
==== Front
Int J Pharm X
Int J Pharm X
International Journal of Pharmaceutics: X
2590-1567
Elsevier

S2590-1567(24)00048-3
10.1016/j.ijpx.2024.100276
100276
Research Paper
M1 macrophage-membrane-cloaked paclitaxel/β-elemene nanoparticles targeting cervical cancer for enhanced therapy
Wang Yi ab
Wang Jiakun a
Huang Chengbo a
Ding Yang b
Lv Leyao b
Zhu Yuhao b
Chen Nuo b
Zhao Yingyi b
Yao Qing c
Zhou Shengjie a
Chen Mei a
Zhu Qibing a
Li Lifeng a
Chen Fengyun cfy19860530@163.com
a⁎
a Taizhou Women and Children's Hospital of Wenzhou Medical University, Taizhou, China
b The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou 325027, China
c School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China
⁎ Corresponding author. cfy19860530@163.com
13 8 2024
12 2024
13 8 2024
8 10027626 5 2024
8 8 2024
11 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
Cervical cancer is a leading cause of cancer-related mortality in females worldwide, necessitating urgent solutions for effective treatment. Paclitaxel (PTX), a natural diterpene alkaloid compound, has the ability to inhibit mitosis and induce programmed apoptosis in tumor cells. However, its toxicity and drug resistance limit its efficacy in certain cervical cancer patients. β-elemene (β-ELE) can reverse multidrug resistance by inhibiting ATP-binding cassette transporters, thereby enhancing chemotherapy drug retention. Therefore, we propose a combination therapy using PTX/β-ELE to improve chemotherapy sensitivity. To enhance targeted drug delivery, we developed M1-macrophage-membrane-coated nanoparticles (M1@PLGA/PTX/β-ELE) for co-delivery of PTX&β-ELE. Through both in vitro and in vivo cervical cancer models, we demonstrated that M1@PLGA/PTX/β-ELE effectively suppressed tumor progression and polarization of tumor-associated macrophages. Furthermore, H&E staining confirmed the high therapeutic biosafety of M1@PLGA/PTX/β-ELE as there was no significant damage observed in major organs throughout the entire therapeutic process. Overall, this study presents a targeted biomimetic nanoplatform and combinatorial strategy that synergistically enhances chemosensitivity in malignant tumors.

Graphical abstract

Unlabelled Image

Keywords

Biomimetic nanoparticles
Macrophage membrane coating
β-Elemene
PTX
Cervical cancer
==== Body
pmc1 Introduction

Carcinoma of the cervix, ranking as the fourth most prevalent neoplasm in women globally, accounts for 12% of all female cancers (Siegel et al., 2020; Small Jr. et al., 2017). The standard management approach for cervical cancer patients involves radiotherapy or chemotherapy. Despite recent advancements in therapeutic options that have enhanced patient survival rates (Hegde et al., 2023), prolonged use of chemotherapeutic agents can lead to treatment resistance and failure (Yu et al., 2022). Paclitaxel, a highly potent and broad-spectrum antineoplastic drug derived from natural sources, holds promise as a viable option for solid tumor treatment, including cervical cancer. The mechanism underlying PTX-induced programmed cell apoptosis is not only governed by survival signaling pathways (such as PI3K/Akt), but also influenced by modulation of pro-apoptotic factors or tumor suppressors (Deng et al., 2020). Resistance to chemotherapy and cytotoxicity towards normal cells remain significant obstacles in achieving successful outcomes in cervical cancer treatment.

Multidrug resistance (MDR) frequently arises as a consequence of clinical chemotherapy and is characterized by the failure of cancer cells to respond to previously effective drugs (Lee, 2010). The predominant mechanism underlying resistance involves the active efflux of cytotoxic drugs mediated by overexpressed ATP-binding cassette (ABC) transporters. Many MDR modulators currently available in the market often exhibit side effects or significantly impair fundamental metabolism and elimination processes (Bates et al., 2001; Kou et al., 2018b). In contrast, several Chinese medicinal herbs have demonstrated remarkable potential in reversing MDR with minimal adverse effects, while also exhibiting superior anti-cancer efficacy (Gu et al., 2022; Kong et al., 2023a; Kong et al., 2023b; Tian et al., 2023; Wang et al., 2022b). Notably, β-ELE, a plant-derived flavonoid, has been reported for its therapeutic effectiveness against specific solid tumors and malignancies (Guo et al., 2014; Liu et al., 2011). Therefore, we selected β-ELE as our model drug for this study due to its ability to enhance the chemotherapy efficiency of PTX through suppression of ABCB1 transporter-mediated drug efflux (Tang et al., 2018), thereby providing multifaceted benefits for cervical cancer therapy.

Nanotechnology-based drug delivery systems have been extensively developed over the years, and several commercial products, such as Doxil, Abraxane, and Liporaxel, have been successfully applied in clinical settings (Karahmet Sher et al., 2024; Liang et al., 2023; Patra et al., 2018; Sahu et al., 2021; Shen et al., 2024; Wu et al., 2024; Xu et al., 2024). Nanomedicines exhibit tremendous potential for anticancer drug delivery due to their significant advantages in improving drug solubility and stability, reducing side effects, and enhancing delivery efficiency (Chen et al., 2024; Jiang et al., 2023c; Kou et al., 2020a; Kou et al., 2019; Yao et al., 2017). Poly lactic-co-glycolic acid (PLGA) polymers have demonstrated significant potential as drug delivery carriers due to their robust physical properties, high biocompatibility, and biodegradability. Moreover, numerous studies have shown that PLGA can be easily formulated into controlled drug delivery systems for expected delivery efficiency (Kou et al., 2022b; Kou et al., 2017; Lee et al., 2024; Yao et al., 2024a). Moreover, nanomedicines can further enhance their performance by incorporating target ligands on the nanoparticle surface (Chen et al., 2023a; Kou et al., 2018a; Kou et al., 2020d; Yao et al., 2019) or responding to specific microenvironments to increase drug exposure at tumor sites (Kou et al., 2020c; Yao et al., 2020; Yao et al., 2018). Recently, biomimetic nanoparticles have gained increasing attention due to their superior delivery efficiency, improved biocompatibility, and retained bioactivity of components (Huang et al., 2024; Jiang et al., 2023b; Yao et al., 2023). Among various biomimetic nanoparticle strategies employed, membrane camouflage strategy has demonstrated exceptional tumor adhesion and accumulation capabilities (Fang et al., 2023). Macrophages possess innate inflammation-directed chemotactic ability with a particular emphasis on LPS-stimulation (Kou et al., 2022a; Zhang et al., 2018). M1-type macrophages that overexpress PD-1 protein may respond and home to chronic inflammatory tissue extracellular matrix (Hu et al., 2020; Yin et al., 2022).

