M1 macrophage membrane-engineered PLGA nanoparticles reprogram M2 tumor-associated macrophages to enhance anti-tumor immunity in breast cancer.

Introduction
Cancer immunotherapy is a promising treatment that engages the immune system to recognize and destroy cancer cells, offering a more targeted and less toxic alternative to traditional treatments such as chemotherapy. One of its most transformative strategies is reprogramming the tumor microenvironment (TME), specifically converting a cold tumor to a hot one [1–3], characterized by increased immune cell infiltration and activation of adaptive immune responses. This approach is particularly critical for enhancing the success of cancer immunotherapy, specifically for triple-negative breast cancer (TNBC) [4].
Macrophages are abundant in the TME and often exist as tumor-associated macrophages (TAMs). TAMs mostly exhibit an M2 phenotype and contribute to tumor progression, immune evasion, and metastasis [5]. Exposure to secreted factors in TME, such as IL-4, IL-10, TGF-β1, and lactic acid, drives macrophages to polarize into the M2 phenotype [6, 7]. In contrast, M1 macrophages inhibit tumor growth by releasing pro-inflammatory factors (nitric oxide, IL-12, TNF-α), degrading stromal tissue, and recruiting cytotoxic T cells. Therefore, strategies that deplete or reprogram TAMs from the M2 to M1 phenotypes can be used in cancer immunotherapy [8, 9].
Nanoparticles (NPs) have gained considerable attention as a means of drug delivery for cancer treatment and TME reprogramming [10, 11]. PLGA (poly(lactic-co-glycolic acid)) NPs provide an efficient platform due to their biodegradability, biocompatibility, and FDA-approved features. They provide controlled release and minimal toxicity [12], reduced immunogenicity, and enhanced cellular uptake compared to free agents [13, 14]. While these NPs are often recognized and rapidly cleared by the immune system through opsonization [15], various strategies have been developed to improve their performance. To address this challenge, a recent practical approach suggested developing biomimetic NPs by coating synthetic cores with natural cell membranes for various applications [16, 17]. Cell membranes, a thin, semipermeable layer made of lipids, proteins, and carbohydrates, play critical roles in cell recognition, communication, and immune modulation [18]. These hybrid systems preserve the core physicochemical properties of the synthetic NPs while harnessing the complex functions of the natural membranes, such as extending circulation time, enabling tumor targeting, and improving therapeutic delivery [16, 19, 20]. Various cell membranes of stem cells [21], leukocytes [22], platelets [23], bacteria [24], or cancer [25] have been explored for NP coating, showing the ability to target tumors. Among these, macrophage membrane-coated NPs are particularly promising due to their intrinsic ability to interact with tumor cells via adhesion molecules and integrin [26, 27], reduce immune clearance, and extend circulation time [7, 8, 28].
Incorporating immunomodulatory agents into these systems enhances their therapeutic potential. Toll-like receptor (TLR) agonists, particularly TLR7/8 agonists such as R848, have shown significant promise in polarizing M2 TAMs into the M1 phenotype [5]. R848 stimulates the production of nitric oxide and pro-inflammatory cytokines and restores anti-tumor immunity [29]. Moreover, cyclic dinucleotide (CDN) agonists, which activate the stimulator of interferon genes (STING) pathway, enhance the innate immune response by promoting type I interferon production, activating dendritic cells, and recruiting cytotoxic T cells to the tumor site [30]. STING activation is also critical for overcoming the immunosuppressive nature of cold tumors and fostering a pro-inflammatory microenvironment [31].
Based on the role of TAMs in breast cancer development and the limitations of current reprogramming strategies, we hypothesized that PLGA NPs encapsulating the TLR7/8 agonist R848, coated with M1 macrophage cell membrane with a CDN agonist, which is termed PLGA-CM1-CDN-R848 NPs, would provide a targeted delivery system to reprogram M2 TAMs toward an anti-tumor M1 phenotype. PLGA-CM1-CDN-R848 NPs influenced reprogramming of the M2 TAMs and simultaneously activated immune pathways (TLR7/8 and STING), resulting in enhanced anti-tumor activity against 4T1 breast cancer cells.

