Cooperative nanomaterial system to sensitize, target, and treat tumors

Abstract

A significant barrier to the clinical translation of systemically administered therapeutic nanoparticles is their tendency to be removed from circulation by the mononuclear phagocyte system. The addition of a targeting ligand that selectively interacts with cancer cells can improve the therapeutic efficacy of nanomaterials, although these systems have met with only limited success. Here, we present a cooperative nanosystem consisting of two discrete nanomaterials. The first component is gold nanorod (NR) "activators" that populate the porous tumor vessels and act as photothermal antennas to specify tumor heating via remote near-infrared laser irradiation. We find that local tumor heating accelerates the recruitment of the second component: a targeted nanoparticle consisting of either magnetic nanoworms (NW) or doxorubicin-loaded liposomes (LP). The targeting species employed in this work is a cyclic nine-amino acid peptide LyP-1 (Cys-Gly-Asn-Lys-Arg-Thr-Arg-Gly-Cys) that binds to the stress-related protein, p32, which we find to be upregulated on the surface of tumor-associated cells upon thermal treatment. Mice containing xenografted MDA-MB-435 tumors that are treated with the combined NR/LyP-1LP therapeutic system display significant reductions in tumor volume compared with individual nanoparticles or untargeted cooperative system.

Fig. 1.

Characterization of the components of cooperative nanosystems. (A) Schematic showing the components of the two cooperative nanomaterials systems used in this study. The first component consists of gold nanorods (NR), which act as a photothermal sensitizer. The second component consists of either magnetic nanoworms (NW), or doxorubicin-loaded liposomes (LP). Irradiation of the NR with a NIR laser induces localized heating that stimulates changes in the tumor environments. The NW or LP components decorated with LyP-1 tumor targeting peptides bind to the heat-modified tumor environments more efficiently than to the normal tumor environments. Transmission electron microscope images of all three components are shown. Scale bars indicate 50 nm. (B) Temperature changes induced by localized laser irradiation (+L) of mice injected with NR alone (no NW or LP). Tumor-bearing mice were injected intravenously with either PEGylated NRs (NR) or saline (saline). Trace labeled “NR-L” is a control where NRs were injected but the tumor was not irradiated. Data and images obtained 72 h postinjection; infrared thermographic maps of average tumor surface temperature were obtained after laser exposure for the indicated times. Scale bar indicates 1 cm. (C) Effect of heating time on p32 expression in MDA-MB-435 xenograft tumor. Tumor in an athymic (nu/nu) mouse was heated at 45 °C for 30 min in a water bath. Images at left show cell surface p32 immunostaining of tumor sections 6 h posttreatment. Symbols + and - indicate with and without heating, respectively. Scale bar indicates 50 μm. At right are Western blot results for p32 relative to β-actin control. * indicates P < 0.05 for 0 h and 6 h intensity ratio (n = 3 ∼ 4). Brightness and contrast have been adjusted across the whole image. (D) Fluorescence microscope images of C8161 or MDA-MB-435 cells probing in vitro cellular binding and internalization of LyP-1-conjugated Cy5.5-labeled magnetic nanoworms (LyP1NWs, in green) upon heating to 45 °C. Samples were incubated for 20 min at 37 °C (-) or 45 °C (+) and then held at 37 °C for an additional 2 h. Cell nuclei and p32 stained with 4^′-6-diamidino-2-phenylindole (DAPI, Blue), and anti-p32 antibody followed by Alexa Fluor® 594 goat antirabbit IgG antibody (Red), respectively. Scale bar indicates 50 μm. All error bars indicate standard deviations from ≥3 measurements. Brightness and contrast have been adjusted across the whole image.

Fig. 2.

Temperature-induced amplification of in vivo tumor targeting. (A) Fluorescence intensity from Cy7-labeled LyP-1-conjugated magnetic nanoworms (LyP1NW) and Cy7-labeled control nanoworms (NW) in MDA-MB-435 tumor as a function of externally applied heat (30 min). Heated at (45 °C) and unheated (37 °C) samples indicated with (+) and (-), respectively. The tissues were collected from the mice 24 h postinjection; NIR fluorescence images use Cy7 channel. * indicates P < 0.05 (n = 3 ∼ 4). (B) Fluorescence image of major organs from the mice in (A). T+, T-, Li, Sp, K, and Br indicate heated tumor, unheated tumor, liver, spleen, kidney, and brain, respectively. (C) Histological analysis of LyP1NW or NW distribution in MDA-MB-435 tumors with (+) or without (-) application of external heat. Green indicates NWs (labeled with Cy 5.5). Cellular stains same as in Fig. 1D, blood vessels stained with CD31 followed by Alexa Fluor® 594 goat antirat IgG. Arrowhead indicates a lymphatic vessel structure displaying a signal from the labeled LyP1NWs. Scale bar is 100 μm. Error bars indicate standard deviations from ≥3 measurements.

Fig. 3.

Heat-mediated cytotoxicity of targeted therapeutic nanoparticles in vitro. (A and B) Temperature-induced cytotoxicity of various therapeutic molecule or nanoparticle formulations toward MDA-MB-435 human carcinoma cells by MTT assay. The cells were treated with free DOX, control DOX-containing liposomes (LP), or LyP-1-conjugated, DOX-containing liposomes (LyP1LP) with the indicated concentrations of DOX. Samples incubated at 37 °C (A) or 45 °C (B).

Fig. 4.

Successful antitumor therapy using cooperative nanosystem, demonstrated in mice bearing MDA-MB-435 tumors. (A) Quantification of in vivo accumulation of DOX in tumors as a function of NR-mediated laser heating of LyP-1-conjugated liposomes (LyP1LP) or control liposomes that contain no targeting peptide (LP). NR + L and NR - L indicate mice containing gold nanorods that were or were not subjected to laser treatment, respectively. Amount of DOX present quantified by fluorescence microscopy to yield a percentage of injected dose per tissue mass. * indicates P < 0.05 (n = 3 ∼ 4). (B) Histological analysis of DOX distribution in tumors from the mice in (A) who were subjected to NR-mediated thermal therapy showing the distribution of nanoparticles (Alexa Fluor® 488 label on control liposome and 5(6)-carboxyfluorescein (FAM) label on LyP-1, Green) and DOX (Red). Nuclei stained with DAPI (Blue). Scale bar is 100 μm. (C) Change in tumor volume of different treatment groups containing bilateral MDA-MB-435 xenograft tumors. 72 h postinjection of gold nanorods (NR, 10 mgAu/kg), mice were injected with a single dose of saline, control liposomes (LP), and LyP-1-conjugated liposomes (LyP1LP). “+H (Hyperthermia)” denotes one of the two tumors in the animal that was irradiated with the NIR laser. The tumor not irradiated is indicated as “-H”. Tumor volumes monitored every 3 d postirradiation. Error bars indicate standard deviations from ≥3 measurements. * indicates P < 0.05 and ** indicates P < 0.02 for +H + LyP1LP sample and all other treatment sets (n = 4 ∼ 6). (D) Survival rate in different treatment groups after a single dose (3 mgDOX/kg) into mice (n = 6) containing single MDA-MB-435 xenograft tumors. Error bars indicate standard deviations from ≥3 measurements.