Genetically engineered macrophage membrane-coated nanoparticles for enhanced tumor targeting and synergistic cancer therapy

doi:10.21203/rs.3.rs-2074299/v1

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Genetically engineered macrophage membrane-coated nanoparticles for enhanced tumor targeting and synergistic cancer therapy

https://doi.org/10.21203/rs.3.rs-2074299/v1

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The poor targeting capability of systemically administered drugs is a major hurdle in designing effective therapies with minimal off-target side effects. Here, as a proof of concept, we propose a genetic engineering strategy to embed a prostate-specific membrane antigen (PSMA)-targeting antibody fragment (gy-1) in the macrophage membrane, which is then coated onto core-shell Fe₃O₄@Au nanoparticles to produce a biomimetic nanoplatform, Magic. The results indicate that Magic exhibits not only high specificity and affinity towards prostate tumor cells in vitro and in vivo, but also effective immunomodulatory capability. Upon further encapsulation with the anti-tumor drug DM1, Magic shows superior synergistic effects in highly targeted chemo-photothermal therapy and potent immunotherapy, eliciting significant therapeutic efficacy against tumor growth, micro-metastasis and concomitant damage without overt toxicity. These findings reveal that Magic may provide a promising platform for enhancing cancer treatment by overcoming undesired drug delivery barriers and the tumor immune microenvironment.

The past few decades have witnessed a boom in the field of cancer nanomedicine, which has emerged as a promising candidate to overcome the limitations of conventional therapeutic drugs, such as poor pharmacokinetics and unwanted non-target accumulation; thereby, providing opportunities to improve the balance between therapeutic efficacy and adverse effects.^1–4 To achieve satisfactory therapeutic outcomes, different strategies have been explored to ensure that nanomedicines can be specifically transported to the tumor tissue at an effective concentration.^5,6 Passive targeting via enhanced permeability and retention (EPR) effect or modification of nanomaterial surfaces with active targeting ligands, such as aptamers and peptides, has been widely exploited to facilitate the preferential tumor accumulation of nanomaterials.^1,7,8 Despite extensive research and progress, the targeted delivery of nanomedicine to its site of action still faces significant challenges. Serious hurdles, including low biocompatibility, rapid clearance by the mononuclear phagocytes system (MPS), and high off-target effects, prevent nanomedicines from reaching the tumor site.^9,10 An analysis revealed that only 0.7% of the administered nanoparticles are delivered to the solid tumor owing to the complexity and heterogeneity of the tumor microenvironment.¹¹ As a result, the therapeutic benefits of most cancer nanomedicines remain far below expectations, particularly in clinical practice. Moreover, solid tumors establish an immunosuppressive environment suitable for tumor progression and metastasis, which greatly compromises the anti-cancer effects of nanomedicines.^12,13 Indeed, the development of nanomedicines capable of specifically targeting tumors and remodeling the immunosuppressive tumor microenvironment would be preferable in the fight against cancer.

Unlike modifications with synthetic materials, cell membrane-camouflaged nanomaterials have garnered much attention owing to their unique properties inherited from source cells, such as high biocompatibility, extended blood circulation, immune evasion, and homotypic binding capabilities.^14,15 Using natural cell membranes derived from erythrocytes,^16,17 immune cells,^18–21 platelets,^22,23 cancer cells,^24–26 and stem cells,^27,28 diverse biomimetic nanoformulations have been fabricated for customized biomedical applications. The targeting ability of these biomimetic nanoparticles is often mediated by proteins that are expressed on the source cells, which endows nanoparticles with the capability to specifically interact with various disease substrates. Macrophages are among the most abundant immune cells in the tumor microenvironment and play essential roles in cancer progression, metastasis, and therapy failure.²⁹ In addition to the common features shared by other cells used for nanomaterial coating, macrophages possess inherent tumor-homing ability and dual functions in tumor immunomodulation by either promoting multiple aspects of tumorigenesis or less frequently by enhancing anti-tumor responses.^30–33 However, most macrophage membrane-based nanomedicines only utilize the natural homing property of macrophages to migrate towards tumor sites, leading to insufficient targeting ability and migration efficiency. There have been a few attempts to introduce exogenous tumor-targeting moieties (e.g., small molecules, aptamers, peptides, and antibodies) on macrophage surfaces via lipidized ligand insertion or chemical coupling.³⁴ However, a host of uncontrollable factors, including a tedious modification process, unstable coupling linkers, prevailing membrane protein denaturation, and unpredictable binding sites, and unreasonable packing density of ligands, often negatively impact the targeting efficiency and pose additional challenges towards the translation of nanomedicines into clinical practice.³⁵

Instead of relying on post-fabrication approaches to incorporate additional functionality, we herein propose a novel genetic engineering strategy to stably transmembrane (TM)-express a tumor-specific ligand on macrophages to increase the active targeting efficiency and therapeutic efficacy of macrophage membrane-based nanomedicines (Scheme 1). Prostate cancer (PCa) was used as a proof of concept to demonstrate the effectiveness of our strategy. Prostate-specific membrane antigen (PSMA) is a well-established target for PCa diagnosis and treatment.³⁶ We previously reported that a PSMA extracellular domain-specific single-chain antibody fragment (scFv) (termed gy-1) obtained from a large yeast-display naive human scFv library can specifically bind to PSMA with a high binding affinity and be efficiently internalized into PCa^PSMA+ cells.^37–39 Encouraged by the findings with gy-1, we envisioned that the incorporation of gy-1 into macrophages would facilitate their migration to prostate tumors with enhanced targeting ability. Thus, the prognostic significance of PSMA in PCa was evaluated using bioinformatics first. Then, we successfully TM-expressed gy-1 on macrophages (termed M^gy−1) by lentiviral infection and harvested the engineered membrane to encapsulate multifunctional core-shell Fe₃O₄@Au nanoparticles (FeAuNPs). By combining the advantages of the Fe₃O₄ core and Au shell, FeAuNPs can be used for drug carrier and photothermal therapy (PTT), and also enable both magnetic resonance imaging (MRI) and X-ray computed tomography (CT) to evaluate the tumor-targeting performance from multiple perspectives. The resulting M^gy−1 membrane-coated FeAuNPs were termed Magic. Benefiting from the M^gy−1 membrane coating, Magic exhibited not only prolonged circulation time, but also remarkable PSMA recognition-mediated tumor targeting and tumor cell-specific internalization. In addition, Magic inherited cytokine neutralization-mediated immunomodulation from source cells and was able to reprogram macrophages from the pro-tumor M2 phenotype to an anti-tumor M1 phenotype. After loading with a microtubule-targeted cytotoxic agent, DM1,^40,41 highly targeted chemotherapy, PTT, and immunomodulation of Magic acted synergistically to exert amplified therapeutic efficacy against tumor growth, micro-metastasis and concomitant damage. Thus, Magic could be a promising cancer-targeting platform with a bright future for enhanced cancer treatment.