Materials and methods

Preparation of PLGA NPs
PLGA NPs encapsulating the TLR7/8 agonist (R848) were prepared using an emulsion solvent evaporation method as previously described [32]. Briefly, PLGA NPs and R848 were dissolved in dichloromethane. For the uptake study, coumarin-6 dye was also added at this stage [33]. This organic solution was then emulsified in an aqueous polyvinyl alcohol solution via sonication. The resulting emulsion was stirred to evaporate the solvent and facilitate NPs formation. Finally, the NPs were collected by centrifugation (20,00 × g, 20 min).

Differentiation of RAW264.7 cells into M1 and M2 cell subtypes
RAW264.7 monocyte/macrophage-like cell line (C639, Pasteur Institute of Iran, Iran; ATCC TIB-71) was used for polarization into M1 and M2 phenotypes [34]. Cells were cultured in RPMI-1640 medium (Biowest, France) containing 10% heat-inactivated FCS (56 °C for 30 min), 2 mM L-glutamine, and 1% antibiotics (penicillin, streptomycin) at 37 °C and 5% CO2. M1 and M2 macrophages were polarized as described in [34], with modifications. In brief, M1 macrophages were generated by treating cells with 100 ng/ml LPS and 20 ng/ml IFN-γ for 24 h. Conversely, M2 macrophages were induced by 20 ng/ml of IL-4 for 48 h.
Then, M1 and M2 macrophage subtypes were confirmed by determining the relative expression of genes il6, tnfa, and inos for M1 and il10 and arginase (arg) for M2 via qPCR. All data were normalized to the reference gene hprt (Table 1) [35]. For this purpose, first, total RNA was extracted from undifferentiated (M0) and differentiated cells (M1, M2) using the Total RNA Extraction Kit (Pars Tous, Iran). After determining the concentration and quality of the extracted RNA, cDNA was synthesized using the Easy cDNA Synthesis Kit (Pars Tous, Iran). Then, the expression of different genes was evaluated by specific primers according to the MIQE guidelines [36]. RT-qPCR was performed by one μl of sample DNA, 5 μL of a RealQ Plus Master Mix SYBR Green (Amplicon, Denmark), 10 pM of primers, and 2 μL of nuclease-free water by a Rotor-Gene 6000 thermocycler (Corbett Research, Australia). The amplification conditions were 95°C for 15 min, then 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 15 s with 40 times repetitions. The gene expression level was determined by the 2^–ΔΔCt method [37].

Isolation of the M1 macrophage membrane
The membrane of M1 macrophage cells was isolated according to Cao et al., with some modifications [20]. Approximately 1 × 108 of M1 cells were cultured as described in Section 2.2, harvested using 0.25% trypsin–EDTA, and washed three times with 0.15 M PBS (pH 7.4) by centrifugation at 500 × g for 10 min each. Subsequently, the resulting cell pellet was washed twice with Tris–magnesium buffer (10 mM Tris-HCl, 1 mM MgCl₂, 1 mM PMSF, pH 7.4), resuspended in 10 ml of the same buffer, and sonicated for 30 seconds. The homogenized cells were mixed with 1 M sucrose to a final concentration of 0.25 M and centrifuged (2000 × g, 10 min, 4 °C). The supernatant from this step was then centrifuged at 12800 × g for 35 min at 4 °C. The resulting supernatant was ultimately subjected to ultracentrifugation at 50,000 × g for 60 min at 4 °C to pellet the M1 macrophage cell membrane. The isolated M1 macrophage membrane was resuspended in PBS and stored at 4 °C. Its protein content was then measured by a bicinchoninic acid (BCA) assay kit (Pars Tous, Iran, A101252) according to the manufacturer’s guidelines.