In this study, we developed a M1-macrophages membrane-camouflaged nanoparticle (M1@PLGA/PTX/β-ELE) to exert the synergistic effects of PTX and β-ELE in the context of chronic inflammatory tumor microenvironment for enhanced treatment, while also targeting the PI3K/Akt survival signal axis (Scheme 1). Moreover, our study provides direct insights into the combined potency and regulatory mechanisms of paclitaxel and β-ELE in human cervical cancer. The utilization of a targeted delivery system in combination with these agents may offer a promising strategy for improving cancer management.Scheme 1 Schematic representation of the preparation of Macrophage-Membrane-Coated nanoparticles loaded with paclitaxel and β-elemene (M1@PLGA/PTX/β-ELE) and the in vivo application. (A) M1@PLGA/PTX/β-ELE was synthesized using a phacoemulsification and film coating method. (B) M1 macrophage membrane facilitated targeted drug delivery of M1@PLGA/PTX/β-ELE to the tumor microenvironment, while simultaneously releasing paclitaxel and β-elemene for synergistic anti-tumor effects by suppressing PI3K/AKT pathways and modulating the tumor microenvironment.

Scheme 1

2 Materials and methods

2.1 Materials

β-elemene (>98%) was purchased from Dalian Huali Jingang Pharmaceutical Co. Ltd. (Dalian, Liaoning, China). Paclitaxel and poly (D, L-lactic-co-glycolic acid) (PLGA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Coumarin 6 was bought from Sigma-Aldrich (USA). Tunel Apoptosis Detection Kit (Green fluorescence, C1086), Cell Counting kit-8 (CCK-8), DiO (Cell Membrane Green Fluorescent Probe), hematoxylin and eosin (H&E) staining kit and BCA protein assay kit were acquired from Beyotime Biotechnology (California, USA). DiR (MB12482) was purchased from Dalian Meilun Biotechnology Co., LTD (Dalian, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin and trypsin were purchased from Gibco Corporation (South America). Lipopolysaccharide (LPS) were from Peprotech (USA). Primary antibodies such as GAPDH (D16H11, 5174) Akt (pan, 4685), Phospho-Akt (Ser473, 4060), Bcl-2 (D17C4, 3498), Bax (D2E11, 5023), HIF-1α (E1V6A, 48,085), Ki-67 (8D5, 9449) and Tubulin (D65A4, 5666) were obtained from Cell Signaling Technology. Multidrug efflux transporter P glycoprotein (MDR1/P-gp) ligand screening (ab284553) was obtained from Abcam Biotechnology (USA). APC anti-mouse CD279 (PD-1) Antibody, PE anti-human CD206 (MMR) Antibody and APC anti-human CD86 Antibody were purchased from BioLegend Co., Ltd. Horseradish peroxidase (HRP)-conjugated secondary antibody and Propidium iodide (PI)/Annexin V-FITC kit were bought from BD Biosciences (Mountain View, CA, USA).

2.2 Cell lines and culture

Hela cells, CaSki cells (human cervical cancer cell lines) and RAW 264.7 cells (macrophage cell line) were purchased from the Cell Bank of Shanghai Chinese Academy of Sciences. The cervical cancer cell lines and RAW 264.7 cells were maintained in DMEM with 10% FBS, and streptomycin (100 units/mL)-penicillin (100 units/mL). The cells culture maintained at 37 °C in a humidified atmosphere of 5% CO2.

2.3 Encapsulation of macrophage membranes

The cell membrane of macrophages was extracted using a previously reported method. (Gong et al., 2020; Zhang et al., 2021). M1-polarized macrophages were produced after LPS (1 μg/mL) stimulated to RAW264.6 cells for 24 h. Untreated were considered as M0 macrophages. After washing with PBS, the macrophages were harvested gently with a rubber scraper, and were resuspended in a hypotonic lysing buffer to lysate at 4 °C overnight. Then, the mixture was collected and centrifuged at 1000 ×g for 5 min. The supernatant was collected, and the macrophage membranes supernatant was obtained by centrifuged at 14000 ×g for 60 min. The purified cell membranes were collected and stored at −80 °C until use. The protein content of the macrophage membranes was determined by a BCA protein assay, which was calculated at about 2 mg per 1 × 108 cells.