Bioinformatics analysis

Single-cell sequencing data and PSMA-IHC staining of PCa tissues were retrieved from HPA database. Macrophage related tumor-promoting cascades were analyzed via TCGA dataset (https://portal.gdc.cancer.gov). Briefly, tumor-infiltrating immune cells were quantified by CIBERSORT method and LM22 signature matrix. The algorithm ran at 100 permutations with a threshold of P < 0.05 to select eligible patients. Pearson correlation coefficient test was used for correlation analysis. The impacts of macrophage on clinicopathological features were analyzed with Wilcox test. Prognostic value of macrophage-related cytokines was retrieved from Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn).

Patient specimens

Tumor tissues were obtained from patients with PCa who underwent radical prostatectomy at Xijing Hospital. The resected tissues were fixed with 4% paraformaldehyde for morphological staining. The present study was approved by the Medical Ethics Committee of the First Affiliated Hospital of the Fourth Military Medical University (approval number: KY20203274-1) and all of the participating patients gave their informed written consent.

Section staining

4-µm-thick tissue sections were applied to antigen retrieval in citrate solution. Being blocked with goat serum for 1 h at room temperature, slices were incubated with primary antibodies: anti-CD68 (Abcam, ab53444; ab955, UK), anti-iNOS (Abcam, ab178945, UK), anti-CD206 (Abcam, ab64693, UK), anti-ECAD (Proteintech, 20874-1-AP, USA), anti-Vimentin (Proteintech, 10366-1-AP, USA), anti-VEGFR2 (Proteintech, 26415-1-AP, USA), and anti-CD105 (Proteintech, 10862-1-AP, USA) in a humidified box overnight at 4°C. Slices were then incubated with Alexa Fluor 488 (Abcam, ab150157; ab150113, UK) and Alexa Fluor 594 (Abcam, ab150080, UK) conjugated secondary antibodies for 2 h at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Digital images were acquired using laser confocal microscope (Olympus, FV31s-sw, Japan). Quantitative analysis was performed using ImageJ software.

Paraffin-embedded PCa tissue microarray (Outdo Biotech, HProA150CS01, China) was deparaffinized, rehydrated, and antigen-recovered before staining. PSMA was stained with horseradish peroxidase (HRP)-conjugated mouse monoclonal antibody (MXB, MAB-0575, China) according to the manufacturer’s instructions. Digital images were acquired using a Nikon microscope (TE2000-S, Japan).

Cell culture and lentivirus infection

Murine macrophage Raw264.7 and human PCa cell lines (i.e., PC3^PSMA−, PC3^PSMA+ and LNCaP) were maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM), DMEM/F12 and RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, 10099-141, USA) and 1% penicillin/streptomycin (Invitrogen, 15140-122, USA). Cells were cultured at 37°C with 5% CO₂ in a humidified incubator. Lentivirus used for Raw264.7 infection was synthesized based on GV218 vector (GeneChem, China). Three days after lentivirus infection (MOI = 30), M^gy−1 with stable gy-1 TM-expression was screened with 3 µg mL^− 1 puromycin. Then, M^gy−1 was harvested, washed, and re-suspended in fluorescence-activated cell sorting (FACS) buffer at the density of 1×10⁶ cells mL^− 1 for flow cytometry analysis (Beckman Coulter, USA).

Cellular ELISA

To evaluate binding ability of M^gy−1 to PSMA, 2×10³ M^gy−1 was seeded in 96-well plate and incubated overnight. Then, recombinant PSMA protein with His tag (Sino Biological, 15877-H07H, China) was added and incubated for 1 h at 4°C. Subsequently, cells were washed with PBS and incubated with HRP-conjugated anti-6×His antibody (Abcam, ab1187, UK) for 1 h at 4°C. 3,3′,5,5′-tetramethylbenzidine (TMB, Beyotime, P0209, China) was used for visualization, and absorbance was measured using a microplate reader at 450 nm (Infinite 200, Tecan, Switzerland).

Membrane derivation

Plasma membrane of M^gy−1 was collected according to the previously published protocol.⁴² Briefly, 1×10⁷ M^gy−1 was washed with PBS three times and resuspended in 1 mL hypotonic lysing buffer for ultrasonic fragmentation. The homogenized solution was centrifuged at 3500 g for 5 min at 4°C, and resulting supernatant was centrifuged again at 20000 g for 30 min at 4°C. The precipitates were washed with DNase free/RNase free water and stored at -80°C for subsequent studies.

Preparation of Magic

Core-shell FeAuNPs was prepared by reducing gold chloride trihydrate (HAuCl₄·3H₂O, 16961-25-4, Aladdin, China) in the presence of pre-synthesized magnetite nanoparticles.⁴³ The resulting FeAuNPs were collected magnetically and washed three times with deionized water to remove excess reagents. Then, 200 µg FeAuNPs and 400 µg M^gy-1 membranes were dissolved in 1 mL DNase free/RNase free water. The mixture was subjected to ultrasonic treatment in an ice-bath with a FS30D ultrasonic instrument (Fisher Scientific, USA) at a frequency of 42 kHz and a power of 100 W for 5 min. The resulting suspensions were extruded serially through 400 nm and 200 nm membrane filter (Whatman, UK) using a mechanical extruder for 10 times as previously reported.⁴⁴

Characterization of Magic

TEM images were taken on a FEI TECNAI Spirit transmission electron microscope operating at an acceleration voltage of 120 kV. SEM images were taken on a Philips XL30 scanning electron microscope operating at an acceleration voltage of 3 kV. The hydrodynamic size and zeta potential of nanoparticles were measured by dynamic light scattering (DLS) using a Nano Zetasizer (Malvern, UK).

