Advancing Targeted Combination Chemotherapy in Triple Negative Breast Cancer: Nucleolin Aptamer-Mediated Controlled Drug Release

doi:10.21203/rs.3.rs-4133693/v1

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Advancing Targeted Combination Chemotherapy in Triple Negative Breast Cancer: Nucleolin Aptamer-Mediated Controlled Drug Release

https://doi.org/10.21203/rs.3.rs-4133693/v1

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published 30 Jun, 2024

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Triple negative breast cancer (TNBC) poses a significant challenge due to its aggressive nature and limited treatment options. While scheduled treatment with paclitaxel and fluorouracil has shown efficacy, their uncontrolled distribution remains challenging. To address this issue, we designed a dual chemo-loaded aptamer with redox-sensitive caged paclitaxel for rapid release and non-cleavable caged fluorouracil for slow release. The nucleolin aptamer significantly improved tumor-targeting, enhancing the effectiveness of the conjugated drugs in TNBC cells. Through nucleolin-mediated endocytosis, the drugs achieved scheduled release, resulting in improved antitumor activity and reduced toxicity in vitro and in vivo. These findings offer new possibilities for developing targeted combination chemotherapy in TNBC.

AS1411

Fluorouracil

Paclitaxel

Redox-responsive linker

Triple negative breast cancer

Scheduled drug release

Triple-negative breast cancer (TNBC) accounts for 15%~25% of all breast cancers[1], which is characterized by clinical features such as strong invasiveness, high likelihood of relapse, high metastatic potential, and poor prognosis. TNBC lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 gene (HER2). Due to the abnormal phenotype, TNBC patients cannot benefit from widely used HER2-targeted therapy or hormone therapy[2]. Currently, chemotherapeutics remains the reference treatment of TNBC patients[3]. Paclitaxel (PTX) is a tetracyclic diterpene compound that impedes cell mitosis by tubulin inhibition[4], whereas 5-fluorouracil (5FU) disrupts DNA/RNA synthesis in tumors. Both PTX and 5FU are FDA-approved frontline drugs for breast cancer (BC) treatment[5, 6]. However, both PTX and 5-FU exhibit limited target specificity, leading to systemic side-effects such as myelosuppression, neurotoxicity, and gastrointestinal irritation[7, 8]. Indeed, the use of either PTX or 5FU as monotherapy has demonstrated ineffective outcomes in the treatment of TNBC[6]. Combining drugs with diverse therapeutic mechanisms is a fundamental strategy in tumor chemotherapy[9]. Significant findings from clinical studies have revealed that incorporating capecitabine (5FU prodrug) as adjuvant chemotherapy after PTX-based standard treatment significantly prolonged the progression-free survival (DFS) of TNBC patients[10-13]. It's worth noting that there is a fixed sequence of administration for PTX and 5FU: PTX must be administered before initiating long-term administration of 5FU[14-16]. Interestingly, the administration of PTX prior to 5FU showed a synergistic effect, because PTX could enhance the sensitivity of tumor cells to 5FU by reducing thymidylate synthase expression[17]. Conversely, the administration of 5FU prior to PTX showed an antagonistic effect, possibly by preventing tumor cells from entering G₂-M phase[18]. Nevertheless, due to the substantial difference in pharmacokinetic performance between PTX and 5FU in vivo[19, 20], it is challenging to precisely co-deliver PTX and 5FU to tumor tissue, and then to perform scheduled drug release (PTX prior to 5FU) in the clinic[21].

In recent years, attention is shifting from conventional drug to targeted drug in the realm of cancer therapy. Researchers have been exploring new targeted therapies that that focus on different molecules overexpressed in TNBC. In 2021, U.S. Food and Drug Administration (FDA) approved the first antibody (Trop2-targeting)-drug (SN38) conjugate (named sacituzumab govitecan) for the treatment of TNBC patients who have previously received at least 2 chemotherapies with advanced disease[22]. However, approximately 70% TNBC patients do not respond to sacituzumab govitecan treatment[23]. On the one hand, heavily modified antibody embodies the risk of difficult transmembrane, strong immunogenicity, rigid storage requirements, reduced target affinity, altered pharmacokinetics and increased heterogeneity[24, 25]. On the other hand, the pH-responsive carbonate linker (named CL2A) in sacituzumab govitecan was not sufficiently stable in plasma[26, 27], restricting its pharmacokinetics and pharmacodynamics properties. Thus, it is crucial to develop more targeted options for precise TNBC treatment.