2.4 Preparation of M1@PLGA/PTX/β-elemene

First, the PLGA/PTX/β-elemene nanoparticles were prepared via the method of ultrasonic emulsification. Briefly, PTX and β-elemene with a ratio of 1 to 10 (mol/mol) were added to the dichloromethane solution containing PLGA (20 mg/mL) to get a fully clarified solution. The mixture solution was then added to the 1% (v/v) PVA solution and rapidly followed by sonication with a probe sonicator (50 W, 50 Hz, 5 min) for obtaining the nanoparticles (PLGA/PTX/β-elemene). The dichloromethane solvent was then removed by stirring under room temperature for 12 h. The unencapsulated drugs were removed by centrifugation at 3000 rpm for 5 min. The PLGA/PTX/β-elemene in solution were collected by centrifugation at 13,000 rpm for 30 min and washed with double-distilled water for three times. Above all, M1@PLGA/PTX/β-elemene was prepared by co-extrusion. Freshly prepared PLGA/PTX/β-elemene were mixed with macrophage membranes at the ratio of 10:1 (mg/mg) under ultrasound (20 W, 20 Hz, 5 min). The mixed liquid was squeezed through a 200 nm polycarbonate membrane for 20 times with an extruder to generate M1-macrophages membrane-camouflaged nanoparticle (M1@PLGA/PTX/β-elemene). C6-labeled was regarded as M1@PLGA/C6 which prepared by following a forementioned procedure. Non-polarized M0m coated on PLGA/C6 were regarded as M0@PLGA/C6. To verify the coating of membranes on nanoparticles, we further performed co-localization assay. PLGA/DiR were prepared via the above-mentioned method except using DiR to replace PTX and β-elemene. The staining of the cell membrane was obtained by co-incubation of the membrane with the fluorescent marker. In briefly, 2 μg/mL DiO was incubated with macrophage membranes for 30 min at room temperature. After centrifugation at 14000 ×g for 60 min, the supernatant was removed. The samples were washed with PBS for three times to remove the unbound dye, and the membranes were collected for preparing M1@PLGA nanoparticles as the above-mentioned method. The prepared nanoparticles were further visualized by a fluorescence microscope.

2.5 Characterization of M1@PLGA/PTX/β-elemene

The hydrodynamic diameter and surface zeta potential of M1@PLGA/PTX/β-elemene were evaluated by dynamic light scattering (DLS, Nano-ZS90, Malvern Instruments, Ltd., UK). The size and microstructures of the PLGA/PTX/β-elemene and M1@PLGA/PTX/β-elemene were characterized by transmission electron microscopy (TEM, 120KV, JEM-2100, JEOL Ltd., Japan). The stability of M1@PLGA/PTX/β-elemene was reflected by the size distribution changes in PBS (pH 7.4) at room temperature for 7 days.

To quantify PTX and β-elemene loaded in nanoparticles, we employed excess acetone to disrupt nanoparticles and dissolve the loaded PTX and β-elemene within them. The quantification of loaded drugs was then performed using an HPLC analysis system (Agilent, Agilent 1260). For PTX quantification, we used the following conditions: mobile phase - acetonitrile −0.01 M sodium acetate buffer solution (42/58, v/v); flow rate - 1 mL/min; wavelength - 227 nm. The concentrations of β-elemene were measured by HPLC under these conditions: mobile phase - acetonitrile-water (90:10, v/v); flow rate - 1 mL/min; wavelength - 210 nm.

The drug loading efficiency (DL) and entrapment efficiency (EE) of PTX and β-elemene were calculated by the following formula:Encapsulation efficiencyEE=weight of the loaded drug/weight of the drug in feed×100%

Drug loading efficiency (DL) = (weight of the loaded drug)/ (total weight of NPs) × 100%.

The in vitro drug release profiles of paclitaxel and β-elemene were evaluated by a dialysis method. Briefly, PTX/β-elemene, PLGA/PTX/β-elemene and M1@PLGA/PTX/β-elemene were placed in dialysis bag (MWCO 3.5 kDa) and immersed in 20 mL of phosphate buffer solutions (PBS, pH 7.4) containing 0.1% tween 80 (w/v) and stirred in shaker at 400 rpm. At predetermined time intervals, 2 mL of the aliquot solution was removed, and 2 mL of fresh supplemented media was put back to remain a constant volume. The supernatant was isolated, measured and calculated for PTX and β-elemene release behavior.

The biocompatibility of NPs was assessed with fresh blood cells. The 2% erythrocyte suspension was obtained by repeated resuspension and centrifugation for hemolytic evaluation. PTX/β-elemene, PLGA/PTX/β-elemene and M1@PLGA/PTX/β-elemene were added for co-incubation in a 37° water bath. PBS was used as a negative control and the water-treated group was used as a positive control. After 12 h, the samples were centrifuged at 2000 rpm for 5 min and photographed for recording. The supernatant was extracted, and the absorbance was measured at 545 nm using a UV spectrophotometer (Shimadzu, Japan).

Fluorescence confocal microscopy was used for co-localization to determine whether the membrane was successfully coated with PLGA nanoparticles. DiO-labeled macrophage membranes were used as M1@DiO and DiR-labeled PLGA nanoparticles were regarded as PLGA/DiR. M1@DiO/PLGA/DiR was obtained by ultrasonic extrusion method and observed by fluorescence confocal microscope.

2.6 CCK-8 assays

The standard cell counting kit-8 (CCK-8) assay was used to examine the cytotoxicity of PTX and β-elemene in single drug or combination. Hela cells and CaSki cells (both 5 × 103 cells/well) were seeded in 96-well plates for allowing adhere overnight. Subsequently, at a series different drug of concentration gradients were administrated to each well and further incubated for 24 h. And then, the CCK-8 assay was carried out. The cell viability and the inhibition ratio, and the IC50 value were also estimated by GraphPad Prism 8.0.1 software.

To explore the synergistic effect of paclitaxel and β-elemene, the combination index (CI) was calculated based on the following equation:

CI=Dp/Dpx+De/Dex

Dp or De represents the combined concentration for paclitaxel or β-elemene to achieve X% cytotoxicity, and Dpx or Dex indicates the concentration of single drug to achieve X% cytotoxicity.

