| Backgrounds There is currently an increasing need for early detection and treatment of cancer before anatomic anomalies are visible. Nanotechnology has advanced greatly in recent years is becoming a promising approach for cancer diagnosis and treatment. One of the current challenges in biomedicine is to develop safe and effective nanoprobes for tumor targeting and selective therapy. Gold nanorods (GNRs) are strongly absorbing in at near-infrared wacelength and have excellent potential for photothermal therapy.Objective Here, we prepared unique tumor targeting nanoprobes by conjugating dendrimer-modified GNRs with RGD peptides, with the aim of developing a novel method for early detection, diagnosis and imaging of melanoma.Methods First, gold nanorods were prepared by seed-mediated surfactant directed approach and controlling the different reaction synthetic conditions.Then, we used the partially thiolated polyamidoamine (PAMAM) dendrimer to replace CTAB molecules on the surface of GNRs, with the aim of improving the biocompatibility of prepared GNRs. Finally, we prepared unique tumor targeting nanoprobes by conjugating dendrimer-modified GNRs with RGD peptides. Prepared RGD-dGNR nanoprobes were characterized by high-resolution-transmission-electron microscopy (HR-TEM), atomic force microscopy (AFM), 1H Nuclear magnetic resonance (1H-NMR) and UV-vis absorbance spectroscopy.HUVEC cell line with overexpression ofαvβ3 integrin was used as positive control group. The MCF-7 cell line with lower expression of integrinαvβ3 was selected as the negative control group. The melanoma A375 cell line with over-expression of integrin αvβ3 was selected as the test group. The Cell Counting Kit-8 assay was used to measure cytotoxicity of synthesized nanoprobes following the instruction of the kit. Then we evaluated the specificity and sensitivity of RGD-dGNR nanoprobes for tumor cell targeting using the dark field microscope. Parallel competition inhibition control experiments were set up. Cells incubated with and without RGD-dGNR nanoprobes were exposed to NIR laser irradiation, with a wavelength of 808 nm and varying intensities, from 30 mW (4 W/cm2) to 150 mW (20 W/cm2) in increments of 40 mW. The laser spot size is 1.0 mm in diameter, and the exposure time is 4 min. The cell viability was tested by both 0.4% trypan blue and Calcein-AM staining.Melanoma A375 cells (5×106) were injected subcutaneously into the right rear flank area of nude mice. When tumors grew to 5 mm in diameter, 200μg of RGD-dGNR nanoprobes was injected into mice via tail vein. For the blocking experiment, ten mice were injected with the mixture of 0.5 mg of RGD peptides and 200μg of RGD-dGNR nanoprobes. Mice were respectively sacrificed at 3 h, 6 h, 9 h, and 12 h. Blood and organs were collected and kept in liquid nitrogen. The amount of RGD-dGNRs was measured by inductively coupled plasma mass spectrometry.Mice loaded with tumors were randomly divided into three groups: test group (200μg of nanoprobes plus NIR laser irradiation); sham control group (10 mice) (PBS plus NIR laser irradiation), and blank control (untreated). The nude mice were injected with 200μg of prepared nanoprobes in PBS via tail vein. At 6 h after injection, the mice were anesthetized and irradiated with a NIR laser with a wavelength of 808 nm at a power density of 24 W/cm2 and a spot size of 5 mm diameter for 5 min. Tumors were irradiated for four times per month, once every week. Real time reflectance confocal micrscopy (RCM) was used to collect images of tumors from test group and control group and to assess the histological and blood flow changes. Hematoxylin and eosin staining was used to examine tumor histological changes. Tumor size and mice survivability were monitored for 7 weeks.Results The original gold nanorods (GNRs-CTAB) are about 42 nm in length, and 10 nm in width. Dendrimer-modified GNRs exhibit better dispersion in water solution than GNRs-CTAB. GNRs-CTAB has two absorption bands, a weak short-wavelength band around 520 nm and a strong long-wavelength band around 821 nm. RGD-dGNRs exhibit an absorption band red-shift (about 3nm). We observed that RGD-dGNRs exhibited good stability and dispersibility in water or organic solution with different pH conditions. Initial evaluation of the cytotoxicity of RGD-dGNR nanoprobes showed both the dendrimer-modified GNRs and RGD-dendrimer-modified GNRs were biologically nontoxic within the concentration of 