Immunoliposome co-delivery of bufalin and anti-CD40 antibody adjuvant induces synergetic therapeutic efficacy against melanoma

Abstract

Liposomes constitute one of the most popular nanocarriers for improving the delivery and efficacy of agents in cancer patients. The purpose of this study was to design and evaluate immunoliposome co-delivery of bufalin and anti-CD40 to induce synergetic therapeutic efficacy while eliminating systemic side effects. Bufalin liposomes (BFL) conjugated with anti-CD40 antibody (anti-CD40-BFL) showed enhanced cytotoxicity compared with bufalin alone. In a mouse B16 melanoma model, intravenous injection of anti-CD40-BFL achieved smaller tumor volume than did treatment with BFL (average: 117 mm(3) versus 270 mm(3), respectively); the enhanced therapeutic efficacy through a caspase-dependent pathway induced apoptosis, which was confirmed using terminal deoxynucleotidyl transferase-mediated dUTP-Fluorescein nick end labeling and Western blot assay. Meanwhile, anti-CD40-BFL elicited unapparent body-weight changes and a significant reduction in serum levels of tumor necrosis factor-α, interleukin-1β, interleukin-6, interferon-γ, and hepatic enzyme alanine transaminase, suggesting minimized systemic side effects. This may be attributed to the mechanism by which liposomes are retained within the tumor site for an extended period of time, which is supported by the following biodistribution and flow cytometric analyses. Taken together, the results demonstrated a highly promising strategy for liposomal vehicle transport of anti-CD40 plus bufalin that can be used to enhance antitumor effects via synergetic systemic immunity while blocking systemic toxicity.

Keywords: anti-CD40; bufalin; chemoimmunotherapy; liposomes.

Figure 1

Preparation schematic and characterization of PEGylated liposomes containing bufalin with or without anti-CD40. Notes: (A) Schematic illustration of the surface coupling of anti-CD40 to PEGylated liposomes via maleimide–thiol reaction. (B) Particle size distribution of BFL and conjugated with anti-CD40 antibody (anti-CD40-BFL) in the range of 150–200 nm, as measured by dynamic light scattering. (C) TEM images of BFL and anti-CD40-BFL, with a uniform spherical shape. Abbreviations: BFL, bufalin liposomes; DTT, dithiothreitol; Mal-thiol, maleimide–thiol; SH, sulfhydryl; TEM, transmission electron microscope.

Figure 2

In vitro release profiles of bufalin and anti-CD40 from combination liposomes by dialysis against phosphate buffered saline, compared to the dialysis of freely soluble bufalin and anti-CD40. Notes: The results are a representative of three independent experiments. (A) Release of encapsulated bufalin from combination liposomes was measured by high-performance liquid chromatography and occurred more slowly than release of freely soluble bufalin. (B) Release of conjugated anti-CD40 from combination liposomes was measured by enzyme-linked immunosorbent assay and reached a plateau after 2 days. Abbreviation: BFL, bufalin liposomes.

Figure 3

In vitro cytotoxicty of blank liposome, free anti-CD40, bufalin, BFL, and anti-CD40-BFL against B16 melanoma cells. Notes: The diagrams of B16 melanoma cell viability at various drug concentrations (A) after 6 hours of treatment and (B) after 24 hours of treatment. Bufalin showed a higher cytotoxicity on B16 cells than liposomes after 6 hours, due to the controlled release of liposome. After 24 hours, the cell viability was adverse. The cell viabilities of BFL and anti-CD40-BFL were similar, attributable to the absence of CD40 surface expression on B16 cells. Data are presented as mean ± standard deviation (n=6). Abbreviation: BFL, bufalin liposomes.

Figure 4

Therapeutic efficacy on C57/BL6 mice carrying subcutaneous B16 melanoma tumors. Notes: (A) Average tumor growth curves from a representative experiment. Mice were given tail-vein injections on day 9, day 11, day 13, and day 15 following tumor cell inoculation with phosphate-buffered saline, soluble anti-CD40, bufalin, BFL, or anti-CD40-BFL at an equivalent dose of 1 mg/kg or 8 mg/kg of anti-CD40 per injection. (B) Body-weight changes of the mice over the experimental period. (C) Photographs of melanoma tumors in each group. (D) Average tumor weight at the endpoint of study. P-values were determined by paired Student’s t-tests: *P<0.05, **P<0.01 (n=6, mean ± standard error). Abbreviation: BFL, bufalin liposomes.

