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30 January 2026

27 Pages

Taming the Tumor Stroma: A Two-Stage Targeted Nanocapsule for Potent Deep Chemo-Immunotherapy in Triple-Negative Breast Cancer

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1
School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China
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State Key Laboratory of Component-Based Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China
3
Engineering Research Center of Modern Chinese Medicine Discovery and Preparation Technique, Ministry of Education, Tianjin University of Traditional Chinese Medicine, Tianjin 301617, China
4
Haihe Laboratory of Modern Chinese Medicine, Tianjin 301617, China
Pharmaceutics2026, 18(2), 184;https://doi.org/10.3390/pharmaceutics18020184
This article belongs to the Section Drug Targeting and Design
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Abstract

Background: The tumor microenvironment (TME) poses significant challenges to effective therapy, with cancer-associated fibroblasts (CAFs) playing a key role in tumor progression and drug resistance in triple-negative breast cancer (TNBC). Herein, a TME responsive nanocapsule, NPC-ABS/FDS, was developed utilizing baicalein, a CAFs modulator, and the cytotoxic drug doxorubicin to selectively target CAFs and tumor cells, respectively, in a stepwise manner. Methods: NPC-ABS/FDS was designed with CD13-mediated primary targeting for tumor accumulation and secondary targeting via σ-receptor binding (ABS nanoparticles) for CAFs and folate modification (FDS nanoparticles) for cancer cells. Physicochemical properties were assessed using TEM, particle size, and ζ-potential analyses. Fluorescence imaging evaluated tumor retention, while cellular uptake and TME modulation were analyzed in vitro and in vivo. Results: The successful preparation of NPC-ABS/FDS was demonstrated by its uniform morphology, stable characteristics, charge reversal, and increased particle size. Fluorescence imaging confirmed prolonged peritumoral retention. Cellular uptake increased 2.5-fold for baicalein in CAFs and 4.3-fold for doxorubicin in cancer cells. NPC-ABS/FDS downregulated α-SMA and FAP, reducing CAFs activation, improving intratumoral drug penetration, and enhancing CD8+ and CD4+ T cell infiltration while decreasing regulatory T cells. Conclusions: NPC-ABS/FDS effectively modulates multiple TME components, including CAFs and immune cells, and improves drug delivery in TNBC. These findings may support the development of improved therapeutic approaches for TNBC.

1. Introduction

Cancer-associated fibroblasts (CAFs) are the most abundant stromal cell population within tumor-associated matrices (TAMs). They are widely present across different tumor types, exhibit substantial subtype heterogeneity, and possess complex biological functions, all of which are strongly associated with tumor initiation and progression. The primary sources of CAFs include tissue-resident fibroblasts in a quiescent state, stellate cells from the pancreas or liver, and mesenchymal stem cells derived from the bone marrow [1]. In triple-negative breast cancer (TNBC), cancer cells can promote the conversion of normal fibroblasts into CAFs by secreting various cytokines [2]. Likewise, CAFs profoundly influence the development and progression of TNBC through multiple mechanisms. Specifically, they secrete various factors, exosomes, and metabolites that regulate tumor angiogenesis, immune responses, and metabolic processes [1]. For example, CAFs secrete transforming growth factor-β (TGF-β) and other cytokines that promote regulatory T cells (Tregs) and M2 macrophage infiltration while suppressing effector T-cell activity, thus facilitating tumor immune escape [3]. In addition, activated G protein-coupled estrogen receptors in CAFs can upregulate glutamine synthetase, thereby supplying glutamine to TNBC cells and enhancing their proliferation, invasion, and chemoresistance [4]. Under hypoxic conditions, CAFs-derived colony-stimulating factor 3 activates downstream signaling pathways and induces glycolytic reprogramming in TNBC cells, providing essential energy sources [5]. Second, CAFs remodel the structural properties of the tumor matrix. Through dense deposition of extracellular matrix (ECM) proteins such as collagen and fibronectin within a confined stromal space, CAFs elevate interstitial fluid pressure, compress blood vessels, and tightly pack cancer cells. The resulting physical barrier restricts the penetration of cytotoxic T cells and nanomedicines into distal avascular tumor regions, ultimately leading to therapeutic resistance and chemoresistance [6,7,8].
Because of their unique role within the TAMs, CAFs drive tumor progression and influence patient prognosis, making them a highly promising therapeutic target for TNBC [1]. Inhibiting CAFs activation not only disrupts the physical stromal barrier and improves the penetration and efficacy of chemotherapeutic agents [9] but also modulates the tumor immune microenvironment, restores normal cellular function, and converts “cold” tumors into “hot” tumors, thereby facilitating responses to immune checkpoint inhibitors [10]. Studies have shown that blocking CAFs in tumor tissues can suppress tumor growth and reduce tumor volume by 30.6–64.0% [1]. Current approaches to CAFs inhibition mainly include the following: (1) eliminating CAFs, (2) inhibiting key steps in CAFs activation, and (3) reprogramming CAFs into a quiescent state [11,12,13]. A large body of research has focused on targeting and removing CAFs to enhance drug delivery efficiency in solid tumors [11]. However, recent studies have shown that complete CAFs depletion may promote cancer stem cell dissemination and enhance tumor invasiveness and metastasis [14,15]. Moreover, fibroblast-induced binding-site barriers may lead to non-specific uptake of nanomedicines by CAFs, inducing stress–response signals that promote cancer cell invasion, proliferation, metastasis, and acquired chemoresistance [16]. Therefore, current research is shifting away from extreme strategies aimed at complete CAFs elimination, and reprogramming activated CAFs toward a quiescent state is becoming increasingly attractive [17,18].
Baicalein (BAE), a natural active compound, has shown favorable therapeutic effects in fibrosis. In our preliminary studies, we found that BAE likewise exerts inhibitory effects on CAFs, reducing fibroblast activation protein (FAP) expression to levels close to those of quiescent fibroblasts, suggesting significant therapeutic potential. Nanocarriers—known for their precise targeting, high specificity, and multifunctionality [19]—provide a platform for delivering therapeutic agents into TAMs and precisely regulating CAFs [1,10,15]. However, because tumor microenvironments are highly adaptive and plastic, monotherapies that solely target CAFs may activate alternative protumor pathways, resulting in therapeutic resistance in TNBC [9]. Therefore, combining CAFs-targeted therapy with chemotherapy or immunotherapy has become a necessary strategy for combating TNBC.
In this study, we designed a two-stage targeted nanocapsule system to deliver BAE and doxorubicin (DOX) to CAFs and cancer cells, respectively, with the aim of regulating CAFs while simultaneously killing cancer cells. BAE and DOX were separately loaded into cationic solid lipid nanoparticles (BAE-SLN and DOX-SLN). To achieve targeted delivery, BAE-SLN was modified with aminoethyl-aniline amide (AEAA) to form AEAA-BAE-SLN (ABS), which selectively binds σ-receptor on CAFs [12]. DOX-SLN was modified with folic acid (FA) to form FA-DOX-SLN (FDS), allowing selective binding to folate receptors on 4T1 cells and reducing DOX toxicity toward CAFs [20]. Given that physical barriers in TNBC hinder drug penetration, ABS and FDS were co-encapsulated within carboxymethyl chitosan (CMCS), enabling tumor accumulation and pH-responsive release while minimizing off-target effects and systemic toxicity. Additionally, NGR peptides were conjugated to CMCS via an SCM-PEG2000-SCM linker to form NGR-PEG2000-CMCS (NPC), enabling selective recognition and binding of CD13 on tumor neovascular endothelial cells [13] and promoting nanocapsule accumulation through active targeting. Our findings indicate that this two-stage targeted system suppressed CAF activation and reprogramed them toward a quiescent state, while enhancing deep chemotherapeutic penetration and inducing immune activation, providing a rationale for combining CAF modulators with chemotherapeutic agents for TNBC treatment.