Aptamers as targeting-components are a promising modality for cancer treatment, conferring further advantages such as fast screening, rapid cell penetration, low immunogenicity as well as easy synthesis, modification and industrialization[28, 29]. In approximately 80% of TNBC cases, nucleolin has been reported to be overexpressed[30, 31]. The highly expressed nucleolin is related to TNBC metastasis and tumor relapse[32], which is an attractive target for TNBC treatment[33]. ACT-GRO-777 (also known as AS1411) is a nucleolin-targeting aptamer entering clinical phase II study (NCT00740441)[34]. AS1411 could mediate the conjugated cargo internalization into TNBC cells through clathrin-dependent endocytosis[35], and exhibit a slow degradation kinetic to release guanine-based degradation products[36]. Thus, AS1411 conjugated fluorouracil by a non-cleavable linker, such as a phosphodiester bond, could significantly enhance the tumor-targeting ability, and then perform a slow release of fluorouracil.

Since the antitumor efficacy of PTX and 5FU is highly schedule-dependent, the scheduled release of PTX and 5FU is critical for effective TNBC treatment. Redox species significantly contribute to the development of tumor microenvironment (TME)-activating prodrugs[37]. On the one hand, the concentration of glutathione (GSH) in cancer cells can reach 2-10 mM, which is 7–10 times greater than that found in normal tissues[38, 39]. On the other hand, the concentration of H₂O₂ in cancer cells can reach 5–1000 μM, which is also much higher than that in normal cells (0.001-0.7 µM)[40]. However, the antitumor efficacy of redox-responsive prodrug with single reduction-responsivity may be restricted by heterogeneous redox microenvironment in tumors. H₂O₂ and GSH dual-activated prodrugs enable to address the tumor heterogeneity concerns[41]. Thus, AS1411 conjugated paclitaxel by a redox-dual stimuli cleavable linker, such as a thioether bond, could significantly enhance the tumor-targeting ability, and then perform a rapid release of paclitaxel.

In the study, we designed and synthesized various redox-responsive floxuridine modified AS1411-paclitaxel conjugates with the aim of selective delivery and scheduled release of paclitaxel and 5FU in TNBC. Pharmacologically, fluorouracil modified AS1411-PTX conjugate with a thioether linker (FASP) significantly improved antitumor activity and reduced toxicity in vitro and in vivo. Mechanistically, fluorouracil modification at site 6 facilitated the modified AS1411 enhancing its binding ability for higher specificity. Upon nucleolin-mediated endocytosis, the paclitaxel and fluorouracil performed scheduled drug release and combination antitumor effects. Our findings provided new possibilities for the development of TNBC targeted combination chemotherapy.

Different redox-responsive aptamer-paclitaxel conjugates were synthesized. The synthesis of compound 4/5 commenced with compound 1 and 1,4-oxathiane-2,6-dione to afford compound 2, accompanied by moderate pyridine (Py) as an activator. Compound 3 was then prepared through treating compound 2 with N,N’-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS). Without further purification, the activated carboxyl group in compound 3 reacted with 3′-amino DNAs [AS1411 or Negative control (NC)] in the present of NaHCO₃(pH=8.35), yielding crude compound 4 (Scheme S1). Additionally, compound 6 was constructed by oxidizing propanethiol in the present of iodine (I₂) and triethylamine (TEA), followed by refluxing in acetyl chloride to obtain compound 7. After reacting with paclitaxel, compound 8 was then activated and reacted with 3′-amino DNAs in the phosphate buffer (pH=8.0) to produce compound 9 (Scheme S2). Furthermore, compound 10 was gained through the reaction of propanethiol and acetone. Then, compound 10 was directly conjugated to PTX, the afforded compound 11 reacted with 3’-amino DNAs in the present of equal amount of DCC, hexafluorophosphate benzotriazole tetramethyl uronium (HBTU), N,N-diisopropylethylamine (DIPEA) to afford compound 12 (Scheme S3). The structures of critical intermediates were confirmed using ¹H-NMR, ¹³C-NMR, and high-resolution mass spectrometry (Attachment figures). Finally, the products were purified and confirmed by mass spectrometry (Table S1, Attachment figures)

