Abstract
Background
Triple-negative breast cancer (TNBC) remains highly aggressive and lacks effective molecularly targeted therapies. Programmed death-ligand 1 (PD-L1) represents a potential cell-surface target for selective drug delivery.
Objective
To develop and evaluate a PD-L1 aptamer–functionalized mesoporous silica nanoparticle system loaded with doxorubicin (Apt-PD-L1-MSN/DOX) for targeted DOX delivery in TNBC.
Methods
Apt-PD-L1-MSN/DOX was synthesized and characterized by TEM, DLS, zeta-potential measurement, FT-IR, and UV–Vis analysis. DOX release was evaluated at pH 7.4, 6.5, and 5.0. Cellular targeting, cytotoxicity, and aptamer-blocking effects were assessed in MDA-MB-231 and MCF-10A cells. In vivo fluorescence distribution, antitumor efficacy, and short-term safety were evaluated in MDA-MB-231 xenograft-bearing mice.
Results
Aptamer functionalization increased the hydrodynamic diameter from approximately 95 to 106 nm, with an estimated surface-associated aptamer loading of ~6.65 nmol/mg. DOX release was pH dependent, remaining below 40% at pH 7.4 but exceeding 80% at pH 5.0 after 96 h. Apt-PD-L1-MSN/DOX showed enhanced cellular targeting and the lowest IC50 in MDA-MB-231 cells (~0.8 μg/mL vs ~1.6 μg/mL for MSN/DOX and ~2.0 μg/mL for free DOX). Aptamer pre-blocking attenuated its cytotoxic effect. In vivo, Apt-PD-L1-MSN/DOX showed stronger tumor-associated fluorescence, greater tumor-growth inhibition, increased apoptosis, and prolonged survival, without overt short-term hepatic or renal toxicity.
Conclusion
Apt-PD-L1-MSN/DOX shows promise as a PD-L1 aptamer-guided platform for targeted DOX delivery in the evaluated TNBC model.
Keywords: PD-L1 aptamer, mesoporous silica nanoparticles, triple-negative breast cancer, targeted drug delivery, doxorubicin
Introduction
Triple-negative breast cancer (TNBC) accounts for 15–20% of all breast cancers and is characterized by absence of estrogen receptor, progesterone receptor, and HER2 expression.1,2 Patients often present with high proliferative indices, early visceral metastasis, and notably poor overall survival compared with other breast cancer subtypes.3 Conventional cytotoxic chemotherapy remains the backbone of systemic treatment; however, the lack of actionable molecular targets limits further improvement in clinical outcomes.4 Immunotherapy has shown benefit in subsets of TNBC, largely driven by programmed death-ligand 1 (PD-L1) expression, yet durable responses remain inconsistent, and immune-related toxicity remains a concern.5–7
Mesoporous silica nanoparticles (MSNs) have emerged as versatile drug-delivery platforms owing to their high surface area, tunable pore structure, chemical stability, and high loading capacity. These properties enable efficient encapsulation and controlled release of therapeutic cargos, while surface functionalization allows active tumor targeting and improved intratumoral drug accumulation.8,9
Aptamers are short single-stranded oligonucleotides generated by systematic evolution of ligands by exponential enrichment (SELEX) and exhibit high target specificity with low immunogenicity. Compared with monoclonal antibodies, aptamers offer advantages including smaller size, superior stability, ease of chemical modification, and reduced production cost, making them attractive targeting ligands for nanomedicine applications.10,11
PD-L1 is an immune-checkpoint ligand that contributes to tumor immune evasion through engagement of PD-1 on T cells.7 PD-L1 expression has been documented in TNBC and has been investigated for its association with treatment response and clinical outcome.12–14 Importantly, its accessibility on the tumor-cell surface makes PD-L1 an appealing molecular target for ligand-directed drug-delivery strategies.15,16 However, PD-L1 is heterogeneous within TNBC and is not tumor-exclusive, which may limit targeting uniformity and increase potential off-target interactions. Recent nanomedicine studies have explored PD-L1 aptamer-functionalized nanoplatforms and related immune-modulating nanosystems, further supporting the feasibility of nanoscale tumor-targeted therapeutic strategies.17,18 Compared with prior PD-L1 aptamer-based or related nanomedicine systems, our study focuses on a DOX-loaded MSN platform for targeted TNBC therapy. The PD-L1 aptamer used in this study was adapted from a previously reported high-affinity PD-L1-binding sequence and was terminally carboxyl-modified to enable EDC/NHS-mediated coupling to amine-functionalized MSNs.19 Thus, the aptamer sequence itself was not newly selected in the present study. Rather, the study focused on integrating this PD-L1-targeting ligand with a DOX-loaded mesoporous silica nanoparticle system and evaluating its functional performance in a TNBC model.
Based on these considerations, we developed a PD-L1 aptamer–functionalized mesoporous silica nanoparticle system loaded with doxorubicin (Apt-PD-L1-MSN/DOX) for selective PD-L1-associated drug delivery. The overall design and proposed targeting and drug-release process are illustrated in Figure 1. We systematically evaluated nanoparticle characterization, pH-dependent DOX release, PD-L1-associated cellular targeting, aptamer-blocking effects, in vitro cytotoxicity, fluorescence distribution, and in vivo antitumor efficacy.