2. Materials and Methods

2.1. Materials

Baicalein was purchased from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). Doxorubicin was purchased from Shanghai Mairel Biotechnology Co., Ltd. (Shanghai, China). Soy lecithin was purchased from A.V.T. Pharmaceutical Co., Ltd. (Shanghai, China). DOTAP was provided by CordenPharma (Liestal, Switzerland). NGR, SCM-PEG2000-SCM, and DSPE-PEG2000-FA were provided by Xi’an Ruixi Biotechnology Co., Ltd. (Xi’an, China). Carboxymethyl chitosan was obtained from Shanghai yuanye Bio-Technology Co., Ltd. (Shanghai, China). FAP ELISA kit and α-smooth muscle actin Elisa kit were obtained from Jiangsu Enzyme Immunoassay Industry Co., Ltd. (Nanjing, China). α-Smooth Muscle Actin XP Rabbit mAb was obtained from Cell Signaling Technology, Inc. (Boston, MA, USA). Rabbit Anti-FAP antibody was obtained from Bioss (Beijing, China). Anti-CD4 antibody, Anti-CD8 alpha antibody, and Anti-FOXP3 antibody were obtained from Abcma (Shanghai, China). FITC Plus Anti-Mouse CD31 was obtained from Proteintech Group, Inc. (Chicago, IL, USA). TGF beta 1 protein, Rat/Mouse, recombinant was purchased from Sino Biological (Beijing, China). All other materials and reagents were of analytical reagent grade.

2.2. Cells

Human umbilical vein endothelial cells (HUVECs), mouse embryonic fibroblast cells (NIH3T3), and mouse breast cancer cells (4T1) were purchased from Wuhan Shanen Biotechnology Co., Ltd. (Wuhan, China). All cells were cultured in DMEM medium supplemented with 10% fetal bovine serum at 37 °C, incubated with 5% CO2.

2.3. Animals

Female Balb/c and nude Balb/c (age: 6−8 weeks) mice were supplied by Spiff (Beijing) Biotechnology Co., Ltd. (Beijing, China). All animal experiments were conducted in accordance with the principles of animal experimentation.

2.4. Preparation of NPC-ABS/FDS

BAE-SLN and DOX-SLN were prepared by thin-film hydration [21]. Soy lecithin, DOTAP, BAE, or DOX, and PEG2000 at a molar ratio of 120:3:10:10 were dissolved in 15 mL of mixed solvents (methanol/dichloromethane = 1:1) in a round-bottom flask. Rotary evaporation (15 min, 50 °C; Tokyo Rikakikai Co., Ltd., Tokyo, Japan) was employed to concentrate the drug and copolymer into a solid film. Subsequent vacuum treatment (2 h) preceded the hydration step, wherein the film was dispersed in 5 mL of 0.1% Poloxamer 188 aqueous solution at 50 °C under ultrasonic treatment. Nonencapsulated drug was removed by filtering through 0.22 μm filters. ABS was obtained using DSPE-PEG2000-AEAA instead of PEG2000. FDS was obtained using DSPE-PEG2000-FA instead of PEG2000. After preparing the core ABS and FDS, a 1:1 mixture was introduced dropwise into an equal volume of NPC solution (3 mg/mL) under constant stirring to facilitate encapsulation. Through electrostatic interaction, ABS/FDS spontaneously encapsulated within the NPC, thereby forming the surface-modified NPC-ABS/FDS. The non-targeting ligands modified CMCS-BAE/DOX-SLN was obtained using CMCS, BAE-SLN, and DOX-SLN instead of NPC, ABS, and FDS. For synthesis of DSPE-PEG2000-AEAA and NPC, refer to the Supplementary Data.

2.5. Characterization of NPC-ABS/FDS

Dynamic light scattering (DLS) using a Nano ZS system (Malvern Instruments) was employed to assess the hydrodynamic diameter, zeta potential, and size distribution of the NPs. Morphological analysis was conducted via transmission electron microscopy (TEM) (JEM1200EX, Tokyo, Japan). Additionally, the payloads of BAE and DOX were quantified by HPLC at wavelengths of 280 nm and 254 nm, respectively, allowing for the calculation of encapsulation efficiency (EE) and drug loading (DL) according to the following formulas:
EE% = Wdrug/Wtotal drug × 100%
DL% = Wdrug/(Wdrug + Wcarrier) × 100%
Wdrug represents the drug determined, Wcarrier represents the carrier of the NPs, and Wtotal drug represents the drug of the NPs.

2.6. Stability Evaluation

The physical stability of NPs was evaluated at 4 °C for 7 days (n = 3) [22]. The stability of NPs co-incubation with saline and 10% serum was performed at 37 °C for 48 h (n = 3). The variations in zeta potential and the mean hydrodynamic size were characterized via dynamic light scattering (DLS).

2.7. Hemolysis Testing

A 2% (v/v) suspension of rat erythrocytes was prepared by centrifugation and subsequently resuspended in normal saline (NS) [23]. Following this, various volumes of NPC-ABS/FDS (0.1, 0.2, 0.3, 0.4, or 0.5 mL) were incubated with 1.5 mL of the erythrocyte suspension for 4 h at 37 °C. Additional groups involving other targeted co-delivery systems are listed in Table S1.

2.8. In Vitro Release of NPC-ABS/FDS

A dialysis approach was employed to assess the release kinetics of BAE and DOX from the NPC-ABS/FDS formulation [22]. DOX solution (DOX-Sol), BAE-SLN, DOX-SLN, ABS, and FDS were set as controls. One milliliter samples of DOX-Sol, BAE-SLN, DOX-SLN, ABS, and FDS were sealed in dialysis membranes (MWCO 3500 Da) and immersed in 19 mL of PBS (pH 7.4) supplemented with 0.1% Tween-80. The release medium was maintained at 37 °C with continuous magnetic stirring at 150 rpm. To ensure consistent BAE/DOX content, the volume of NPC-ABS/FDS was 2 mL, and PBS was 18 mL (pH 7.4, 6.5). Two milliliters of the released substrate were removed at a predetermined time point, and then fresh 37 °C release media were added. All samples were performed three times. The concentration of BAE and DOX released was measured by HPLC.

2.9. First-Level Targeting Effect of NPC-ABS/FDS In Vitro

Cellular uptake in HUVECs was carried out to evaluate active targeting to tumor neovascularization of NPC-ABS/FDS [22]. Drugs were replaced by Coumarin 6 (C6) to form NPC-C6-SLN and CMCS-C6-SLN. HUVECs were plated in 12-well culture plates at a density of 1.0 × 105 cells per well and allowed to adhere for 12 h. Subsequently, the cells were exposed to fresh medium containing NPC-C6-SLN (pH 7.4 or 6.5), CMCS-C6-SLN, C6-SLN, or free C6 (C6-Sol, 50 ng) for incubation periods of 1 and 4 h. For competitive inhibition assays, HUVECs were pretreated with free NGR peptide (1 mg/mL) for 1 h prior to incubation with NPC-C6-SLN. After treatment, the cells were stained with Hoechst 33342 and washed with PBS. Fluorescence images were acquired using a confocal microscope (SPE, Leica, Wetzlar, Germany). Quantitative analysis of cellular uptake was performed by resuspending cells in 200 μL of PBS and measuring with a BD FACSymphony™ A1 flow cytometer (BD Biosciences, San Jose, CA, USA). The excitation and emission wavelengths for C6 were set at 488 and 530 nm, respectively.

2.10. Second-Level Targeting Effect of NPC-ABS/FDS In Vitro

Cellular uptake of C6-loaded C6-SLN, FA-C6-SLN (FCS), and NPC shell-coated NPC-FCS in 4T1 cells was carried out to evaluate active targeting to the cancer cells of NPC-ABS/FDS [22]. Cellular uptake of Nile red (Nr)-loaded Nr-SLN, AEAA-Nr-SLN (ANS), and NPC shell-coated NPC-ANS in CAFs was carried out to evaluate active targeting to CAFs of NPC-ABS/FDS. 4T1 cells were plated in 12-well plates at a density of 1.0 × 105 cells per well and cultured for 12 h to allow adhesion. Fresh medium (pH 7.4 or 6.5) containing NPC-FCS, FCS, C6-SLN, or free C6 solution (C6-Sol, 50 ng) was then added, with incubation durations of 1 and 4 h. To assess targeting specificity, competitive inhibition assays were performed by pretreating 4T1 cells with free folic acid (FA, 1 mg/mL) for 1 h prior to exposure to FCS. Following incubation, cells were stained with Hoechst 33342 and washed with PBS, and fluorescence images were acquired using confocal microscopy under the previously described conditions. For quantitative analysis of cellular internalization, cells from each group were resuspended in 200 μL PBS and analyzed by flow cytometry. C6 fluorescence was measured at the same excitation/emission wavelengths as specified earlier. The uptake of NPC-ANS by cancer-associated fibroblasts (CAFs) at varying pH levels was evaluated following a protocol analogous to that used for NPC-FCS in 4T1 cells, with free Nile red (Nr-Sol) at a concentration of 100 ng. Nile red was detected at excitation and emission wavelengths of 532 nm and 576 nm, respectively.

