Dual targeting of l-carnitine-conjugated nanoparticles to OCTN2 and ATB0,+ to deliver chemotherapeutic agents for colon cancer therapy

Abstract

l-Carnitine, obligatory for oxidation of fatty acids, is transported into cells by the Na⁺-coupled transporter OCTN2 and the Na⁺/Cl^--coupled transporter ATB^0,+. Here we investigated the potential of L-carnitine-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticles (LC-PLGA NPs) to deliver chemotherapeutic drugs into cancer cells by targeting the nanoparticles to both OCTN2 and ATB^0,+. The cellular uptake of LC-PLGA NPs in the breast cancer cell line MCF7 and the colon cancer cell line Caco-2 was increased compared to unmodified nanoparticles, but decreased in the absence of co-transporting ions (Na⁺ and/or Cl^-) or in the presence of competitive substrates for the two transporters. Studies with fluorescently labeled nanoparticles showed their colocalization with both OCTN2 and ATB^0,+, confirming the involvement of both transporters in the cellular uptake of LC-PLGA NPs. As the expression levels of OCTN2 and ATB^0,+ are higher in colon cancer cells than in normal colon cells, LC-PLGA NPs can be used to deliver chemotherapeutic drugs selectively into cancer cells for colon cancer therapy. With 5-fluorouracil-loaded LC-PLGA NPs, we were able to demonstrate significant increases in the uptake efficiency and cytotoxicity in colon cancer cells that were positive for OCTN2 and ATB^0,+. In a 3D spheroid model of tumor growth, LC-PLGA NPs showed increased uptake and enhanced antitumor efficacy. These findings indicate that dual-targeting LC-PLGA NPs to OCTN2 and ATB^0,+ has great potential to deliver chemotherapeutic drugs for colon cancer therapy. Dual targeting LC-PLGA NPs to OCTN2 and ATB0,+ can selectively deliver chemotherapeutics to colon cancer cells where both transporters are overexpressed, preventing targeting to normal cells and thus avoiding off-target side effects.

Keywords: ATB0,+; OCTN2; colon cancer; l-Carnitine; nanoparticles.

Figure 1.

(A) Particle size and size distribution of PLGA NPs and LC-PLGA NPs, (n = 3); (B) In vitro release profiles of free 5-FU, 5-FU-loaded PLGA NPs and LC-PLGA NPs (n = 3); (C) TEM image of PLGA NPs; (D) TEM image of 10% LC-PLGA NPs.

Figure 2.

(A) Expression of OCTN2 (SLC22A5) and ATB^0,+ (SLC6A14) proteins in MB231, MCF7 and Caco-2 cells, with β-actin as an internal control; (B) Uptake of coumarin 6 from bare nanoparticles (PLGA NPs) and L-carnitine conjugated nanoparticles (LC-PLGA NPs) in these three cell lines; (C) Effect of Na⁺ and Cl^– on the uptake of coumarin 6 from LC-PLGA NPs; (D) Effect of specific inhibitors (α-MT and glycine for ATB^0,+, L-carnitine for OCTN2) on the uptake of coumarin 6 from LC-PLGA NPs. Data are shown as mean ± SD, n = 3. *, p < .05, **, p < .01, ***, p < .001, compared to uptake in NaCl buffer (C, D).

Figure 3

, Fluorescent images of the colocalisation for LC-PLGA NPs and OCTN2/ATB^0,+ in MCF7 cells (A, B, E, F) and Caco-2 cells (C, D, G, H). Coumarin 6 (green) was used to label LC-PLGA NPs, and Alexa Fluor^® 594 (red) conjugated secondary antibody was used to mark OCTN2 or ATB^0,+, and DAPI (blue) was used to show the nucleus. A, B, C, D for 15 min incubation, and E, F, G, H for 30 min incubation. In MCF7 cells, A and E show the colocalisation of LC-PLGA NPs with ATB^0,+, and B and F show the colocalisation of LC-PLGA NPs with OCTN2; In Caco-2 cells, C and G show the colocalisation of LC-PLGA NPs with ATB^0,+, and D and H show the colocalisation of LC-PLGA NPs with OCTN2. Arrowheads show the colocalisation of LC-PLGA NPs and transporters (yellow, green + red). Dashed lines mark the cell borders.

Figure 4.

(A) Expression of OCTN2 (SLC22A5) and ATB^0,+ (SLC6A14) in the normal colon cell line (CCD841) and colon cancer cell lines (Caco-2, HCT116, HT29, LS174T); (B) Uptake of coumarin 6 from LC-PLGA NPs with different ligand density (0 to 10%) in colon cells. Data are shown as mean ± SD, n = 3. *, p < .05, **, p < .01, ***, p < .001, compared to uptake in normal colon cells (CCD841).

Figure 5.

The MTT assay for 5-FU, 5-FU-loaded PLGA NPs and 5-FU-loaded LC-PLGA NPs in CCD841 (A); Caco-2 (B); HCT116 (C); HT29 (D); and LS174T (E); (F), the calculated IC50 values. Data are shown as mean ± SD, n = 3. *, p < .05, **, p < .01.

Figure 6.

(A) Nanoparticle penetration of spheroids. Z-stack images taken by confocal microscopy showing penetration of coumarin 6-labeled PLGA NPs and LC-PLGA NPs in HCT116 spheroids. Green color indicates coumarin 6-labeled nanoparticles; (B) Corrected coumarin 6 flourescence intensity represents the nanoparticles in spheroids from periphery to the inner layer (n = 3); (C) Morphological change in HCT116 spheroids during 10-day treatment with 10 µg/mL of free 5-FU, 5-FU-loaded PLGA NPs and 5-FU-loaded LC-PLGA NPs; (D) Compared to control group, the size change of HCT116 spheroids treatment with 1 µg/mL and 10 µg/mL of free 5-FU, 5-FU-loaded PLGA NPs and 5-FU-loaded LC-PLGA NPs (n = 3).