2.7 In vitro cellular uptake

Fluorescent confocal microscopy was utilized to visualize cellular uptake process of PLGA/C6, M0@PLGA/C6 and M1@PLGA/C6 (2.5 μg/mL, calculated as C6). Briefly, CaSki cells were seeded at a density of 1 × 105 cells/well on coverglass in 12-well plates overnight and subsequently co-incubated with different drug groups for 1 h, 4 h and 12 h. After treatment, cells were washed three times with cold PBS and the slides were sealed using an anti-fluorescence quencher containing DAPI.

Multiple endocytosis inhibitors were pretreated with CaSki cells to determine the endocytosis pathway of M1@PLGA/C6 in vitro. After treating with 10 μM colchicine (APEXBIO, A3324) or 10 μM filipin (Mellonbio, MB1848) at 37 °C for 60 min, or untreatment at 4 °C for 60 min, M1@PLGA/C6 was added for 4 h, respectively.

2.8 In vitro macrophage polarization by M1@PLGA/PTX/β-elemene

The macrophages RAW264.7 were seeded in upper chamber of 24-well transwell, and the bottom chamber contained the CaSki cells to simulate the tumor microenvironment. After 24 h incubation, different drugs were added in the bottom wells for another 24 h of treatment. Afterward, the RAW264.7 cells were collected and labeled with fluorescently labeled PE anti-human CD206 antibody and APC anti-human CD86 antibody and then analyzed by flow cytometry. To investigate the polarization of macrophages, RAW264.7 cells were collected and analyzed by western blot.

2.9 Cell apoptosis by flow cytometry

Apoptosis was analyzed using an annexin V-FITC/PI apoptosis detection kit. After treating with different groups, including PTX, β-elemene, PLGA/PTX/β-elemene and M1@PLGA/PTX/β-elemene. The treated CaSki cells were incubated with Annexin V FITC (10 μL) and PI (5 μL) in the dark according to the instructions, after which all samples were examined by flow cytometry (Beckman Coulter, CytoFLEX S).

2.10 Colony formation assay

To evaluate the cell proliferation inhibition rate, CaSki cells were inoculated onto 6-well plates at a density of 1000 per well. After overnight incubation, the medium containing PTX, β-elemene, PLGA/PTX/β-elemene and M1@PLGA/PTX/β-elemene were incubated with the cells for 7 days. After that, the cells were washed with PBS and stained with Giemsa. The images were recorded by digital camera and then lysed colony and measured absorbance at 450 nm.

2.11 Western blot analysis

Western blot analysis was performed to determine whether M1@PLGA/PTX/β-elemene drown-regulated the expression of proliferation correlated proteins. Briefly, cells were plated in 6-well plate at a density of 4 × 106 cells/mL. The medium was then replaced with fresh medium containing therapeutic drugs. After 24 h, all cells were washed twice with ice-cold PBS. Proteins were extracted from the cells using RIPA buffer cocktail according to the manufacturer's instructions. The final protein concentration of the cells was determined by the BCA assay. We electrophoresed the same amount of proteins using 12% SDS-PAGE gel and transferred the proteins to the PVDF membrane. The membrane was blocked with 5% nonfat dry milk in TBST for 2 h, which was then incubated overnight at 4 °C with diluted primary antibodies. The membranes were washed quickly for three times and incubated with diluted anti-rabbit secondary antibody (1:3000) for 1 h at room temperature. Finally, PTG ECL chemiluminescence detection kit was used for color development.

2.12 MDR1/P-gp fluorescent quantitative analysis

The CaSki cells were seeded approximately 3 × 104 cells/well in a white-walled 96-well plate (with clear bottom). After incubation with medium containing therapeutic drugs for 12 h, the cells were washed and add 100 μL fresh Efflux Assay Buffer to each well. Assays for the inhibitory capacity of efflux proteins were performed according to the MDR1/P-gp ligand screening kit instructions. The fluorescence intensity (Ex/Em = 488/532 nm) of the wells used the spectrofluorometer.

2.13 Anti-tumor effect in CaSki cells bearing mouse model

All female BALB/c nude mice (6–8 weeks) were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and were reared in a freedom environment. After one week, CaSki cells (1 × 106) were subcutaneously injected into the right flank of mice under anaesthetic. The mice were freely divided into 5 groups (n = 4), and caudal intravenous administration was initiated when the tumors grew to 100mm3. Saline only, free PTX, PTX/β-elemene, PLGA/PTX/β-elemene and M1@PLGA/PTX/β-elemene was scheduled intravenously at an equivalent PTX dose of 2 mg/kg every two days. The body weight and tumor sizes of tumor-bearing mice were monitored throughout an electronic scale and digital calipers, respectively. The tumor volume was estimated by the following formula: Tumor volume (mm3) = Long diameter × (short diameter)2/2.

After 18 days later, all the mice were euthanasia. All the tumors and major organs were fixed and sectioned into slices, followed by histological analysis with H&E and Tunel fluorescence staining. Toxicity evaluation in vivo was performed by blood biochemical analysis.

2.14 In vivo tumor accumulation capacity

The CaSki cells bearing female BALB/c nude mice were administered PTX/β-elemene, PLGA/PTX/β-elemene and M1@PLGA/PTX/β-elemene by injection via the tail vein. After 12 h, sacrificed and their organs (heart, liver, lung, spleen, and kidney) and tumor were harvested for further analysis. Briefly, tissues of the same weight were taken and ultrasonic crushed in acetonitrile. After centrifugation (1500 rpm/20 min), the supernatant containing the PTX and β-elemene was taken and analyzed by liquid chromatography-mass spectrometry (LC-MS).

2.15 Statistical analysis

All data were recorded as mean ± standard deviation (SD) with statistical analysis by GraphPad Prism 8.0.1 software. Statistical analysis of two group comparisons were determined by two-tailed Student's t-test. Mean fluorescence intensity of cell internalization was quantified manually using ImageJ densitometry software. Comparisons between multiple groups were analyzed using one-way analysis of variance (ANOVA). (*P < 0.05, ** P < 0.01, and *** P < 0.001).