2.11. Study on Tumor Accumulation In Vivo

To evaluate tumor accumulation and targeting efficiency of the nanocapsule system, in vivo fluorescence imaging was performed using female nude Balb/c mice (6–8 weeks old) [24]. A CAFs-positive TNBC xenograft model was established by subcutaneous injection into the axillary region of each mouse with a mixed cell suspension containing 1 × 106 CAFs and 2 × 106 4T1 cells in 0.1 mL PBS. Tumor-bearing mice were used when tumor volumes reached approximately 100 mm3. To visualize biodistribution, BAE and DOX were replaced with cy5, generating the following formulations: cy5-Sol (free cy5); CMCS-cy5-SLN; NPC-cy5-SLN; NPC-AEAA-cy5-SLN; NPC-FA-cy5-SLN. Mice were randomly assigned to five groups (n = 3 per group) and received a single intravenous injection of the corresponding formulation (cy5 dose: 0.25 mg/kg). Whole-body fluorescence imaging was performed at 2, 4, 8, 12, and 24 h post-injection using an IVIS imaging system (Maestro, Caliper). Mice were anesthetized with isoflurane during imaging. After the final time point, mice were euthanized, and tumors and major organs (heart, liver, spleen, lung, and kidney) were harvested for ex vivo fluorescence imaging. cy5 fluorescence was detected at an excitation wavelength of 635 nm and an emission wavelength of 670 nm. Quantitative analysis was conducted using Maestro v5.0 software. Primary outcome measures included the following: in vivo tumor fluorescence intensity over time; ex vivo fluorescence distribution in tumors and major organs. Investigators performing fluorescence acquisition and quantitative analysis were not blinded to group allocation.

2.12. Inhibition Efficacy on CAFs Mediated by NPC-ABS/FDS In Vitro and In Vivo

In vitro evaluation of CAFs inhibition NIH3T3 fibroblasts stimulated with TGF-β1 (10 ng/mL) were used as an in vitro CAFs model [12]. Cells were seeded into 12-well plates at a density of 1.0 × 105 cells per well and allowed to attach for 12 h. Cells were then treated with one of the following formulations (BAE concentration: 2.0 μg/mL) for 24 h: NPC-ABS; ABS; BAE-SLN; Blank NPC-AEAA-SLN; Free BAE (BAE-Sol). After treatment, cells were collected, and the expression levels of FAP and α-smooth muscle actin (α-SMA) were quantified using ELISA kits according to the manufacturers’ protocols. Each experiment was performed in triplicate.
TNBC-bearing mice were established as described in 2.11 above. When tumors reached approximately 100 mm3, mice were randomly divided into five groups (n = 6 per group): NS; Blank NPC-AEAA-SLN; BAE-SLN; ABS; NPC-ABS. The BAE dose was 2.0 mg/kg, administered intravenously once daily for 3 consecutive days. On day 4, mice were euthanized, and tumors were excised. For each group: three tumors were processed for immunofluorescence staining; three tumors were used for masson’s trichrome staining; immunofluorescence staining was performed using FITC-labeled antibodies for α-SMA and rhodamine-labeled antibodies for FAP. Masson staining was used to assess collagen deposition within tumor tissues. Primary outcome measures included the following: Expression levels of FAP and α-SMA in tumor sections; Collagen deposition evaluated by Masson staining; Quantitative image analysis was conducted using Image J software (version 1.52). Investigators performing histological analysis were not blinded to treatment groups.

2.13. Study on Enhanced Deep Chemotherapy Mediated by NPC-ABS/FDS In Vitro and In Vivo

Three-dimensional tumor spheres were utilized to investigate the deep penetration capability of NPC-ABS/FDS in vitro [23]. The detailed preparation process was provided in the Supplementary Data. Specifically, CAFs and 4T1 mixed cells (ratio 2:1, total 1.5 × 104 cells per well) were seeded in 96-well plates and incubated for 3 days to form 3D tumor spheres. C6-loaded NPC-FCS (50 ng) was employed to trace the position of NPC-ABS/FDS. The following formulations were added, respectively: NPC-FCS; BAE-Sol (2.0 or 5.0 μg/mL) plus NPC-FCS; ABS plus NPC-FCS; NPC-ABS plus NPC-FCS (NPC-ABS/FCS); blank NPC-AEAA-SLN plus NPC-FCS. After 4 h of incubation, the 3D tumor spheres were washed 3 times with PBS, fixed with 4% histocyte fixation solution for 30 min, and subjected to Z-axis scanning using a laser confocal microscope (scanning interval 10 μm). The excitation and emission wavelengths for C6 were as described above.
The TNBC model was established as described in 2.11 above. cy5 loaded NPC-FA-cy5-SLN (0.25 mg/kg) was used to trace the position of NPC-ABS/FDS in tumors. When tumors reached approximately 100 mm3, mice were randomly divided into five groups (n = 3 per group): NS; blank NPC-AEAA-SLN; BAE-SLN; ABS; NPC-ABS. The dosage of BAE was 2.0 mg/kg, administered intravenously once daily for 3 consecutive days. On the fourth day, each group was intravenously injected with NPC-FA-cy5-SLN. Eight hours post-injection, the mice were euthanized, and tumor tissues were harvested. After dehydration, clearing, waxing, embedding, and sectioning, tumor vessels were stained with FITC-CD31 and mounted with DAPI-containing medium. Tissue sections were observed and photographed under a 20× confocal laser microscope. Quantitative image analysis was conducted using Image J software. Investigators conducting image analysis were not blinded to group allocation.

2.14. Study on Immune Activation Mediated by NPC-ABS/FDS In Vivo

To evaluate immune activation within the tumor microenvironment following treatment, immunohistochemical analysis was performed [25]. A TNBC xenograft model was established as described in 2.11 above. When tumor volumes reached approximately 100 mm3, mice were randomly assigned into five groups (n = 3 per group): NS; Blank NPC-AEAA-SLN; BAE-SLN; ABS; NPC-ABS. The experimental unit was a single animal. All formulations were administered via intravenous injection once daily for 3 consecutive days, with a BAE dose of 2.0 mg/kg. On day 4, mice were euthanized, and tumor tissues were excised and fixed in 4% paraformaldehyde for 48 h. Tumor sections were processed for immunohistochemical staining, including paraffin embedding, deparaffinization, antigen retrieval using EDTA buffer, endogenous peroxidase blocking, BSA blocking, primary antibody incubation, secondary antibody incubation, DAB chromogenic development, and hematoxylin nuclear counterstaining. Immunohistochemical images were acquired under a light microscope, and quantitative analysis was performed using Image J software. Investigators conducting image analysis were not blinded to group allocation.

2.15. The Antitumor Efficacy of NPC-ABS/FDS In Vitro and In Vivo

The cytotoxicity of NPC-ABS/FDS was evaluated using a CCK-8 assay [23]. 4T1 cells were seeded into 96-well plates at a density of 5 × 103 cells per well and incubated for 12 h. Cells were then treated with the following formulations at DOX-equivalent concentrations of 0.5, 1.0, 2.0, and 5.0 μg/mL: DOX-Sol; DOX-SLN; FCS; NPC-FCS; Blank SLN; Blank NPC-FA-SLN. After 24 h incubation, CCK-8 solution was added and incubated for an additional 0.5 h. Absorbance was measured at 490 nm using a microplate reader (Spark, TECAN). All experiments were performed in triplicate.
TNBC-bearing mice were established as described in 2.11 above. When tumors reached approximately 100 mm3, mice were randomly divided into nine treatment groups (n = 5 per group) [23]: NS; Blank NPC-AEAA/FA-SLN; DOX-Sol; Positive control (DOX®-Sol); BAE/DOX-SLN; ABS/FDS; NPC-ABS/FDS; NPC-FDS; NPC-ABS. BAE and DOX were administered at doses of 2.0 mg/kg each, via intravenous injection every 2 days for a total of 10 days. Tumor volumes and body weights were recorded every 2 days. Tumor volume was calculated using standard formulas. On day 11, the mice were euthanized, and tumors were excised, weighed, and photographed for further analysis. Investigators assessing tumor growth were not blinded to group allocation.

2.16. Statistical Analysis

Data were presented as mean ± standard deviation (SD). Statistical significance was evaluated using Student’s t-test, with p < 0.05 considered indicative of a significant difference.

