Advancing targeted combination chemotherapy in triple negative breast cancer: nucleolin aptamer-mediated controlled drug release

Abstract

Background: Triple-negative breast cancer (TNBC) is a recurrent, heterogeneous, and invasive form of breast cancer. The treatment of TNBC patients with paclitaxel and fluorouracil in a sequential manner has shown promising outcomes. However, it is challenging to deliver these chemotherapeutic agents sequentially to TNBC tumors. We aim to explore a precision therapy strategy for TNBC through the sequential delivery of paclitaxel and fluorouracil.

Methods: We developed a dual chemo-loaded aptamer with redox-sensitive caged paclitaxel for rapid release and non-cleavable caged fluorouracil for slow release. The binding affinity to the target protein was validated using Enzyme-linked oligonucleotide assays and Surface plasmon resonance assays. The targeting and internalization abilities into tumors were confirmed using Flow cytometry assays and Confocal microscopy assays. The inhibitory effects on TNBC progression were evaluated by pharmacological studies in vitro and in vivo.

Results: Various redox-responsive aptamer-paclitaxel conjugates were synthesized. Among them, AS1411-paclitaxel conjugate with a thioether linker (ASP) exhibited high anti-proliferation ability against TNBC cells, and its targeting ability was further improved through fluorouracil modification. The fluorouracil modified AS1411-paclitaxel conjugate with a thioether linker (FASP) exhibited effective targeting of TNBC cells and significantly improved the inhibitory effects on TNBC progression in vitro and in vivo.

Conclusions: This study successfully developed fluorouracil-modified AS1411-paclitaxel conjugates with a thioether linker for targeted combination chemotherapy in TNBC. These conjugates demonstrated efficient recognition of TNBC cells, enabling targeted delivery and controlled release of paclitaxel and fluorouracil. This approach resulted in synergistic antitumor effects and reduced toxicity in vivo. However, challenges related to stability, immunogenicity, and scalability need to be further investigated for future translational applications.

Keywords: AS1411; Fluorouracil; Paclitaxel; Redox-responsive linker; Scheduled drug release; Triple negative breast cancer.

Fig. 1

AS1411-paclitaxel conjugate with thioether linker exhibited high anti-proliferation ability against triple negative breast cancer cells and its targeting ability was further improved through fluorouracil modification. (A) Anti-proliferation effect of AS1411-paclitaxel conjugates with different redox-responsive linkers on 4T1 cells, MDA-MB-231 cells and MIHA cells. The concentration of AS1411-paclitaxel conjugates was 250 nM. Data were expressed as mean ± standard deviation from at least three replicates, followed by one-way ANOVA with Tukey’s post-hoc test. ns P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 versus PTX group. (B) Drug release of AS1411-paclitaxel conjugates in phosphate buffered saline (PBS, 0.1×), reduction microenvironment (DTT, 10 mM), oxidation microenvironment (H₂O₂, 1 mM), respectively. ApDCs (0.15 nmol) were incubated in different buffers at 37 °C and sampled every three hours. The normalized intact ApDCs at 0 h was treated as 100%. Data were presented as mean ± standard deviation from three replicates. (C) Binding ability of fluorouracil modified AS1411s to nucleolin. The X-axis represents modified AS1411s. The Y-axis was the normalized absorbance at 450 nm, which represented the relative binding ability of the modified aptamer to nucleolin. The absorbance of unmodified AS1411 was treated as 100%. Data were expressed as mean ± standard deviation from at least three replicates, followed by one-way ANOVA with Tukey’s post-hoc test. * P < 0.05; ** P < 0.01; **** P < 0.0001 versus unmodified AS1411 group. Sequence 2 was fluorouracil modified at site 6 in AS1411 (named FA). (D) Binding affinity of fluorouracil modified AS1411-paclitaxel conjugates to nucleolin. A represented AS1411 treatment. ASP represented AS1411-paclitaxel conjugate with thioether linker treatment. ASSP represented AS1411-paclitaxel conjugate with disulfide linker treatment. ATKP represented AS1411-paclitaxel conjugate with thioketal linker treatment. FASP represented fluorouracil modified AS1411-paclitaxel conjugate with thioether linker treatment. RU represented resonance units. The concentrations of the ApDCs and aptamers ranged from 0.156 µM to 10 µM