3 Results and discussion

3.1 Optimization of combination therapy with paclitaxel/β-elemene

Paclitaxel is a mitosis inhibitor that induces tumor apoptosis through chromosome breakage. To investigate the synergistic therapy of paclitaxel and β-elemene on human cervical cancer cells, Hela and CaSki cells were separately co-incubated with varying concentrations of drugs for 24 h. Consistent with previous studies(Faried et al., 2006), Hela cells exhibited high sensitivity to paclitaxel (IC50 = 2.4 μg/mL). In contrast, CaSki cells showed greater resistance, displaying a 48.1% decrease at 3.6 μg/mL upon treatment with paclitaxel (Fig. 1A). Both Hela and CaSki cells demonstrated similar results in the CCK8 assay after treatment with β-elemene, exhibiting almost 50% inhibition in cell viability at concentrations of 0.09 mg/mL and 0.18 mg/mL, respectively (Fig. 1B). To further explore the ability of β-elemene to enhance chemo-sensitivity in cervical cancer cells towards paclitaxel, three ratios of paclitaxel to β-elemene (1:5, 1:10, and 1:20) were chosen to evaluate their combined anticancer potency on human cervical cancer cell lines (Fig. 1C&D). Lethality was measured to optimize the combination ratio of paclitaxel&β-elemene. Compared to PTX alone, the combination ratio of 1:20 exhibited significant killing ability in Hela cells (P < 0.05). In PTX-insensitive CaSki cell lines, suppression efficiency was significantly enhanced when using a ratio of paclitaxel:β-ELE = 1:5 (P<0 0.05), as well as at ratios of both 1:10 and 1:20 (P<0 0.05). We calculated the combination index (CI) for the combination of paclitaxel and β-elemene in different ratios (Fig. 1G&H). The results showed that the CI values for the ratio of 1:20 were smaller than 0.9 in both Hela cells and CaSki cell lines, strongly indicating the synergism of paclitaxel and β-elemene in this ratio. Based on these results, a synergistic ratio of 1:20 (PTX: β-ELE, mol/mol) was selected from an economic standpoint, with CaSki cells used as the cellular model.Fig. 1 β-elemene enhances chemo-sensitivity of cervical cancer cells to paclitaxel by CCK8 assay. The effect of paclitaxel (A) and β-elemene (B) on the growth inhibition of Hela and CaSki cells. Cell growth inhibition of (C) HeLa and (D) CaSki after treatment with various ratio of PTX/β-ELE for 24 h. The value of 50% inhibiting concentration (IC50) with different treat groups in (E) HeLa and (F) CaSki cells. The combination index (CI) of PTX and β-ELE in (G) HeLa and (H) CaSki cells. (I) Effect of different PLGA concentration on the PLGA/PTX/β-ELE nanoparticle size. (J) Effect of different drug/PLGA ratio on the PLGA/PTX/β-ELE nanoparticle size. Data are represented as mean ± SD. ns, not significant, *P < 0.05, ***P < 0.001 representing significance compared to PTX group.

Fig. 1

3.2 Characterization of M1@PLGA/PTX/β-ELE

The phacoemulsification method successfully facilitated the loading of Paclitaxel and β-elemene into PLGA nanoparticles, as confirmed by dynamic light scattering (DLS) results (Fig. 1I&J). Subsequently, a formulation consisting of 20% PLGA and 20% PTX/β-ELE was selected for the preparation of PLGA/PTX/β-ELE nanoparticles (NPs). The resulting PLGA NPs exhibited a spherical morphology with a size distribution of approximately 140 nm (Fig. 2A&C). There was no significant difference in particle size distribution observed for macrophage membrane-modified NPs (M1@PLGA/PTX/β-ELE) compared to unmodified NPs (Fig. 2B). Specifically, SDS-PAGE results showed similar protein patterns between M1@PLGA and M1 macrophage membrane, indicating the successful coating of M1 macrophage membrane on PLGA nanoparticles (Fig. 2E). Additionally, fluorescence colocalization analysis confirmed successful loading of M1m onto the core-shell structure formed by DiO-labeled cell membranes and DiR-labeled PLGA in M1@PLGA/PTX/β-ELE NPs (Fig. 2F). Transmission electron microscopy revealed an evident core-shell structure where membrane of M1 effectively coated onto PLGA/PTX/β-ELE to form M1@PLGA/PTX/β-ELE (Fig. 2D). Furthermore, M1@ PLGA/PTX/β-ELE displayed negative ζ-potential which contributed to avoiding reticuloendothelial system phagocytosis (Fig. 2G).Fig. 2 Characterization of M1@PLGA/PTX/β-ELE. (A) Analysis of size distribution of PLGA/PTX/β-ELE and (B) M1@PLGA/PTX/β-ELE by dynamic light scattering. TEM images of (C) PLGA/PTX/β-ELE and (D) M1@PLGA/PTX/β-ELE. Scale bar: 100 nm. (E) SDS-PAGE of the samples with equivalent protein contents (25 μg). (F) Representative CLSM images and colocalization analysis of M1/DiO (green)-PLGA/DiR (red). Scale bar: 100 μm. (G) Average ζ potentials of PLGA, PLGA/PTX/β-ELE and M1@PLGA/PTX/β-ELE. (H) Stability evaluation of PLGA/PTX/β-ELE and M1@PLGA/PTX/β-ELE by DLS. Drug release profile of (I) PTX and (J) β-ELE detected by HPLC. Data are represented as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

The mean diameter of PLGA/PTX/β-ELE and M1@PLGA/PTX/β-ELE remained unchanged for 7 days in mimic in vivo serum-containing medium, indicating their excellent stability (Fig. 2H). Moreover, the low hemolysis rate confirmed the safety of these formulations for systemic intravenous administration (Fig. S1). The encapsulation efficiency and drug loading capacity of PTX and β-ELE were evaluated in Fig. S2, demonstrating successful co-encapsulation of them into nanoparticles. Compared to the free PTX/β-ELE group, both PLGA/PTX/β-ELE and M1@PLGA/PTX/β-ELE exhibited sustained release profiles over a period of 96 h.