3. Results

3.1. Preparation and Characterization of NPC-ABS/FDS

NPC-ABS/FDS nanocapsule was engineered to enable the co-delivery of BAE and DOX to CAFs and tumor cells, respectively. Initially, specific targeting ligands were prepared to facilitate accumulation within tumor tissue and the distinct delivery of the two payloads. Specifically, DSPE-PEG2000-AEAA was synthesized to target CAFs, capitalizing on the high affinity of AEAA for σ-receptors [12]. In triethylamine, the amino group of one end of diethylenetriamine reacts with the carbonyl group of DSPE-PEG2000-NHS to form DSPE-PEG2000-diethylenetriamine, and the amino group at the other end of diethylenetriamine reacts with anisyl chloride to obtain DSPE-PEG2000-AEAA. Figure 1A and Figure S2 show the synthesis route and 1H-NMR spectrum of DSPE-PEG2000-AEAA, indicating that DSPE-PEG2000-AEAA was successfully synthesized. The NPC was developed to direct itself toward tumor neovascular endothelial cells via the NGR-CD13 binding interaction [13]. SCM-PEG2000-SCM was initially functionalized using 4-dimethylaminopyridine, followed by reaction with the amino groups of NGR and CMCS. Figure 1B shows the synthesis route of NPC. Figure S3 displayed the 1H-NMR spectra for CMCS, SCM-PEG2000-SCM, NGR, and NPC. The appearance of NGR’s resonance signals in the 1.4–1.8 ppm range within the NPC spectrum confirmed its presence. In Figure S4, FTIR spectroscopy shows that at 1100 cm−1, NPC shows the characteristic fingerprint peak of NGR. Collectively, these findings verified the successful synthesis of the targeting ligand.
Figure 1. Synthesis routes of (A) DSPE-PEG2000-AEAA and (B) NPC. (Red color represents the reactive groups to be reacted, and blue color represents the groups after the reaction).
Cationic BAE-SLN and DOX-SLN were prepared by thin-film hydration dispersion method [21]. Targeting cationic SLNs (ABS and FDS) were functionalized, respectively, with DSPE-PEG2000-AEAA and DSPE-PEG2000-FA to enable distinct cellular targeting. Subsequently, the NPC-ABS/FDS was formulated via electrostatic interaction between these cationic cores (ABS: FDS = 1:1) and the anionic polymer NPC, aiming to induce TME-triggered disassembly and improve in vivo tumor retention [23]. DLS and TEM were employed to evaluate the mean particle sizes, zeta potentials, and morphological features. According to Table S2 and Figure 2A,B, ABS exhibited 92.81 ± 0.66 nm and 18.40 ± 0.70 mV, which were similar to those of FDS (91.55 ± 0.29 nm and 20.70 ± 0.80 mV), and they were of analogous spherical shape. Due to the presence of DOTAP, the drug-loaded SLNs were all positively charged. Electrostatic adsorption of the anionic polymer NPC onto the dual cationic cores (ABS and FDS) was employed to generate the NPC-ABS/FDS. As shown in Table S2 and Figure 2C, NPC-ABS/FDS exhibited a spherical morphology with an average diameter of 175.43 ± 3.69 nm and possessed a Zeta potential of −8.19 ± 0.88 mV. When viewed under a microscope with enhanced magnification, ABS and FDS appeared distinctly in spherical configurations, and it became unequivocally evident that they were enclosed within NPC, resulting in the formation of NPC-ABS/FDS (Figure S5A–C). The properties of BAE-SLN, DOX-SLN, and CMCS-BAE/DOX-SLN were similar to those of ABS, FDS, and NPC-ABS/FDS, respectively. The observed variations in particle size and zeta potential indicated that both ABS and FDS were effectively encapsulated by NPC. Additionally, the DL and EE of ABS and FDS are shown in Table S2. The ABS, FDS, and NPC-ABS/FDS were stable at 4 °C for 7 days (Figure 2D,E) and remained stable at 37 °C for 48 h after incubation with NS (Figure S5D,E) and 10% serum (Figure 2F,G).
Figure 2. Characterizations of NPC-ABS/FDS. Property characterization, including size, zeta potential, and TEM morphology of (A) ABS, (B) FDS, and (C) NPC-ABS/FDS. Variations in size and zeta potential for ABS, FDS, and NPC-ABS/FDS (D,E) monitored over 7 days at 4 °C (Mean ± SD, n = 3). Stability profiles (F,G) following incubation with 10% serum at 37 °C for 48 h (Mean ± SD, n = 3). Cumulative drug release of BAE (H) and DOX (I) in vitro (Mean ± SD, n = 3, ** p < 0.01, *** p < 0.001). (J) Hemolysis assays of different preparations (serial numbers 1–22 representative preparations are shown in Table S1).
Once NPC-ABS/FDS accumulated in tumor tissue, protonation of NPC’s amino groups [22] facilitated its disassembly and the release of ABS and FDS. To investigate this, pH-responsive release was assessed in vitro at pH 6.5 and 7.4. As shown in Figure 2H,I, the cumulative release of BAE and DOX at pH 6.5 (40.6% and 75.9% over 24 h) was significantly greater (p < 0.01) than that at pH 7.4 (36.0% and 65.9%), consistent with disassembly triggered by the acidic TME. Table S3 shows the detailed preparations of the hemolysis assay, suggesting that NPC-ABS/FDS remained stable for 4 h after incubation with 2% red blood cell suspension (Figure 2J).
The NGR, attached to the NPC, exhibited tumor neovascularization targeting [22]. We hope to achieve first-level targeting to the tumor site through NPC, so that drugs will be enriched at the tumor site. Then, NPC releases ABS and FDS in the acidic environment, and achieves second-level targeting under the interaction of FA and AEAA with receptors [12,20], that is, the different targeting of ABS and FDS to CAFs and cancer cells, respectively. Next, we will prove our conjecture.

3.2. NPC-ABS/FDS Exhibited First-Level Targeting to Tumor Neovascularization

The first-level targeting delivery properties of NPC-ABS/FDS were studied using CD13-positive HUVECs in vitro through cellular uptake experiments. As shown in Table S3, the properties of C6-loaded NPs modified with either targeting or non-targeting ligands were similar to those of the drug-loaded NPs. Flow cytometric analysis and confocal laser scanning microscope (CLSM) images showed that signals of NPC-C6-SLN were significantly increased compared with those of C6-Sol, C6-SLN, and CMCS-C6-SLN in HUVECs, as illustrated in Figure 3A,B. It could be observed that after 1h incubation, the nano-prescriptions loaded with C6 rapidly enter HUVECs; the fluorescence intensity of NPC-C6-SLN (pH 7.4) was 1.3 times, 1.9 times, and 15.8 times that of CMCS-C6-SLN (pH 7.4), C6-SLN, and C6-Sol, respectively (p < 0.05). Additionally, NPC-C6-SLN exhibited a higher internalization rate under acidic conditions (pH 6.5) than at physiological pH (7.4). Specifically, the uptake increased by 1.2-fold at 1 h and 1.1-fold at 4 h (p < 0.05). The results show that NPC exhibits better delivery of inner cargoes under acidic conditions of TME due to acid degradation characteristics. In addition, the cellular uptake of NPC-C6-SLN was suppressed by pretreatment of cells with free NGR both in 1 h and in 4 h (p < 0.05), proving that the active targeting of NPC was mediated by NGR [22]. Compared with incubation for 1 h, the uptake rate of C6-loaded nano-prescriptions was not significantly improved within 4 h, but the uptake rate of NPC-C6-SLN (pH 6.5) was still about seven times higher than that of C6-Sol. These results indicated that NPC-ABS/FDS could accumulate in tumor neovascularization in a short time, through NGR-mediated first-level targeting.
Figure 3. NPC-ABS/FDS exhibited tumor neovascularization targeting. (A) Confocal microscopy images, and (B) flow cytometry quantification of cellular uptake after treatment with different groups in HUVECs (Mean ± SD, n = 3, * p < 0.05, *** p < 0.001, scale bar = 25 μm).

