Comparison of Micelles Single- and Dual-Targeted with Folic Acid and Biotin as the Delivery System of Docetaxel─The Influence of the Type and Amount of the Ligand on Morphology, Physicochemical Properties, and Cytotoxicity

Abstract

Nanoparticles (NPs) dual-targeted with folic acid (FA) and biotin (BIO) are developed to overcome problems associated with conventional chemotherapy and tumor heterogeneity. Although some preliminary studies have been conducted to develop dual-targeted micelles for delivery of docetaxel (Dtx), a more detailed analysis is needed to discover their pharmaceutical potential. Therefore, this manuscript is focused on a comprehensive analysis of the influence of the type and density of targeting ligand on micelles' morphology, physicochemical properties, drug loading and release, cell internalization, cytotoxicity in vitro against several kinds of cells and in vivo on two mice cancer models. For this purpose, three kinds of micelles were obtained: decorated with one type of ligand (FA or BIO) and dual-targeted. The micelles contained different amounts of FA- and/or BIO-functionalized polymer (10, 25, and 50%). Additionally, the dual-targeted micelles were compared with a mixture of two kinds of single-targeted micelles. The study showed that the cytotoxic effect of micelles strongly depends on the type of cancer cells and their surface characteristics. The micelles with a higher density of ligands may have higher efficiency against cells with lower expression of cell surface receptors (SK-BR-3 and MCF-7). Conversely, micelles with lower ligand density may be advantageous against cancer cells with overexpression of surface receptors (HeLa, 4T1). Interestingly, the drug delivered in FA-targeted micelles showed higher cytotoxicity compared to dual-targeted micelles in which two ligands are present on the same NP. However, Dtx delivered in a mixture of micelles single-targeted with FA and BIO showed the highest cytotoxic effect, which was confirmed in vitro and in vivo on the mice model.

Keywords: PLA−PEG; active targeting; biotin; docetaxel; dual-targeted micelles; folic acid; nanoparticles.

1

Cryo-TEM images of various kinds of micelles: FA10 (A), FA25 (B), FA50 (C), BIO10 (D), BIO25 (E), BIO50 (F), FABIO10 (G), FABIO25 (H), and FABIO50 (I).

2

Comparison of the in vitro cumulative release profile of Dtx from micelles functionalized with FA (FA10, FA25, and FA50) and micelles dual-functionalized with FA and BIO (FABIO10, FABIO25, and FABIO50). Data shown as a mean ± SD (n = 3).

3

Microscopic observations of the in vitro internalization of micelles with the fluorescent FITC dye nonfunctionalized, functionalized with folic acid (FA10), biotin (BIO10), and dual-functionalized with FA and BIO (FABIO10) by 4T1, B16-F10, HeLa, and WI-38 cells. Nucleus stained with Hoechst 33342. Images were taken using a confocal microscope with a 20× objective magnification.

4

Viability of WI-38 cells in the presence of various types of drug-free micellar formulations: micelles functionalized with folic acid (FA10, FA25, and FA50), micelles functionalized with biotin (BIO10, BIO25, and BIO50), and micelles dual-functionalized with FA and BIO (FABIO10, FABIO25, and FABIO50). The results of the SRB assay are shown as mean ± SD; p < 0.05 compared with the negative control.

5

Viability of SK-BR-3 cells (left row) and MCF-7 cells (right row) in the presence of various formulations of Dtx: from the top, free drug (Dtx), Dtx in micelles functionalized with folic acid (FA10, FA25, and FA50), Dtx in micelles functionalized with biotin (BIO10, BIO25, and BIO50), and Dtx in micelles dual-functionalized with FA and biotin (FABIO10, FABIO25, and FABIO50). The results of the SRB assay are shown as mean ± SD; *p < 0.05 compared with negative control; **p < 0.05 compared between groups with various ligand contents.

6

Viability of HeLa cells (left row) and 4T1 cells (right row) in the presence of various formulations of Dtx: from the topfree drug (Dtx), Dtx in micelles functionalized with folic acid (FA10, FA25, and FA50), Dtx in micelles functionalized with biotin (BIO10, BIO25, and BIO50), and Dtx in micelles dual-functionalized with FA and biotin (FABIO10, FABIO25, FABIO50, and FA10 + BIO10). The results of the SRB assay are shown as mean ± SD; *p < 0.05 compared with the negative control; **p < 0.05 compared between groups with various ligand contents.

7

Effect of 1.28 μM of Dtx encapsulated in FABIO10 or a mixture of FA10 + BIO10 micelles on the morphology (A) and mitochondrial transmembrane potential (B) of 4T1 cells. Morphological changes in 4T1 cells were observed under the light inverted microscope at 40× magnification. The bar graph presents changes in the proportion of cells with polarized mitochondria and late-apoptotic 4T1 cells. Data are expressed as % of the controls; mean values ± SD (n = 9), *p < 0.5, and **p < 0.05.

8

Cytometric analysis of cell cycle distribution in 4T1 cells treated with 1.28 μM of Dtx in FABIO10 or a mixture of FA10 + BIO10 micelles: cell cycle phases (A), DNA fragmentation (B), changes in Annexin V/PI intensity (C), and cellular GSH (D). Data are expressed as mean values ± SD, **p < 0.05.

9

Internalization of different micelles (without ligand, FA10, BIO10, FABIO10, FA10 + BIO10) with FITC in tumor tissue-4T1 breast cancer (A), B16-F10 melanoma (B). Fluorescence of collected tissues was visualized using IVIS, indicated as radiant efficiency. The results are the mean value of 5 in each group and are shown as means ± SD.

10

Inhibition of tumor growth after administration of the tested Dtx-loaded micelles (without ligands, FA10, BIO10, FABIO10, FA10 + BIO10) and free Dtx. Each formulation was administered 3 times every other day to mice bearing well-developed tumors, as indicated by pink arrows. All mice were administered docetaxel at a dose of 10 mg/kg body weight. Experiments were performed on 4T1 mammary carcinoma (A) and B16-F10 melanoma (B). Each group consisted of 5 mice. The tumor volume is shown as mean + SEM; *p < 0.05, **p < 0.01.