Western blotting

To confirm the retention of membrane proteins during Magic fabrication, samples were prepared for protein electrophoresis and Coomassie blue staining. For Western blotting analysis, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, ISEQ00010, USA). After blocking with 5% bovine serum albumin (BSA) solution for 1 h at room temperature, membrane was incubated with antibodies against F4/80 (CST, 70076, USA) and eGFP (Abcam, ab184601, UK). Subsequently, membrane was incubated with HRP-conjugated secondary antibodies and visualized using an enhanced chemiluminescence reagent (Millipore, WBKLS0500, USA).

Specific binding assay

PC3^PSMA−, PC3^PSMA+ and LNCaP were harvested, washed, and re-suspended in FACS buffer at the density of 1×10⁶ cells mL^–1. Then, cells were incubated with 200 µg mL^–1 MVs^gy-1 or Magic for 30 min at 4°C under darkness. After washing with FACS buffer, cells were analyzed by flow cytometry (Beckman Coulter, USA).

Immune evasive assay

Rho-PEG-SH (Ruixi Biotech, China) and FeAuNPs were mixed in stoichiometric ratio of 2:1. The reaction mixture was stirred for 2 h at room temperature for Au-S bonds-mediated Rho loading. Rho-NPs and Rho-Magic were incubated with Raw264.7 cells for 24 h in incubator. After washing with PBS, cells were fixed with 4% paraformaldehyde and visualized using laser confocal microscope (Olympus, FV31s-sw, Japan). Image Pro Plus software was employed for quantification.

Cytokine neutralization and macrophage polarization

200 µg mL^–1 Magic was pre-incubated with M2 macrophage-related cytokines (i.e., TGF-β, IL-4, and IL-10, Sino Biological, China) at 37°C for 2 h. Subsequently, cytokine solutions were centrifuged with a speed of 14000 g for 20 min at 4°C to separate Magic and neutralized cytokines. The supernatant was collected, and residual cytokines were quantified with ELISA kits (Proteintech, USA). To investigate cytokine neutralization-mediated polarization regulation, Magic pre-treated IL-4 solution (20 ng mL^–1) was used for Raw264.7 cell culture. Three days later, cells were stained with APC-conjugated CD206 polyclonal antibody (PA5-46879, Invitrogen, USA) for flow cytometry detection.

Animal model construction

Animal experiments reported in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University (approval number: 20201207). All applicable institutional guidelines for the care and use of animals were followed. Male BALB/c nude mice, purchased from the Laboratory Animal Center of the Fourth Military Medical University (Xi’an, China), were housed in a temperature-controlled room with free access to food and water. To construct subcutaneous tumor-bearing mice, 5×10⁶ luciferase-expressing PC3^PSMA+ cells were subcutaneously injected in right flank. When tumor volume reached 200 mm³, animals were used for follow-up experiments. For bone metastasis model, 1×10⁶ PC3^PSMA+ cells were injected into medullary cavity of right tibia via knee joint. Two weeks later, animals were subjected to further studies.

In vivo fluorescence imaging

After tail-vein-injection of 4 mg mL^–1 ICG-Magic at the dose of 3 µL g^–1, its distribution in vivo was monitored using a Xenogen IVIS Kinetic imaging system. Following the last monitoring point, animals were sacrificed, and major organs were collected for imaging. To clarify the pathological property of micro-metastasis in bone metastasis model, the resected tumor was exposed to H&E and PSMA-IHC staining (Abcam, ab133579, UK). The distribution of Magic in tumor sections was detected using laser confocal microscope (Olympus, FV31s-sw, Japan).

IC50 determination

To evaluate DM1-induced cytotoxicity and Au-mediated PTT, DM1-Magic or Magic were incubated with PC3 cells for 24 h. Then, plate was washed with PBS and irradiated with 1.5 W cm^− 2 laser for 5 min. Cell viability was measured with CCK-8 assay (Beyotime, C0038, China) according to the user manual. Results were recorded using a microplate reader (Infinite 200, Tecan, Switzerland) at 450 nm. IC50 was derived from the variable slope model.

Tubulin staining

1×10⁵ PC3^PSMA+ cells were seeded into confocal dishes for overnight incubation. Subsequently, medium was replaced with fresh medium containing DM1-Magic (200 µg mL^–1 Magic loaded with 1.2 µg mL^–1 DM1). After 24 h incubation, cells were washed with PBS, fixed with 4% paraformaldehyde, and blocked with primary antibody dilution buffer. After incubating with α-tubulin rabbit polyclonal antibody (Proteintech, 11224-1-AP, USA) overnight at 4°C, dishes were incubated with Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Fisher Scientific, R37118, USA) for 2 h at room temperature. Digital images were obtained using Zeiss laser confocal microscope (LSM 800, Germany). ImageJ software was adopted for quantification.

Integrated anti-tumor effects in vivo

PC3^PSMA+ xenograft mice were constructed and randomly divided into five groups as mentioned above. For treatment group, 4 mg mL^− 1 Magic or DM1-Magic (loaded with 24 µg mL^− 1 DM1) were tail-vein-injected every 3 days at the dose of 3 µL g^− 1, so that their blood concentration could reach the calculated IC50 in vitro (blood volume of mice was reported as 58.5 µL g^− 1). 24 h later, animals in PTT group were irradiated with 808 nm laser for 5 min (power density = 1.5 W cm^− 2, irradiation distance = 2 cm). Tumor growth was monitored with vernier caliper and bioluminescent imaging.

Tibial micro-structure analysis

After treatment experiment, tumor-metastatic tibias were collected. The coronal plane of tibia was placed parallel to examination bed in the test chamber. Parameter setting was scanning voltage = 45 kV; scanning current = 200 µA; spatial resolution = 8 µm; and bone mineral density > 80. NRecon software and CTvox software was adopted for 3D reconstruction and rendering, respectively. Data Viewer software was used for image visualization. CTan software was employed for region of interest selecting and bone metrological analysis.

