Tea nanoparticle, a safe and biocompatible nanocarrier, greatly potentiates the anticancer activity of doxorubicin

Abstract

An infusion-dialysis based procedure has been developed as an approach to isolate organic nanoparticles from green tea. Tea nanoparticle (TNP) can effectively load doxorubicin (DOX) via electrostatic and hydrophobic interactions. We established an ABCB1 overexpressing tumor xenograft mouse model to investigate whether TNP can effectively deliver DOX into tumors and bypass the efflux function of the ABCB1 transporter, thereby increasing the intratumoral accumulation of DOX and potentiating the anticancer activity of DOX. MTT assays suggested that DOX-TNP showed higher cytotoxicity toward CCD-18Co, SW620 and SW620/Ad300 cells than DOX. Animal study revealed that DOX-TNP resulted in greater inhibitory effects on the growth of SW620 and SW620/Ad300 tumors than DOX. In pharmacokinetics study, DOX-TNP greatly increased the SW620 and SW620/Ad300 intratumoral concentrations of DOX. But DOX-TNP had no effect on the plasma concentrations of DOX. Furthermore, TNP is a safe nanocarrier with excellent biocompatibility and minimal toxicity. Ex vivo IHC analysis of SW620 and SW620/Ad300 tumor sections revealed evidence of prominent antitumor activity of DOX-TNP. In conclusion, our findings suggested that natural nanomaterials could be useful in combating multidrug resistance (MDR) in cancer cells and potentiating the anticancer activity of chemotherapeutic agents in cancer treatment.

Keywords: biocompatibility; drug delivery; multidrug resistance; tea nanoparticle; tumor xenograft.

Figure 1

(A) Concentration-response curves of human normal colon fibroblast cell line CCD-18Co treated with doxorubicin (DOX), DOX-loaded Tea nanoparticles (DOX-TNP) and TNP. (B) Concentration-response curves of human colon cancer cell line SW620 treated with DOX, DOX-TNP and TNP. (C) Concentration-response curves of SW620/Ad300 cells treated with DOX, DOX-TNP and TNP. (D) Schematic drawing of the electrostatic and hydrophobic conjugation of TNP and DOX. Each cell line was incubated with different concentrations of DOX, DOX-TNP or TNP for 72 h. Cell survival rate was determined by the MTT assay. Points with error bars represent the mean ± RSD. Each above figure is a representative of three independent experiments, each done in triplicate. (E) Immunofluorescence assay showing the expression level and localization of ABCB1 in SW620 and SW620/Ad300 cells. (F) Western blot analysis showing the expression level of ABCB1 in SW620 and SW620/Ad300 cells.

Figure 2. The effect of DOX, TNP and DOX-TNP on the growth of SW620 tumors in nude athymic mice

(A) The images of excised SW620 tumors implanted subcutaneously in athymic NCR nude mice (n = 8) that were treated with vehicle, TNP, DOX and DOX-TNP, at the end of the 18-day treatment period. (B) The changes in tumor volume over time following the implantation. Data points represent the mean tumor volume for each treatment group (n = 8). Error bars, SEM. *P < 0.01 versus the vehicle group; ^#P < 0.01 versus the DOX group. (C) The mean weight (n = 8) of the excised SW620 tumors from the mice treated with vehicle, TNP, DOX and DOX-TNP, at the end of the 18-day treatment period. Error bars, SEM. *P < 0.01 versus vehicle group; ^#P < 0.01 versus the DOX group.

Figure 3. The effect of DOX, TNP and DOX-TNP on the growth of SW620/Ad300 tumors in nude athymic mice

(A) The images of excised SW620/Ad300 tumors implanted subcutaneously in athymic NCR nude mice (n = 8) that were treated with vehicle, TNP, DOX and DOX-TNP, at the end of the 18-day treatment period. (B) The changes in tumor volume over time following the implantation. Data points represent the mean tumor volume for each treatment group (n = 8). Error bars, SEM. *P < 0.01 versus the vehicle group; ^#P < 0.01 versus the DOX group. (C) The mean weight (n = 8) of the excised SW620/Ad300 tumors from the mice treated with vehicle, TNP, DOX and DOX-TNP, at the end of the 18-day treatment period. Error bars, SEM. *P < 0.01 versus vehicle group; ^#P < 0.01 versus the DOX group.

Figure 4. The effect of DOX, TNP and DOX-TNP on the body weight, white blood cells and platelets in nude athymic mice

(A) The changes in mean body weight of mice (n = 8) before and after the treatment. NS, not statistically significant (P > 0.05). (B) The changes in mean white blood cells in nude mice (n = 8) at the end of the 18-day treatment period. (C) The changes in mean platelets in nude mice (n = 8) at the end of the 18-day treatment period.

Figure 5

(A) The effect of DOX, TNP and DOX-TNP on the levels of cardiac troponin I in nude athymic mice. The standard curve illustrating the relationship between the absorbance value (A₄₅₀) and the levels of cardiac troponin I (pg/ml). (B) The changes in mean levels of cardiac troponin I in nude mice (n = 8) at the end of the 18-day treatment period. *P < 0.05 versus the vehicle group; NS, not statistically significant (P > 0.05). (C) Plasma doxorubicin concentrations in nude athymic mice at 10, 30, 60, 120, 240 min following administration of DOX or DOX-TNP (n = 8). (D) Intratumoral doxorubicin concentrations in SW620 (n = 8) and SW620/Ad300 tumors (n = 8) after 240 min following administration of DOX or DOX-TNP. Columns and error bars represent mean ± SEM. ^#P < 0.05 versus the DOX group; ^##P < 0.01 versus the DOX goup.

Figure 6. Ex vivo immunohistochemistry (IHC) analysis of SW620 tumor sections (A) and SW620/Ad300 tumor sections (B)

In H & E staining, nuclei are stained blue, and extracellular matrix and cytoplasm are stained red. In ABCB1, Caspase-3, PARP and CD4 staining, nuclei are stained blue, and ABCB1, Caspase-3, PARP and CD4 are stained brown.