Fig. 2

Fluorouracil modified AS1411-paclitaxel conjugate with thioether linker could target to MDA-MB-231 cells, resulted in significantly improved anticancer activity in vitro. (A) Anti-proliferation effect of FUDR, FT, FA, PTX, TSP, ASP, FTSP and FASP on MDA-MB-231 cells. The concentrations of each sample ranged from 0.12 nM to 500 nM. Data were expressed as mean ± standard deviation from at least three replicates, followed by one-way ANOVA with Tukey’s post-hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001. (B) The apoptosis of MDA-MB-231 cells after 72 h treatment of Vehicle, FUDR, FT, FA, PTX, TSP, ASP, FTSP and FASP. The concentration of each sample was 200 nM. The percentage of live cells, early apoptosis, later apoptosis, and dead cells were analyzed by flow cytometry using TransDetect^® Annexin V-FITC/PI Cell Apoptosis Detection Kit. (C) Drug release of fluorouracil modified AS1411-paclitaxel conjugates (0.15 nmol) in phosphate buffered saline (PBS, 0.1×), reduction microenvironment (DTT, 10 mM), oxidation microenvironment (H₂O₂, 1 mM), respectively. Data were presented as mean ± standard deviation from three replicates. (D) The cellular endocytosis of Vehicle, AS1411, ASP, FA and FASP in MDA-MB-231 cells were evaluated by confocal microscopy. The concentration of each sample was 50 nM. The representative images showed the cells (light). The AS1411, ASP, FA and FASP were visualized by Cy5 fluorescein (red). The nucleus was stained with Hoechst 33,342 (blue). Scale bar: 20 μm. (E) The cellular uptake of AS1411, ASP, FA and FASP in MDA-MB-231 cells evaluated by flow cytometry using APC channel. The concentration of each sample was 50 nM. Control represents no treatment; FUDR represents fluorouracil prodrug treatment; FT represents fluorouracil modified at site 6 in negative control aptamer treatment; TSP represents negative control aptamer-paclitaxel conjugate with thioether linker treatment; FTSP fluorouracil modified negative control aptamer-paclitaxel conjugate with thioether linker treatment. Vehicle represents PBS treatment

Fig. 3

Fluorouracil modified AS1411-paclitaxel conjugate with thioether linker could accumulate in tumors, resulting in significantly improved anticancer activity in vivo. (A) The relative tumor volume of the MDA-MB-231 xenografted tumor treated with FASP, ASP, PTX, FTSP, FUDR and Vehicle (30 nmol, twice per week), respectively. The tumor volumes were the normalized to the mean tumor volume at day 0. (B) The tumor weights of the xenografted tumor after 23 days of different treatments from the groups indicated. (C) The relative body weight of the MDA-MB-231 inoculated mice of different treatments from the groups indicated. The body weights were the normalized to the mean body weight at day 0. (D) The distribution of MDA-MB-231 inoculated mice of different treatments from FASP group and FTSP group after 4 h. The groups indicated were visualized by Cy5 fluorescein. After mice sacrificed, the tissues, including heart, liver, spleen, lung, kidney, and tumor were isolated and photographed. (E-J) The kidney and liver functions of the MDA-MB-231 inoculated mice of different treatments from the groups indicated. Notes: AST indicated aspartate aminotransferase, DBIL indicated direct bilirubin, TBIL indicated total bilirubin, CREA indicated creatinine, CK indicated creatine kinase, GLDH indicated glutamate dehydrogenase. Data were expressed as mean ± standard deviation from seven replicates (n = 7), followed by one-way ANOVA with Tukey’s post-hoc test. * P < 0.05; ** P < 0.01; *** P < 0.001 versus Vehicle group. All statistical analyses were performed using GraphPad Prism 8.0.1 software

Fig. 4

Insight into the binding mode of fluorouracil modified AS1411 to nucleolin target. (A) The secondary structures of AS1411, FA, PTX and FT in PBS evaluated by CD spectra. The concentration of each sample was 0.2 mM. (B) The RMSD curves of AS1411 and FA evaluated by molecular dynamics simulation. (C) The total energy curves of AS1411 and FA evaluated by molecular dynamics simulation. (D) The AS1411-NCL BRD complex structure predicted by molecular docking. (E) The FA-NCL BRD complex structure predicted by molecular docking