Coating of PLGA NPs with M1 macrophage membrane (CM1) and CDN
First, PLGA R848-encapsulated NPs (1 mg/ml) were co-incubated with 1 mg/ml of isolated plasma membranes from M1-polarized macrophages [38] with or without 3 µg/ml of the STING agonist 2′3′-cGAMP (cyclic [G(2’,5’)pA(3’,5’)p]; InvivoGen, San Diego, CA, USA) [39]. This mixture was then subjected to sonication (42 kHz, 100 W) for 2 min to form the final product (PLGA-CM1-CDN-R848 NPs). To ensure the coating of the NPs with these components, the NPs were placed on the rocker for one night at 4 °C [40]. Finally, the NPs were centrifuged at 20,000 × g for 20 min at 4 °C to remove the uncoated cell membranes and CDN, washed, and finally dissolved in deionized water, freeze-dried, and stored at 4 °C until use.

Characterization of NPs
The morphology of NPs was visualized by transmission electron microscopy (TEM) by depositing a dilute sample onto a carbon-coated copper grid [41].
Physical properties of NPs (size, polydispersity index (PDI), and zeta potential (ζ)) were determined by dynamic light scattering (DLS) using a Zetasizer (Nano ZS, Malvern, Worcestershire, UK) [42].
The encapsulation efficiency (EE%) and loading efficiency (LE%) were determined by an indirect method [43]. Briefly, following synthesis, the NPs were centrifuged (20,000 × g, 4 °C, 15 min). The concentrations of R848 and CDN were quantified in the supernatant using triplicate assays by spectrophotometry at 327 nm for R848 [44] and at 260 nm for CDN [30]. Standard curves for quantification were generated using serial dilutions of R848 and CDN (0.1–5 μg/ml) [45].
The release and stability assays were done according to our previous study [43]. Briefly, the release of R848 and CDN was determined by incubation of a certain amount of dissolved PLGA NPs in PBS on a shaker (150rpm) at 37 °C. At various time points up to 72 h, 1 ml of the solution was centrifuged (1300 × g, 10 min at 37 °C). Then, the amounts of R848 and CDN in the supernatant were calculated as mentioned above. PLGA NPs were used as a control.
For determining the stability of NPs, PLGA NPs, PLGA-CM1-R848 NPs, and PLGA-CM1-CDN-R848 NPs were dissolved in PBS and incubated at 4 °C. Particle size, PDI, and zeta potential were evaluated weekly by DLS over a period of three weeks.

Evaluation of the cytotoxicity effect of synthesized NPs
The cytotoxicity of different NPs was assessed in RAW264.7 monocyte/macrophage-like cells. RAW264.7 cells were incubated with NPs concentrations ranging from 0.0078 to 1 mg/mL for 24 h. The untreated cells (100% viable cells, control) and treated cells with empty PLGA NPs were also used. The cytotoxicity was evaluated as previously described [43].

Assessment of NPs uptake efficiency
The fluorescent coumarin-containing-NPs were incubated with M2 macrophages, and the cellular uptake of them was evaluated after 8 and 24 h using a Partec PAS III flow cytometry device (Partec GmbH, Germany) in the FL1 channel and then analyzed with FlowJo software 10.9 (Tree Star, Ashland, OR, USA). In addition, changes in the mean fluorescence intensity (MFI) following their uptake by M2 macrophages were examined using fluorescence microscopy (Ceti Inverso TC-100, Chalgrove, Oxfordshire) at these times, too. The images were analyzed using ImageJ 1.54j (National Institutes of Health) software.

Determining the polarization ability of M2 macrophages after incubation with NPs
For further evaluating the effect of NPs on polarization, M2 macrophages were treated with NPs for 24 h. Untreated M2 and M1 macrophages were considered controls. To investigate the polarization of M2 macrophages, the gene expression of il6, tnfa, inos, il10, arg and hprt was examined by qPCR. Additionally, the expression of the ifnb gene was evaluated to investigate the activation of the STING pathway.