ELISA assay for macrophage-related cytokines

The resected tumors were weighed and homogenized in lysis buffer supplemented with proteinase inhibitor using a homogenizer device (Leica, Germany). The supernatant was collected after centrifugation at 14000 g for 20 min at 4°C. ELISA kits (Proteintech, USA) were employed for cytokines detection according to the manufacturer’s instructions. The absorbance was measured by using a microplate reader at 450 nm (Infinite 200, Tecan, Switzerland).

Statistical analysis

Student t-test (twotailed), Pearson correlation coefficient test, Wilcox test, and repeated-measures analysis of variance (ANOVA) were used for data analysis. Data were presented as mean ± SD from at least triplicate measurements. Statistical analyses were performed using SPSS 20.0 (SPSS, Chicago) or R software (https://www.r-project. org). P < 0.05 was considered as statistically significant.

High PSMA expression and M2 macrophages infiltration in PCa

Because Magic is designed to specifically bind to PSMA, we first checked the expression of PSMA at the protein level based on the samples in the Human Protein Atlas (HPA) database (https://www.proteinatlas.org). PSMA was found to be exclusively expressed in prostatic glandular cells (Fig. 1a), and its expression level was positively correlated with the histological grade of PCa and, thus, tumor aggressiveness and therapeutic outcome (Fig. 1b and Supplementary Fig. 1), indicating that PSMA is not only a promising biomarker in the diagnosis and therapy of PCa, but also useful in determining the prognosis of patients with PCa.

Considering that M2 macrophages contribute to the pro-tumoral environment and are associated with adverse clinical outcomes in many types of cancers, we next investigated the involvement of M2 macrophages in PCa progression. CIBERSORT was applied to statistically estimate the relative proportions of immune cell subsets among different grades of PCa samples from The Cancer Genome Atlas (TCGA) dataset. As shown in Fig. 1c and Supplementary Fig. 2, the infiltration levels of macrophages were higher in tumor tissues than in the normal prostate tissues. Notably, M2 macrophages not only accounted for a large proportion of tumor-infiltrating immune cells, but also were significantly enriched in high-grade compared with low-grade PCa tissues, suggesting that they may play a key role in the progression of PCa. Immunofluorescence staining of tumor tissues from patients with PCa showed consistent results. Compared with adjacent normal tissues, the number of CD68⁺ macrophages, especially CD68⁺/CD206⁺ M2 macrophages, was markedly increased in tumors and positively correlated with advanced tumor stage, confirming the prognostic value of M2 macrophages in PCa (Fig. 1d and Supplementary Fig. 3,4). Notably, PSMA expression also increased in parallel with tumor progression, as shown by immunohistochemistry (IHC) testing performed on tumor tissues (Fig. 1e). Further analysis revealed a positive correlation between M2 macrophage infiltration and PSMA expression (Fig. 1f and Supplementary Fig. 5a), suggesting that it might be possible to combine PSMA-targeted therapy with immunoregulation of the tumor microenvironment to promote enhanced anti-PCa treatment based on PSMA recognition. In addition, M2 macrophages have been reported to participate in epithelial-mesenchymal transition (EMT) and tumor angiogenesis by secreting the tumor-promoting cytokines, transforming growth factor-β (TGF-β) and vascular endothelial growth factor C (VEGFC), resulting in enhanced tumor invasion and metastasis.⁴⁵ Therefore, we analyzed the expression levels of TGF-β and VEGFC in PCa. The results shown in Fig. 1g clearly demonstrate that both TGF-β and VEGFC are positively correlated with M2 macrophage infiltration in PCa. Patients with high TGF-β or VEGFC expression appeared to have much poorer disease-free survival (DFS) than those with low TGF-β or VEGFC expression (Fig. 1h). Consistently, reduced E-cadherin (ECAD) expression (a hallmark of EMT) and Endoglin (ENG, CD105) overexpression (a marker of tumor angiogenesis) were significantly associated with shorter DFS in patients with PCa (Supplementary Fig. 5b). All these results indicate that M2 macrophages are probably involved in PCa progression via tumor-promoting cascades (i.e., M2 macrophage → cytokines → phenotypes → prognosis). Therefore, depletion or re-education of M2 macrophages may be a promising modality for immunomodulatory anti-PCa treatment.

Preparation and characterization of Magic

Inspired by the importance of PSMA in PCa treatment, and in light of the high affinity of gy-1 towards PSMA,^37–39 we envisioned that a macrophage-based platform with enhanced PSMA-targeting ability might be obtained through the integration of gy-1 with macrophages. To confirm this, we genetically engineered Raw264.7 cells to stably TM-express gy-1 through lentiviral infection, resulting in M^gy−1. The lentiviral vector used and preparative procedures of M^gy−1 are schematically shown in Fig. 2a and Scheme 1a. Briefly, the extracellular segment of gy-1 acting as the PSMA-targeting moiety and cytoplasmic domain of the enhanced green fluorescent protein (eGFP) as a reporter were anchored onto the macrophage membrane via a rational intracellular protein synthesis process with the help of the CD8 hinge domain and CD8 TM region. High level (near 100%) expression of the gy-1-eGFP fusion protein can be easily detected by confocal laser scanning microscopy (CLSM), flow cytometry, and western blotting measurements, confirming that gy-1 was efficiently expressed on the macrophage membrane (Fig. 2b and Supplementary Fig. 6). Moreover, as depicted by the enzyme-linked immunosorbent assay (ELISA) results in Fig. 2c, the introduction of gy-1 significantly enhanced the binding affinity of Raw264.7 to PSMA, further demonstrating the successful fabrication of M^gy−1. This is a prerequisite for endowing macrophage membrane-based nanomedicines with improved PCa targetability.