200μg/mL. GNR-CTAB concentration of 100μg/ml exhibited marked cytotoxicity.Under dark field microscopy, melanoma A375 cells incubated with RGD-dGNR nanoprobes exhibit a strong golden color; the melanoma A375 cells incubated with dGNRs and preincubated with free RGD peptides did not exhibit a golden color, and similar negative results also were observed for MCF-7 cells incubated with dGNRs or RGD-dGNRs, which highly suggests that free RGD peptides can block the binding of RGD-dGNR nanoprobes with integrinαvβ3 over-expressed in the tumor cells, and RGD-dGNR nanoprobes can specifically target melanoma A375 cells.A375 cells incubated with RGD-dGNR nanoprobes exhibited destruction within the laser spots after exposure to the laser at 70 mW. When the laser energy reached 110 mW, the amount of destroyed tumor cells increased accordingly. Few damaged cells were observed for the A375 cells treated with dGNRs or NIR light alone. Similarly, no dead cells were observed for MCF-7 cells treated with RGD-dGNR nanoprobes. These results fully showed that RGD-dGNR nanoprobes were only employed to kill cancer cells with overexpression of integrinαvβ3 under NIR irradiation. Under the condition of 110 mW laser irradiation for 4 min, as the amount of RGD-dGNR nanoprobes in the medium increased, the amount of destroyed cells also increased accordingly. 100μg/ml RGD-dGNR nanoprobes was considered as the optimal photothermal therapeutic concentration for in vitro cancer cells.The distribution of RGD-dGNR nanoprobes was examined in the whole body of mouse models, 17% of the RGD-dGNR nanoprobes accumulated in local tumor tissues at 6 h after injection. Nanoprobes in the tumor tissues increased gradually as time increased, which fully suggests that the prepared RGD-dGNR nanoprobes were targeting tumor tissues. Six hours after injection was selected as the optimal time to begin the NIR laser irradiation on the tumor locations. For the competition inhibition experiment group, few RGD-dGNR nanoprobes accumulated in the tumor location, highly indicating the free RGD peptides can first bind with integrinαvβ3 on the surface of tumor cells, and block the specific binding of RGD-dGNR nanoprobes with the tumor cells and vasculature endothelial cells in vivo.Under the NIR laser irradiation, the average tumor size in the test group was markedly smaller than those in the control groups. Interestingly, we observed, the tumor tissues in four mice almost completely disappeared in three weeks or so. We observed that the median survivability of mice in the control group without any treatment was three weeks, in the control group treated with PBS plus NIR laser was four weeks, and in the test group was more than seven weeks. The survivability of mice in the test group was markedly stronger than the one in the control group, P= 0.006. The Kaplan-Meier curve suggests that the therapy based on nanoprobe injection plus NIR laser irradiation can markedly increase the survival time of mice with tumors.We observed that the blood flow inside tumor tissues in the test group began to be blocked at 3 h after NIR laser irradiation using real time reflectance confocal microscopy (RCM). In the control group, no obvious dynamic blood flow alteration was observed. Additionally, after seven weeks of treatment, RCM images of tumors in the test group clearly displayed numerous gossamer-like collagen bundles and branchlike collagens. Numerous reflective melanoma cells were still distributed throughout the tumor tissues in the control group. Accordingly, histological analysis showed that the tumors in test group after seven weeks of treatment exhibited a scarlike structure containing numerous collagen bundles. A lot of tumor cells were observed in those tumors untreated and treated with the PBS plus laser irradiation.Conclusion In conclusion, our study confirms that the RGD-conjugated dGNR nanoprobes are not cytotoxic, can specifically target tumor cells and vascular cells inside tumor tissues, and exhibit selective destructive effects on the melanoma cells under NIR laser irradiation. Compared with presently available reports, the RGD-dGNR nanoprobe-based targeting therapy possesses some major advantages, which can extend to various tumors with overexpressed integrinαvβ3. The RGD-dGNR-based therapy strategy has great potential in applications such as tumor targeting imaging and selective photothermal therapy.
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