Novel vinyl-modified RGD conjugated silica nanoparticles based on photo click chemistry for in vivo prostate cancer targeted fluorescence imaging

Abstract

Molecular imaging is a powerful tool for non-invasive visualization of tumors that plays an important role in their diagnosis and treatment. The specificity of molecular imaging probes for cancer cells is important for accurate tumor visualization, with antibody and polypeptide nanoprobe conjugates having often been used as targeting agents for tumor detection. However, many traditional chemical conjugation methods employ complex conjugation reactions that result in poor efficiency and poor bioactivity. Herein, we describe the use of photo click methodology for the rapid synthesis of nanoprobes comprised of silica nanoparticles functionalized with RGD targeting units (SiO₂@T1-RGDk NPs) (∼80 nm) for in vivo prostate cancer fluorescent imaging applications. These SiO₂@T1-RGDk NPs exhibit a maximum absorption wavelength of 380 nm in their UV absorption spectra with a maximum fluorescence emission wavelength of 550 nm. Confocal immunofluorescent imaging reveal that SiO₂@T1-RGDk NPs exhibit excellent targeting ability for visualizing cancer cells, with in vivo fluorescence imaging intensity in a subcutaneous tumor model of prostate cancer reaching a maxima after 4 h. Biosafety assessments showed that SiO₂@T1-RGDk NPs demonstrate no obvious toxicity in mice, thus demonstrated that these novel NPs may prove to be promising fluorescent imaging agents for the accurate detection and treatment of tumors.

Scheme 1. Synthesis of SiO₂@T1-RGDk NPs. (A) Synthesis schematic of SiO₂@T1-RGDk NPs; (B) the photo click reaction between tetrazole compound T1 and RGD.

Fig. 1. Characterization of SiO₂@T1-RGDk NPs. (A) TEM images of SiO₂ NPs; (B) size distribution of SiO₂ NPs determined through DLS; (C) UV absorption spectra of T1, SiO₂ NPs, SiO₂@T1 NPs and SiO₂@T1-RGDk NPs; (D) UV absorption and fluorescence spectra of T1-RGDk.

Fig. 2. (A) Confocal immunofluorescent images of SiO₂@T1-RGDk NPs showing their localization in PC-3 cells. (B) Confocal immunofluorescent images of SiO₂@T1-RGDk NPs showing their localization in PC-3 cells in the presence of RGD-Ack blocking agent. Cells stained with DAPI are colored blue. Cells containing SiO₂@T1-RGDk NPs are colored red.

Fig. 3. Cell viability of PC-3 cells incubated with different concentrations of SiO₂@T1-RGDk NPs, SiO₂ NPs, T1 and RGD-Ack.

Fig. 4. Acute toxicity results of SiO₂@T1-RGDk NPs in BALB/c mice; (A) survival rates of SiO₂@T1-RGDk NPs in BALB/c mice; (B) H&E staining of major organs (including heart, liver and kidneys) from mice 14 days after injected with SiO₂@T1-RGDk NPs. Healthy mice treated with saline were used as control.

Fig. 5. (A) In vivo fluorescent imaging of SiO₂@T1-RGDk NPs after 12 h. (B) Changes in fluorescent intensity of SiO₂@T1-RGDk NPs that occur during in vivo imaging studies over time.

Fig. 6. Fluorescent images of frozen tumor cross-sections, shown by good superposition of blue and green fluorescence signals that correspond to DAPI and dSiO₂@T1-RGDk NPs, respectively.