Subsequently, multifunctional FeAuNPs were synthesized according to previous report⁴³ and then wrapped with M^gy−1 membranes to yield Magic. The transmission electron microscopy (TEM) images shown in Fig. 2d (upper panel) clearly reveal that the FeAuNPs were highly uniform spheres with an average diameter of 101.30 ± 7.29 nm. The core-shell structure of the FeAuNPs was identified by elemental mapping analysis (Supplementary Fig. 7). In agreement with the changes in the diameter and zeta potential (Supplementary Fig. 8a,b), the scanning electron microscopy (SEM) images in Fig. 2d (bottom panel) clearly show that the FeAuNPs core was homogeneously coated with the M^gy−1 membrane, confirming the formation of Magic. Western blotting results indicate that the composition of the membrane proteins and their biological activities were well retained during Magic synthesis (Fig. 2e). Moreover, the hydrodynamic size of Magic remained nearly unchanged after lyophilization, indicating its excellent stability (Supplementary Fig. 8c).

Cellular targeting and internalization of Magic

Having successfully fabricated Magic, we next investigated whether Magic with TM-expressed gy-1 could inherit the prominent PSMA-targeting ability and tumor cell-specific internalization of gy-1 (Fig. 2f). Magic or M^gy−1-derived vesicles (MVs^gy−1, Fig. 2d) were incubated with PC3^PSMA+, PC3^PSMA−, or LNCaP (PSMA+) cells for 30 min. The binding affinity to PSMA was analyzed using flow cytometry. The results show that both Magic and MVs^gy−1 were able to specifically bind to PSMA-positive PCa cells (PC3^PSMA+ and LNCaP) rather than PSMA-negative PC3^PSMA− (Fig. 2g and Supplementary Fig. 9–11), probably because of the higher binding efficiency of Magic and MVs^gy−1 to PSMA in tumor cells. Furthermore, Magic rapidly bound to PC3^PSMA+ cells within 15 min and was internalized into the cells within 30 min (Supplementary Fig. 12). However, internalization was almost completely inhibited by pretreatment of PC3^PSMA+ cells with a PSMA antibody (PSMAb) with the same binding site as gy-1, demonstrating the effect of TM-expressed gy-1 on enhanced tumor targeting and cellular uptake of Magic (Supplementary Fig. 13). For better comparison, the conventional macrophage membrane-coated FeAuNPs (M-NPs) were labeled with the lipophilic dye 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) to yield the M^DiO-NPs. In contrast to the superior PSMA-targeting capability of Magic, M^DiO-NPs failed to distinguish PC3^PSMA+ cells from PC3^PSMA− cells, suggesting that TM-expressed gy-1 contributed to enhanced PSMA recognition of Magic (Supplementary Fig. 14).

In vitro performance evaluation of Magic

After confirming the prominent targeting performance of Magic, we conducted multiple experiments to explore its other functions. To verify whether the M^gy−1 membrane coating could endow Magic with the capacity to avoid immune clearance, Rhodamine (Rho)-PEG-SH-modified Magic (Rho-Magic) and Rho-PEG-SH-modified FeAuNPs (Rho-NPs) were fabricated to compare their macrophage-mediated phagocytosis. The CLSM images in Fig. 2h,i shows that Rho-Magic escaped phagocytosis by macrophages more effectively than PEGylated Rho-NPs. Flow cytometry measurement further confirmed that 97.53% of Rho-NPs were phagocytized by Raw264.7 versus 11.87% for Rho-Magic (Supplementary Fig. 15), firmly demonstrating that the M^gy−1 membrane coating did confer Magic enhanced immune evasion capability, which would be helpful to prolong the retention time of Magic in blood circulation and further benefit its tumor targeting.

Furthermore, the immunomodulatory potential of Magic was investigated. To verify whether Magic could re-educate M2 macrophages by neutralizing tumor-promoting cytokines secreted by M2 macrophages, ELISA was used first to measure the concentrations of representative cytokines including TGF-β, interleukin (IL)-4, and IL-10 after pretreatment with Magic for 2 h. As shown in Fig. 2j, Magic pretreatment substantially decreased the amounts of TGF-β, IL-4, and IL-10, indicative of the cytokine-neutralization capability of Magic endowed by the M^gy−1 membrane coating. As a result, cytokine-induced M2 macrophage polarization was greatly inhibited, as illustrated in Fig. 2k,l. Collectively, our results suggest that Magic has great potential to effectively ameliorate the immunosuppressive tumor microenvironment, which is crucial for improving anti-tumor efficacy.

Encouraged by the modulatory effect of Magic on the immune microenvironment, we next investigated the possibility of using it as a drug carrier for chemotherapy. The Au-S bond-mediated drug loading capacity and release property of the FeAuNPs were first examined using fluorescein as a drug model. To this end, the FeAuNPs were modified with fluorescein-polyethylene glycol 2000-thiol (FITC-PEG-SH). As shown in Supplementary Fig. 16, FeAuNPs exhibited high drug loading capacity and glutathione (GSH)-responsive drug release within tumor cells, suggesting that FeAuNPs can act as promising carriers of chemotherapeutic drugs to maximize therapeutic effectiveness while minimizing adverse effects. Next, we evaluated the Au-mediated photothermal conversion capability of Magic under continuous laser irradiation (Supplementary Fig. 17). Magic displays the concentration- and power-dependent photothermal behavior and excellent photothermal stability, making it favorable for tumor PTT applications. Taken together, Magic could act as a potential therapeutic agent with multiple favorable features for tumor therapy. In addition, Magic exhibited good MRI and CT capabilities in vitro (Supplementary Fig. 18), which provided the opportunity to evaluate the targeting performance of Magic through multimodality imaging.

In vivo biodistribution of Magic after systematic injection

The above in vitro results encouraged us to assess the biodistribution of Magic in xenograft mice. Indocyanine green (ICG)-PEG-SH functionalized Magic (ICG-Magic) was first intravenously injected into normal BALB/c nude mice to evaluate the pharmacokinetics of Magic. ICG-PEG-SH-functionalized FeAuNPs (ICG-NPs) were used as a control. Subsequently, 20 µL of blood was collected from the mice at different time points, and the fluorescence intensity of ICG in the plasma was measured. As depicted in Supplementary Fig. 19a, circulatory half-life of ICG-Magic was significantly extended to 10 h, in sharp contrast to 3.3 h for the ICG-NPs, indicating that macrophage membrane coating did prevent Magic from being cleared from the blood. Furthermore, in vivo fluorescence images were acquired after the intravenous injection of ICG-Magic or ICG-NPs in normal mice. Distinct from the high accumulation of ICG-NPs in the liver and spleen owing to clearance by MPS, ICG-Magic effectively avoided MPS recognition and elimination (Supplementary Fig. 19b), which was confirmed by hematoxylin and eosin (H&E) staining (Supplementary Fig. 19c).

