Hyaluronic acid-decorated liposomal nanoparticles for targeted delivery of 5-fluorouracil into HT-29 colorectal cancer cells

Abstract

The use of liposomes as drug carriers improves the therapeutic efficacy of anticancer drugs, while at the same time reducing side effects. Hyaluronic acid (HA) is recognized by the CD44 receptor, which is overexpressed in many cancer cells. In this study, we developed HA-modified liposomes encapsulating 5-fluorouracil (5-FU) and tested them against a CD44 expressing colorectal cell line (HT29) and a non-CD44 expressing hepatoma cell line. The average size of 5-FU-lipo and 5-FU-lipo-HA nanoparticles were 112 ± 28 and 144 ± 77 nm, respectively. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay showed selective cancer cell death depending on the CD44 expression in a time-dependent manner. Apoptosis assays and cell-cycle analysis indicated that G0/G1 arrest occurred. The colony formation study revealed that cells treated with 5-FU-lipo and 5-FU-lipo-HA had reduced colony formation. Quantitative reverse-transcription polymerase chain reaction study showed that the oncogenic messenger RNA and microRNA levels were significantly reduced in the 5-FU-lipo-HA-treated group, while tumor suppressors were increased in that group. We suggest that optimal targeted delivery and release of 5-FU into colorectal cancer cells, renders them susceptible to apoptosis, cell-cycle arrest, and decreased colony formation.

Keywords: 5-fluorouracil; CD44; apoptosis; cell cycle; clonogenicity; colorectal cancer; hyaluronic acid; liposomes; microRNA.

Figure 1.

Shows the FTIR spectrum for DOPE (a), HA (b) and DOPE-HA (c).

Figure 2.

DLS obtained particle size for 5-FU-lipo (A), zeta potential of 5-FU-lipo (B), particle size of 5-FU-lipo-HA (C), and zeta potential of 5-FU-lipo-HA (D).

Figure 3.

SEM visualized the morphology of (A) 5-FU-lipo and (B) 5-FU-lipo-HA nanoparticles

Figure4.

The in vitro release of 5-FU from 5-FU-lipo and 5-FU-lipo-HA nanoparticles at pH = 7.4 and pH = 5.5.

Figure 5.

Fluorescence microscopy images of HT29 (CD44^high expressed) cancer cells treated with lipo/RhoB and lipo-HA/RhoB nanoparticles (with 1μM concentration of RhoB in each nanoparticle) showing the cellular uptake at 6 and 12 h incubation times (A-B). Uptake of lipo/RhoB and lipo-HA/RhoB nanoparticles measured by flow cytometry (C). Blue and red fluorescence are referred to the DAPI and RhoB respectively (magnification 40x). *p<0.001 versus lipo/RhodB. Data represent mean ± SD (n=3). MFI: Mean fluorescence intensity.

Figure 6.

Fluorescence microscopy images of HEPG2 (CD44^low expressed) cancer cells treated with lipo/RhoB and lipo-HA/RhoB nanoparticles (with 1μM concentration of RhoB in each preparation) showing the cellular uptake at 6 and 12 h incubation times (A-B). Uptake of lipo/RhoB and lipo-HA/RhoB nanoparticles measured by flow cytometry (C). Blue and red fluorescence are related to the DAPI and RhoB respectively (magnification 40x). The data of each group compared to lipo/RhodB. Data represent mean±SD (n=3). MFI: Mean fluorescence intensity.

Figure 7.. Cell toxicity of 5-FU loaded liposomes in colorectal cancer cells.

Represents in vitro cell viability results of blank liposomes, free 5-FU, 5-FU-lipo and 5-FU-lipo-HA in HT-29 and HepG2 cell lines over 24, 48, and 72 h (A-G). The data represent mean±SD (n = 3); *P < 0.05, versus blank liposome treated cells.

Figure 8.. Cell apoptosis induced by free 5-FU, 5-FU-lipo and 5-FU-lipo-HA nanoparticles in colorectal cancer cells.

Annexin V/PI staining in free 5-FU, 5-FU-lipo and 5-FU-lipo-HA over 48 h showed the high rate of apoptosis in 5-FU-lipo-HA group (8A and B). Fluorescent images of treated HT-29 cells stained with DAPI (8C). The data represent mean±SD (n = 3); **P < 0.01, ***P < 0.001, and ****P < 0.0001, versus cells with blank liposomes.

Figure 9.. 5-FU-lipo-HA could arrest the cell cycle in the G0/G1 phase in colorectal cancer cells.

Blank liposomes, free 5-FU, and 5-FU-lipo and 5-FU-lipo-HA treated cells were prepared and stained with the DAPI. The percentage of the cell population in the blank liposomes, free 5-FU, 5-FU-lipo and 5-FU-lipo-HA groups were evaluated in each cell cycle phase. The data represent mean±SD (n = 3); **P < 0.01, and ***P < 0.001 versus blank liposome treated cells.

Figure 10.. 5-FU loaded liposome suppressed the clonogenicity in HT-29 colorectal cancer cells.

Free 5-FU and 5-FU-lipo and 5-FU-lipo-HA decreased the formation of the colorectal cancer cell colonies (5A and B). Data are presented as means ± SD. (n=3);* p<0.05, and **** p<0.0001versus blank liposome treated cells.

Figure 11.. 5-FU-lipo-HA changes the expression of mRNAs and miRNAs in colorectal cancer cells.

Relative ALDH, KLF4, HMGA2, cMyc, Nanog, CD44, Kras mRNAs, miR-181a, miR-181b, Let-7, miR-21, miR-155, mir-200c and miR-34c were evaluated by the qRT-PCR assay. Data are presented as means ± SD. (n=3);** p<0.01, and **** p<0.0001 versus blank liposome treated cells.