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Paclitaxel-Loaded Macrophage Membrane Camouflaged Albumin Nanoparticles for Targeted Cancer Therapy

Authors Cao X, Tan T, Zhu D, Yu H, Liu Y, Zhou H, Jin Y, Xia Q

Received 5 January 2020

Accepted for publication 10 March 2020

Published 19 March 2020 Volume 2020:15 Pages 1915—1928

DOI https://doi.org/10.2147/IJN.S244849

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr Mian Wang

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Xi Cao,1,2 Tingfei Tan,1,2 Dongchun Zhu,1,2 Haixia Yu,1,2 Yaru Liu,1,2 Haiyun Zhou,1,2 Yong Jin,3,4 Quan Xia1,2

1Department of Pharmacy, The First Affiliated Hospital of Anhui Medical University, Hefei, People’s Republic of China; 2The Grade 3 Pharmaceutical Chemistry Laboratory of State Administration of Traditional Chinese Medicine, Hefei, People’s Republic of China; 3Key Laboratory of Anti-Inflammatory and Immune Medicines, Ministry of Education, School of Pharmacy, Anhui Medical University, Hefei, People’s Republic of China; 4The Key Laboratory of Major Autoimmune Diseases, Anhui Province, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei, People’s Republic of China

Correspondence: Quan Xia
Tel +86 551 62922154
Email [email protected]

Background: Melanoma is the most common symptom of aggressive skin cancer, and it has become a serious health concern worldwide in recent years. The metastasis rate of malignant melanoma remains high, and it is highly difficult to cure with the currently available treatment options. Effective yet safe therapeutic options are still lacking. Alternative treatment options are in great demand to improve the therapeutic outcome against advanced melanoma. This study aimed to develop albumin nanoparticles (ANPs) coated with macrophage plasma membranes (RANPs) loaded with paclitaxel (PTX) to achieve targeted therapy against malignant melanoma.
Methods: Membrane derivations were achieved by using a combination of hypotonic lysis, mechanical membrane fragmentation, and differential centrifugation to empty the harvested cells of their intracellular contents. The collected membrane was then physically extruded through a 400 nm porous polycarbonate membrane to form macrophage cell membrane vesicles. Albumin nanoparticles were prepared through a well-studied nanoprecipitation process. At last, the two components were then coextruded through a 200 nm porous polycarbonate membrane.
Results: Using paclitaxel as the model drug, PTX-loaded RANPs displayed significantly enhanced cytotoxicity and apoptosis rates compared to albumin nanoparticles without membrane coating in the murine melanoma cell line B16F10. RANPs also exhibited significantly higher internalization efficiency in B16F10 cells than albumin nanoparticles without a membrane coating. Next, a B16F10 tumor xenograft mouse model was established to explore the biodistribution profiles of RANPs, which showed prolonged blood circulation and selective accumulation at the tumor site. PTX-loaded RANPs also demonstrated greatly improved antitumor efficacy in B16F10 tumor-bearing mouse xenografts.
Conclusion: Albumin-based nanoscale delivery systems coated with macrophage plasma membranes offer a highly promising approach to achieve tumor-targeted therapy following systemic administration.

Keywords: macrophage membrane, albumin nanoparticles, melanoma, paclitaxel, tumor-targeted therapy

Introduction

Melanoma is a malignancy deriving from melanocytes, and the most common symptom is aggressive skin cancer.1,2 In recent years, the incidence of malignant melanoma has continued to increase, and it has become a serious health concern worldwide.3,4 Additionally, the metastasis rate of malignant melanoma remains high, and it is highly difficult to cure with the currently available treatment options.5 With the rapid advances in cancer diagnostics and treatment options, early stage melanoma is often curable after surgery, while advanced melanoma remains challenging to treat.4,6 Ipilimumab, dabrafenib, trametinib, and vemurafenib are the first-line therapeutic drugs for advanced melanoma; however, side effects are commonly observed, and the cost of treatment remains exceedingly high.7 Thereby, alternative treatment options are in great demand to improve the therapeutic outcome against advanced melanoma.