Figure 1.
Schematic illustration of the construction and proposed targeting process of Apt-PD-L1-MSN/DOX. DOX is loaded into amine-functionalized mesoporous silica nanoparticles (MSN-NH2), followed by surface functionalization with a PD-L1 aptamer to generate Apt-PD-L1-MSN/DOX. The PD-L1 aptamer facilitates association with PD-L1-expressing tumor cells and subsequent cellular uptake, followed by enhanced DOX release under acidic intracellular conditions.
Abbreviations: MSN, mesoporous silica nanoparticle; DOX, doxorubicin; PD-L1, programmed death-ligand 1.
Methods
Reagents and Materials
Doxorubicin hydrochloride (DOX·HCl) was obtained from Dalian Meilun Biotech (Dalian, China). Analytical‐grade tetraethyl orthosilicate (TEOS), cetyltrimethylammonium bromide (CTAB), ammonium hydroxide (NH4OH), EDC, NHS, and 3-aminopropyltriethoxysilane (APTES) were purchased from Sigma-Aldrich (USA). All chemicals were of analytical grade and used as received. A carboxyl-modified PD-L1 aptamer (5′-ACGGGCCACATCAACTCATTGATAGACAATGCGTCCACTGCCCGT(T)10-COOH-3′), based on the previously reported high-affinity apt-PD-L1 sequence,19 was synthesized and HPLC-purified by Sangon Biotech (Shanghai, China). A (T)10 spacer and terminal carboxyl group were incorporated to facilitate EDC/NHS-mediated coupling to the amino-functionalized MSN surface. Ultrapure water (18.2 MΩ·cm) was used for all aqueous preparations.
Cells and Animals
MDA-MB-231 TNBC cells and MCF-10A nonmalignant mammary epithelial cells were obtained from the Cell Center of the Chinese Academy of Medical Sciences (Beijing, China). MDA-MB-231 cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. MCF-10A cells were maintained in DMEM/F12 supplemented with 5% horse serum, epidermal growth factor, insulin, hydrocortisone, and cholera toxin under standard culture conditions. MCF-10A cells were used as a nonmalignant PD-L1-low comparator rather than as a tumor cell line. Baseline PD-L1 expression in MDA-MB-231 and MCF-10A cells was confirmed by Western blotting before the targeting experiments. Human L-02 and 293T cells were maintained under the culture conditions used for the corresponding cell lines and were included only for in vitro biosafety evaluation.
Female BALB/c nude mice (4–5 weeks old) were purchased from the Guangxi Laboratory Animal Center and housed under specific pathogen–free conditions. All animal procedures were approved by the Institutional Animal Care and Use Committee of Guilin Medical University (GLMU202517013) and performed in accordance with institutional guidelines, the Guide for the Care and Use of Laboratory Animals, and the ARRIVE guidelines.20 For anesthesia, mice received inhalational isoflurane (2–3% induction, 1–2% maintenance), with depth confirmed by loss of pedal reflex; euthanasia was performed by gradual CO2 inhalation (10–30%/min) followed by cervical dislocation, with death confirmed before tissue collection.
Preparation of Apt-PD-L1-MSN/DOX
MSNs were synthesized using a CTAB-templated sol–gel method as previously described.21,22 Briefly, TEOS was added dropwise to a CTAB/NaOH solution at 80 °C under stirring to form mesoporous silica, followed by template removal via acidic ethanol reflux. The obtained MSNs were aminated with APTES to introduce surface –NH2 groups.
DOX was loaded by incubating MSN–NH2 with aqueous DOX solution for 24 h in the dark, and excess drug was removed by centrifugation. For aptamer coupling, the carboxyl-modified PD-L1 aptamer was first activated with EDC/NHS in MES buffer and subsequently reacted with the surface amino groups of MSN/DOX overnight at 4 °C to promote amide-bond formation. After extensive washing to remove unbound aptamer, the Apt-PD-L1-MSN/DOX formulation was collected.
Characterization of Apt-PD-L1-MSN/DOX
Nanoparticle structure was analyzed by X-ray diffraction (XRD) and nitrogen adsorption–desorption isotherms to determine mesoporous architecture, surface area (BET), and pore-size distribution (BJH). Hydrodynamic size and zeta potential were measured by dynamic light scattering, and morphology was examined by transmission electron microscopy (TEM). Surface modification was evaluated by FT-IR spectroscopy. The amount of nanoparticle-associated aptamer after washing was estimated indirectly from the decrease in aptamer concentration in the collected supernatants using UV absorbance at 260 nm. Because this depletion-based method cannot distinguish covalently coupled aptamer from strongly adsorbed aptamer, the resulting value was reported as the apparent surface-associated aptamer loading rather than as the covalent conjugation efficiency. DOX loading was determined by UV–Vis spectroscopy at 480 nm.
DOX Release from Apt-PD-L1-MSN/DOX
DOX release from Apt-PD-L1-MSN/DOX was evaluated using a dialysis method at pH 7.4, 6.5, and 5.0 to simulate physiological, tumor, and lysosomal conditions. Samples were incubated at 37 °C with gentle shaking, and released DOX was quantified at predetermined time points by UV–Vis spectrophotometry.