3.3. NPC-ABS/FDS Exhibited Second-Level Targeting of DOX to Cancer Cells and BAE to CAFs

In order to verify the second-level targeting, the different-targeting capability of DOX toward cancer cells and BAE toward CAFs was validated via cellular uptake assays. Specifically, C6-loaded NPC-FCS was tracked in 4T1 cells, while Nr-loaded NPC-ANS was evaluated in CAFs. NPs labeled with C6 or Nr showed similar size and zeta potential profiles regardless of the presence of targeting ligands, matching the properties observed for drug-loaded nanoparticles (Table S3).
The FA-mediated active targeting to cancer cells (4T1) was demonstrated using cellular uptake studies of C6-loaded FCS. C6-Sol and C6-SLN were employed as references for FCS, and competitive inhibition assays were included to evaluate the active targeting capability. As shown in Figure 4A,B, flow cytometric analysis and CLSM images show that the fluorescence intensity of FA-modified FCS was greater than those of the unmodified nano-prescription groups and the C6-Sol group, both at 1 h and 4 h. After incubating for 1 h, the signal intensity of FCS in 4T1 cells was 1.1 times than that of C6-SLN and 7.0 times than that of C6-Sol. After 4 h of incubation, the signal intensity was 1.1 times and 4.3 times that of C6-SLN and C6-Sol, respectively (p < 0.01). In addition, the cellular uptake of FCS was suppressed by pretreatment of cells with free FA both in 1 h and in 4 h (p < 0.001), proving that active targeting mediated by the specific binding of FA and folate receptor, highly expressed in 4T1 cells, increased the uptake of inner FDS in the targeted co-delivery system by 4T1 cells. Notably, the fluorescence signal of NPC-FCS in 4T1 cells was markedly amplified under acidic conditions (pH 6.5) relative to physiological pH (7.4), which was ascribed to the structural disintegration of NPC and the release of FA-decorated FCS (p < 0.001).
Figure 4. NPC-ABS/FDS exhibited different-targeting delivery efficiency of DOX to tumor cells. (A) Confocal microscopy images, and (B) flow cytometry quantification of cellular uptake after treatment with different groups in 4T1 (Mean ± SD, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, scale bar = 25 μm).
Cellular uptake of Nr-loaded ANS was assessed to confirm AEAA-driven active targeting to CAFs, with TGF-β1-induced NIH3T3 cells used as the CAF model [12]. Nr-Sol and Nr-SLN were included as controls, and competitive inhibition assays were carried out to substantiate the targeting efficiency. The uptake behavior of Nr-loaded NPC-ANS and ANS in CAFs at various pH conditions mirrored that of C6-loaded NPC-FCS and FCS in 4T1 cells (Figure 4). As shown in Figure 5A,B, flow cytometric analysis and CLSM images show that the fluorescence intensity of AEAA-modified ANS was greater than that of the unmodified nano-prescription groups and the Nr-Sol group, both at 1 h and 4 h. After incubating for 1 h, the signal intensity of ANS in CAFs was 1.7 times and 2.9 times that of Nr-SLN and Nr-Sol, respectively, and 1.6 times and 2.5 times, respectively, after 4 h of incubation (p < 0.001). Furthermore, the cellular uptake of ANS was suppressed by pretreatment of cells with free AEAA (p < 0.001), proving that active targeting mediated by specific binding of AEAA and σ-receptor, specifically expressed in CAFs, increased the uptake of inner ABS in targeted co-delivery system by CAFs [26]. Similar to NPC-FCS, the fluorescence signal of Nr-loaded NPC-ANS in CAFs was stronger at pH 6.5 than at pH 7.4, due to the disassembly of NPC and the release of AEAA-modified ANS (p < 0.001).
Figure 5. NPC-ABS/FDS exhibited different-targeting delivery efficiency of BAE to CAFs. (A) Confocal microscopy images, and (B) flow cytometry quantification of cellular uptake after treatment with different groups in CAFs (Mean ± SD, n = 3, *** p < 0.001, scale bar = 25 μm).
Taken together, the results showed that mediated by the interaction of targeted peptides and receptors, NPC-ABS/FDS exhibited a second-level targeting effect on the precise guidance of different cell types in the TME. Specifically, FDS and ABS targeted cancer cells and CAFs, respectively, with better effect than drug-carrying SLN without targeted peptide modification.

3.4. NPC-ABS/FDS Exhibited Enhanced Tumor Accumulation via the Two-Stage Targeting Process

The accumulation of NPC-ABS/FDS in tumor tissues was evaluated using a CAFs-positive TNBC xenograft model as described in 2.11. The properties of NPs loaded with cy5 and modified with either targeting or non-targeting ligands were similar to those of the drug-loaded counterparts (Table S3). As shown in Figure 6A, the fluorescence intensity of cy5-loaded nano-prescriptions was highest at 12 h. The fluorescence intensity was as follows: NPC-FA-cy5-SLN > NPC-AEAA-cy5-SLN > NPC-cy5-SLN > CMCS-cy5-SLN ≈ cy5-Sol. As shown in Figure 6B,C, the fluorescence intensity of cy5-loaded NPC-cy5-SLN at the tumor site was 1.4-fold and 1.9-fold higher than that of cy5-loaded CMCS-cy5-SLN and cy5-Sol, respectively (p < 0.05). This result confirmed that NGR-mediated first-level targeting significantly enhanced the delivery of the cargo by NPC to the tumor region. The reason for the non-statistical significance of fluorescence intensity in CMCS-cy5-SLN and cy5-Sol may be that CMCS did not significantly enhance the uptake of cy5 in the tumor site without NGR-mediated active targeting. To verify the second-level targeting, cy5-loaded NPC-AEAA-cy5-SLN and NPC-FA-cy5-SLN were administered. On the basis of first-level targeting, the second-level targeting further improved delivery efficiency. Specifically, AEAA-mediated targeting increased cargo delivery by 1.3-fold, while FA-mediated targeting increased it by 1.9-fold, both compared to the NPC-cy5-SLN group (p < 0.05). The above results suggested that NPC shell first co-delivered the inner cargoes to the tumor site, and rapidly degraded and released the inner cargoes under acidic conditions. Then, second-level targeting mediated by AEAA and FA further improved the targeting efficiency of NPC-ABS/FDS to heterotargeted cells in the TME. The combined effect of first-level and second-level targeting significantly enhanced the accumulation of NPC-ABS/FDS at the tumor site.
Figure 6. NPC-ABS/FDS show improved tumor targeting efficiency. (A) Representative in vivo images of mice after receiving different treatments. (B) Ex vivo imaging of excised tissues at the 8 h time point, and (C) quantitative analysis of their relative fluorescence signals. (a. cy5-Sol; b. CMCS-cy5-SLN; c. NPC-cy5-SLN; d. NPC-AEAA-cy5-SLN; e. NPC-FA-cy5-SLN. Mean ± SD, n = 3, * p < 0.05, ** p < 0.01).
The intravenous administration of nanocarriers frequently results in significant hepatic accumulation, primarily due to clearance by the mononuclear phagocyte system [27]. Therefore, high accumulation of NPs in the liver was observed. The elevated pulmonary uptake may be likely attributed to the physical entrapment of slightly aggregated particles within the pulmonary capillary [28]. The fluorescence response of NPs in the kidneys reflected both the metabolic clearance of the dissociated dye and the significant tumor accumulation comparable to the main metabolic organs, thereby confirming the targeting effect [29]. The low uptake in the spleen may also be attributed to the size-dependent filtration mechanism. Our glycoconjugated NPs (approximately 180 nm) are more likely to pass through the intercellular gaps between the endothelium than larger particles (>200 nm), thereby minimizing non-specific accumulation [30].