Investigation of apoptosis and necrosis in 4T1 breast cancer cells
To understand the apoptosis and necrosis in 4T1 cells after polarization of macrophages, first, M2 macrophages were incubated with PLGA NPs and PLGA-CM1-CDN-R848 NPs for 24 h. Then, macrophages were co-cultured with 4T1 breast cancer cells (C604, Pasteur Institute of Iran, Iran) in complete medium at 37 °C and 5% CO2 for 24 h. Untreated M2 macrophage served as the control. After incubation, the cells were detached using 0.25% Trypsin and washed with PBS [46]. To analyze cell death specifically in cancer cells, the cells were stained with an anti-CD11b-PE antibody (BioLegend, 101207). Apoptosis and necrosis were assessed within the gated CD11b-negative population (4T1 cells) using a FITC Annexin V Apoptosis Detection Kit with propidium iodide (PI) (Padza Padtan Pajooh, Iran, PDZK-01). Finally, the percentages of cells in early apoptosis (AnnexinV + ), late apoptosis (AnnexinV+ PI + ), and necrosis (PI + ) were measured and analyzed using a Partec PAS III flow cytometer and FlowJo software 10.9, respectively.

Cell cycle distribution in 4T1 breast cancer cells
After treatment of M2 macrophages with PLGA NPs or PLGA-CM1-CDN-R848 NPs, they were incubated with 4T1 cancer cells for 24 h, and the cell cycle distribution was analyzed using a previously described method with minor modifications [47]. The cells were collected and washed. After cell fixation, 20 μg/ml of RNase A (Bio Basic, RB0473) and 50 μg/ml of PI (Sigma-Aldrich, 25535-16-4) were added to cells. 4T1 cells were separated and evaluated as described in a previous section.

Statistical analysis
Data analysis was done by one- or two-way analysis of variance (ANOVA) followed by Dunnett’s or Sidak’s multiple comparisons test using GraphPad Prism 10.3.0.507 software (GraphPad Software Inc. 2024, San Diego, California, USA). All results were shown as the mean + standard deviation (SD) of three replicates (n = 3). The statistically significant and non-significant (ns) data were shown as follows: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns (p > 0.05).

Results

Macrophage polarization
RAW264.7 macrophage cell (M0) polarization into M1 and M2 phenotypes was evaluated based on gene expression patterns using qPCR. M0 macrophage was considered an unpolarized cell (control). The M1 phenotype was characterized by significantly elevated levels of M1 macrophage-related genes (il6, tnfa, inos), while the M2 phenotype exhibited increased expression of genes related to M2 macrophages (il10, arg) compared to M0 (****p < 0.0001) (Fig. 1). These results showed the effective M1 and M2 macrophage polarization, so they can be used for nanoparticle coating, evaluation of treatments, and as controls in the next steps.

Characterization of synthesized PLGA NPs
The physicochemical properties of various PLGA NPs formulations were determined by the DLS method. As shown in Table 2, PLGA NPs showed an average size of 135 nm, which increased by 32 nm after R848 loading and M1 macrophage membrane coating (CM1) and 43 nm after R848 loading and CM1 and CDN coating. Furthermore, the zeta potential changed from –2.6 mV to –9.56 mV and –10.3 mV in PLGA-CM1-R848 and PLGA-CM1-R848-CDN NPs, respectively, indicating a more negative outer membrane surface. The encapsulation efficiency of R848 and CDN was higher than 90%. The loading efficiency and coating efficiency for both nanoparticles were between 1.22% and 2.3%. Entirely, the increase in size and negative charge confirmed the coating of the M1 macrophage membrane on PLGA-CM1-R848 and PLGA-CM1-CDN-R848 NPs.
The TEM microscopy images of PLGA-CM1-R848 NPs and PLGA-CM1-CDN-R848 NPs showed a spherical structure with a hollow core and a uniform outer membrane layer surrounding the nanoparticle core (Fig. 2B, C) compared to PLGA NPs that lack an outer membrane but have a spherical structure with no pores (Fig. 2A). The presence of a uniform membrane layer surrounding the nanoparticle core indicated the successful coating of both NPs.
In general, the results of TEM microscopy and DLS showed the presence of a uniform M1 macrophage membrane layer coating on PLGA-CM1-R848 NPs and PLGA-CM1-CDN-R848 NPs.
In vitro release of R848 and CDN from PLGA NPs was determined during 72 h (Fig. 3). R848 showed a slow release from the synthesized NPs over time. The release rate seems to reach 90% around 72 h. CDN exhibits a similar release pattern, but the initial release rate appears to be slightly slower than R848.
DLS confirmed the stability of NPs over three weeks, with no significant changes in size distribution (Fig. 4), PDI (~0.1), or zeta potential (~ –10 mV).