In vitro Targeting Ability of Apt-PD-L1-MSN/DOX
For confocal fluorescence microscopy, MDA-MB-231 and MCF-10A cells were seeded on sterile coverslips in 24-well plates at 5.0 × 104 cells/well in 500 μL of complete culture medium and allowed to adhere overnight. Cells were then incubated with MSN/DOX or Apt-PD-L1-MSN/DOX at an aptamer-equivalent concentration of 200 nM in a final treatment volume of 500 μL/well for 30 min at 37 °C. After treatment, cells were washed three times with 500 μL PBS, fixed with 500 μL of 4% paraformaldehyde for 15 min, washed again, and counterstained with DAPI (1 μg/mL, 500 μL/well, 5 min). Cells were subsequently examined by confocal fluorescence microscopy (Nikon DS-Ri1). DOX autofluorescence (red) and nuclear staining (blue) were used to assess intracellular DOX-associated fluorescence.
For flow-cytometric analysis, MDA-MB-231 and MCF-10A cells were seeded in 6-well plates at 3.0×105 cells/well in 2 mL of complete medium and cultured overnight. Cells were incubated with MSN/DOX or Apt-PD-L1-MSN/DOX at an aptamer-equivalent concentration of 200 nM in a final volume of 2 mL/well for 30 min at 4 °C to minimize active endocytosis. Cells were then washed twice with 1 mL ice-cold PBS, detached with 0.5 mL trypsin-EDTA, collected by centrifugation, and resuspended in 500 μL PBS for flow-cytometric analysis (Beckman Coulter Epics XL). DOX fluorescence intensity in the FL2 channel was quantified as a measure of cell-associated fluorescence. Untreated cells were used as blank controls to establish baseline autofluorescence and flow-cytometric gating but were not included as treatment groups in the comparative quantitative analysis. All experiments were independently repeated three times.
In vitro Cytotoxicity Evaluation of Apt-PD-L1-MSN/DOX
MDA-MB-231 and MCF-10A cells were seeded in 96-well plates at 1.0 × 104 cells/well in 100 μL of complete medium and incubated overnight. Cells were then treated with free DOX, MSN/DOX, or Apt-PD-L1-MSN/DOX at the indicated DOX-equivalent concentrations in a final volume of 100 μL/well for 24 h at 37 °C.
After treatment, 20 μL of MTT solution (5 mg/mL) was added to each well and incubated for 4 h. The culture medium was then removed, and the resulting formazan crystals were dissolved in 150 μL DMSO. Absorbance was measured at 570 nm. Dose-response curves were generated, and IC50 values were calculated by nonlinear regression.
For blocking experiments, MDA-MB-231 cells were seeded in 96-well plates at 1.0 × 104 cells/well in 100 μL of complete medium and incubated overnight. Cells were preincubated with 2 μM free PD-L1 aptamer (10-fold molar excess relative to the 200 nM aptamer-equivalent concentration used for Apt-PD-L1-MSN/DOX) for 30 min at 37 °C, followed by treatment with Apt-PD-L1-MSN/DOX for 24 h. Cell viability was subsequently determined using the same MTT procedure described above.
In vivo and ex vivo Fluorescence Distribution Analysis
To evaluate the in vivo and ex vivo DOX-associated fluorescence distribution, MDA-MB-231 tumor-bearing BALB/c nude mice were intravenously injected with MSN/DOX, Apt-PD-L1-MSN/DOX, or Apt-PD-L1-MSN/DOX following pre-administration of excess free PD-L1 aptamer (blocking group).
Whole-body fluorescence imaging was performed at predetermined time points (0, 0.5, 1, 6, and 12 h post-injection) using an in vivo imaging system. Fluorescence signals were recorded under identical acquisition settings, and tumor regions were defined as regions of interest (ROIs) for quantitative analysis of fluorescence intensity.
For ex vivo fluorescence distribution analysis, mice were euthanized at 12 h post-injection, and major organs including tumor, heart, lung, liver, kidney, and spleen were excised and imaged immediately. Fluorescence intensity in each organ was quantified using ROI analysis and normalized for comparison among groups. Tumor-to-organ fluorescence ratios were calculated to assess tumor selectivity.
In vivo Antitumor Efficacy of Apt-PD-L1-MSN/DOX
In vivo antitumor efficacy was evaluated using a subcutaneous MDA-MB-231 xenograft model in female BALB/c nude mice (4–5 weeks old). MDA-MB-231 cells (5 × 106) suspended in PBS/Matrigel (1:1) were injected subcutaneously into the right flank. When tumors reached approximately 100 mm3, mice were randomly assigned to four treatment groups (n = 5 per group): PBS, free DOX, MSN/DOX, and Apt-PD-L1-MSN/DOX. Treatments were administered intravenously at a DOX-equivalent dose of 5 mg/kg once every three days for 14 days. Tumor dimensions and body weight were measured every two days, and tumor volume was calculated as V = (length × width2)/2. At the end of the 14-day efficacy experiment, mice in this cohort were euthanized, and tumors were excised, photographed, weighed, and collected for histological analysis.