Figure 5

Apoptosis in control and tumor cells. Notes: (A) Typical fluorescence microscopic images of TUNEL staining of tumor cytosections treated with saline, free anti-CD40, bufalin, BFL, or anti-CD40-BFL; scale bars denote 100 μm. Blue: cell nuclei, DAPI. Green: apoptosis cells. (B) H&E-stained tumor paraffin sections from mice at the endpoint of the study after treatment with saline, free anti-CD40, bufalin, BFL, or anti-CD40-BFL. Original magnification: 100×. (C) Histogram of mean apoptotic index in each group. (D) Western blot analysis of cytochrome c, caspase-3, and caspase-9 protein in tumor tissues after treatment with saline, free anti-CD40, bufalin, BFL, or anti-CD40-BFL. P-values were determined by paired Student’s t-tests: *P<0.05, **P<0.01 (n=6, mean ± standard error). Abbreviations: BFL, bufalin liposomes; CYTC, cytochrome c; DAPI, 4,6-diamidino-2-phenylindole; H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling.

Figure 6

Serum circulating inflammatory marker levels. Notes: Serum circulating levels 24 hours after the first dose of saline, free anti-CD40, bufalin, BFL, or anti-CD40-BFL. (A) Hepatic enzyme ALT. Inflammatory cytokines (B) TNF-α, (C) IL-6, (D) IL-1β, and (E) IFN-γ. P-values determined by unpaired Student’s t-test: **P<0.01. Abbreviations: ALT, alanine transaminase; BFL, bufalin liposomes; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

Figure 7

Immunofluorescence analysis of frozen tissue sections. Notes: Immunofluorescence analysis of frozen tissue sections shows fluorescence rhodamine-labeled liposomes (red), anti-CD40 (green), and merged (yellow) images. Scale bars denote 100 μm. Liposomes were retained at a high level within the representative cryosections of tumors, spleens and lymph nodes for 24–48 hours postinjection, and anti-CD40 still remained coupled to the liposome in vivo. Meanwhile, green fluorescence was barely observed following an injection of soluble anti-CD40 therapy after 24 hours and nearly disappeared after 48 hours. Abbreviation: BFL, bufalin liposomes.

Figure 8

Bar graph summary of the flow cytometer analysis of excised tumors, spleens, and lymph nodes. Notes: (A) Percentage of dendritic cells (CD45⁺CD11c⁺) in tumors, spleens, and lymph nodes after injected saline, free anti-CD40, bufalin, BFL, or anti-CD40-BFL for 24 hours or 48 hours. (B) Percentage of CD4⁺ T cells (CD3⁺CD4⁺) in tumors, spleens, and lymph nodes after injected saline, free anti-CD40, bufalin, BFL, or anti-CD40-BFL for 24 hours or 48 hours. (C) Percentage of CD8⁺ T cells (CD3⁺CD8a⁺) in tumors, spleens, and lymph nodes after injected saline, free anti-CD40, bufalin, BFL, or anti-CD40-BFL for 24 hours or 48 hours. Abbreviation: BFL, bufalin liposomes.

Figure 9

Schematic illustration of immunoliposome co-delivery of bufalin and anti-CD40 antibody adjuvant induce synergetic therapeutic efficacy. Notes: After anti-CD40-BFL is injected, liposomes enter tumor cells via endocytosis. The bufalin and anti-CD40 are released after liposome fusion. Bufalin induces apoptosis via a caspase-dependent pathway. On the other hand, the anti-CD40 stimulation is delivered by CD40 ligand-expressing activated CD4⁺ T cells. CD40 ligand binding to CD40 adhesion molecules on the surface of antigen-presenting cells, such as dendritic cells, stimulates the production of cytokines, further induces sufficient cytotoxic T lymphocytes, and produces a systemic immune response. Abbreviations: BFL, bufalin liposome; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.