3.3 Cellular uptake of M1@PLGA/PTX/β-ELE

The cellular internalization behavior of M1@PLGA/PTX/β-ELE was evaluated in the CaSki cell line using fluorescence imaging (Fig. 3A). After a 4 h incubation, the highest C6-labeled fluorescence signal was observed in the M1@PLGA/PTX/β-ELE group. Furthermore, at 12 h, there was a further enhancement in the fluorescence intensity of C6, indicating time-dependent uptake behavior. Compared to PLGA/C6, the fluorescence intensity of M0@PLGA/C6 and M1@PLGA/C6 was increased by approximately 1.42 and 2.14 fold, respectively (Fig. 3D). These results suggested that overexpression of proinflammatory cytokines on M1@PLGA/PTX/β-ELE nanoparticles enhanced their internalization efficiency of tumors, thereby promoting their antitumor efficacy. The specific mechanism underlying the internalization of M1@PLGA/C6 nanoparticles was investigated using various endocytosis inhibitors including colchicine (macropinocytosis-mediated pathway), filipin complex (caveolae-mediated pathway), and ice incubation (ATP-dependent pathway). Representative confocal laser scanning microscopy images demonstrated that chloroquine inhibition resulted in a reduction of up to 35.60% in nanoparticle internalization. Similarly, energy inhibition led to a decrease of approximately 29.60%. These results suggested that macropinocytosis-dependent pathways and ATP-dependent pathways were primarily responsible for the internalization process of M1@PLGA/C6 nanoparticles while caveolae-mediated pathways play a minor role (Fig. 3C).Fig. 3 The uptake profiles of M1@PLGA/C6 in CaSki cells. (A) Representative CLSM images showing the cell internalization of PLGA/C6, M0@PLGA/C6 and M1@PLGA/C6 after coincubation with CaSki cells at 1 h, 4 h and 12 h. (B) Representative CLSM images of the cell internalization of M1@PLGA/C6 after treatments with various inhibitors. Scale bar: 100 μm. (C) Quantitative analysis of fig. B. (D) Quantitative analysis of fig. A. Data are represented as mean ± SD. *P < 0.05, ***P < 0.001 representing significance compared to the control group or as indicated.

Fig. 3

3.4 Anti-proliferation of M1@PLGA/PTX/β-ELE on tumor cells

In vitro pharmacodynamic studies, colony-forming assays, and apoptosis tests were conducted to investigate the antitumor activity against CaSki cells and their underlying mechanism. The untreated cells served as the control in the colony formation assay (Fig. 4A&C). The PTX-treated group exhibited a minor anti-proliferative effect on CaSki cells, while the combination of PTX/β-ELE treatment displayed the strongest synergistic effect (25.40 ± 0.67%). However, PLGA/PTX/β-ELE showed weaker anti-proliferation efficacy compared to M1@PLGA/PTX/β-ELE due to enhanced intracellular penetration of NPs triggered by PD-L1. To further elucidate the antitumor efficacy, we evaluated apoptosis in CaSki cells treated with PTX, PTX/β-ELE, PLGA/PTX/β-ELE, and M1@PLGA/PTX/β-ELE using Annexin V-FITC and PI staining (Fig. 4B). Flow cytometry was used for quantitative analysis of cell activity stages. As depicted in Fig. 4D, negligible cell death was observed in the PTX-treated group compared to the PTX/β-ELE group due to their combined cytotoxicity efficiency. Moreover, membrane-coated preparations such as M1@PLGA/PTX/β-ELE induced significant apoptosis by causing massive amounts of cell death. To gain deeper insights into cell death mechanisms after different drug treatments, changes in related proliferation and apoptosis signaling proteins were analyzed. Akt-mediated survival signals represent one of the most crucial pathways involved in promoting cell survival or inducing apoptosis signaling cascades (Kim et al., 2007). As shown in Fig. 4E, our findings revealed that p-AKT expression and anti-apoptotic protein Bcl-2 were down-regulated by PTX treatment while pro-apoptotic protein Bax expression was increased. Recent reports have suggested that serine/threonine protein kinase Akt (p-Akt) is implicated in chemo-resistance mechanisms (Gao et al., 2020; Peng et al., 2022; Zheng, 2017); therefore, inhibiting Akt could enhance paclitaxel-induced apoptotic efficacy.Fig. 4 Anticancer mechanisms of M1@PLGA/PTX/β-ELE were investigated in CaSki cells. (A) Colony formation assay of CaSki cells was cultured under the different preparation groups. (B) The flow cytometry analysis of apoptosis induction. (C) Quantitative analysis of colony numbers. (D) Quantitative analysis of early and late apoptosis. (E) Western blotting image of p-Akt, Akt, Bcl-2, Bax and tubulin expressions. Data are represented as mean ± SD. **P < 0.01, ***P < 0.001 representing significance compared to the control group.

Fig. 4

The development of chemoresistance is primarily attributed to the active efflux of drugs, such as P-glycoprotein (P-gp, ABCB1) and multidrug resistance-associated protein 1 (MRP, ABCC1). As depicted in Fig. S3, the intracellular accumulation of hydrolysis products from a fluorogenic substrate of P-glycoprotein was upregulated in the presence of β-elemene, providing evidence for the inhibition of P-gp transporter efflux function. Western blot analysis revealed a significant suppression in the expression levels of p-Akt and Bcl-2 after treatment with M1@PLGA/PTX/β-ELE, indicating an excellent synergistic effect between PTX and β-ELE. We hypothesized that β-elemene may enhance PTX sensitivity through two mechanisms: by increasing intracellular accumulation of PTX via attenuation of P-gp protein efflux function and by directly modulating Akt signaling pathway expression. These findings suggest that M1@PLGA/PTX/β-ELE could serve as a potent suppressor of proliferation and inducer of apoptosis mediated through the PI3K/Akt pathway.