A separate independent survival cohort of tumor-bearing mice was randomized to the same four treatment groups (n = 5 per group) and received the same 14-day treatment regimen. These animals were not used for terminal tumor-weight or histological analyses and were monitored separately for survival for up to 80 days. Survival was analyzed using the Kaplan–Meier method and Log rank test.
For TUNEL analysis, tumors harvested from the 14-day efficacy cohort were fixed, paraffin-embedded, sectioned, and stained using a TUNEL assay to assess apoptosis. TUNEL-positive cells were quantified in representative microscopic fields to evaluate treatment-associated tumor-cell apoptosis.
Cytotoxicity of MSNs
The biosafety of blank MSNs was evaluated in L-02, 293T, and MCF-10A cells using an MTT assay. Cells were seeded in 96-well plates (1.0 × 104 cells/well), allowed to adhere overnight, and then incubated with MSNs (25–100 μg/mL) for 24 or 48 h. After treatment, MTT solution (20 μL, 5 mg/mL) was added for 4 h, followed by dissolution of formazan crystals in DMSO. Absorbance at 570 nm was measured to determine cell viability.
Hemolysis Assay
Hemocompatibility was assessed using a standard red blood cell (RBC) hemolysis assay. RBCs were isolated from fresh mouse blood by centrifugation (1500 rpm, 10 min), washed three times with PBS, and diluted to 2% (v/v). PBS and 0.5% Triton X-100 served as negative and positive controls, respectively. RBC suspensions were incubated with PBS, free DOX, MSN/DOX, or Apt-PD-L1-MSN/DOX at 37°C for 2 h. After centrifugation, supernatant absorbance was measured at 540 nm, and hemolysis percentages were calculated relative to the positive control.
In vivo Biosafety and Serum Biochemistry Analysis
Systemic biosafety was evaluated by monitoring body weight, serum biochemical parameters, and histopathological changes in tumor-bearing mice. Body weight was recorded every two days during treatment. At day 14, blood samples were collected for serum analysis of ALT, AST, BUN, and creatinine to assess hepatic and renal function. Major organs (heart, liver, spleen, lung, and kidney) were harvested, fixed in paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) for histological evaluation.
Statistical Analysis
Data are presented as mean ± SD. Comparisons between two groups were performed using two-tailed Student’s t-tests after verification of normality and variance homogeneity. For multiple-group comparisons, one-way ANOVA followed by Tukey’s post hoc test was applied. Longitudinal data were analyzed using repeated-measures ANOVA, and survival was assessed by Kaplan–Meier analysis with Log rank tests. IC50 values were calculated by nonlinear regression. Statistical analyses were conducted using GraphPad Prism 9.0, with P < 0.05 considered statistically significant.
Results
Baseline PD-L1 Expression in Cell Lines
To validate endogenous PD-L1 expression in the cell lines used for targeting studies, Western blot analysis was performed. As shown in Supplementary Figure 1A, PD-L1 protein expression was markedly higher in MDA-MB-231 cells than in MCF-10A cells. Densitometric analysis normalized to GAPDH further confirmed significantly elevated PD-L1 levels in MDA-MB-231 cells (Supplementary Figure 1B), supporting their classification as PD-L1–positive versus PD-L1–low cells and providing a molecular basis for subsequent targeting experiments.
Characterization of Apt-PD-L1-MSN/DOX
X-ray diffraction showed a distinct low-angle peak characteristic of ordered hexagonal mesoporous silica (Figure 2A). Nitrogen adsorption–desorption analysis revealed a typical type-IV isotherm (Figure 2B). BET analysis indicated a high specific surface area (~1400 m2/g), while BJH analysis (Figure 2B) demonstrated a narrow pore-size distribution centered at 4–6 nm with a pore volume of approximately 1.8–2.4 cm3/g, supporting efficient drug loading.
Figure 2.
Structural characterization of MSNs. (A) Small-angle XRD pattern of MSN showing the characteristic diffraction peaks of mesoporous silica. (B) Nitrogen adsorption–desorption isotherms of MSN. (Inset) Corresponding BJH pore size distribution derived from the adsorption branch.
TEM showed that both MSN/DOX and Apt-PD-L1-MSN/DOX maintained uniform spherical morphology and well-ordered mesoporous channels after drug loading and aptamer modification (Figure 3A). Dynamic light-scattering analysis revealed an increase in hydrodynamic diameter from ~95 nm to ~106 nm following PD-L1 aptamer functionalization (Figure 3B and C). Zeta potential shifted from −29.6 mV to −36.3 mV (Figure 3D), consistent with surface oligonucleotide modification. FT-IR spectra showed the expected Si–O–Si bands (~1103 and ~798 cm−1), a DOX-associated C=O signal (~1696 cm−1), and a spectral feature in the amide region (~1655 cm−1) after the coupling procedure (Figure 3E), consistent with altered surface chemistry following aptamer modification. However, FT-IR alone was not considered definitive evidence of covalent amide-bond formation. UV–Vis depletion analysis yielded an estimated surface-associated aptamer loading of ~6.65 nmol/mg after washing (Figure 3F). This value represents aptamer retained with the nanoparticles after the coupling and washing procedure and should not be interpreted as a direct quantitative measurement of covalent amide bonds.
Figure 3.