We further evaluated the targeting ability of Magic to PCa tumors in vivo using M-NPs functionalized with ICG (termed M-ICG-NPs) as a control. ICG-Magic and M-ICG-NPs were injected intravenously into PC3^PSMA+ subcutaneous tumor-bearing mice (n = 3 for each group). As shown in Fig. 3a, the tumors could be clearly delineated with strong fluorescence even at 96 h post-injection of Magic, indicating its appreciable tumor specificity and blood circulation. In contrast, the signal was barely visible at the tumor site for the M-ICG-NPs. The excellent tumor recognition capability of Magic could also be confirmed by ex vivo imaging of the tumors and major organs collected from mice that were sacrificed 96 h post-injection (Fig. 3b and Supplementary Fig. 20). Quantitative analysis clearly revealed a 12-fold accumulation of Magic at the tumor site compared to that of M-ICG-NPs (Fig. 3c), which is consistent with the in vivo results. Collectively, M^gy-1 membrane ingeniously combines the advantages of the macrophage membrane and gy-1, endowing Magic with enhanced immune evasion and tumor targeting. In addition to the fluorescence imaging results, Fe₃O₄-based MRI and Au-based CT imaging consistently demonstrated that Magic is capable of imaging prostate tumors with remarkable specificity and selectivity both in vivo and ex vivo (Supplementary Fig. 21) owing to the presence of TM-expressed gy-1.

Active targeting ability of Magic to PCa metastasis

Having demonstrated the high targeting capability of Magic to primary prostate tumors, we further explored its feasibility for actively targeting PCa bone metastases, which are the leading cause of morbidity and mortality in patients with metastatic PCa (Fig. 3d and Supplementary Fig. 22a).⁴⁶ ICG-Magic and M-ICG-NPs were intravenously injected into PC3^PSMA+ metastatic tumor-bearing mice (n = 3 for each group). As shown in Fig. 3e, for the M-ICG-NPs group, the fluorescence intensity in the tumor decreased rapidly 1 h post-injection, suggesting low tumor accumulation of M-ICG-NPs. In contrast, the fluorescence at the tumor site after injection with ICG-Magic gradually intensified at 1 h and 12 h, indicative of efficient enrichment of ICG-Magic in tumor tissue, which could be attributed to higher PSMA-mediated tumor targeting of ICG-Magic (Supplementary Fig. 22b). Surprisingly, a micro-metastasis with a diameter of 3 mm in the groin next to the bone metastasis could also be effectively identified by Magic instead of M-ICG-NPs after excluding the interference from urine signal. Such striking metastasis targeting was confirmed by ex vivo imaging of the tibia with metastatic tumors and micro-metastatic lesions resected from mice sacrificed 12 h post-injection (Fig. 3f,g). Compared to the fluorescence signals of M-ICG-NPs, Magic could lead to a more than 7- and 9-fold increase in signals at the tibia metastatic and micro-metastatic foci, respectively (Supplementary Fig. 22c,d).

It is worth noting that the micro-metastases were further confirmed to be metastatic lymph nodes by H&E and PSMA-IHC staining (Fig. 3h). TM-eGFP-labeled Magic was specifically internalized into PC3^PSMA+ cells rather than germinal centers of metastatic lymph nodes (Fig. 3i and Supplementary Fig. 22e), demonstrating that Magic with superior PSMA-binding affinity specifically promoted its internalization into PCa^PSMA+ cells in vivo, which is a prerequisite for targeted tumor therapy.

In vitro evaluation of the anti-tumor effects of Magic-based nanomedicine

DM1 is a potent inhibitor of microtubule assembly with an anti-mitotic effect at sub-nanomole concentrations (median effective dose, ED50 = 10^–5–10^–4 µg mL^–1), making it promising for tumor chemotherapy.⁴⁷ Although DM1 has been widely explored in cancer treatment, the risk of systemic toxicity remains a significant concern.^40,41 Thus, the possibility of using Magic as a drug carrier for the targeted delivery of DM1 was investigated. Thiol-containing DM1 can be incorporated into Magic through Au-S bonds (DM1-Magic), and the cytotoxicity of DM1-Magic towards PC3^PSMA+ and PC3^PSMA− cells was evaluated using the Cell Counting Kit-8 (CCK-8) assay. As shown in Fig. 4a and Supplementary Fig. 23a, DM1-Magic showed highly targeted cytotoxicity against PC3^PSMA+ cells with a half reduction in cell viability (IC50) of 1.131 µg mL^− 1 and significantly reduced non-specific cytotoxicity of free DM1, indicating that Magic could act as a potent carrier to enable PSMA-targeted DM1 delivery. As a result, tubulin was severely damaged and mitosis of PC3^PSMA+ cells was inhibited, as depicted in the CLSM results (Fig. 4b-d). Additionally, Magic displayed notable photothermal ablation efficacy on PC3^PSMA+ cells (Supplementary Fig. 23b). Collectively, Magic could improve the targeting capability of DM1 to ensure high anti-tumor activity while minimizing side effects. Meanwhile, the immunomodulatory effects and PTT capability of Magic synergistically sensitize chemotherapy, thus further enhancing the killing of cancer cells.

Anti-tumor effect of Magic-based nanomedicine in vivo

Having demonstrated the potential of Magic for targeted DM1 delivery and synergistic anti-PCa benefits in vitro, we further verified the therapeutic efficacy of Magic in luciferase-expressing PC3^PSMA+ xenograft mice. Tumor-bearing BALB/c nude mice were randomly divided into five groups (n = 5 per group): (1) intravenous injection with phosphate-buffered saline (PBS) as a control, (2) intravenous injection with Magic without laser irradiation, (3) intravenous injection with Magic followed by 808 nm laser irradiation, (4) intravenous injection with DM1-Magic without irradiation, and (5) intravenous injection with DM1-Magic followed by 808 nm laser irradiation. Tumor growth was monitored using a Xenogen IVIS in vivo imaging system for the detection of bioluminescence or by measuring the average tumor size. In contrast to the continuous tumor growth in the PBS group, all other four groups showed significant inhibition of tumor growth (Fig. 4e-g, and Supplementary Fig. 24). It is noteworthy that the injection of Magic alone substantially suppressed tumor progression. Compared to other treated groups, the integration of DM1 with PTT exerted stronger anti-tumor efficacy during the entire treatment period, strongly demonstrating the competence of the Magic-based platform for highly specific treatment of PCa through its prominent PSMA-mediated tumor targeting. Moreover, no evident changes in mice body weight were observed, revealing the good biosafety of Magic-based formulations.

