Enhanced docetaxel therapeutic effect using dual targeted SRL-2 and TA1 aptamer conjugated micelles in inhibition Balb/c mice breast cancer model

Abstract

Effective targeting and delivery of large amounts of medications into the cancer cells enhance their therapeutic efficacy through saturation of cellular defensive mechanisms, which is the most privilege of nano drug delivery systems (NDDS) compared to traditional approaches. Herein, we designed dual-pH/redox responsive DTX-loaded poly (β-amino ester) (PBAS) micelles decorated with a chimeric peptide and TA1 aptamer. In vitro and in vivo results demonstrated that the designed nanoplatform possessed an undetectable nature in the blood circulation, but after exposure to the tumor microenvironment (TME) of 4T1 breast cancer, it suddenly changed into dual targeting nanoparticles (NPs) (containing two ligands, SRL-2 and TA1 aptamer). The dual targeting NPs destruction in the high GSH and low pH conditions of the cancer cells led to amplified DTX release (around 70% at 24 h). The IC50 value of DTX-loaded MMP-9 sensitive heptapeptide/TA1 aptamer-modified poly (β-amino ester) (MST@PBAS) micelles and free DTX after 48 h of exposure was determined to be 1.5 µg/ml and 7.5 µg/ml, respectively. The nano-formulated DTX exhibited cytotoxicity that was 5-fold stronger than free DTX (Pvalue˂0.001). Cell cycle assay test results showed that following exposure to MST@PBAS micelles, a considerable rise in the sub G1 population (48%) suggested that apoptosis by cell cycle arrest had occurred. DTX-loaded MST@PBAS micelles revealed significantly higher (Pvalue ˂ 0.001) levels of early apoptosis (59.8%) than free DTX (44.7%). Interestingly, in vitro uptake studies showed a significantly higher TME accumulation of dual targeted NPs (6-fold) compared to single targeted NPs (Pvalue < 0.001) which further confirmed by in vivo biodistribution and fluorescent TUNEL assay experiments. NPs treated groups demonstrated notable tumor growth inhibition in 4T1 tumor bearing Balb/c mice by only 1/10th of the DTX therapeutic dose (TD) as a drug model. In conclusion, cleverly designed nanostructures here demonstrated improved anticancer effects by enhancing tumor targeting, delivering chemotherapeutic agents more accurately, promoting drug release, reducing the therapeutic dosage, and lowering side effects of anticancer drugs.

Keywords: DTX; Delivery; Drug resistance; GSH/pH responsive; SRL-2; Smart; TA1 aptamer.

Scheme 1

Schematic illustration of the work which showed biologically transformable NPs (aptamer and peptide-modified DTX-loaded pH and glutathione (GSH) dual responsive polymeric micelles) with altered performance after exposure to overexpressed MMP-9 in breast cancer TME. The synthesized NPs generally exhibit relatively neutral biological activity in the bloodstream and show minimal interaction with blood proteins (especially opsonins), as noted in the earlier article. However, upon entering the tumor microenvironment, and following the breakdown of the fusogenic peptide, the hidden aptamers (TA1) between these peptides, along with the remaining peptide sequence (SRL-2), suddenly become exposed. These aptamers alongside of SRL-2 peptide (10 amino acid remaining after cleavage of fusogenic peptide) then synergistically recognize the CD44 and LRP-1, respectively on cancer cells, demonstrating a significantly high cellular uptake efficiency. Essentially, our initial nanoparticles function like a shuttle, as depicted, that upon reaching the tumor boundary (illustrated by the gray bubble around the tumor in the schematic), loses part of its structure. The remaining part of the structure, resembling a missile cap with dual targeting ends, identifies the target cell receptors and effectively delivers the chemotherapeutic drug with high efficacy (at 1/10th of the therapeutic concentration) to the cancer cells.

Fig. 1

(A) The transmission electron microscopy (TEM) image of DTX-loaded MST@PBAS NPs. (B) The in vitro drug release profile of DTX from MST@PBAS micelles over 120 h at different conditions: pH 6 with 10 mM GSH (purple), pH 6 (green), pH 7.4 with 10 µM GSH (red), and pH 7.4 (blue).

Fig. 2

In vitro cellular uptake results. (A) Qualitative uptake of different decorations of DTX-loaded nano-micelles; T@PBAS (A–C), MS@PBAS (D–F), and MST@PBAS (G–I) at concentration of 0.625 µg/ml and exposure time of 3 h in 4T1 cells by the fluorescent microscopy. (B) Concentration dependent uptake of different decorations of DTX-loaded T@PBAS, MS@PBAS, and MST@PBAS micelles (2.5, 1.25, 0.625 µg/ml) in 4T1 cell line in constant exposure time of 3 h by the flow cytometer.

Fig. 3

Cell cycle, apoptosis, and in vitro cytotoxicity assay. (A) The viability of 4T1 cells was assessed using the MTT assay by treating them with different doses (2.5, 1.25, 0.625, 0.312, 0.156, and 0.078 µg/ml) of free DTX and DTX-loaded MST@PBAS NPs for 24 and 48 h. (B) Flow cytometry was used to analyze cell cycle arrest. The effects of blank micelles, free DTX, and DTX-loaded MST@PBAS micelles on the sub G1, G0/G1, S, and G2/M phases were evaluated. (C) The apoptosis assay was performed in different formulations and measured by a flow cytometer. The percentages of necrosis, late apoptosis, early apoptosis, and healthy cells are shown in Q1, Q2, Q3, and Q4, respectively. (D) The viability of blank micelles in HUVEC and HFFF2 cell lines. mean ± SD, n = 3, **p < 0.01; ***p < 0.001.

Fig. 4

The DTX-loaded micelles’ anticancer properties in vivo. (A) Evaluation of the MST@PBAS micelles’ ability to penetrate deep into 3D in vitro tumors. (B) Illustration of tumor tissues from 4T1 tumor-bearing mice that were given saline, free DTX, or MST@PBAS NPs that were DTX-loaded. (C) A comparison of the tumor responses in tumors treated with saline, free DTX, and MST@PBAS NPs loaded with DTX. (D) Assessment of tumor apoptosis using the TUNEL assay after different therapeutic interventions. Mean ± SD, n = 3, **p < 0.01; ***p < 0.001.

Fig. 5

On day 21 following implantation, key organs (heart, kidney, liver, lung, spleen, and brain) and samples of the 4T1 breast cancer tumor were obtained. With the use of a light microscope (Olympus-CH30, Japan), the tissue samples were examined. The histology findings are shown, with each group given the appropriate label: A control-established group, a saline group, a group without DTX, a group with DTX, and a group of DTX-loaded MST@PBAS NPs. The observed features in the tissue sections included metastases (M), necrosis (N), and congestion (C).