Characterization of nanomaterials. (A) Representative TEM image of Apt-PD-L1-MSN/DOX. (B and C) Particle size distribution of MSN/DOX (B) and Apt-PD-L1-MSN/DOX (C). (D) Zeta potential of Apt-PD-L1, MSN/DOX, and Apt-PD-L1-MSN/DOX. (E) FT-IR spectra of MSN, MSN/DOX, and Apt-PD-L1-MSN/DOX. (F) UV–Vis spectra of Apt-PD-L1 and Apt-PD-L1-MSN/DOX.
pH-Dependent Release of DOX
DOX release from Apt-PD-L1-MSN/DOX exhibited clear pH dependence. Under physiological conditions (pH 7.4), cumulative DOX release remained below 40% over 96 h, whereas release increased progressively under acidic conditions and exceeded 80% at pH 5.0 after 96 h (Figure 4A). In mildly acidic conditions (pH 6.5), the release rate increased noticeably. MSN/DOX showed a similar pH-dependent release pattern but exhibited comparatively greater DOX release at neutral pH (Figure 4B). These results demonstrate greater DOX retention under neutral conditions and enhanced release under acidic conditions.
Figure 4.
In vitro pH-dependent DOX release profiles of DOX from Apt-PD-L1-MSN/DOX and MSN/DOX. (A) Cumulative release of DOX from Apt-PD-L1-MSN/DOX at pH 7.4, 6.5, and 5.0. (B) Cumulative release of DOX from MSN/DOX under the same pH conditions. Data are presented as mean ± SD from three independent experiments (n = 3).
Enhanced PD-L1-Associated Cellular Targeting and Intracellular DOX Fluorescence
Confocal fluorescence microscopy showed weak intracellular DOX fluorescence in PD-L1–low MCF-10A cells treated with either MSN/DOX or Apt-PD-L1-MSN/DOX (Figure 5A). In contrast, PD-L1–positive MDA-MB-231 cells treated with Apt-PD-L1-MSN/DOX exhibited markedly stronger intracellular DOX fluorescence compared with non-targeted MSN/DOX, consistent with their higher PD-L1 expression. Flow cytometry performed at 4 °C further demonstrated substantially greater cell-associated DOX fluorescence in MDA-MB-231 cells exposed to Apt-PD-L1-MSN/DOX than to MSN/DOX (approximately 64 vs 13–14 arbitrary units), whereas considerably weaker signals were observed in MCF-10A cells (Figure 5B and C). These findings support enhanced PD-L1-associated cellular targeting of the aptamer-functionalized formulation.
Figure 5.
In vitro cellular targeting of MSN/DOX and Apt-PD-L1-MSN/DOX. (A) Confocal fluorescence microscopy images of MCF-10A and MDA-MB-231 cells incubated with MSN/DOX or Apt-PD-L1-MSN/DOX at 37 °C. Cell nuclei were stained with DAPI (blue), and DOX fluorescence is shown in red. Confocal imaging at 37 °C was used to assess intracellular DOX-associated fluorescence. Scale bar, 50 μm. (B) Flow-cytometric analysis of cell-associated DOX fluorescence after incubation at 4 °C to minimize active endocytosis. (C) Quantification of cell-associated DOX fluorescence intensity. Untreated cells were used to establish baseline autofluorescence and flow-cytometric gating and were not included in the comparative fluorescence quantification. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. ***P < 0.001 for the indicated comparisons.
Targeting-Associated Cytotoxicity
MTT assays demonstrated dose-dependent cytotoxicity of all DOX-containing formulations in MDA-MB-231 cells. Apt-PD-L1-MSN/DOX produced the greatest reduction in cell viability across the tested DOX-equivalent concentrations, with lower viability than that observed with free DOX or MSN/DOX (Figure 6A). In MCF-10A cells, Apt-PD-L1-MSN/DOX showed substantially lower cytotoxicity than free DOX and MSN/DOX, with cell viability remaining above 70% even at the highest tested DOX-equivalent concentration, whereas viability decreased to approximately 30–40% with free DOX or MSN/DOX (Figure 6B).
Figure 6.
In vitro cytotoxicity evaluation of Apt-PD-L1-MSN/DOX. (A) Viability of MDA-MB-231 cells after treatment with free DOX, MSN/DOX, or Apt-PD-L1-MSN/DOX at the indicated DOX-equivalent concentrations for 24 h. (B) Viability of MCF-10A cells under the same treatment conditions. (C) IC50 values of free DOX, MSN/DOX, and Apt-PD-L1-MSN/DOX in MDA-MB-231 and MCF-10A cells. (D) Aptamer-blocking experiment in MDA-MB-231 cells. Cells were treated with free PD-L1 aptamer (Apt-PD-L1), preincubated with excess free PD-L1 aptamer followed by Apt-PD-L1-MSN/DOX (aptamer-blocking group), or treated with Apt-PD-L1-MSN/DOX alone. Cell viability was determined after 24 h. Data are presented as mean ± SD from three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison test. **P < 0.01 and ***P < 0.001 for the indicated comparisons.