AS1411-paclitaxel conjugate with thioether linker exhibited high anti-proliferation ability against TNBC cells and its targeting ability was further improved through fluorouracil modification. To evaluate the cytotoxic effects of AS1411-paclitaxel conjugates with different redox-stimuli responsive linkers, cell counting kit-8 (CCK-8) assay was performed on TNBC cells (MDA-MB-231, 4T1) and normal liver cells (MIHA), respectively. Our in vitro data demonstrated that AS1411-paclitaxel conjugate with thioether linker (ASP) exhibited higher anti-proliferation ability against TNBC cells compared to that with either disulfide linker (ASSP) or with thioketal (ATKP) linker. Additionally, all AS1411-paclitaxel conjugates showed reduced toxicity against normal liver cells (Figure 1A). It indicated that AS1411-paclitaxel conjugate with thioether linker could improve anti-TNBC activity and reduce toxicity in vitro. To validate the oxidation-reduction dual-activated drug release, high performance liquid chromatography (HPLC) assay was performed to measure the relative intact ApDCs after incubation in physiological buffer (PBS), reduction (DTT) or alternative oxidation (H₂O₂) buffers. Our in vitro data demonstrated that ASP sustained high stability after incubation in physiological buffer at 37 °C for 24 hours. Notably, ASP could be cleaved either in reduction buffer or in oxidation buffer within hours (Figure 1B), suggesting in a rapid release of paclitaxel. However, our surface plasmon resonance (SPR) data showed that the binding affinity of ASP to nucleolin (K_d=253.2 nM) was 3.7 folds lower than that of AS1411 (K_d=69.1 nM) (Figure 1D). It could be explained by the fact that the steric hindrance of PTX impacted the interactions between aptamer to target protein.

To reverse the suppressive effect of PTX on AS1411 in binding affinity, post-SELEX modification strategies were conduct for AS1411 affinity optimization, uridine (U), 5-indole-uridine (Indole-U) and 5-fluoro-uridine (5FU) modifications were introduced into AS1411, individually (Table S2). Structure-activity relationship investigations by enzyme-linked oligonucleotide assay (ELONA) showed that deletion of 5-methy in thymidine (i.e. U) significantly reduced the binding affinity of modified AS1411, indicating the vital roles of 5-methy in thymidine. Furthermore, replacement of 5-methy to hydrophobic 5-indole (i.e. Indole-U) also reduced the binding affinity of modified AS1411, indicating the large steric hindrance group would impact their interactions. Noteworthily, replacement of 5-methy to 5-fluorine (i.e. 5FU) significantly improved the binding affinity of modified AS1411, especially in site 6 (Figure S1). Importantly, a single 5FU modification could facilitate the highest binding affinity of the modified AS1411, whereas multiple 5FU modification would mitigate the binding affinity of the modified AS1411 (Figure 1C). To validate the high affinity effect of 5FU in aptamer, the binding affinity of 5FU modified AS1411 (FA), 5FU modified AS1411-paclitaxel conjugate with thioether linker (FASP), negative control sequence (T), 5FU modified negative control sequence (FT), negative control sequence-paclitaxel conjugate with thioether linker (TSP) were determined by SPR assay. It was found that the binding affinity of FA (K_d=24.8 nM) was improved for 2.8 folds. There is no significant difference in binding affinity parameter between AS1411 (K_d=69.1 nM) and FASP (K_d=61.7 nM) (Figure 1D, S2). It indicated that 5FU modification indeed reverse the suppressive effect of PTX on AS1411 in binding affinity. In contrast, either T, FT or TSP showed no binding affinity to nucleolin (Figure S2). It suggested that the conjugated 5FU and PTX would not cause nonspecific binding.

Fluorouracil modified AS1411-paclitaxel conjugate with thioether linker could target to TNBC cells, resulted in significantly improved anticancer activity in vitro. After confirming the high binding affinity of fluorouracil modified AS1411-paclitaxel conjugate with thioether linker (FASP), the anti-proliferative ability of floxuridine (FUDR, 5FU prodrug), FT, FA, PTX, TSP, ASP, FTSP and FASP was performed on MDA-MB-231 cells. Our in vitro data demonstrated that FTSP group exhibited higher anti-proliferative ability compared to PTX group, indicating a superimposed effect of 5FU and PTX against TNBC cells. Additionally, ASP group exhibited higher anti-proliferative ability compared to PTX group, indicating that AS1411 could facilitate the tumor-targeting ability of PTX. Furthermore, FASP group showed the highest anti-proliferative ability among all groups, which was much higher than either ASP group or FTSP group (Figure 2A). Consistently, FASP group characterized superior apoptosis induction compared to either ASP group or FTSP group in vitro (Figure 2B). Collectively, it indicated that AS1411 significantly enhanced the tumor-targeting ability of the conjugated paclitaxel and fluorouracil in TNBC cells, resulting in improved antitumor activity. To validate the oxidation-reduction dual-activated drug release, HPLC assay was performed to measure the relative intact ApDCs after incubation in PBS, DTT or H₂O₂ buffers (Figure 2C). Our in vitro data demonstrated that FASP sustained high stability after incubation in physiological buffer at 37 °C for 24 hours. Notably, FASP could be cleaved either in reduction buffer or in oxidation buffer within hours, suggesting that PTX could be rapid released in redox tumor microenvironment.

