The Preparation Of Activatable Cell Penetrating Peptide Modified Fe₃O₄ /Au Shell Nanocomposites And Their Applications In Multimode Bioimaging And Photothermal Therapy

Objective:In recent years, multifunctional nanoparticles?NPs? with various components had garnered significant research interest because they showed superior properties than that of the individual component. Nanocomposites containing both magnetic and optically active plasmonic components have attracted intensive attention due to their potential biomedical applications as drug-delivery vehicles, imaging contrast agents, photothermal therapeutic agent etc. Many previous studies showed that the enhanced permeability and retention?EPR? effect is one of main tumor-targeting mechanisms of the multifunctionalized nanoparticles, but it is still difficult to obtain the optimal accumulation of nanoparticles in tumor site only via EPR effect. In this work, an activatable cell penetrating peptides?ACPP? modified superparamagnetic Fe₃O₄/Au shell nanocomposites were prepared to realize simultaneous multimode bioimaging with tumor selectivity and photothermal therapy. Contents:The superparamagnetic Fe₃O₄-silica composite nanoparticles coated with Au nanoshells?SSCNs@Au NSs? were prepared and subsequently modified with conjugations of polyethylene glycol?PEG? and activatable cell penetrating peptide?ACPP? on their surface to form SSCNs@Au NSs-PEG-ACPP nanocomposites. Their performances as contrasts of magnetic resonance?MR? imaging and computed tomography?CT? and photothermal therapy of tumor were investigated via a serials of characterizations in vitro and in vivo.Methods :?1? The preparations and characterizations of nanocomposites. Fe₃O₄ nanoparticles were prepared by a chemical coprecipitation method and subsequently surface modified with olic acid to form an oil phase. Fe₃O₄ aggregations were prepared via an emulsion method with SDS as an emulgator and then coated with SiO₂ by a St?ber method and further modified with-SH on the surface. SSCN@Au nanoshells?SSCN@Au NSs? core-shell nanocomposites were then prepared via a seed-mediated growth method by reducing HAu Cl4 in the alkaline environment with Na BH4 and L-ascorbic acid as reductants in order. Finally, the conjugations of PEG and ACPP were modified on the surface of SSCN@Au NSs via S-Au coordination interactions to form SSCN@Au NSs-PEG-ACPP nanocomposites. Then transmission electron microscopy?TEM?, dynamic light scattering?DLS?, Fourier translation infrared spectrometer?FT-IR?, Ultraviolet-visible-near infrared region absorption spectrometer?UV-vis-NIR?, atomic absorption spectroscopy?AAS? and inductively coupled plasma mass spectrometry?ICP-MS? were used to characterize the surface morphology, size and size distributions, chemical structures and metal element contents of the nanoparticles generated from every step. The photothermal properties of SSCNs@Au NSs-PEG-ACPP nanocomposites were detected by measuring the temperatures of their solutions with different concentrations under the irradiation of NIR laser using an electronic thermometer. T2-weighted MR imaging and relaxation time of SSCNs@Au NSs-PEG-ACPP at different concentrations were recorded by a 1.2 T nuclear magnetic resonance?NMR? image system to evaluate their property as MR imaging contrast agents. The X-ray absorption property of SSCNs@Au NSs-PEG-ACPP nanocomposites was measured via a clinical CT scanning system.?2? Characterizations of nanocomposites in vitro. The cell toxicity and photothermal therapy property of SSCN@Au NSs-PEG-ACPP nanocomposites were investigated with MCF-7 cells by a MTT assay. The cellular uptake of SSCNs@Au NSs-PEG-ACPP nanocomposites was observed by TEM.?3? Characterizations of nanocomposites in vivo. The human breast MCF-7 tumor bearing nude mouse