Toxicity evaluation of cordycepin and its delivery system for sustained in vitro anti-lung cancer activity

Abstract

In the previous study, we have found that the cordycepin which was extracted from Cordyceps mycelia produced by growing Cordyceps militaris on the dead larva of Bombyx mori silkworms showed the anti-proliferative effect toward lung cancer cells without toxicity to non-cancer cells. In this work, the cordycepin was tested for its in vitro mutagenicity and in vivo toxicity. From the Ames test and subacute toxicity test using oral administration in a rat model, the cordycepin was proved to be a non-mutagenic and non-toxic compound. The hematology and blood chemistry as well as the microanatomical characteristic of the tissues of rats fed with cordycepin every day for consecutive 30 days were comparable to those of the normal ones. Then, the cordycepin was incorporated in gelatin type A (GA) and gelatin type B (GB) nanoparticles aimed to sustain its release and activity. The cordycepin incorporated in both GA and GB nanoparticles showed the sustained release profiles. GA nanoparticles could encapsulate cordycepin at higher encapsulation efficiency due to the attractive electrostatic interaction between the positive-charged GA and the negative-charged cordycepin. However, GA nanoparticles released cordycepin at the higher amount possibly because of the large surface area of small size nanoparticles. Comparing to GB nanoparticles, the higher amount of cordycepin released from GA nanoparticles showed the higher anti-proliferative and anti-migratory effects on A549 lung cancer cells. In conclusion, GA nanoparticles were suggested as a suitable carrier for the sustained release of cordycepin. The GA nanoparticles releasing cordycepin could be an effective and non-invasive material for the treatment of lung cancer cells.

Keywords: Cordycepin; Gelatin; Lung cancer; Nanoparticles; Sustained release; Toxicity.

Figure 1

Hematology values in the blood of rats fed with cordycepin every day for consecutive 30 days. Groups: (filled square) 0.25 mg/mL, (empty square) 1 mg/mL, (gray square) control. *ANOVA p value, p < 0.05. WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean cell hemoglobin; PLT, platelet; RDW, red cell distribution width; PDW, platelet distribution width; MPV, mean platelet volume; PCT, plateletcrit; N, neutrophil; L, lymphocyte; E, eosinophil; B, basophil; M, monophil.

Figure 2

Clinical chemistry in the blood of rats fed with cordycepin every day for consecutive 30 days. Groups: (filled square) 0.25 mg/mL, (empty square) 1 mg/mL, (gray square) control. *ANOVA p value, p < 0.05. BILT, total bilirubin; ALP, alkaline phosphatase; TP, total protein; ALB, albumin; BUN, blood urea nitrogen; CRE, creatinine; ALT, alanine transaminase; AST, aspartate aminotransferase; UA, uric acid; GLU, glucose; TRI, triglyceride; GLO, globulin.

Figure 3

Histology of tissue collected from different organs. The liver, spleen, kidney, testicle, and brain of the rats fed with cordycepin every day for consecutive 30 days.

Figure 4

In vitro cumulative release of cordycepin from GA and GB nanoparticles. After incubated in phosphate buffered saline (pH 7.4) at 37°C for different periods.

Figure 5

In vitro cytotoxicity of the GA and GB nanoparticles. Releasing cordycepin against SAEC after being cultured for 24 h; (mean ± SD, n = 4), control = cordycepin solution (5 μM).

Figure 6

In vitro cytotoxicity of the GA and GB nanoparticles and morphology of A549 cells. (A) In vitro cytotoxicity of the GA and GB nanoparticles releasing cordycepin against A549 cells after being cultured for 24 h. (mean ± SD, n =4), control = cordycepin solution (5 μM). (B) Morphology of A549 cells after treating with GA nanoparticles releasing 40 μM cordycepin for 24 h, observed on TEM (arrow: disrupted cell membrane).

Figure 7

In vitro migration of A549 cells incubated with GA and GB nanoparticles. Releasing cordycepin for 24 h; (mean ± SD, n = 4), control = cordycepin solution (5 μM).