Design and Application of pH-Responsive Liposomes for Site-Specific Delivery of Cytotoxin from Cobra Venom

Abstract

Background: Current immunotherapies with unexpected severe side effects and treatment resistance have not resulted in the desired outcomes for patients with melanoma, and there is a need to discover more effective medications. Cytotoxin (CTX) from Cobra Venom has been established to have favorable cytolytic activity and antitumor efficacy and is regarded as a promising novel anticancer agent. However, amphiphilic CTX with excellent anionic phosphatidylserine lipid-binding ability may also damage normal cells.

Methods: We developed pH-responsive liposomes with a high CTX load (CTX@PSL) for targeted acidic-stimuli release of drugs in the tumor microenvironment. The morphology, size, zeta potential, drug-release kinetics, and preservation stability were characterized. Cell uptake, apoptosis-promoting effects, and cytotoxicity were assessed using MTT assay and flow cytometry. Finally, the tissue distribution and antitumor effects of CTX@PSL were systematically assessed using an in vivo imaging system.

Results: CTX@PSL exhibited high drug entrapment efficiency, drug loading, stability, and a rapid release profile under acidic conditions. These nanoparticles, irregularly spherical in shape and small in size, can effectively accumulate at tumor sites (six times higher than free CTX) and are rapidly internalized into cancer cells (2.5-fold higher cell uptake efficiency). CTX@PSL displayed significantly stronger cytotoxicity (IC₅₀ 0.25 μg/mL) and increased apoptosis in than the other formulations (apoptosis rate 71.78±1.70%). CTX@PSL showed considerably better tumor inhibition efficacy than free CTX or conventional liposomes (tumor inhibition rate 79.78±5.93%).

Conclusion: Our results suggest that CTX@PSL improves tumor-site accumulation and intracellular uptake for sustained and targeted CTX release. By combining the advantages of CTX and stimuli-responsive nanotechnology, the novel CTX@PSL nanoformulation is a promising therapeutic candidate for cancer treatment.

Keywords: cobra venom cytotoxin; liposomes; pH-responsive; targeted delivery; tumor microenvironment.

Figure 1

Self-assembly and release process of pH-sensitive nanoparticle loaded with CTX.

Figure 2

Characterization, Stability and drug release of CTX@PSL. (A) Morphology of CTX@PSL, the scale bar = 200 nm. (B) Particle size distribution of CTX@PSL. (C) CTX@PSL stability examination. (D) Effect of pH on the cumulative release of CTX@PSL in vitro.

Figure 3

Cellular uptake efficacy of Dir@CTX loaded nanoparticles using flow cytometry, n = 3. All the data are presented as mean ± SD, *p<0.05, **p<0.01.

Figure 4

In vitro cytotoxicity assay. (A and B) FACS analysis of apoptosis induced by CTX in different liposome in B16-F10 cells (Mean ± SD, n = 4), **P < 0.01, ***P < 0.005. (C) Cellular inhibition of B16-F10 cells after 24h treatment (Mean ± SD, n = 6).

Figure 5

In vivo distribution. (A) Time-volume relationship of CTX@PSL in the targeting distribution of experimental melanoma growth in mice. (B) Distribution of CTX and CTX@PSL in tumor-bearing mice at 0.5h, *p < 0.05.

Figure 6

Therapeutic efficacy in a melanoma tumor model. (A) Melanoma growth in mice (some mice); (B) Melanoma masses exfoliated from mice; (C) Body weight gain curve of mice during treatment; (D) Inhibitory effect of CTX@PSL on the growth of melanoma in mice. All the data are presented as mean ± SD (n=10), ^##p<0.01, ***p<0.001; (E) Histological sections of ICR mice. Heart: red arrows indicate enhanced cytoplasmic eosinophilia and nuclear fixation of cardiomyocytes; Liver: red arrows indicate granular degeneration of hepatocytes and balloon-like degeneration; Lungs: red arrows indicate a small amount of eosinophilic granules visible in the bronchial lumen, mild thickening of the alveolar wall and inflammatory cell infiltration; Kidney: red arrows indicate tubular epithelial cell edema and interstitial stasis of the kidney; Spleen: red arrows indicate multinucleated giant cells visible in the red pulp, in addition to a few granulocytes. HE, scale bar = 100 μm.