Over the past few decades, nanoscale delivery systems have become a popular research topic for the targeted therapy of solid tumors. Nanoparticles have high drug-loading capacity, good storage stability, enhanced cytotoxicity, and may circumvent the multidrug resistance in tumor cells.811 With a well-designed nanoparticle shell, drug-loaded nanoparticles are indicated to prolong the circulation of small molecule drugs in the blood circulation, which could facilitate the extravasation and passive targeting to the tumor site with an enhanced permeability and retention effect.11,12 To achieve tumor-targeted drug delivery, a common strategy is to modify nanoparticles with targeting ligands such as antibodies and peptides.1315 These targeting ligands could bind with the receptors specifically overexpressed in cancer cells and mediate targeted delivery. Chen et al fabricated anisamide-modified cationic liposomes to systemically deliver small interfering RNA to treat melanoma,16 and the anisamide could bind with Sigma 1 receptors associated with melanoma cells to mediate the specific delivery of cargos to the tumor cells. However, problems such as the complex chemistry involved in the modification step remain to be explored, as well as how nanoparticles can escape from the rapid clearance by the reticuloendothelial system and achieve prolonged circulation.

Recently, increasing attention has been focused on the nanocarrier delivery system coated with different membranes for targeted disease therapy.1720 For example, cancer cell membrane-coated nanoparticles have been used for anticancer vaccination and drug delivery.21 Erythrocyte-platelet hybrid membrane-coated nanoparticles successfully prolonged the circulation time of nanoparticles in vivo.22 Moreover, emtansine liposomes coated with macrophage membranes were demonstrated to improve delivery to cancer metastatic sites.20 With the increasing knowledge of cancer biology, some inflammatory factors have been recognized as associated with malignant growth, which may further promote and enhance tumor progression.23 Neutrophils can be recruited to the inflammation site in response to a granulocyte colony-stimulating factor in the inflammatory microenvironment of the tumor.24,25 A recent study demonstrated that neutrophil-mimicking nanoparticles could effectively inhibit the formation of cancer metastatic niches by targeting the circulating cancer stem cells.26 Moreover, Xue et al reported that viable neutrophils carrying paclitaxel-loaded liposomes successfully suppressed postoperative glioma recurrence.27 However, there are no reports available on the treatment of melanoma using such strategies.

Here, we reported albumin nanoparticles coated with macrophage plasma membranes as a means to achieve targeted therapy against malignant melanoma. Among a number of nanocarriers, albumin nanoparticles have received great attention in both academia and industry due to their noticeable advantages as a pharmaceutical carrier, including biodegradability, biocompatibility, high chemical stability, and versatility and nonimmunogenicity (human source).2830 In particular, albumin nanoparticles could preferentially accumulate in tumor sites due to their remarkable targetability to tumors.31 They permeate leaky blood vessels via an efficient extravasation process.32 Thus, whether the coating of plasma membrane can improve the uptake efficiency and targeting therapy of albumin nanoparticles remains to be explored. Paclitaxel (PTX) is a white crystalline powder isolated from the Pacific Yew (Taxus brevifolia). It is one of the most effective chemotherapeutics and is mainly used to treat lung cancer, ovarian cancer, and breast cancer;3335 however, its high toxicity and insolubility in aqueous solution limit its applications. In this study, albumin nanoparticles loaded with PTX (ANPs/PTX) were fabricated and decorated with macrophage membranes (RANPs/PTX). The cellular uptake efficiency of RANPs in different kinds of tumor cells, including B16F10 cells, MCF-7 cells, 4T1 cells, and A549 cells, was characterized. The uptake efficiencies were significantly improved among these cells, and murine B16F10 melanoma was chosen as the disease model to explore the treatment efficiency of the preparations. The in vitro antitumor effect of RANPs/PTX was evaluated in terms of cytotoxicity, cell cycle, and apoptosis rate in B16F10 cells. To evaluate the tumor-specific accumulation of RANPs, a biodistribution study in a tumor-bearing mouse xenograft model was also performed. Moreover, we preliminarily explored the antitumor efficacy against melanoma in the B16F10 tumor-bearing mouse xenografts.