The cytotoxicity of NPs
The cytotoxicity of PLGA NPs, PLGA-CM1-R848 NPs, and PLGA-CM1-CDN-R848 NPs on the RAW264.7 cell line after 24 h of incubation with different concentrations of NPs (0.0078-1 mg/ml) was evaluated by the MTT method. The untreated RAW264.7 cell was also used as a control. As shown in Fig. 5, NPs had no significant effect on cell viability in all concentrations, which confirms the non-toxicity of these NPs.

Uptake of NPs by M2 macrophages
PLGA NPs, PLGA-CM1-R848 NPs, and PLGA-CM1-CDN-R848 NPs were incubated with M2 macrophages, and their relative uptake rate was evaluated after 8 and 24 h by flow cytometry (Fig. 6 and Supplementary Fig. S1). The relative uptake of PLGA-CM1-R848 NPs did not change significantly compared to PLGA NPs (control) at 8 and 24 h (p > 0.05), while the uptake of PLGA-CM1-CDN-R848 NPs was significantly higher compared to the control at both time points (p < 0.01 and p < 0.001, respectively). In addition, the uptake of PLGA-CM1-CDN-R848 NPs was significantly increased after 24 h vs. 8 h (p < 0.05). Altogether, the results showed the preferential uptake of PLGA-CM1-CDN-R848 NPs compared to the other NPs by M2 macrophages at both times.
In addition, the mean fluorescence intensity of NPs after uptake with M2 macrophages by fluorescent microscopy confirmed the selective uptake of PLGA-CM1-CDN-R848 NPs by M2 macrophages at 8 and 24 h (Fig. 7).

Polarization of M2 to M1 macrophages after treatment with NPs
For evaluating the re-polarization of M2 to M1 macrophages, the relative expression of genes (il6, tnfa, inos, il10, arg, ifnb, and hprt) was assessed after treatment with NPs using qPCR for 24 h (Fig. 8). PLGA-CM1-R848 NPs significantly induced the expression of IL-6 and TNF-α compared to untreated M2 macrophages and PLGA NPs controls, respectively. For IL-6, the significance was p < 0.0001 versus untreated and p < 0.001 versus PLGA NPs (Fig. 8A). For TNF-α, the significance was p < 0.05 versus untreated and p < 0.001 versus PLGA NPs (Fig. 8B). Besides, M2 macrophages treated with PLGA-CM1-CDN-R848 NPs showed significantly increased expression levels of IL-6 (p < 0.0001, Fig. 8A), TNF-α (p < 0.0001, Fig. 8B), as well as iNOS (p < 0.05, Fig. 8C), compared to untreated control M2 macrophages and PLGA NPs controls. Although there was no significant difference in the level of il10 and arg gene expression between NPs-treated groups, the level of these genes was decreased significantly in PLGA-CM1-R848 NPs and PLGA-CM1-CDN-R848 NPs compared to untreated M2 macrophages (p < 0.0001 and p < 0.05, respectively) (Fig. 8D, E). In addition, the level of IFN-β expression also increased significantly in the PLGA-CM1-CDN-R848 NPs compared to the other treated and untreated groups (p < 0.001) (Fig. 8F). Interestingly, PLGA-CM1-CDN-R848 NPs showed significantly increased expression levels of IL-6 (p < 0.0001), TNF-α (p < 0.01), iNOS (p < 0.0001), and IFN-β (p < 0.0001) compared to PLGA-CM1-R848 NPs.
Collectively, the PLGA-CM1-CDN-R848 NPs treated M2 macrophages showed a significantly elevated expression of key M1 markers or pro-inflammatory cytokines (IL-6, TNF-α, iNOS) and IFN-β compared to the other treated groups, indicating dual activation of the TLR7/8 and STING pathways.