To enable targeted drug delivery, we conducted further investigations to assess the cellular uptake ability of AS1411, ASP, FA, and FASP using confocal microscopy and flow cytometry. To visualize their uptake in cells, we labeled the aptamers with Cy5 fluorescence (red) and stained the nucleus with Hoechst 33342. The confocal microscopy images revealed a robust Cy5 fluorescence signal surrounding the nucleus in MDA-MB-231 cells treated with the FASP group. Conversely, no Cy5 fluorescence was detected in MDA-MB-231 cells treated with the vehicle group (Figure 2D). Consistently, the flow cytometry images revealed a strong Cy5 fluorescence signal in MDA-MB-231 cells treated with the FASP group. However, neither the TNBC cells (MDA-MB-231 cells) treated with the vehicle group nor the normal breast cells (MCF10A cells) treated with the FASP group exhibited any Cy5 fluorescence signal (Figure 2E). Collectively, these findings indicated that the cellular uptake of FASP occurred through a target cell-specific ligation approach.

Fluorouracil modified AS1411-paclitaxel conjugate with thioether linker could accumulate in tumors, resulting in significantly improved anticancer activity in vivo. To evaluate the inhibitory effect of FASP on tumor growth in vivo, the preliminary pharmacodynamics, safety and distribution studies were conducted to measure the tumor size, tumor weight, body weight, kidney and liver function parameters, drug distribution in MDA-MB-231 inoculated Balb/c-nu mice. The mice were subcutaneously administrated (twice per week, 3.5 weeks) with FASP, ASP, PTX, FTSP, FUDR and Vehicle. For tumor volume and tumor weight (Figure 3A-B), FASP group exhibited higher antitumor growth ability compared to PTX group and ASP group, indicating a superimposed effect of 5FU and PTX against TNBC tumor growth in vivo. For body weight, there were no significant differences of different treatments from the groups indicated (Figure 3C). For drug distribution, FASP group exhibited higher tumor accumulation compared to FTSP group, indicating an enhanced tumor-targeting ability (Figure 3D). For kidney and liver functions, PTX group or FTSP group modestly increased aspartate aminotransferase (AST), direct bilirubin (DBIL), total bilirubin (TBIL), creatinine (CREA), creatine kinase (CK) and glutamate dehydrogenase (GLDH), whereas FASP group restored the above parameters to wide-type level (Vehicle group) (Figure 3E-J). It indicated that compared to free chemotherapeutics, FASP group exhibited better safety with reduced toxicity. Collectively, our in vivo data demonstrated that FASP could accumulate in tumors, resulting in significantly improved anticancer activity and reduced toxicity in MDA-MB-231 inoculated mice.

In summary, we simultaneously incorporated fluorouracil and paclitaxel into a nulceolin aptamer by a non-cleavable linker (phosphodiester bond) and a redox-stimuli cleavable linker (thioether bond), respectively. Our established fluorouracil modified AS1411-paclitaxel conjugates with thioether linker exhibited efficient recognition of TNBC cells, enabling targeted delivery and scheduled release of PTX and 5FU, resulting in superimposed antitumor effects and reduced toxicity in vivo. Our findings provided new possibilities for the development of TNBC targeted combination chemotherapy.

Synthesis of aptamer-paclitaxel conjugates: The AS1411-PTX were synthesized as described in supporting information. Briefly, redox-responsive di-acids were constructed firstly. Then, the di-acids were conjugated to PTX, to afford carboxylic acid derivatives. Subsequently, the carboxylic acid derivatives obtained were further conjugated to amino-DNA. This conjugation process resulted in the formation of diverse types of aptamer-paclitaxel conjugates (ApDCs).