Materials and Methods

Materials

Paclitaxel (PTX) was purchased from Xi’an Haoxuan Biotechnology Co., Ltd. (Xi’an, China). Human serum albumin was obtained from Yuanda Shuyang Pharmaceutical (Chengdu, China). Soybean oil was obtained from BASF (Ludwigshafen, Germany). 4ʹ,6-Diamidino-2-phenylindole (DAPI) was purchased from Beyotime (Haimen, China). 1,1ʹ-Dioctadecyl-3,3,3ʹ,3ʹ-tetramethylindodicarbocyanine perchlorate (Did) was provided by Sigma-Aldrich (St. Louis, MO, USA). All other reagents and chemicals used were of analytical grade.

Cell Culture and Animals

Cell Culture

Murine B16F10 melanoma cells, murine 4T1 breast cancer cells, human MCF-7 breast cancer cells, murine A549 lung cancer cells, dendritic cells (DC), and macrophage cells (RAW 264.7) were obtained from the Cell Bank of Chinese Academy of Sciences (CAS, Shanghai, China). All the cells were cultured in RPMI 1640 with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin. The cells were kept at 37°C in a humidified atmosphere containing 5% CO2.

Animals

Female C57BL/6 mice (18–22 g) were purchased from Anhui Medical University (Hefei, China) and maintained under standard housing conditions. All animal protocols were approved by the Ethics Committee of Anhui Medical University (Laboratory animal-General guidelines for health monitoring T/CALAS 3–2017).

Preparation of Macrophage Membrane-Coated ANPs (RANPs)

To prepare RANPs, purified macrophage cell membranes were first collected. We used RAW 264.7 mouse macrophage cells as the model cell line. Membrane derivations were achieved by using a combination of hypotonic lysis, mechanical membrane fragmentation, and differential centrifugation to empty the harvested cells of their intracellular contents. The collected membrane was then physically extruded through a 400 nm porous polycarbonate membrane to form macrophage cell membrane vesicles. Concurrently, albumin nanoparticles were prepared through a well-studied nanoprecipitation process.36 In brief, human serum albumin (HSA) was dissolved in water. PTX and soybean oil were dissolved in dichloromethane and ethyl acetate. These solutions consisting of aqueous and organic phases were gently mixed and evaporated. To encapsulate the albumin nanoparticles within the macrophage cell membrane, the two components were then coextruded through a 200 nm porous polycarbonate membrane.

To determine the encapsulation efficiency, unentrapped PTX was separated by filtering through a 0.22 um membrane filter (We). The amount of PTX encapsulated within nanoparticles was determined by LC-MS/MS as previously reported.37 An equal amount of micelles were dissolved in ethanol and subjected to LC-MS/MS analysis to determine the total amount of PTX (Wt). One milliliter of micelle was dried under vacuum to obtain the total weight of micelles (Wd). Encapsulation efficiency (EE) was calculated according to the equations as follows:

Cellular Uptake and Internalization Pathways

B16F10 cells, 4T1 cells, MCF-7 cells, and A549 cells were cultured in 12-well plates (2 × 105 cells per well) for 24 hrs. Did solution, ANPs/Did, and RANPs/Did at an equivalent dose of 5 μg/mL of Did were added into each well and coincubated with B16F10 cells for 0.5 hr, 1 hr, and 2 hrs. At the given time points, the cells were washed three times with PBS. After centrifugation, cells were analyzed by flow cytometry (Beckman Coulter, USA). Then, the cells were fixed in 4% paraformaldehyde (20 min) and permeabilized in 0.5% Triton X-100 (20 min), which was followed by DAPI staining (5 min); the cells were then subjected to observation under confocal laser scanning microscope (CLSM) (Leica, Wetzlar, Germany).

To elucidate the internalization pathways of ANPs and RANPs in B16F10 cells, both nanoparticles were preincubated with selected endocytosis inhibitors, including amiloride (25 μM), chlorpromazine (20 μM), verapamil (6.5 mM) and colchicine (100 mM) for 1 hr at 37°C before the uptake study. Next, B16F10 cells were subjected to incubation at 37°C for another 1 hr after addition of ANPs/Did and RANPs/Did. The cells were then collected and subjected to flow cytometry analysis to determine the uptake efficiency.