The excellent anti-PCa efficacy of Magic-based nanomedicine is further supported by Ki67 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end (TUNEL) staining. It can be seen clearly that all treatment modalities, even with Magic only, strongly suppressed tumor cell proliferation and efficiently induced cell apoptosis (Fig. 4h,i and Supplementary Fig. 25). The synergistic treatment using DM1 and PTT showed dramatically improved anti-tumor efficacy, consistent with the in vivo results described above.

Anti-tumor immunomodulation of Magic in vivo

To gain deeper insight into the inhibitory mechanism of pure Magic in vivo, we further investigated whether it could exert anti-tumor effects by blocking M2 macrophage-related tumor-promoting cascades, considering that Magic neutralized tumor-promoting cytokines secreted by M2 macrophages in vitro (Fig. 5a). To verify our hypothesis, ELISA was first performed to detect representative cytokines secreted by M1 and M2 macrophages in tumor tissues collected from mice after treatment with Magic. As shown in Fig. 5b, untreated tumors exhibited greater amounts of the pro-tumoral cytokines IL-4, IL-10, and TGF-β, which are mainly produced by M2 macrophages, than tumors treated with Magic. Meanwhile, those of M1-related anti-tumor cytokines IL-1β , IL-6, and tumor necrosis factor-alpha (TNF-α) were elevated in Magic-treated tumors. These results indicate that Magic effectively reprogramed macrophages from the M2 phenotype to the M1 phenotype by neutralizing pro-tumoral cytokines to improve anti-tumor immunomodulation. Further immunofluorescence staining of macrophages in tumor tissues revealed that Magic-treated tumors displayed a clear reversal in the ratio of M1/M2, which is in agreement with the ELISA results described above (Fig. 5c,d).

As mentioned previously, M2 macrophages stimulate endothelial cell proliferation-based tumor angiogenesis and EMT-mediated tumor metastasis by secreting vascular endothelial growth factor C (VEGFC) and TGF-β. Inspired by the excellent capability of Magic to neutralize TGF-β and downregulate VEGFC (Supplementary Fig. 26a), we further investigated the potential of Magic to inhibit the EMT process and tumor angiogenesis. The expression levels of EMT-associated proteins (vimentin and ECAD) and markers of tumor angiogenesis (VEGF receptor 2, VEGFR2, and CD105) were evaluated by immunofluorescence staining. Compared to the PBS control, Magic significantly reversed EMT and inhibited tumor angiogenesis, indicating the strong potential of Magic to effectively prevent tumor progression (Fig. 5e,f and Supplementary Fig. 26b-d). Collectively, Magic not only specifically delivered therapeutic drugs to prostate tumors for improved anti-cancer treatment, but also exerted immunomodulatory effects to promote the polarization of M2 to M1, thus blocking M2 macrophage-mediated tumor-promoting cascades and tumor progression.

Anti-tumor effects of Magic-based nanomedicine in PCa bone metastasis mode

Active targeting-mediated anti-tumor effects were observed in PCa bone metastasis models (Fig. 6a,b and Supplementary Fig. 27). Consistently, synergistic treatment with DM1 chemotherapy, PTT, and immunomodulation of the tumor microenvironment resulted in optimal therapeutic outcomes, and the metastatic tumors were completely eliminated after 21-d treatment. Furthermore, tumor-induced tibial destruction was significantly improved after effective treatment of metastases, as illustrated by the Micro-CT results in Fig. 6c and Supplementary Fig. 28. The three synergistic effects not only recovered the mineral content of the tibia and the microstructure of bone trabeculae, but also enhanced the mechanical properties of the tumor-metastatic tibia by more than 50% (Fig. 6d and Supplementary Fig. 29). Finite element analysis further verified that Magic-mediated tumor treatment was able to reduce the risk of fracture compared to the PBS group (Fig. 6e-g). Therefore, Magic benefited from its impressive PSMA targetability and exhibited great potential for the successful management of primary prostate tumors, micro-metastases, and concomitant damages.

In addition, neurotoxicity, peripheral organ toxicity, and embryotoxicity of Magic have been comprehensively evaluated. As shown in Supplementary Fig. 30–32, no obvious physiological abnormalities or systemic toxicity were observed in any of the treated mice, demonstrating that Magic can be safely applied as an excellent platform for tumor therapy.

Precise targeting is the major prerequisite for effective cancer therapies with minimal off-target side effects. Despite recent advances in nanomaterial-based strategies, simple surface functionalization of nanoparticles with synthetic materials or additional targeting ligands is still insufficient to cope with the complex physiological and pathological barriers in the tumor microenvironment.^9–11 Moreover, the therapeutic benefits of multiple nanomedicines can also be greatly dampened by the immunosuppressive tumor environment.^12,13 Therefore, the development of targeted nanomedicines capable of reversing the immunosuppressive tumor microenvironment is urgently needed.