Drug release assays: ApDCs (0.15 nmol) was dissolved in PBS (0.1×, 60 µL), DTT (10 mM, 60 µL) and H₂O₂ (1 mM, 60 µL), respectively. The mixture was incubated at 37 °C. At timed intervals, the intact ApDCs were assayed utilizing an Agilent 1290 HPLC system along with UV detector set at 260 nm. Phase A was ACN, and phase B was TEAA (50 mM). The gradient was run from 2% to 55% of phase A in 30 minutes for the YMC-Triart C18 column (3 mm, 150 mm×4.6 mm) at a flow rate of 0.5 mL·min^-1with ambient column temperature. The percentage of intact ApDCs was normalized with the initial amount treated as 100%.

CCK-8 assay for cell viability: To identify the most efficient ApDC in tumor cells, the cell viability assay will be conducted using a Cell Counting Kit-8 (CCK8, Dojindo). According to the provided protocol, MDA-MB-231, 4T1, SKOV-3, MIHA, MCF7, MCF10A cells will be seeded in 96-well plates with approximately 5,000 cells in each well and incubated overnight for adherence. Solutions of ApDCs and controls will be made up in medium. The media of cells will be removed, and the solutions will be added for 72 h incubation at 37 °C. At the end of the incubation, 100 µL of culture medium containing 10% of CCK8 solution will be added to each well. The plates will be read after additional 2 h incubation by a spectrometer at 450 nm.

Cellular apoptosis assays: To analyze the different phases of apoptosis, cells were seeded in 6-well plates and treated with ApDCs and corresponding controls for 48 h, respectively. Following the treatment period, cells were harvested, rinsed with cold PBS, and resuspended in 100 μL 1× Annexin-V binding buffer. Next, 5 μL Annexin-V–FITC (fluorescein isothiocyanate) and 5 μL PI (propidium iodide) were added to the cell suspension and incubated at room temperature for 15 min in the dark with gentle vertexing. Quantitative determination was performed using a flow cytometer. By analyzing the fluorescence signals obtained from the flow cytometer, we were able to assess the proportion of cells undergoing early apoptosis (Annexin-V positive, PI negative), late apoptosis (Annexin-V positive, PI positive), and necrosis (PI positive) in response to the treatment with ApDCs and respective controls.

ELONA for relative binding ability: To analyze the binding ability of the modified AS1411 to nulceolin,160 ng nucleolin protein was coated to 96-well microtiter plate in SELEX B&W buffer (1 mM MgCl₂ and 0.05% Tween 20 in 1×PBS, 100 µL) by incubating at 4 °C overnight. The plate was then blocked with blocking buffer (0.1% Tween 20 and 1% BSA in 1×PBS) for 1 h at room temperature and washed with SELEX B&W buffer for 4 times. Then, biotinylated aptamers (1 μM) in SELEX B&W buffer (100 µL) were added into each well and incubated for 45 min at room temperature with continuous gentle shaking. After binding, the plate was washed with SELEX B&W buffer for 4 times to remove non-specific and very weak binding. 0.01% streptavidin-HRP (100 µL) was added to each well and incubated for 30 min and washed with washing buffer (1 mM MgCl₂, 0.1% Tween 20 and 0.1% BSA in 1×PBS, 100 µL) for 4 times. TMB (50 µL) was added to each well and incubated for 20 min. The reaction was stopped by adding H₂SO₄ (2 M, 50 µL). Absorbance at 450 nm was measured with microplate reader. The absorbance of modified AS1411 was normalized to unmodified AS1411 with the initial amount treated as 100%.

SPR assay for binding affinity: The nucleolin protein was immobilized onto a CM5 chip according to manufacturer′s protocol. Briefly, the surface of chip was activated using a mixture of 0.5 M N-hydroxysuccinimide (NHS) and 0.1 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in a 1:1 volume ratio, followed by addition of nucleolin diluted in 10 mM sodium acetate (pH=5.0). For the interaction analysis between ApDCs and nucleolin, ApDCs were diluted with 1×PBS ranged from 0.156-10 mM. Seven consecutive injections of equal volume were performed to ensure the same binding signals, followed by regeneration using 50 mM sodium hydroxide (NaOH). The sodium hydroxide (K_d) was determined using a GE Biacore X100 SPR System.