IC50 analysis further demonstrated differential cytotoxicity between the two cell models. In MDA-MB-231 cells, Apt-PD-L1-MSN/DOX exhibited the lowest IC50 (~0.8 μg/mL), compared with MSN/DOX (~1.6 μg/mL) and free DOX (~2.0 μg/mL) (Figure 6C). In contrast, the corresponding IC50 values in MCF-10A cells were approximately 3.8 μg/mL for free DOX, 5.0 μg/mL for MSN/DOX, and approximately 10 μg/mL for Apt-PD-L1-MSN/DOX. These findings indicate greater cytotoxic selectivity of the aptamer-functionalized formulation toward MDA-MB-231 cells under the tested conditions.
Aptamer-blocking experiments further supported a contribution of PD-L1-associated targeting to the cytotoxic effect of Apt-PD-L1-MSN/DOX. Free Apt-PD-L1 alone showed limited cytotoxicity, with cell viability remaining at approximately 84%. Treatment with Apt-PD-L1-MSN/DOX reduced cell viability to approximately 22%, whereas preincubation with excess free PD-L1 aptamer statistically significantly increased cell viability to approximately 43% (Figure 6D). Cell viability in the free Apt-PD-L1 group was also significantly higher than that in the Apt-PD-L1-MSN/DOX group. Taken together, these findings support a substantial contribution of PD-L1-associated targeting to the enhanced cytotoxicity of Apt-PD-L1-MSN/DOX.
Biosafety of Blank MSNs
Blank MSNs exhibited minimal cytotoxicity in L-02, 293T, and MCF-10A cells at 24 and 48 h (Supplementary Figure 2A–C). In addition, hemolysis testing showed negligible red blood cell lysis for MSN/DOX and Apt-PD-L1-MSN/DOX compared with the 0.5% Triton X-100 positive control, supporting favorable hemocompatibility (Supplementary Figure 2D).
In vivo and ex vivo Fluorescence Distribution
In vivo fluorescence imaging was performed in MDA-MB-231 tumor-bearing mice. As shown in Figure 7A, Apt-PD-L1-MSN/DOX produced stronger and more sustained tumor-associated DOX fluorescence than MSN/DOX, with tumor-region fluorescence detectable from 0.5 to 12 h after injection. Pre-administration of excess free PD-L1 aptamer markedly reduced the tumor-associated fluorescence signal. Quantitative ROI analysis showed that tumor-region fluorescence in the Apt-PD-L1-MSN/DOX group peaked at approximately 490 arbitrary units at 6 h, compared with approximately 65 arbitrary units for MSN/DOX and approximately 100 arbitrary units in the blocking group (Figure 7B).
Figure 7.
In vivo and ex vivo fluorescence distribution of Apt-PD-L1-MSN/DOX in MDA-MB-231 tumor-bearing mice. (A) Representative whole-body fluorescence images of MDA-MB-231 tumor-bearing mice at 0, 0.5, 1, 6, and 12 h after intravenous administration of MSN/DOX, Apt-PD-L1-MSN/DOX, or Apt-PD-L1-MSN/DOX following pre-administration of excess free PD-L1 aptamer (blocking group). Dashed circles indicate the tumor regions. (B) Quantification of tumor-associated DOX fluorescence intensity at the indicated time points. (C) Representative ex vivo fluorescence images of excised tumors and major organs, including the heart, lung, liver, kidney, and spleen, collected at 12 h after administration. (D) Quantification of ex vivo DOX-associated fluorescence intensity in tumors and major organs. Data are presented as mean ± SD (n = 5 mice per group). For panel B, tumor-associated fluorescence over time was analyzed using two-way repeated-measures ANOVA followed by Tukey’s multiple-comparison test. For panel D, tumor fluorescence intensity among the three treatment groups was analyzed using one-way ANOVA followed by Tukey’s post hoc test. ***P < 0.001 for the indicated comparisons.
Ex vivo imaging at 12 h showed stronger DOX-associated fluorescence in excised tumors from the Apt-PD-L1-MSN/DOX group than from the MSN/DOX and blocking groups (Figure 7C). Quantitative analysis showed tumor fluorescence intensities of approximately 430, 90, and 120 arbitrary units for Apt-PD-L1-MSN/DOX, MSN/DOX, and the blocking group, respectively (Figure 7D). Fluorescence signals were also detected in normal organs, particularly the liver, consistent with systemic distribution of the DOX-associated signal.
Collectively, these fluorescence-imaging results are consistent with enhanced tumor-associated localization following PD-L1 aptamer functionalization, while the fluorescence measurements do not distinguish nanoparticle-bound from released DOX.
In vivo Antitumor Efficacy of Apt-PD-L1-MSN/DOX
In the MDA-MB-231 xenograft model, tumors in the PBS group grew rapidly, while free DOX and MSN/DOX produced partial growth inhibition. In contrast, Apt-PD-L1-MSN/DOX achieved the greatest tumor-growth suppression. At day 14, mean tumor volume was approximately 120 mm3 in the Apt-PD-L1-MSN/DOX group, compared with approximately 280 mm3 for MSN/DOX, 380 mm3 for free DOX, and 650 mm3 for PBS (Figure 8A). In a separate survival cohort, Kaplan–Meier analysis showed prolonged survival in mice treated with Apt-PD-L1-MSN/DOX compared with those receiving PBS, free DOX, or MSN/DOX (Figure 8B). Consistently, final tumor weight was approximately 130 mg in the Apt-PD-L1-MSN/DOX group, compared with approximately 290 mg for MSN/DOX, 330 mg for free DOX, and 650 mg for PBS (Figure 8C). Representative tumor images visually supported these differences (Figure 8D).
