| Current clinical therapy investigation has shown that thermo-chemotherapy therapy could result in additionally enhanced anticancer efficacy, because hyperthermia can promote drug delivery into tumor and increase the drug toxicity. However, for traditional treatment techniques, the synergistic effects of thermo-chemotherapy are difficult to realize in vivo because co-delivery of chemotherapeutic agents together with hyperthermia sources to the target tissues is still challenging. Fortunately, the rapid development of nanotechnologies provides a new impetus for cancer therapy due to its unique physical properties.Herin, we developed GNRs@mSiO2nanomaterials as NIR-light-absorbing agents and a drug delivery system for in vivo chemo-photothermal destruction of tumor. In order to target the tumor and increase the therapeutic efficacy, RGD peptides were conjugated on the terminal groups of poly(ethylene glycol)(PEG) on GNRs@mSiO2. DOX, a classic anticancer drug, was also successfully loaded into pGNRs@mSiO2-RGD nanomaterials. The results proved that the synergistic effect of DOX-pGNRs@mSiO2-RGD for the efficacious treatment of A549(Human lung adenocarcinoma epithelial cell line) tumor were better than that of the chemotherapy or photothermal therapy alone.The first part described the construction and characterization of DOX-pGNRs@mSiO2-RGD. CTAB-coated GNRs were firstly synthesized by the seed-mediated method. The TEM image of the obtained CTAB-coated GNRs showed that the average length and width were51.6±2.1and13.2±1.2nm, respectively (about3.9:1aspect ratio). The GNRs@mSiO2were synthesized via a single step coating method, and CTAB molecules on the surface of GNRs served as an organic template for the deposition of a mesoporous silica coating. The GNRs@mSiO2were centrifuged and washed with NH4NO3/ethanol solutions to remove the CTAB residual in the mesoporous channels. The TEM image of the obtained GNRs@mSiO2core-shell nanomaterials indicated that the GNRs were coated with uniform mesoporous silica shells (ca.25nm thickness). The GNRs@mSiO2nanomaterilas have two SPR bands; the longitudinal SPR band is at800nm, whereas the transverse plasmon band is at530nm. The N2adsorption-desorption isotherms and pore size distribution of the GNRs@mSiO2showed the characteristic of mesopores. The pore size distribution exhibited a sharp peak centered at the mean value of2.9nm, indicating a uniform mesopore. The BET surface area and total pore volume were calculated to be348m2/g and0.51cm3/g, respectively. In order to enhance nanomaterials delivery into target cells, RGD peptides were conjugated on the terminal groups of PEG on pGNRs@mSiO2nanomaterials.Our as-prepared GNRs@mSiO2could be used as effective thermal generators owing to the GNRs ingredients. The photothermal effects depend on GNRs@mSiO2concentration, laser illumination intensity and laser illumination time. DOX was readily loaded into the GNRs@mSiO2, pGNRs@mSiO2and pGNRs@mSiO2-RGD at pH of7.0with a28.1%±1.4%,25.2±1.5%and25.5±1.1%loading capacity. The rates and amounts of DOX released from the nanocarries were strongly dependent on the pH of the medium and the releasing time. The DOX release from nanocarries could be controlled by using NIR laser. After the NIR laser irradiation, the cumulative release of DOX could increase.The second part described the uptake of pGNRs@mSiO2-RGD and tumor targeting properties. The uptake efficiencies of pGNRs@mSiO2and pGNRs@mSiO2-RGD nanomaterials by A549cells were compared. TEM demonstrated that the uptake amount of pGNRs@mSiO2-RGD in A549cells were significantly more than pGNRs@mSiO2. Meanwhile, DOX-loaded pGNRs@mSiO2and pGNRs@mSiO2-RGD were also employed to investigate cellular uptake characteristics by mean of fluorescent images