3.5 The effect of M1@PLGA/PTX/β-ELE on macrophages polarization

Tumor-associated macrophages, comprising heterogeneous subsets including M1 and M2, have been implicated in the progression of cervical cancer. To investigate the impact of M1@PLGA/PTX/β-ELE in vitro, co-cultured cervical cells were subjected to flow cytometry analysis for surface marker expression on different macrophage subsets (Fig. S4A). Notably, M1 macrophages exhibited a significantly higher percentage of CD86+ cells, while M2 macrophages showed a significantly higher percentage of CD206+ cells. Moreover, the mean fluorescence intensity of CD206+ subset levels in tumor culture medium containing M1@PLGA/PTX/β-ELE was markedly lower compared to an untreated cervical cancer microenvironment. Quantitative analysis revealed that the addition of β-elemene resulted in a significantly higher percentage of CD86+ subsets compared to PTX alone (Fig. S4B). In summary, our findings demonstrate that M1@PLGA/PTX/β-ELE can effectively modulate the cervical cancer microenvironment by regulating macrophage polarization.

3.6 In Vivo anticancer performance of M1@PLGA/PTX/β-ELE

The M1@PLGA/PTX/β-ELE exhibited remarkable therapeutic efficacy in vitro, prompting us to further investigate its potential anti-tumor activity in vivo. To evaluate the quantitative distribution profiles of different drug-loading formulations, we initially administered them via intravenous injection. Among these formulations, M1@PLGA/PTX/β-ELE demonstrated the highest enhancement of nanoparticle accumulation at the tumor site in CaSki tumor-bearing mice. Moreover, following M1m coating, there was a 1.22-fold increase in PTX and β-ELE concentration at the tumor site compared to PLGA/PTX/β-ELE alone, confirming targeting and selective delivery. Additionally, despite lacking membrane coating, PLGA/PTX/β-ELE exhibited greater tumor accumulation than free PTX/β-ELE solution due to the enhanced permeability and retention (EPR) effect of nanoparticles. By achieving precise drug delivery to tumors, M1@PLGA/PTX/β-ELE enhanced the chemotherapeutic efficacy of PTX combined with β-ELE while reducing the risk of off-target adverse reactions on other organs (Fig. S5).

Encouraged by the efficient accumulation of drugs at tumor sites, the anticancer efficacy of M1@PLGA/PTX/β-ELE was further evaluated in CaSki tumor-bearing mice. The treatment procedure is illustrated in Fig. 5A. Female BALB/C nude mice were randomly divided into five groups (n = 5) and received intravenous injections of saline, PTX alone, free PTX/β-ELE, PLGA/PTX/β-ELE or M1@PLGA/PTX/β-ELE every two days. Tumor volumes and body weights were monitored every other day during the 14-day treatment period. Compared to the PTX-only group and saline group, treatment with PTX/β-ELE moderately delayed tumor progression, most likely due to increased synergy and sensitivity to chemotherapy with β-ELE. As shown in Fig. 5C, the M1@PLGA/PTX/β-ELE nanoparticles group exhibited the most pronounced therapeutic effect, demonstrating enhanced antitumor activity attributed to facilitated accumulation and cellular internalization. Morphology and weight of tumors were observed after 18 days of treatment (Fig. 5B). Significant suppression in size of isolated tumor tissues in the M1@PLGA/PTX/β-ELE group was observed compared to the others (Fig. 5D).Fig. 5 In vivo therapeutic efficacy of M1@PLGA/PTX/β-ELE. (A) Schematic representation of the treatment for tumor eradication; (B) Digital photos of tumor size on day 18; (C) Average tumor growth curves; (D) tumor weight during PTX, PTX/β-ELE, PLGA/PTX/β-ELE and M1@PLGA/PTX/β-ELE treatment on day 18. (E) Body weight variation of tumor-bearing rats during drug treatment (n = 5). The plasma biochemical analysis of AST/ ALT (F), ALP (G) and creatinine (E) at the endpoint. Data are represented as mean ± SD. *P < 0.05, and **P < 0.01 represents significance compared to the control group or as indicated.

Fig. 5

To further validate the antitumor effects, tumor sections were subjected to hematoxylin and eosin (H&E) staining, transferase-mediated dUTP nick end labeling (Tunel) fluorescence staining, HIF-1α, and Ki-67 antigen staining after sacrificing the mice. As depicted in Fig. 6A H&E staining results revealed evident destruction of cancer cells and nuclear shrinkage upon treatment with M1@PLGA/PTX/β-ELE. In comparison to the PTX-only group, these nanoparticles exhibited significant induction of cell apoptosis at an isodose injection due to the synergistic effect of β-ELE and PTX. The therapeutic efficacy of M1@PLGA/PTX/β-ELE was further confirmed by Ki67 (Fig. 6B) and Tunel assays (Fig. 6D). Over the course of 18 days of treatment, there was a substantial reduction in HIF-1α expression levels following treatment with M1@PLGA/PTX/β-ELE compared to the untreated group (P < 0.01). This decrease in HIF-1α expression may be attributed directly or indirectly to the inhibition of PI3K/Akt pathway activation. Therefore, M1@PLGA/PTX/β-ELE demonstrated remarkable suppression of tumor growth by promoting apoptosis and regulating the tumor microenvironment.Fig. 6 Histological analysis of tumor in CaSki-tumor-bearing mice treated with various treatments. (A) Histochemical analysis in tumors treated with M1@PLGA/PTX/β-ELE. Micrographs of H&E, Ki67, HIF-1α and Tunel fluorescence stained from different groups on day 18. Scale bar: 100 μm. Quantitative analysis of (B) Ki67, (C)HIF-1α amount and (D) Tunel fluorescence intensity. Data are represented as mean ± SD. *P < 0.05, **P < 0.01 representing significance compared to the control group.