3.5. NPC-ABS/FDS Inhibited CAFs Activation and Removed Restrictive Physical Barrier

The inhibitory effect on CAF activation and the removal of the restrictive physical barrier by NPC-ABS/FDS were verified through elisa assay, immunofluorescence assay, and masson staining. FAP and α-SMA are the “gold standard” for verifying CAF activation [31,32]. As shown in Figure 7A,B, compared with normal NIH3T3, the expressions of FAP and α-SMA in CAFs were significantly increased by 19.93% and 39.05%, respectively (p < 0.05), indicating that CAFs were activated. After treatment with different groups of nano-prescriptions containing BAE, the expression levels of the two proteins were significantly reduced and tended towards the level of normal NIH3T3. The content of FAP in CAFs treated with NPC-AEAA-SLN, BAE-Sol, BAE-SLN, ABS, and NPC-ABS decreased by 11.59%, 17.07%, 22.81%, 31.35%, and 11.07%, respectively, and the content of α-SMA decreased by 15.05%, 19.17%, 26.59%, 31.46%, and 20.91%, respectively. However, there were no statistically significant differences in the level of FAP compared with normal NIH3T3. Except for NPC-AEAA-SLN and BAE-Sol, there were no statistical significances in the level of α-SMA between the other groups and the normal NIH3T3 group. These results suggested that the inhibition of BAE on CAFs was a mild action, but did not cause the side effect of excessive inhibition or killing CAFs, which was consistent with our previous research (Figure S1). Immunofluorescence assay also showed that NPC-ABS/FDS could inhibit the expression of FAP and α-SMA in tumor tissues in vivo. As shown in Figure 7C,D, in the mouse model of TNBC, the expressions of FAP and α-SMA after intravenous injection of NPC-ABS were lower than those in the NS and NPC-AEAA-SLN groups (p < 0.05). After masson staining of the tumor tissues, we found that the restrictive barrier caused by ECM deposition in TME was greatly weakened. Collagen is the main component of ECM [33]. As shown in Figure 7E, blue represented collagen matrix and collagen fiber. A large amount of ECM deposition was observed in NS and NPC-AEAA-SLN groups, and the tumors showed a dense fibrotic matrix. After intravenous injection of BAE-Sol and different groups of nano-prescriptions containing BAE, collagen was significantly reduced. In conclusion, the above studies suggested that NPC-ABS/FDS effectively inhibited the activation of CAFs in vitro and in vivo, reduced ECM deposition caused by overexpression of FAP and α-SMA in CAFs, and eliminated the physical barrier.
Figure 7. NPC-ABS/FDS inhibited CAFs activation and eliminated the restrictive physical barrier. Content of (A) α-SMA and (B) FAP after treatment with different groups in CAFs (Mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.01 compared with Normal NIH3T3 group; a. normal NIH3T3, b. CAFs, c. NPC-AEAA-SLN, d. BAE-sol, e. BAE-SLN, f. ABS, and g. NPC-ABS.). (C) Representative photomicrographs of FITC-labeled α-SMA and rhodamine-labeled FAP in mouse tumors after administration of different preparations (a. normal saline, b. NPC-AEAA-SLN, c. BAE-SLN, d. ABS, and e. NPC-ABS, scale bar = 100 μm), and (D) their relative fluorescence intensity (Mean ± SD, n = 3. * p < 0.05 compared with Normal NIH3T3 group of α-SMA and # p < 0.05 compared with Normal NIH3T3 group of FAP). (E) Representative photomicrographs of collagen in mouse tumors after administration of different preparations (a. normal saline, b. NPC-AEAA-SLN, c. BAE-SLN, d. ABS, and e. NPC-ABS. Blue color represented positive cells; scale bar = 100 μm).

3.6. NPC-ABS/FDS Mediated Deep Chemotherapy and Immune Activation

The effects of NPC-ABS/FDS on deep chemotherapy and enhanced tumor immunotherapy were verified by three-dimensional tumor spheres penetration assay, intratumoral tracing, and immunohistochemistry of tumor tissues. We demonstrated that the removal of the restrictive barrier increased the penetration of NPs into the deep avascular tissue of tumors through the 3D tumor spheres penetration assay. Three-dimensional tumor spheres were established according to Figures S6 and S7 in the Supplementary Data. The DOX in NPC-ABS/FDS was replaced by C6 for better imaging. Figure 8A,B illustrate the effects of different nano-prescriptions on the penetration of 3D tumor spheres. As shown in Figure 8A, NPC-FCS was able to penetrate a distance of 30 μm rather than attach to the surface of the 3D tumor spheres, possibly due to the targeting effect of FA [23], but still did not penetrate deep into the 3D tumor spheres. NPC-AEAA-SLN did not enhance the penetration of NPC-FCS to 3D tumor spheres; on the contrary, BAE-Sol and BAE-containing nano-prescriptions enhanced this penetration effect to varying degrees, suggesting that the penetration-enhancing effect was derived from BAE. The penetration ability was as follows: NPC-ABS/FCS > ABS+NPC-FCS > BAE-Sol+NPC-FCS > NPC-AEAA-SLN+NPC-FCS ≈ NPC-FCS, once again demonstrating the importance of AEAA-targeted modification and acid-degradable NPC shell. The reason why the penetration effect of NPC-ABS/FCS was better than that of ABS + NPC-FCS might be due to the principle of reducing excessive positive charges to minimize non-specific adhesion. Studies have shown that strongly positively charged NPs have a strong affinity for negatively charged membranes, which often leads to “binding-site blocking”, causing the NPs to be irreversibly trapped at the edge of the membrane and thus limiting their deep penetration [34]. In contrast, the NPC-ABS/FCS complex regulated the charge potential through encapsulation to a suitable range, reducing strong non-specific binding and preventing particles from being trapped at the edge, thereby promoting their deep penetration into the core of the sphere [35]. NPC-ABS/FCS penetrated deeper into the 3D tumor spheres (60 μm) with twice the penetration effect of NPC-FCS. This penetration effect was inhibited by AEAA (source of NPC-AEAA-SLN), indicating that targeted peptides increased the uptake of BAE by CAFs, thus producing a stronger inhibitory effect on CAFs [12,26]. Interestingly, the increase in BAE concentration did not result in better penetration enhancement to 3D tumor spheres (Figure 8B). Similarly, we also found that BAE had no dose–response relationship in inhibiting CAFs. We will continue to investigate the causes of these two strange phenomena in our future work.
Figure 8. NPC-ABS/FDS mediated deep chemotherapy and induced immune activation. (A,B) Confocal microscopy images in 3D tumor spheres after treated with different groups (a. NPC-FCS, b. NPC-FCS+NPC-AEAA-SLN, c. NPC-FCS+BAE-Sol, d. NPC-FCS+ABS, and e. NPC-ABS/FCS. Z-axis scan spacing = 10 μm. BAE = 2 μg/mL in A and BAE = 5 μg/mL in B, respectively; the magnification was 10×). (C) Representative photomicrographs of tumor biopsies treated with different groups (a. normal saline, b. NPC-AEAA-SLN, c. BAE-SLN, d. ABS, and e. NPC-ABS). Tumor blood vessels were labeled with FITC-CD31; the magnification was 20×). Relative expression quantity of immune-associated cells in mouse tumors after administration of different preparations of (D) CD4+ T cells, (E) CD8+ T cells, and (F) Tregs (a. normal saline, b. NPC-AEAA-SLN, c. BAE-SLN, d. ABS, and e. NPC-ABS. Mean ± SD, n = 3. ** p < 0.01, *** p < 0.001. Blue color represented positive cells; the magnification was 10×).
In the intratumoral tracing assay, cy5 was used to replace DOX in order to obtain better imaging results. As shown in Figure 8C, green represented FITC-labeled tumor blood vessels. In the NS and blank NPC-AEAA-SLN group, nano-prescriptions of red fluorescence tightly stayed near the blood vessels and did not spread to the surrounding area, which was caused by the dense restrictive physical barrier. After the nano-prescriptions containing BAE were administered, the ECM deposition was weakened, and the restrictive physical barrier was removed, and the targeted co-delivery system loaded with cy5 infiltrated into the avascular tumor region. In addition, the penetration effect of AEAA-containing peptides was better than that of the non-targeted peptides group. Combined with the above experimental results, it was verified that NPC-ABS/FDS inhibited the activation of CAFs, removed the barrier caused by ECM deposition, and enhanced the penetration of NPs into tumor tissues, thus achieving an effect on deep chemotherapy.
Studies have shown that inhibiting CAFs contributed to the infiltration of immune-associated cells in tumor tissue [1,36]. CD4+ and CD8+ are two representative cytotoxic T cells, which play an important role in antitumor immunity [37,38]. Immunohistochemical assessment revealed a marked increase in the tumor infiltration of CD4+ and CD8+ T cells following physical barrier disruption, although the magnitude of the increase varied. As shown in Figure 8D,E, yellow-brown represented positive cells (CD4+ or CD8+), and the infiltration of CD4+ or CD8+ T cells was almost invisible in the NS and the blank NPC-AEAA-SLN group. After intravenous injection of BAE-SLN, ABS and NPC-ABS, the level of CD4+ T cell in tumor tissues was 1.3 times, 1.7 times, and 1.6 times than that of in NS group, respectively (p < 0.01), and the level of CD8+ T cell was 1.7 times, 2.5 times, and 2.4 times than that of in NS group, respectively (p < 0.01). After inhibiting CAFs activation, NPC-ABS downregulated the expression of FAP, α-SMA, and collagen deposition, opening pathways for cytotoxic T cell infiltration into tumor tissues. Meanwhile, we found that NPC-ABS inhibited the proliferation of Tregs in tumor tissue (as shown in Figure 8F, p < 0.01). It was reported that a high level of co-inhibitory receptors on the surface of Tregs exerted immunosuppressive functions by interacting with ligands on their target cells, such as CTLA-4 and PD-L1 [39]. Tregs also secreted cytokines such as IL-10 and TGF-β to inhibit T cell functions [40]. We will investigate whether NPC-ABS has more positive significance for immunotherapy in the future.
In summary, NPC-ABS/FDS inhibited CAF activation in vivo and in vitro, specifically by downregulating FAP and α-SMA, thereby inhibiting collagen secretion, removing the physical barrier caused by ECM deposition, and creating conditions for the infiltration of NPs and cytotoxic T cells in the deep avascular tissue of tumors, playing the dual role of deep chemotherapy and enhanced tumor immunotherapy.