Figure 8.
In vivo antitumor efficacy of Apt-PD-L1-MSN/DOX in MDA-MB-231 tumor-bearing mice. (A) Tumor growth curves of mice treated with PBS, free DOX, MSN/DOX, or Apt-PD-L1-MSN/DOX during the 14-day efficacy experiment (n = 5 per group). (B) Kaplan–Meier survival curves from a separate independent survival cohort receiving the same four treatment regimens (n = 5 per group). (C) Final tumor weights at the end of the 14-day efficacy experiment. (D) Representative photographs of excised tumors from the four treatment groups. (E) Representative TUNEL staining of tumor sections collected from the 14-day efficacy cohort. Nuclei are shown in blue and TUNEL-positive cells in green. Scale bar, 100 μm. (F) Quantification of TUNEL-positive cells per microscopic field. Data in (A, C, and F) are presented as mean ± SD (n = 5 mice per group). Tumor-growth curves were analyzed using two-way repeated-measures ANOVA followed by multiple-comparison testing; (C and F) were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Survival was analyzed using the Kaplan–Meier method and Log rank test. ***P < 0.001 for the indicated comparisons.
TUNEL staining revealed extensive apoptosis in tumors from the Apt-PD-L1-MSN/DOX group, whereas fewer TUNEL-positive cells were observed in the comparator groups (Figure 8E). Quantitative analysis showed approximately 225 TUNEL-positive cells per field in the Apt-PD-L1-MSN/DOX group, compared with approximately 125 for MSN/DOX, 120 for free DOX, and 40 for PBS (Figure 8F), which was significantly higher, supporting greater treatment-associated tumor apoptosis.
During the 14-day treatment period, mice receiving Apt-PD-L1-MSN/DOX maintained stable body weight, whereas the free-DOX group showed a progressive decrease of approximately 5% (Supplementary Figure 3A). Serum ALT, AST, BUN, and creatinine levels and H&E examination of major organs revealed no overt short-term treatment-related toxicity under the tested conditions (Supplementary Figure 3B and C).
Discussion
The present study evaluates a PD-L1 aptamer–functionalized MSN platform loaded with doxorubicin (Apt-PD-L1-MSN/DOX) as a targeted drug-delivery strategy for TNBC. The findings collectively suggest that PD-L1 aptamer functionalization is associated with enhanced PD-L1-associated cellular targeting, stronger tumor-associated DOX fluorescence, and improved antitumor effects under the tested conditions.
A major finding of this work is the enhanced PD-L1-associated cellular targeting observed after aptamer functionalization. It is well established that PD-L1 is frequently overexpressed in TNBC and plays a pivotal role in immune evasion by tumor cells.7,14 Importantly, in the context of this study, PD-L1 was primarily exploited as a tumor cell–surface molecular target rather than as an immune checkpoint mediator. By exploiting this cell-surface marker, the Apt-PD-L1-MSN/DOX system showed greater cellular association and intracellular DOX-associated fluorescence in PD-L1–positive MDA-MB-231 cells than in PD-L1–low MCF-10A cells. Confocal microscopy at 37 °C showed stronger intracellular DOX-associated fluorescence in MDA-MB-231 cells, whereas flow cytometry at 4 °C demonstrated greater cell-associated DOX fluorescence. Together with the blocking experiments, these findings support enhanced PD-L1-associated cellular targeting of the aptamer-functionalized formulation. Aptamers therefore offer a distinct advantage over traditional antibody-based targeting:23 they provide enhanced stability under physiological conditions, reduced immunogenicity, and streamlined chemical modification and surface functionalization of nanocarriers.24–26
Beyond improved targeting, the Apt-PD-L1-MSN/DOX formulation exhibited pH-dependent DOX release, with lower cumulative release at pH 7.4 and greater release under acidic conditions. Because the formulation does not contain a dedicated pH-cleavable linker or molecular switch, this behavior should be interpreted as pH-influenced drug release rather than a specifically engineered pH-responsive mechanism. This distinction is important because engineered stimuli-responsive MSN systems generally incorporate gated nanovalves or other stimulus-sensitive elements that regulate cargo release in response to defined physicochemical triggers.27 The observed release profile may nevertheless be favorable for drug retention under neutral conditions and enhanced release in acidic environments.
The observed differences in cellular targeting and intracellular DOX-associated fluorescence, together with pH-dependent DOX release, were associated with enhanced cytotoxicity in vitro. Apt-PD-L1-MSN/DOX reduced the IC50 in MDA-MB-231 cells by more than twofold compared with MSN/DOX or free DOX, suggesting that PD-L1-associated cellular targeting may contribute to the enhanced cytotoxic effect observed in MDA-MB-231 cells. In contrast, lower cytotoxicity was observed in MCF-10A cells under the tested conditions. The reduction in cytotoxicity following aptamer pre-blocking provides additional evidence that PD-L1-associated targeting may contribute to the enhanced cytotoxic effect.