Fig. 6

Moreover, the biological safety of therapeutic drugs was assessed. Following treatment with PTX and β-ELE, a slight decrease in body weight of the mice was observed (Fig. 5E). The changes in AST/ALT blood indices in the PTX/β-ELE group may suggest hepatic dysfunction, whereas no significant alterations were found in plasma biochemistry from mice treated with M1@PLGA/PTX/β-ELE (Fig. 5F). Furthermore, H&E staining revealed no visible tissue damage in major organs (heart, liver, spleen, lung, and kidney), confirming the biosafety and potential application of our membrane-coated formulations (Fig. S6).

4 Discussion and conclusion

In this study, we have developed a macrophage membrane-coated nanoparticle (M1@PLGA/PTX/β-ELE) for efficient tumor-targeted drug delivery and synergistic specific therapy. The cell membrane coating technology enables the replication of unique biological functions, such as homing from macrophages, thereby significantly enhancing tumor microenvironment penetration and tumor accumulation (Hu et al., 2020). Tumor sites secrete cytokines to recruit monocytes, which subsequently differentiate into macrophages at the site of inflammation. Additionally, chemokine receptors play a crucial role in recruiting macrophages to inflammatory sites. Therefore, by coating the macrophage membrane with nanoparticles, it becomes possible to actively target the inflammatory site, resulting in enhanced distribution and therapeutic efficacy. Many studies have demonstrated that macrophage membrane-coated nanoparticles possess exceptional targeting capabilities towards various tumors by improving drug bioavailability and controlling drug release (Rayamajhi et al., 2019; Xiao et al., 2021; Yao et al., 2024b). Wang et al. conducted an extensive investigation on the precise mechanism underlying tumor targeting by macrophage membrane-coated nanoparticles and uncovered the in vivo behavior of these kinds of nanoparticles (Wang et al., 2015). Furthermore, synergistic therapy could also be achieved by these kinds of nanoparticles due to their promising tumor site-targeted delivery (Hu et al., 2020). These findings highlighted the potential of utilizing macrophage membrane-coated nanoparticles for targeted tumor treatment. Our membrane-coated nanoparticle (M1@PLGA/PTX/β-ELE) exhibits desirable properties including uniform distribution, high stability, efficient delivery, and sustained release behavior. Interestingly, there is a difference in tumor tropism ability between M0m and M1m coated nanoparticles due to the upregulation of membrane surface markers (proinflammatory cytokines) after LPS maturation. Based on the alterations of various tumor-associated macrophage markers within the tumor microenvironment (Wang et al., 2022a), we propose that inflammatory chemotaxis may be a key factor contributing to increased targeting efficiency. Mechanistically, we demonstrate that cellular uptake of M1@PLGA/PTX/β-ELE relies on a comprehensive pathway involving macropinocytosis-dependent and ATP-dependent processes.

In vitro pharmacodynamics study, Hela cells (a high sensitivity cell type) and CaSki cells (a resistant cell type) were selected to evaluate the synergistic sensitivity of PTX&β-ELE. The specific anti-tumor mechanism of M1@PLGA/PTX/β-ELE involves multisite suppression of survival signaling proteins, such as Akt and its downstream pathways. Addition of β-elemene effectively increased the sensitivity of human cervical cancer cells to paclitaxel. Our findings are consistent with previous reports that β-ELE mediates multidrug resistance (MDR) by downregulating expression of ABC transporter proteins to inhibit efflux function (Guo et al., 2014). ATP binding transfers ABC transport protein from cytoplasm to extracellular compartment, which is associated with drug-resistant cell types. Meanwhile, M1@PLGA/PTX/β-ELE nanoparticles are transported into the cell via an ATP-mediated pathway, and inhibition of efflux proteins contributes to enhanced retention and antitumor effects. Additionally, regulation of tumor microenvironment also benefits inhibition of tumor progression through suppression of HIF-1α pathway for anti-angiogenesis and reversal immunosuppressive environment via polarization from anti-inflammatory M2 into proinflammatory M1 macrophages.

We have successfully validated the M1@PLGA/PTX/β-ELE nanoplatform, which exhibits enhanced efficacy and biosafety for tumor-specific therapy in CaSki tumor-bearing BALB/c mice through targeted delivery and combination therapy. Therefore, our study presents a combinatorial therapeutic strategy involving sensitizing chemotherapy and a novel codelivery nanoplatform for malignant cancers.

CRediT authorship contribution statement

Yi Wang: Investigation. Jiakun Wang: Methodology. Yang Ding: Methodology. Leyao Lv: Software. Yuhao Zhu: Software. Nuo Chen: Validation. Yingyi Zhao: Visualization. Qing Yao: Writing – review & editing, Conceptualization. Shengjie Zhou: Resources. Mei Chen: Software. Qibing Zhu: Validation. Lifeng Li: Validation. Fengyun Chen: Supervision, Project administration, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

Supplementary material: Characterization, in vivo accumulation, biosafety, and effect on macrophage repolarization and P-gp function of the nanoparticle reported here.

Image 1

Data availability

Data will be made available on request.

Acknowledgments

We appreciate Professor Xueqiong Zhu for her guidance and support in the design of this project as well as valuable suggestions for revising the article. We also thank Scientific Research Center of Wenzhou Medical University for consultation and instrument availability that supported this work.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpx.2024.100276.
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