Apoptosis and necrosis of the 4T1 breast cancer cells
For more evaluation of the co-delivery effect of M1 macrophage membrane with agonists on cancer cells, PLGA-CM1-CDN-R848 NPs were used for subsequent tests. Apoptosis and necrosis of 4T1 breast cancer cells were assessed after incubation with polarized macrophages following 24 h using flow cytometry (Fig. 9 and Supplementary Fig. 2S). Untreated M2 macrophages and treated M2 macrophages with PLGA NPs were used as controls. Both early (AnnexinV + ) and late apoptosis (AnnexinV+ PI + ) were significantly higher in the PLGA-CM1-CDN-R848 NPs group compared to untreated M2 macrophages (p < 0.001 and p < 0.01, respectively) (Fig. 9). In addition, there was no significant difference in necrosis (PI + ) between these two groups (p > 0.05). Early apoptosis as well as necrosis were increased in the PLGA-CM1-CDN-R848 NPs treated group compared to the PLGA NPs (p < 0.01).

Cell cycle arrest of 4T1 breast cancer cells
The distribution of cell cycle phases in 4T1 cells was evaluated after incubation with polarized macrophage cells with PLGA-CM1-CDN-R848 NPs for 24 h by flow cytometry. All groups showed the cell cycle arrest considerably in the G0/G1 phase (Fig. 10). Furthermore, the cell cycle distribution of the PLGA-CM1-CDN-R848 NPs group showed a significant arrest in the G0/G1 and G2/M phases compared to the untreated M2 macrophages and PLGA NPs (p < 0.0001).