These advantages were further confirmed by the in vivo data. In a TNBC xenograft mouse model, Apt-PD-L1-MSN/DOX significantly inhibited tumor progression, reduced final tumor burden, increased apoptosis, and prolonged survival relative to both free DOX and non-targeted MSN/DOX. Tumor tissues from mice treated with Apt-PD-L1-MSN/DOX displayed increased TUNEL-positive staining, consistent with greater treatment-associated tumor-cell apoptosis. In vivo and ex vivo fluorescence imaging showed stronger tumor-associated DOX fluorescence with Apt-PD-L1-MSN/DOX than with non-targeted MSN/DOX, while aptamer pre-blocking reduced the tumor-associated signal. These findings are consistent with enhanced tumor-associated localization following aptamer functionalization. However, because DOX fluorescence does not distinguish nanoparticle-bound from released DOX, these data should not be interpreted as a direct pharmacokinetic measurement of nanoparticle biodistribution. Body weight remained stable throughout treatment, and no overt short-term systemic toxicity was observed under the tested conditions. Although the subcutaneous xenograft model does not fully recapitulate immune interactions or metastatic behavior, it provides a well-established and reproducible platform for evaluating tumor-associated localization, therapeutic efficacy, and short-term systemic safety of targeted nanomedicines. Together, these findings demonstrate that aptamer-guided targeting was associated with improved antitumor efficacy relative to free DOX and non-targeted MSN/DOX in this model.
The present system provides several strategic advantages. MSNs provide high surface area and tunable pore architecture, supporting drug loading and payload retention, features that have been well documented in previous studies.8,9 Recent studies and reviews further emphasize that surface functionalization and active-targeting strategies are central to the development of MSN-based anticancer drug-delivery systems.28,29 Aptamers further enhance the platform by offering a small molecular size, versatile chemical modification, relatively low immunogenicity, and sequence-specific target recognition, making them attractive ligands for targeted cancer drug delivery.10,11,30 The integration of these features provides a stable, ligand-functionalized nanosystem with pH-dependent drug release. In this study, the platform showed enhanced PD-L1-associated cellular targeting, stronger tumor-associated DOX fluorescence, and improved antitumor efficacy compared with non-targeted MSN/DOX. These findings support its potential as a targeted DOX-delivery strategy for TNBC.
Despite these strengths, several limitations should be acknowledged. First, this study used a single TNBC cell line and a subcutaneous xenograft model in immunodeficient mice, which does not recapitulate immune–tumor interactions or metastatic behavior. Moreover, the tumor microenvironment may dynamically influence PD-L1 expression, nanoparticle distribution, and treatment response, which cannot be fully captured by the present subcutaneous immunodeficient xenograft model. Batch-to-batch reproducibility and detailed formulation parameters, including formal encapsulation-efficiency and release sink-condition assessments, were not systematically evaluated. In addition, the limited cellular models and small animal-group size may reduce statistical power and translational generalizability. Second, although expression-dependent cellular association and aptamer blocking support PD-L1-associated cellular targeting at a functional level, quantitative receptor–ligand binding parameters and competitive binding assays were not determined. Third, while pH-dependent DOX release and intracellular efficacy were demonstrated, lysosomal co-localization/trafficking analyses (eg, LysoTracker-based imaging) were not performed. Fourth, pharmacokinetics and systemic exposure parameters (eg, plasma DOX levels, circulation half-life, and AUC), together with long-term safety and clearance beyond the current 14-day observation window, were not evaluated. Such assessments typically require extended follow-up and dedicated biodisposition studies and will be important in later-stage preclinical development. Finally, PD-L1 heterogeneity across TNBC may limit universal applicability, and multi-marker/dual-target strategies could broaden clinical relevance.
Future work may extend this platform toward multimodal imaging, co-delivery of chemotherapeutic and immunomodulatory agents, or dual-aptamer strategies for addressing tumor heterogeneity. Structural optimization to enhance endosomal escape or prolong tumor retention may further improve therapeutic outcomes. Clinical translation would also require scalable manufacturing, further preclinical validation, and detailed regulatory assessment.
In summary, the Apt-PD-L1-MSN/DOX platform showed enhanced PD-L1-associated cellular targeting, pH-dependent DOX release, and improved antitumor efficacy in the evaluated TNBC model. These findings support further investigation of aptamer-guided targeted drug-delivery strategies for TNBC.
Conclusion
This study shows that Apt-PD-L1-MSN/DOX is associated with enhanced PD-L1-associated cellular targeting and greater cytotoxicity in MDA-MB-231 cells under the tested conditions. In vivo, the formulation suppressed tumor growth and prolonged survival compared with free DOX and non-targeted MSN/DOX. Together, these findings support further evaluation of Apt-PD-L1-MSN/DOX as a targeted DOX-delivery strategy for TNBC, with validation in additional models warranted.
Funding Statement
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Data Sharing Statement
The data supporting the findings of this study are available from the corresponding author, Prof. Zhihua Li (E-mail: 13392605973@163.com), upon reasonable request.
Disclosure
The authors report no conflicts of interest in this work.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data supporting the findings of this study are available from the corresponding author, Prof. Zhihua Li (E-mail: 13392605973@163.com), upon reasonable request.