantly lower than that for MCF7 cells(p = 0.004).These results indicated that the cytotoxicity of Anti-Gd-Dox@PEG/PCL nanoparticles to TNBC was significantly enhanced by specifically targeting to it.(5)Gd-Dox@PEG/PCL and Anti-Gd-Dox@PEG/PCL nanoparticles could effectively accumulate in tumor sites and continuously increased within 2 h after injection.The accumulation of Anti-Gd-Dox@PEG/PCL nanoparticles in tumor sites was more efficient than that of Gd-Dox@PEG/PCL nanoparticles.Ex vivo images of major organs and tumors 24 h after injection further confirmed the specific targeting capability of Anti-Gd-Dox@PEG/PCL nanoparticles to tumors.(6)The CNRs continuously increased to peak at 2 h after injection in mice injected with Anti-Gd-Dox@PEG/PCL nanoparticles and Gd-Dox@PEG/PCL nanoparticles,while the CNRs in mice injected with traditional Gd-DTPA reached its peak at merely1 h after injection.Furthermore,the peak CNR of Anti-Gd-Dox@PEG/PCL nanoparticles(3.79 ± 0.21)was significantly higher than that of Gd-Dox@PEG/PCL nanoparticles(3.21 ± 0.08,p = 0.011)or Gd-DTPA(3.05 ± 0.17,p = 0.003).These results indicated that Anti-Gd-Dox@PEG/PCL nanoparticles had longer circulation time and could actively target to TNBC,so as to enhance the accumulation at TNBC.(7)In comparison with untreated control group,significant inhibition of tumor growth was observed in all 3 treated groups(all p < 0.001)during the treatment.Furthermore,the tumor growth inhibition was more obvious in mice treated with AntiGd-Dox@PEG/PCL nanoparticles(p = 0.004)and Gd-Dox@PEG/PCL nanoparticles(p = 0.022)than in those treated with traditional chemotherapeutical drug DOX.Tumor inhibition by Anti-Gd-Dox@PEG/PCL nanoparticles was slightly more effective than by Gd-Dox@PEG/PCL nanoparticles.The proliferation of tumor cells decreased in mice from treated groups,especially in mice treated with Anti-Gd-Dox@PEG/PCL nanoparticles in comparison with untreated control.The apoptosis of tumor cells increased most obviously in mice treated with Anti-Gd-Dox@PEG/PCL nanoparticles.There was no significant damage to major organs such as heart,liver,spleen,kidney,and lung after treatment and no inflammation and abnormal liver and kidney function.Conclusions:(1)ICAM1 could serve as the specific surface target of TNBC.(2)The multifunctional hybrid Anti-Gd-Dox@PEG/PCL nanoparticles had good biocompatibility and biosafety.(3)The multifunctional hybrid Anti-Gd-Dox@PEG/PCL nanoparticles could target to TNBC by binding with ICAM1.(4)The positive and active accumulation of Anti-Gd-Dox@PEG/PCL nanoparticles at TNBC tumor site was enhanced by their prolonged circulation time and the specific targeting capability.(5)Anti-Gd-Dox@PEG/PCL nanoparticles had excellent in vivo T1 contrastenhanced ability and therapeutic efficacy,providing a promising theranostic approach for MRI-guided precise therapy of TNBC.