Discussion
Recently, researchers have focused on macrophages as targets for cancer immunotherapy [48, 49]. The ability to convert M2 macrophages into M1 macrophages using agonists has been extensively researched in immunotherapy [43]. M1 macrophages can hinder tumor growth by producing pro-inflammatory factors such as NO, IL-12, and TNF-α. Thus, strategies in cancer immunotherapy include removing M2 macrophages, converting them into M1 macrophages, or enhancing the tumor-suppressing capabilities of M1 cells in the tumor microenvironment [8, 9].
The significance of PLGA NPs as an efficient drug delivery system has been widely studied due to their unique properties [13, 14]. However, one of the major challenges in immunotherapy is the rapid clearance of NPs by the immune system, which limits their therapeutic efficacy [28, 50]. Protein and cellular component adsorption can alter the function of NPs, leading to their recognition and clearance by the immune system [51]. To address this, cell membrane coating, particularly with macrophage-derived membranes, has emerged as an effective strategy to evade immune detection and enhance tumor targeting [50, 52, 53]. Macrophage- and monocyte-derived membranes have been used to coat synthetic NPs such as PLGA, MSN, and liposomes (28,29). For instance, Cao et al. demonstrated that liposomes coated with RAW264.7 macrophage membranes reduced metastatic lung nodules in breast cancer [20]. Similarly, RAW264.7 macrophage membrane and silica nanoparticle combinations enhanced drug-loading capacity and tumor-specific targeting [54]. In another study, M1 RAW264.7 macrophage-coated PLGA NPs encapsulating doxorubicin and catalase were designed to enhance chemotherapy in hypoxic TME. The NPs exhibited increased uptake in 4T1 tumor cells and a significant reduction in tumor volume, showcasing the effectiveness of M1 macrophage membranes in tumor targeting [55]. More recently, PLGA NPs coated with macrophage membranes and loaded with the TMP195 successfully repolarized TAM toward the M1 phenotype and enhanced PD-1 blockade therapy in a breast cancer model [41].
In line with these findings, our study confirmed successful M1 macrophage membrane integration onto PLGA NP cores, as evidenced by increased size and zeta potential and high biocompatibility under physiological conditions. In addition, TEM microscopy revealed a hollow, spherical structure with a distinct membrane layer in coated NPs, which was absent in uncoated PLGA NPs.
TLR7/8 agonist (R848) selection in our study was based on its documented potency in reprogramming the tumor microenvironment (TME) and polarizing macrophages toward an M1 phenotype [35]. Rodell et al. demonstrated that R848, when loaded into β-cyclodextrin NPs, efficiently targeted TAMs in vivo and promoted their polarization toward the M1 phenotype [56]. Moreover, PLGA NPs loaded with TLR7/8 agonists stimulated nitric oxide (NO) production in macrophages [43]. Wei et al. found that PLGA loaded with R848, absorbed onto Escherichia coli MG1655, and combined with doxorubicin (Dox), improved the efficacy of cancer immunotherapy [57]. Recently, Turco et al. demonstrated that R848, encapsulated in cyclodextrin NPs, induced NO and pro-inflammatory cytokine production, including TNF-α and IL-12 in macrophages within the glioma TME. These NPs reprogrammed immunosuppressive TAM into M1 macrophages, reshaped the TME, and enhanced anti-tumor immune responses independently of adaptive immunity, which led to glioma regression and improved survival outcomes [58]. Furthermore, PLGA NPs encapsulating TLR7/8 agonists reduced metastasis and enhanced immunotherapy effectiveness [59]. It had been shown that PLGA NPs loaded with R837 (a TLR7 agonist) and coated with B16-OVA cancer cell membranes delayed tumor growth [60]. However, simultaneous stimulation of TLR7 and TLR8 by imidazoquinoline agonists has been shown to induce greater cytokine production than activating TLR7 alone [61].
Furthermore, incorporation of a CDN agonist to stimulate the STING signaling pathway was essential for the reprogramming of M2-like TAM macrophages to the M1 phenotype in the TME. One study indicated that liposomal NPs containing a CDN agonist (cGAMP) enhanced antitumor immunity in PD-L1-insensitive TNBC models [62]. Unlike traditional checkpoint inhibitors, which are often ineffective in TNBC, cGAMP-NP activated the intracellular STING pathway, inducing type I interferons and reprogramming TAMs from an M2-like to an M1-like phenotype. This reprogramming promoted pro-inflammatory cytokine production, increased infiltration of cytotoxic CD4+ and CD8+ T cells, and induced apoptosis within the TME [63]. Moreover, studies have indicated that the increased uptake of extracellular vesicles (EVs) loaded with these agonists by M2 macrophages, compared to M1 macrophages, enhances the potency of CDN, preferentially activates antigen-presenting cells within the TME, promotes local Th1 responses, facilitates the recruitment of CD8 + T cells, and generates systemic anti-tumor immunity [39]. Furthermore, STING agonists can induce cytokine (such as IFN-I) and chemokine production, ultimately stimulating innate immune responses in tumor immunotherapy [64, 65].