3.7. NPC-ABS/FDS Enhanced the Anti-TNBC Efficacy In Vivo and In Vitro

The antitumor effect of NPC-FDS on 4T1 cells in vitro was evaluated by cck8 assay. As shown in Figure 9A, the toxicity of FDS to 4T1 cells was slightly higher than that of DOX-Sol and DOX-SLN, although the difference was not statistically significant. The reduced cytotoxicity of NPC-FDS may be attributed to the fact that NPC limited the dissolution of DOX, a finding consistent with the in vitro release profile shown in Figure 2I. In addition, anti-TNBC efficacy was also evaluated on a model of TNBC mice in vivo. The dosages of BAE and DOX were both 2.0 mg/kg. At the cellular level, BAE of 2.0 μg/mL had good inhibition to CAFs (Figure S1). In Figure 9A, we found that DOX of 2.0 μg/mL had a good inhibitory effect on cancer cells. So we prefer a 1:1 ratio of BAE to DOX. Referring to the experimental data of DOX against breast cancer in vivo [20] and the 1:1 principle, the dosage of BAE in mice in this experiment was also 2.0 mg/kg.
Figure 9. NPC-ABS/FDS demonstrates improved therapeutic outcomes in both in vitro and in vivo settings. (A) In vitro cell viability of NPC-FDS in 4T1. Changes in (B) tumor volume and (C) inhibition rate of tumor volume. (D) Photographs of isolated tumors in each group (a. NS, b. blank NPC-AEAA/FA-SLN, c. DOX-Sol, d. DOX®-Sol, e. BAE/DOX-SLN, f. ABS/FDS, g. NPC-ABS/FDS, h. NPC-FDS, and i. NPC-ABS). Changes in (E) weights of isolated tumor, (F) inhibition rate of tumor weight, (G) body weight, and (H) inhibition rate of body weight in different treatment groups (Mean ± SD, n = 5, * p < 0.05, *** p < 0.001). (I) Organ index in different treatment groups (Mean ± SD, n = 5, * p < 0.05, ** p < 0.01). (J) H&E results of excised tumor tissues (a. NS, b. blank NPC-AEAA/FA-SLN, c. DOX-Sol, d. DOX®-Sol, e. BAE/DOX-SLN, f. ABS/FDS, g. NPC-ABS/FDS, h. NPC-FDS, and i. NPC-ABS. Scale bar = 200 μm).
Mice treated with NPC-ABS/FDS exhibited significantly smaller mean tumor volumes than those receiving the NPC-ABS, NPC-FDS, ABS/FDS, BAE/DOX-SLN, DOX-Sol, and positive drug (DOX®-Sol) treatment (Figure 9B). The tumor volume inhibition rate of the NPC-ABS/FDS group was 48%, completely higher than those of the DOX-Sol group (23%), DOX®-Sol group (21%), BAE/DOX-SLN group (24%), ABS/FDS group (32%), NPC-FDS group (18%), and NPC-ABS group (17%) (p < 0.001) (Figure 9C). Following the tumor accumulation study, mice were euthanized on day 11 (12 h post-administration on day 10) to harvest the tumor, heart, kidney, spleen, liver, and lung. The tumors were weighed and photographed. The significantly lower tumor weights in the NPC-ABS/FDS cohort indicate enhanced antitumor efficacy compared to all other treatment groups (Figure 9D,E). The tumor weight inhibition rate of the NPC-ABS/FDS group was 50%, which was higher than that of the DOX-Sol group (20%), DOX®-Sol group (24%), BAE/DOX-SLN group (11%), ABS/FDS group (25%), NPC-FDS group (4%), and NPC-ABS group (13%) (p < 0.001) (Figure 9F). Moreover, the mice in all groups maintained stable body weights without significant reduction (Figure 9G,H).
The antitumor effect of the ABS/FDS group was not significantly improved compared with the BAE/DOX-SLN group, possibly due to the rapid clearance action during systemic circulation, resulting in less active targeting mediated by peptides [41]. Figure 9I shows the organ index in different groups. Only the splenic index was observed, which may be the reason for more T cells entering the tumor and resulting in the positive immunomodulation of T cell immunity [23]. Histological examination of H&E-stained major organs revealed no apparent pathological lesions, with the exception of the DOX-Sol and DOX®-Sol groups (Figure 9J). This indicated a low systemic toxicity of NPC-ABS/FDS. The antitumor effect of the NPC-ABS/FDS indicated that co-delivery of heterotargeted drugs DOX and BAE in cancer cells and CAFs could improve the combined treatment effect.