Our analysis of clinical samples initially revealed that the expression level of PSMA is positively correlated with M2 macrophage-related tumor-promoting cascades, which inspired us to combine PSMA-targeted therapy with immunoregulation of tumor microenvironment for enhanced anti-PCa efficacy. Rather than using conventional post-functionalization approaches, ^34,35 we exploited genetic engineering strategy to directly express a PSMA-specific gy-1 on macrophages membrane, which was then coated onto FeAuNPs to produce a versatile biomimetic nanoplatform, Magic. Benefiting from the M^gy-1 membrane coating, the resulting Magic showed significantly enhanced circulation time, immune evasion, and cell-specific tumor targeting and internalization. The superior PSMA-targeting capacity of Magic causes Magic-based nanomedicine to preferentially accumulate at the tumor site to achieve highly targeted chemotherapy and PTT when loaded with the therapeutic drug DM1. Most importantly, the resulting Magic inherited the biological and physicochemical characteristics of macrophages and elicited M2 macrophage reprogramming to M1, and thus reversing the immunosuppressive tumor microenvironment and blocking M2 macrophages-mediated tumor-promoting cascades (i.e., tumor angiogenesis and EMT process), which further enhanced the anti-tumor efficacy of DM1 and PTT. As a result, synergistic PCa treatment dramatically inhibited tumor growth, micro-metastasis, and concomitant damage without obvious systemic toxicity.

These findings provide novel insights into the integration of targeted treatment and immunomodulation of the tumor microenvironment. Herein, PCa was used as a proof of concept to successfully demonstrate the effectiveness of our strategy. In addition to PSMA, there are many active molecules expressed on the surface of tumor cells, like epidermal growth factor receptor (EGFR),⁴⁸ human epidermal growth factor receptor 2 (HER2),⁴⁹ and Glypican-3 (GPC3).⁵⁰ Therefore, by TM-expressing the corresponding antibodies or specific ligands on macrophages, our strategy could be further expanded for targeting other cancers, such as lung cancer, breast cancer, and hepatocellular carcinoma. Meanwhile, M2 macrophages infiltration contributes to the pro-tumoral environment in many types of cancers,⁴⁵ indicating the universality of our strategy for synergistically treating cancers. Furthermore, with the engineered tumor-specific macrophage membranes, a variety of functional components, such as imaging reagents, photodynamic therapy reagents, chemotherapeutic drugs, and immunotherapy reagents could be flexibly encapsulated within it to meet diverse requirements, which also highlighted the great potential of our system as a versatile platform to enable the targeted tumor imaging and therapies.

This study reports an innovative nanoplatform, Magic, that encapsulates a FeAuNP core with a PSMA-targeting macrophage membrane for the effective treatment of PCa. The resulting Magic exhibited significantly enhanced cell-specific tumor targeting and effective immunomodulatory capability in vitro and in vivo. As a result, Magic reprogramed macrophages from the pro-tumor M2 phenotype to an anti-tumor M1 phenotype, which further enhanced the anti-tumor efficacy of targeted chemo-photothermal therapy. Taken together, we provide novel insights into the integration of precise treatment and immunomodulation of the tumor microenvironment with clinical translation prospects.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82201774, 82202933, 82203633, and 82173204), the Innovation Capability Support Program of Shaanxi (No. 2020PT-021, 2021TD-39, and 2022TD-52), the Natural Science Basic Research Program of Shaanxi (No. 2022JZ-62), the Natural Science Foundation of Shaanxi Province of China (No. 2020PT-020), the Fundamental Research Funds for the Central Universities (No. G2021KY05102), the Youth Innovation Team of Shaanxi Universities, and the Doctoral Project Funding of Fourth Military Medical University (No. 2020d03zky and 2020d11wjw).

ACKNOWLEDGEMENTS

AUTHOR CONTRIBUTIONS

W.J.Q., W.H.W., Z.L.W., R.L.Z., K.Y.Z., and D.H.H. conceived and designed the study. K.Y.Z., D.H.H., J.W.W., Y.L., C.X., X.J.Y., F.Y., S.J.L., X.L.Z., H.J.L., Y.J., S.J.M., J.L.Z., J.H.J., and T.B. performed the experiments. Among them, K.Y.Z., D.H.H., Y.L., and C.X. collected and analyzed the clinical samples. K.Y.Z., D.H.H., J.W.W., and Y.L. helped with nanoplatform preparation and experimental assays. X.J.Y., F.Y., S.J.L., X.L.Z., H.J.L., Y.J., S.J.M., J.L.Z., J.H.J., and T.B. assisted in conducting animal model experiment. Q.Z., R.Z., and A.G.Y. facilitated the data and file processing. All authors discussed the results and commented on the manuscript. K.Y.Z., D.H.H., J.W.W., and Y.L. wrote the manuscript. W.J.Q., W.H.W., Z.L.W., and R.L.Z. further revised and edited the manuscript. All authors have read and approved the article.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supplementary information is available at Cellular & Molecular Immunology’s website. (http://www.nature.com/cmi).

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Scheme 1 is available in the Supplemental Files section.

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SupplementaryMaterials.docx
Scheme1.tif
Scheme 1 Schematic diagram showing Magic-based cancer-targeting platform for PCa precise treatment and immunoregulation. (a) The formation of Magic. (b) Enhanced active tumor-targeting capability. (c) Synergism of immunomodulation, highly targeted chemotherapy, and PTT of Magic-based platform against tumor growth. MVs^gy-1: M^gy-1-derived vesicles.

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Genetically engineered macrophage membrane-coated nanoparticles for enhanced tumor targeting and synergistic cancer therapy

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Abstract

Figures

Introduction

Materials And Methods

Bioinformatics analysis

Patient specimens

Section staining

Cell culture and lentivirus infection

Cellular ELISA

Membrane derivation

Preparation of Magic

Characterization of Magic

Western blotting

Specific binding assay

Immune evasive assay

Cytokine neutralization and macrophage polarization

Animal model construction

In vivo fluorescence imaging

IC50 determination

Tubulin staining

Integrated anti-tumor effects in vivo

Tibial micro-structure analysis

ELISA assay for macrophage-related cytokines

Statistical analysis

Results

High PSMA expression and M2 macrophages infiltration in PCa

Preparation and characterization of Magic

Cellular targeting and internalization of Magic

In vitro performance evaluation of Magic

In vivo biodistribution of Magic after systematic injection

Active targeting ability of Magic to PCa metastasis

In vitro evaluation of the anti-tumor effects of Magic-based nanomedicine

Anti-tumor effect of Magic-based nanomedicine in vivo

Anti-tumor immunomodulation of Magic in vivo

Anti-tumor effects of Magic-based nanomedicine in PCa bone metastasis mode

Discussion

Conclusion

Declarations

References

Scheme 1

Additional Declarations

Supplementary Files

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