Confocal assays for cellular endocytosis: MDA-MB-231 cells were seeded in a 24-well plate at a density of 2 × 10⁵ cells per well and incubate overnight. Then, the cells were incubated with Cy5-labeled AS1411, FA, ASP and FASP, each at a concentration of 250 nM. The cells were then incubated at 37 °C for a duration of 4 hours to allow for aptamer internalization. During the final 15 minutes of the incubation period, a volume of 2 μg·mL⁻¹ Hoechst 33342 (blue) then was added to the cells. Following the incubation, the cells were carefully washed to remove any unbound or extracellular aptamers. Subsequently, the cells were visualized using confocal microscopy.

FCM assays for cellular uptake: MDA-MB-231, MCF7 and MCF10A cells were seeded in a 10 cm dish at a density of 2.0 × 10⁵ cells and incubated overnight. The cells were washed three times with PBS and harvested with Accutase. Then, the cells were treated with Cy5-labeled AS1411, FAS1411, ASP and FASP FASP at a concentration of 250 nM for 3 hours. The cellular uptake level of the FASP and controls were analyzed using flow cytometry in the APC channel.

Animal study for antitumor efficacy in vivo: The Laboratory Animal House of Hong Kong Baptist University provided housing for the mice used in this study. The animal facility maintained a regulated environment with controlled temperature and a 12-hour light/dark cycle, while food and water were freely accessible to the mice throughout the study. Prior to conducting any experiments, a minimum of one week was allocated for the mice to acclimate to their new environment. All in vivo studies were conducted in compliance with ethical guidelines and received approval from the Animal Experimentation Ethics Committee of the Hong Kong Baptist University (REC/22-23/0401).

Eight-week-old female BALB/c nude mice were inoculated subcutaneously with 1 × 10⁷ MDA-MB-231 cells in the armpit. After tumors were observed within 3 weeks, the mice were randomly divided into three groups (seven mice in each group) for further experimentation. The mice were administered with FASP, ASP, PTX, FTSP, FUDR twice a week for three weeks via subcutaneous injection at a dosage of 1.5 µmol·kg⁻¹, and the vehicle group was administered with equivolume of PBS. Tumor size and body weight were measured twice a week, with intervals of 3-4 days. At the end of the treatment, the mice were euthanized, and the tumors were weighted. The blood samples were obtained for biochemical analyses.

Animal study for biodistribution effect in vivo: The biodistribution studies were performed in MDA-MB-231 inoculated BALB/c nude mice. After tumors were observed within 3 weeks, the mice were randomly divided into two groups for further experimentation. The mice were administered with Cy5 labeled FASP and FTSP via subcutaneous injection at a dosage of 0.5 µmol·kg⁻¹. After 4 hours, the mice were euthanized. The heart, liver, spleen, lung, kidney and tumor were obtained for images utilizing Imaging Station Maestro 2 (CRI, MA, USA).

Statistical analysis: All variables were expressed as mean ± standard deviation. One-way ANOVA with Tukey’s post-hoc test was performed to determine the inter-group differences in the study variables. All the statistical data were analyzed by GraphPad Prism, and P < 0.05 was considered to be statistically significant. For the in vivo experiments, the animals were grouped randomly and blindly to researchers.

ASSOCIATED CONTENT

Data is provided within the manuscript or supplementary information files.

ACKNOWLEDGEMENTS

YM has been granted a license to use the BioRender content (IP2669SEKY), and the graphic abstract was created with BioRender.com. This study was supported by Guangdong Basic and Applied Basic Research Foundation (2020A1515110630), the National Key R&D Program of China (2018YFA0800802), Theme-based Research Scheme (T12-201-20R), Interdisciplinary Research Clusters Matching Scheme of Hong Kong Baptist University (RC-IRCs/17-18/02), Guangdong-Hong Kong Technology Cooperation Funding Scheme (GHP/149/21GD), Key-Area R&D Program of Department of Science and Technology of Hunan Province (2022WK2010), and Science and Technology Innovation Commission of Shenzhen Municipality Funds (JCYJ20160229210357960).

AUTHOR CONTRIBUTIONS

Y.M. and D.X. performed the major in vitro and in vivo study. Y.M. and X.S. wrote the manuscript. X.W. assisted with in vitro study. Y.P., Z.C. and F.L. helped in the synthesis of small molecules and aptamer-drug conjugates. A.L. and G.Z. supervised the preparation of the manuscript with the input from all authors.

CONFLICT OF INTEREST

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

DATA AVAILABILITY

All data included in this study are available upon request by contact with the corresponding authors.

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Advancing Targeted Combination Chemotherapy in Triple Negative Breast Cancer: Nucleolin Aptamer-Mediated Controlled Drug Release

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