Combinatorial immunotherapy offers a promising approach to overcoming the therapeutic challenges, specifically in breast cancer. Recent advancements have highlighted the potential of dual nanoparticle systems, such as a macrophage membrane-coated polymer nanoparticle that combines BRD4 and COX-2 inhibitors to reprogram the immunosuppressive TME and activate immune responses in TNBC models [66]. The synergistic effects of TLR and STING agonists in the immune response against tumors further highlight the promise of this approach [67]. Co-administration of STING and TLR7/8 agonists has been demonstrated in cancer vaccines, where co-delivery enhanced antigen-specific CD8+ T cell and NK cell responses, inhibited tumor growth, induced apoptosis, and improved survival in melanoma and bladder cancer models [68].
In the present study, for better understanding of the combination effect of M1 macrophage membrane (CM1) and agonists (R848 and/or CDN) on M2 macrophage polarization in the tumor microenvironment, PLGA-CM1-R848 NPs and PLGA-CM1-CDN-R848 NPs were synthesized. PLGA NPs showed sustained release of both R848 and CDN agonists over three days, with approximately 25% released within 1 h and 90% by 72 h. These release kinetics are consistent with other studies showing that sustained release effectively enhances MHC-I and MHC-II expression and APC activation, which is critical for initiating adaptive immune responses [69–71].
Functionally, we demonstrated that M2 macrophages preferentially uptake PLGA-CM1-CDN-R848 NPs at 8 and 24 h, with no impact on the cell viability of the macrophages, as shown in [12]. This result is consistent with a study, which confirmed the preferential uptake of agonist-loaded vesicles by M2 macrophages over M1 macrophages [39].
In line with the studies mentioned above, our study indicated that general M1 polarization markers (IL-6, TNF-α, iNOS), were upregulated in PLGA-CM1-CDN-R848 NPs treated M2 macrophages, and IFN-β expression specifically increased only in the PLGA-CM1-CDN-R848 NPs treated group, confirming targeted activation of the STING pathway through CDN delivery. Actually, the use of CDN in addition to R848 and CM1 in PLGA-CM1-CDN-R848 NPs was more effective in pro-inflammatory cytokine expression than R848 and CM1 in PLGA-CM1-R848 NPs.
Upon co-culture of these reprogrammed macrophages with 4T1 breast cancer cells, we observed significantly increased early and late apoptosis, as well as G0/G1 and G2/M phase cell cycle arrest in 4T1 cancer cells. TLR7/8 agonists (e.g., Imiquimod and R848) have been shown to induce apoptosis in breast and skin cancer cells and inhibit tumor cell growth [72–74]. Similarly, PEG-PLGA NPs co-delivering CD47 siRNA and R848 enhanced T cell activity, reduced immunosuppressive cell populations, and induced apoptosis in 4T1 tumor cells, confirmed by TUNEL staining [75]. Moreover, STING agonists (3′3′-cGAMP) have demonstrated antitumor activity via apoptosis induction and tumor regression [76]. In addition, the activation of the STING signaling pathway led to the simultaneous production of IFN-β and TNF-α, as well as the induction of necrosis in various cell types [77]. Another study investigated liposomal NPs loaded with cGAMP as a STING pathway activator to induce tumor apoptosis, as evidenced by elevated caspase-3 expression in treated TNBC breast tumor tissues [62]. In our study, while the necrosis levels in 4T1 cancer cells incubated with polarized macrophages did not differ significantly from the control, there was a significant difference compared to the PLGA NPs, suggesting that apoptosis, rather than necrosis, contributed to tumor regression, thereby minimizing inflammatory side effects [78].
The results of this study highlight that PLGA-CM1-CDN-R848 NPs have greater potential than PLGA-CM1-R848 NPs as an innovative platform for cancer immunotherapy. By leveraging the immune-targeting properties of M1 macrophage membranes, the immunostimulatory effects of TLR7/8 and STING agonists, and the controlled release kinetics to reprogram the M2 TME, restore anti-tumor immunity, improve therapeutic outcomes, and overcome the challenges of “cold” tumors. Despite the evidence of macrophage-mediated immune modulation in vitro, these results are primarily aimed at providing proof-of-concept rather than clinical applicability; further in vivo investigations are needed to assess therapeutic efficacy, biodistribution, and potential side effects in tumor models. Integration of this system with existing immunotherapies, such as immune checkpoint inhibitors, could provide synergistic effects and improve outcomes in breast cancer and other malignancies.

Conclusions
This study presents an innovative in vitro platform that addresses the challenges of targeting TAMs and cancer cells by combining nanotechnology with immunomodulatory agents. We developed a nanoparticle-based system consisting of PLGA NPs loaded with R848 and coated with M1 macrophage membranes and CDN agonists. PLGA-CM1-CDN-R848 NPs demonstrated the ability to selectively target and reprogram M2 TAMs into a pro-inflammatory M1 phenotype and enhance immune activity against breast cancer cells. Our findings provide a scientific proof-of-concept for a novel strategy to achieve TAM reprogramming, inducing apoptosis, and triggering cell cycle arrest in 4T1 breast cancer cells.
While this work was conducted in vitro and its clinical translation remains to be established, the findings offer significant scientific value by elucidating a potent mechanistic platform. The study opens promising avenues for developing more targeted and efficient cancer immunotherapy, with the potential to reduce off-target effects, a major limitation of traditional therapies.

Supplementary information