Temperature-dependent cell death patterns induced by functionalized gold nanoparticle photothermal therapy in melanoma cells.

Photothermal therapy (PTT) is a promising approach for cancer targeting therapy. However, the temperature-dependent killing of tumor cells in PTT remains unclear. In this study, we report necroptosis plays a role in the anti-tumor effects observed in gold nanorod (GNR)-mediated PTT in melanoma. We first synthesized gold nanorods with a targeting adaptor FA (GNRs-FA), which achieved high efficacy of targeted delivery to melanoma cells. We further demonstrated PTT, precipitated by GNRs-FA under the induction of near-infrared laser, was temperature-dependent. Furthermore, the photothermal killing of melanoma cells showed different patterns of cell death depending on varying temperature in PTT. In a lower temperature at 43 °C, the percentages of apoptosis, necroptosis and necrosis of tumor cells were 10.2%, 18.3%, and 17.6%, respectively, suggesting the cell killing is ineffective at lower temperatures. When the temperature increased to 49 °C, the cell death pattern switched to necrosis dominant (52.8%). Interestingly, when the PTT achieved a moderate temperature of 46 °C, necroptosis was significantly increased (35.1%). Additionally, GNRs-FA/PPT-mediated necroptosis was regulated by RIPK1 pathway. Taken together, this study is the first to demonstrate that temperature-dependent necroptosis is an important mechanism of inducing melanoma cell death in GNR-mediated PTT in addition to apoptosis and necrosis.

Targeted-gene silencing of BRAF to interrupt BRAF/MEK/ERK pathway synergized photothermal therapeutics for melanoma using a novel FA-GNR-siBRAF nanosystem.

Melanoma is significantly associated with mutant BRAF gene, a suitable target for siRNA-based anti-melanoma therapy. However, a tumor-specific delivery system is a major hurdle for clinical applications. Here, we developed a novel nano-carrier, FA-GNR-siBRAF for safe topical application, which consists of folic acid (FA) as the tumor-targeting moiety, golden nanorods (GNR) providing photothermal capability to kill tumor cells under laser irradiation, and siRNA specifically silencing BRAF (siBRAF). The in vitro and in vivo results revealed that FA-GNR-siBRAF displayed high transfection rates, and subsequently induced remarkable gene knockdown of BRAF, resulting in suppression of melanoma growth due to the interruption of the MEK/ERK pathway. Combinatorial photothermal effects and BRAF knockdown by FA-GNR-siBRAF effectively killed tumor cells through apoptosis, with enhanced efficiency than individual treatments. Therefore, the FA-GNR-siBRAF simultaneously induced BRAF gene silencing and photothermal effects which achieved synergistic efficacy in the treatment of melanoma, paving a new path for developing clinical treatment methods for melanoma.

A novel GNRs-PEI/GNRs-PEI-folate for efficiently delivering siRNA.

RNA interference (RNAi) employs double-stranded RNA or siRNA (small interfering RNA) to silence gene expression in cells. The widespread use of RNAi therapeutics requires the development of clinically suitable, safe and effective delivery vehicles. PEI (Poly(ethylene imine)) carrying the positive charges has attracted considerable attention for siRNA delivery. Gold nanorods (GNRs) exhibit specially localized surface plasmon resonance when excited by the visible and near-infrared laser, which is useful for photothermal therapy. However, the toxicity derived from a large amount of the surfactant cetyltrimethylammonium bromide (CTAB) during GNR synthesis severely limits their medical applications. Here, we report the synthesis of GNRs-PEI/GNRs-PEI-folate to improve biocompatibility, siRNA delivery and photothermal therapy of GNRs. Firstly, GNRs were synthesized according to the seed-mediated template-assisted protocol. The characterization results of GNRs showed: the size was length about 218 nm and width about 26.8 nm; the Zeta potential was +38.1 mV derived from CTAB on their surface; the dipole resonance extinction spectrum peak was 752 nm which is effective for photothermal therapy in vivo. Secondly, we synthesized PEI-MUA (Mercaptoundecanoic acid) and PEI-MUA-folate based on the chemical reaction between amino group of PEI and carboxyl group of MUA or Folate. PEI-MUA or PEI-MUA-folate to replace CTAB on GNRs obtained the GNRs-MUA-PEI system or the GNRs-MUA-PEI-folate system due to the solid conjugation between the thiol group of MUA and GNRs. The products were measured using the FTIR Spectrometer, and the spectra suggest MUA-PEI or PEI-MUA-folate has successfully replaced CTAB on the surface of GNRs. Finally, GNRs-MUA-PEI was incubated with siRNA-Cy3. The unbound siRNA-Cy3 was measured the intensity of fluorescence for calculating the uploaded amount of siRNA by GNRs-MUA-PEI, and the results indicate that the uploaded percentage of siRNA is about 70%. We conclude that the GNRs-MUA-PEI system is an effective siRNA loading vehicle.

Cadmium triggers kidney cell apoptosis of purse red common carp (Cyprinus carpio) without caspase-8 activation.

Caspase-8, the essential initiator caspase, is believed to play a pivotal role in death receptor-mediated apoptotic pathway. It also participates in mitochondria-mediated apoptosis via cleavage of proapoptotic Bid in mammals. However, its role in fish remains elusive in Cadmium-induced apoptotic pathway. In this study, we isolated the caspase-8 gene from common carp, one of the most important industrial aquatic animals in China using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of caspase-8 comprised 475 amino acids, which showed approximately 64.1% identity and 79.8% similarity to zebrafish (Danio rerio) caspase-8, possessed two conserved death effector domains, a large subunit and a small subunit. Phylogenetic analysis demonstrated that caspase-8 formed a clade with zebrafish caspase-8. In kidney, cadmium (Cd) exposure triggered apoptosis and increased caspase-3 and -9 activities, whereas it did not affect caspase-8 activity. Real-time quantitative PCR analysis revealed that caspase-8 transcriptional level was not significantly increased in kidney after exposure to Cd. Using Western blot analysis, no caspase-8 cleaved fragment was detected and no significant alteration of procaspase-8 level was found with the same Cd-treated condition. Moreover, the immunopositive staining was predominantly limited to the cytoplasm of renal tubular epithelial cells and no remarkable changes of immunoreactivities were observed using immunohistochemical detection after Cd treatment. The results reveal that Cd can trigger apoptosis, while it cannot activate caspase-8 in purse red common carp.

Intranasal CpG DNA therapy during allergen exposure in allergic rhinitis.

OBJECTIVE: 1) To estimate the effectiveness of intranasal administration of CpG DNA alone on allergic rhinitis compared with intradermal administration; and 2) to find out how CpG DNA therapy is useful in treatment of allergic rhinitis. STUDY DESIGN: Mice were intraperitoneally sensitized and intranasally challenged with Japanese cedar. Therapy with CpG DNA alone was also performed during challenge, either intranasally or intradermally. Immunologic variables and nasal symptom were studied. RESULTS: Intranasal administration of CpG DNA alone significantly reduced the levels of IgE, IL-5 productions from nasal lymphocytes and splenocytes, nasal eosinophilia, and nasal symptoms, although intradermal administration of CpG DNA alone showed no significant reduction. CONCLUSION: This study demonstrated that CpG DNA has effects not only on splenocytes but also on nasal lymphocytes to attenuate allergic rhinitis, and that intranasal administration, but not intradermal administration, of CpG DNA alone during allergen exposure is useful for control of allergic rhinitis.