4. Discussion

Studies have shown that CAFs participate in tumor inflammation, chemotherapy resistance, and immunosuppression, thereby promoting tumor growth, survival, and maintaining malignancy, particularly in TNBC [3]. In recent years, with the rapid development of multi-omics technologies such as single-cell sequencing and spatial transcriptomics, the understanding of CAFs’ heterogeneity, function, and roles within the TME has entered a new stage. CAFs are no longer regarded as a single type of tumor-promoting stromal cell but are now recognized as a highly heterogeneous and functionally diverse cell population. Their fundamental roles in immune evasion, therapy resistance, and metabolic regulation have been increasingly elucidated. For instance, Huang’s team integrated data from 15 tissue types and more than 2.5 million cells, generating the most comprehensive molecular map of antigen-presenting CAFs (apCAF) to date. Notably, this study revealed that apCAFs consist of two subgroups with distinct origins, spatial distributions, and biological functions. Mesothelial-like apCAFs (M-apCAF) originate from mesothelial cells and are mainly located in tumor core regions, tending to form an immunosuppressive barrier [42]. Fibroblast-like apCAFs (F-apCAF) originate from bone-marrow-derived fibroblasts and concentrate in lymphocyte-rich areas, potentially contributing to immune-cell recruitment [42]. Moreover, Michael et al. [43] found that cancer cells can transfer mitochondria to fibroblasts through tunneling nanotubes, providing metabolic support and reprogramming them into tumor-promoting CAFs. MIRO2, a mitochondrial transport protein, was identified as the key mediator of this process [43]. The inhibition of MIRO2 blocked mitochondrial transfer and suppressed tumor progression, providing a new avenue for CAFs-targeted therapy. Advances in unveiling CAFs heterogeneity and cell–cell interaction networks have not only refined our understanding of TME complexity but have also laid the foundation for developing more effective and selective CAFs-modulating therapeutic strategies.
In our previous work, we found that BAE exhibited notable anti-fibrotic effects and inhibited TGF-β1 signaling during the treatment of pulmonary fibrosis [44]. TGF-β is involved in the regulation of cell growth, proliferation, differentiation, migration, and apoptosis [45]. Meanwhile, TGF-β signaling is also closely associated with immune modulation and angiogenesis [46]. Inhibition of the TGF-β/Smad pathway effectively suppresses CAFs activation and reduces ECM deposition [47]. Previous reports also indicate that BAE exhibits antioxidant activity and significantly reduces intracellular ROS levels [48]. Lower ROS levels may inhibit TGF-β1 and PDGF activation, slowing fibroblast proliferation [49]. Since oxidative stress promotes apoptosis of T cells, the reduction in ROS may help protect tumor-infiltrating T cells [50]. Therefore, the potential of BAE to inhibit CAFs activation provides a promising adjunctive strategy for TNBC therapy. However, the short half-life and poor solubility of BAE substantially restrict its therapeutic potential [51]. Thus, developing an effective approach to prolong the half-life and enhance the bioavailability of BAE is meaningful. In this study, we improved the solubility and bioavailability of BAE by incorporating it into SLNs. We further designed NPC-ABS/FDS to regulate CAFs more precisely at the cellular level while preventing excessive DOX uptake by CAFs that would lead to cell death. Our results demonstrate that this delivery strategy allows low-dose BAE to modulate CAFs toward a normal fibroblast-like phenotype without cytotoxicity and produces significant anti-TNBC efficacy. Future studies will focus on elucidating the detailed mechanisms of this regulatory process. Currently available CAF inhibitors include TGF-β inhibitors, PDGFR inhibitors, and others; however, systemic administration of small-molecule inhibitors often results in off-target effects and considerable toxicity. In contrast, NPC-ABS/FDS can precisely deliver BAE to CAFs. By altering CAFs’ function, it indirectly reduces both the physical barrier and chemical suppression exerted on T cells, thereby “releasing” endogenous antitumor immunity. This represents a more physiological form of immune activation. Therefore, regarding inhibitory efficiency and immunoregulatory capacity, our selectively CAF-modulating NPC-ABS/FDS provides a superior therapeutic strategy.
NPC-ABS/FDS was inspired by the structural design of capsules, in which the outer shell protects the payload and enables controlled release, while different ratios of the inner components exert their intended therapeutic functions. However, some limitations were also observed. The targeted delivery precision of BAE did not reach the ideal level of several-fold enrichment compared with the solution group. This may be attributed to the NPC shell restricting BAE dissolution, as evidenced by the cellular uptake and in vitro release assays. BAE contains three adjacent phenolic hydroxyl groups at positions 5, 6, and 7, which are capable of forming multiple hydrogen bonds with polar components of lipid matrices. Such non-covalent interactions, including hydrogen bonding and hydrophobic associations, have been implicated in the stabilization of flavonoids within nanocarrier systems and contribute to high encapsulation efficiency and uniform particle morphology. This might explain why the release rate of BAE over a 24 h period was around 40%. Specifically, the polar head groups of soy lecithin and the cationic moieties of DOTAP can interact with BAE’s phenolic hydroxyls, leading to stronger drug–carrier affinity compared with compounds lacking such multi-point binding sites. These interactions likely retard the diffusion of BAE out of the lipid matrix, resulting in a comparatively slower release profile relative to DOX, which is less prone to extensive hydrogen bonding. Previous studies on baicalein–phospholipid complexes have demonstrated enhanced drug–carrier association and modulated release behavior, supporting the concept that molecular interactions play a key role in sustained release from lipidic formulations. In addition, similar flavonoid-loaded lipid systems have exhibited prolonged release relative to aqueous suspensions, consistent with strong payload–matrix interactions [52,53]. Nevertheless, such “complexity” in the nanocapsule is an intentional design to achieve programmed and controllable release. In future work, we will consider using alternative shell materials to address the limited release of BAE. Notably, the competitive displacement efficiencies mediated by the targeting ligands varied significantly. Specifically, the displacement efficiency of free AEAA was more pronounced than that of free NGR. This discrepancy may be attributed to the distinct receptor densities, binding kinetics, and internalization behaviors of CD13 and σ receptors. For instance, Riad et al. [54] demonstrated that the σ-2 receptor (σ2R/TMEM97) functions within a complex that modulates the internalization kinetics of ligands like low-density lipoprotein. This suggests a potentially more regulated and slower internalization process compared to the rapid, constitutive internalization observed for receptors like CD13. In endothelial cells with high endocytic activity, such as HUVECs, non-specific uptake may mask the differences in overall cellular internalization. For instance, in HUVECs, the uptake of NGR-modified SLNs was comparable to that of non-targeted SLNs, particularly after 4 h of incubation. Therefore, assessing ligand-mediated targeting via in vivo vascular deposition provides more informative insights than relying solely on absolute in vitro uptake intensity. This conclusion was further corroborated by our in vivo biodistribution results, which demonstrated significantly higher accumulation of NGR-modified nanosystems at tumor sites compared to non-targeted formulations. Additionally, although BAE inhibited the expression of key CAFs-related proteins, this effect did not show a clear dose-dependent trend, and the underlying reasons require further investigation. Previous studies have shown that BAE affects endothelial cells, fibroblasts, and other cells within the TME [1]. This study focused on short-term antitumor efficacy and safety evaluation, and animals were euthanized at a predefined 11th-day endpoint to enable consistent mechanistic and histological analyses. As a result, survival analysis was not performed, which represents a limitation of the current work. Future studies will extend the observation period and define survival based on humane endpoints to better assess long-term therapeutic benefit. Although this study demonstrated the superior efficacy of the combination, the precise combination index was not calculated. Future work will focus on quantifying the synergistic interaction to optimize the drug ratio. As CAF-modulating therapeutic strategies continue to emerge, it is of great importance to clarify whether BAE-based treatments achieve superior outcomes through regulating CAFs or through acting on other stromal populations. Overall, this study provides valuable reference information for clinicians and researchers dedicated to developing novel drug delivery strategies for TNBC therapy.

5. Conclusions

In summary, we have developed a TME-directed delivery system, NPC-ABS/FDS, to enhance BAE-mediated CAF regulation and DOX-based chemotherapy efficacy. The nanocapsule achieved hierarchical targeting through NGR-mediated tumor neovascularization recognition and ligand-guided selective delivery to CAFs and cancer cells. BAE suppressed CAFs activation by downregulating FAP and α-SMA, thereby alleviating stromal barriers and improving the penetration of cytotoxic T cells and nanodrugs. Concurrently, DOX was efficiently internalized by cancer cells via folate-receptor-mediated uptake, resulting in enhanced tumor killing. Compared with DOX solutions, NPC-ABS/FDS exhibited markedly stronger antitumor efficacy while reducing off-target effects and improving BAE bioavailability. Tumors with high CAFs, such as breast cancer, ovarian cancer, melanoma, and colorectal cancer, may have a potentially high therapeutic effect using CAF inactivation. These findings highlight the therapeutic relevance of CAFs modulation for enhancing drug infiltration in desmoplastic tumors and demonstrate the feasibility of ligand-based co-delivery systems capable of simultaneously regulating stromal components and eliminating tumor cells within the TME.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pharmaceutics18020184/s1: Table S1. Sample table of in vitro hemolysis test (Unit: mL); Table S2. Particle parameters of the targeted co-delivery system (mean ± SD, n = 3); Table S3. Particle parameters of C6, Nr, and cy5 loaded targeted co-delivery system; Figure S1. Mildly inhibitory effect of BAE on CAFs. (A) The expression of fibroblast activation protein in CAFs treated with different concentrations of BAE (Mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.01 compared with TGF-β1 group). (B) Cytotoxicity of BAE to NIH3T3 and (C) Cytotoxicity of BAE to CAFs (Mean ± SD, n = 3); Figure S2. 1H-NMR spectra of DSPE-PEG2000-AEAA; Figure S3. 1H-NMR spectra of NPC; Figure S4. FTIR spectra of NPC; Figure S5. Characterizations of NPC-ABS/FDS. The TEM images of (A) ABS, (B) FDS, and (C) NPC-ABS/ABS (scale bar = 500 nm). (D) Particle size and (E) Zeta potential changes in ABS, FDS, and NPC-ABS/FDS after incubation with saline at 37 ± 1 °C for 48 h (Mean ± SD, n = 3); Figure S6. Effect of mixed cell culture with different densities for 72 h; Figure S7. Effect of mixed cells (1.5 × 104 cells/well) cultured continuously for 1 week.

Author Contributions

Conceptualization, B.X. and X.S.; methodology, X.J.; software, X.J.; validation, Y.Z.; formal analysis, X.G. and Z.L. (Zhongyan Liu); investigation, X.G., X.L. and Z.L. (Zhongyan Liu); resources, Y.Z. and Z.L. (Zhidong Liu); data curation, X.L. and Z.L. (Zhongyan Liu); writing—original draft preparation, B.X.; writing—review and editing, X.S.; visualization, X.J.; supervision, Y.Z., X.G. and Z.L. (Zhidong Liu); project administration, X.L.; funding acquisition, Z.L. (Zhidong Liu). All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Administration of Traditional Chinese Medicine Young Qihuang Scholar Project.

Institutional Review Board Statement

All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals and were reviewed and approved by the Ethics Committee of Tianjin University of Chinese Medicine (Approval No. TCM-LAEC2023146, 11 January 2024).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
AEAAAminoethyl Anisidine Amide
CAFsCancer-Associated Fibroblasts
CMCSO-Carboxymethyl Chitosan
DLDrug Loading
DLSDynamic Light Scattering
DSPE1,2-Distearoyl-sn-Glycero-3-Phospho-Ethanolamine
DOTAP(2,3-Dioleoyloxy-Propyl)-Trimethylammonium-Chloride
ECMExtracellular Matrix
EEEncapsulation Efficiency
FAFolic Acid
FAPFibroblast Activation Protein
HPLCHigh-Performance Liquid Chromatography
NGRAsparagine–Glycine–Arginine
NPsNanoparticles
NSNormal Saline
PEG2000Polyethylene Glycol-2000
TAMsTumor-Associated Matrices
TEMTransmission Electron Microscopy
TGF-β1Transforming Growth Factor-β1
TMETumor Microenvironment
TNBCTriple-Negative Breast Cancer
TregsRegulatory T Cells
SLNSolid Lipid Nanoparticle
α-SMAα-Smooth Muscle Actin

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