and flow cytometry of DOX. The fluorescence intensity of A549cells treated with DOX-pGNRs@mSiO2-RGD was considerably higher than that of DOX-pGNRs@mSiO2. Moreover, the cellular uptake of DOX-pGNRs@mSiO2-RGD exhibited time-dependent mode. The quantitative analysis of the cellular uptake by flow cytometry showed that the percent of DOX-positive cells was32.20%after2h incubation with DOX-pGNRs@mSiO2-RGD, but only14.19%with DOX-pGNRs@mSiO2. The results suggested that pGNRs@mSiO2-RGD could act as a transmembrane delivery carrier to increase cell internalization and the DOX intracellular accumulation. With the fluorescent tracking of intracellular pathway, DOX-pGNRs@mSiO2-RGD can enter into the cytoplasm and quickly deliver DOX into the nuclei.To explore their tumor targeting properties, DiR was employed to load with pGNRs@mSiO2and pGNRs@mSiO2-RGD for in vivo fluorescence imaging. The results showed that pGNRs@mSiO2conjugated with RGD could ensure specific delivery and long-time accumulation in tumor tissues through the active tumor targeting. The third part described the pharmacodynamic evaluation. Both DOX-pGNRs@mSiO2and DOX-pGNRs@mSiO2-RGD nanocarries were cytotoxic against A549cells in a dose-dependent manner. The treatment efficacy of chemo-photothermal therapy was higher than the separate therapeutic efficacy of chemo-and photothermal therapy.A pilot chemo-photothermal therapy study in vivo was performed. A549tumor-bearing mice were randomized into5treatment groups (n=8per group):PBS treated (Group Ⅰ), laser treated (Group Ⅱ), DOX treated (6.25mg kg"1, Group Ⅲ), pGNRs@mSiO2-RGD with laser treated (25mg GNRs@mSiO2-equiv./kg, Group IV), DOX-pGNRs@mSiO2-RGD with laser treated (25mg GNRs@mSiO2-equiv./kg, with an equivalent DOX dosage of6.25mg kg-1, Group V). After2h injection, tumors were illuminated with an808-nm NIR laser for30sec (3W/cm2; spot size,5mm) in Group Ⅱ, Ⅳ Ⅴ. During the irradiation, the temperature rapidly increased from31.5to65.9℃in the focal region in Group Ⅳ mouse. In contrast, the maximum temperature of the tumor surface in Group Ⅱ was only about40.1℃. Among them, the antitumor efficiency of Group Ⅴ was particularly prominent and was superior to all the other groups (P<0.05). At23th days, mice were sacrificed and tumors were excised. The tumor weights of Group Ⅴ were the lowest, showing an inhibition rate of66.45%and52.28%compared with Group Ⅲ, Ⅳ. Compared with chemotherapy or photothermal treatment alone, the combined treatment showed a synergistic effect, resulting in higher therapeutic efficacy for in vivo cancer therapy.The results of pharmacodynamic evaluation were in agreement with those of cells uptake and tumor targeting properties. pGNRs@mSiO2-RGD could ensure specific delivery and long-time accumulation in tumor tissues through the active tumor targeting, which facilitated the chemo-photothermal therapy.The fourth part described the biological safety of pGNRs@mSiO2-RGD. Direct irradiation of the cells showed little effect on cell viability. The GNRs@mSiO2, pGNRs@mSiO2and pGNRs@mSiO2-RGD without CTAB residual showed no cells cytotoxicity.The results of hematology analysis showed that the parameters of platelet and hematocrit in pGNRs@mSiO2group appeared to be significant difference in comparison with saline group. The CD68immunohistochemical staining showd that only light toxicity to liver was observed. The results of H&E staining showed that both DOX-pGNRs@mSiO2group and DOX-pGNRs@mSiO2-RGD group presented no significant differences in tissues including brain, liver, spleen, lung and kidney when compared to DOX group. The heart slice of DOX group exhibited myocardial fiber rupture, which was conduced by the cardiac toxicity of DOX. |
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