models were built. The temperature changes of tumor were measured by an infrared thermal camera after injecting intratumorally with SSCNs@Au NSs-PEG-ACPP nanocomposites and the irradiation of NIR laser. The therapeutic effect of tumor was also confirmed via H&E staining of tumor region. The MR and CT imaging properties of SSCNs@Au NSs-PEG-ACPP nanocomposites in vivo were investigated by injecting them intravenously.Results:?1? The SSCNs@Au NSs-PEG-ACPP nanocomposites were prepared successfully from the results of TEM, DLS, UV-vis-NIR and FT-IR. The nanoparticles generated from each step were all spheres with an uniform size and the size of the resultant SSCNs@Au NSs-PEG-ACPP nanocomposites was 187 nm from the observation of TEM. The results of DLS measurement showed the good dispersity and colloidal stability of the nanocomposites. The result of UV-Vis-NIR specturm showed that SSCNs@Au NSs-PEG-ACPP nanocomposites had a relatively strong SPR characteristic peak at about 800 nm, suggesting the formation of Au nanoshell. The typical peaks of amide?and bands can be observed at 1647 and 1542 cm?^-1 in the spectrum of SSCNs@Au NSs-PEG-ACPP, indicating the successful modification of ACPP on the surface of Au nanoshell. The result of photothermal effect detection of the SSCNs@Au NSs-PEG-ACPP nanocomposites showed that their solutions could be easily heated up to above 60 oC at the Au concentration higher than 80 ?g Au m L^-1, which verifying the excellent photothermal efficiency of SSCNs@Au NSs-PEG-ACPP. The T2-weighted MR images of SSCNs@Au NSs-PEG-ACPP revealed a clear concentration-dependent relationship. The relaxation rate of SSCNs@Au NSs-PEG-ACPP was calculated to be 214 m M_Fe^-1 s^-1. The in vitro X-ray absorption of SSCNs@Au NSs-PEG-ACPP increased linearly with the increase of Au concentrations. Therefore, SSCNs@Au NSs-PEG-ACPP showed a good contrast performance of MR and CT.?2? The cytotoxicity assays of SSCNs@Au NSs-PEG-ACPP indicated that MCF-7 cells treated with SSCNs@Au NSs-PEG-ACPP without laser irradiation remained about 84% cell viability at Au concentrations up to 50 ?g Au m L^-1. However, the viability of MCF-7 cells significantly decreased after treatment with SSCNs@Au NSs-PEG-ACPP and laser irradiation, and only 12% of the cells remained viable at an Au concentration of 50 ?g Au m L^-1. The results showed their relatively low cytotoxicity in MCF-7 cells. However, the nanocomposites could kill tumor cells by raising the temperature under irradiation of NIR laser, suggesting their excellent photothermal performance. The cellular uptake of SSCNs@Au NSs-PEG-ACPP determined by TEM showed many nanocomposites in the cytoplasm of MCF-7 cells.?3? Under the NIR laser irradiation, the temperature of tumor region of the MCF-7 tumor-bearing mouse intratumorally injected with SSCNs@Au NSs-PEG-ACPP was raised to 55 oC, a temperature which could ablate the tumor in vivo effectively. Compared with SSCNs@Au NSs-PEG nanocomposites, the SSCNs@Au NSs-PEG-ACPP nanocomposites could be accumulated in tumor site with a large number of after a single tail vein injection from the MR/CT images.Conclusions:The multifunctionalized SSCNs@Au NSs-PEG-ACPP nanocomposites for multimode bioimaging with tumor selecting and photothermal therapy were prepared successfully. SSCNs@Au NSs-PEG-ACPP exhibited good dispersity and colloid stability. Both in vitro and in vivo, SSCNs@Au NSs-PEG-ACPP exhibited excellent photothermal efficiency and high CT- and MR-imaging abilities. Moreover, even at a high concentration of 50 ?g Au m L^-1, SSCNs@Au NSs-PEG-ACPP had no significant cytotoxicity in MCF-7 cells. Thus, we deduced that SSCNs@Au NSs-PEG-ACPP nanocomposites has the excellent properties and exhibits a high potential to be applied in the MR/CT imaging and photothermal therapy.