Lysosomal Staining and Subcellular Distribution

To explore the subcellular localization behavior of nanoparticles, cell endosomes and lysosomes were labeled with LysoTracker Red (ThermoFisher Scientific, USA). Briefly, with the addition of ANPs/Coumarin 6 (C6) and RANPs/Coumarin 6 (C6) (5 μg/mL C6 per well), B16F10 cells were, respectively, incubated for 4 hrs and 24 hrs and were then washed with PBS and stained with LysoTracker Red for 30 min prior to CLSM observation.

Cytotoxicity Assay

Viabilities of B16F10 cells after treatments with blank ANPs, blank RANPs, Taxol, ANPs/PTX, and RANPs/PTX were evaluated by standard MTT assay.39 B16F10 cells were inoculated in 96-well plates (Corning, NY, USA) (1 × 104 cells per well). After 24 hrs, serum-free culture medium containing varying concentrations of blank ANPs, blank RANPs, Taxol, ANPs/PTX, and RANPs/PTX were incubated with cells for another 24 hrs. The mass concentration of albumin in blank ANPs and blank RANPs was kept the same as to the mass concentration of albumin in PTX-loaded ANPs and RANPs. Then, 20 μL of 5 mg/mL MTT solution was added into each well and incubated for 4 hrs in dark at 37°C. The medium was then discarded, and 200 μL of dimethyl sulfoxide was added to dissolve the formazan crystals in each well. The absorbance was measured at 570 nm on a microplate reader (Thermo Scientific, USA).

Tumor-Related Immune Cellular Uptake

Dendritic cells (DC) and RAW 264.7 cells were cultured in 12-well plates (2 × 105 cells per well) for 24 hrs. Did solution, ANPs/Did, and RANPs/Did at an equivalent dose of 5 μg/mL of Did were added into each well and coincubated with B16F10 cells for 1 hr, 2 hrs, and 4 hrs. At given time points, cells were washed three times with PBS. After centrifugation, the cells were analyzed by flow cytometry (Beckman Coulter, USA).

Cell Cycle and Cell Apoptosis and Necrosis

Cell-cycle distributions were assessed by propidium iodide (PI) staining as described in previous studies.38 Briefly, B16F10 cells were seeded in six-well plates at 3 × 105 cells per well and incubated for 48 hrs at 37°C. Cells were then incubated with 2 mL of medium containing different concentrations of Taxol, ANPs/PTX, and RANPs/PTX for another 24 hrs. Subsequently, B16F10 cells were rinsed three times with PBS and fixed in cold 75% ethanol for 30 min at 4°C. After two PBS washes, the cells were incubated with 0.1 mL of 0.1% Triton X-100 for 15 min and 20 μg/mL ribonuclease A at 37°C for another 30 min; then, the cells were stained with 20 μg/mL PI at 4°C for another 30 min. All cell samples were then analyzed by flow cytometry (Cytomics FC500, Beckman Coulter, CA, USA).

B16F10 cells were seeded in six-well plates and cultured until reaching 40–50% confluence. Cells were then incubated with 2 mL of medium containing 1 mg/mL Taxol, ANPs/PTX, and RANPs/PTX for 12 hrs. Subsequently, the cells were rinsed with PBS twice and cultured with serum-free medium for another 48 hrs. The cells were then collected and stained with annexin V-FITC and propidium iodide (PI) according to the manufacturer’s instructions (BD, USA). Stained cells were subjected to flow cytometry analysis to determine the percentages of apoptotic cells.

Biodistribution in Melanoma Xenograft Mice Model

B16F10 tumor-bearing female C57BL/6 mice were randomly divided into three groups (n = 3): Did solution, ANPs/Did, and RANPs/Did at an equivalent dose of 0.015 mg/kg, and saline was used as control. When solid tumors reached ~150 mm3, mice were tail-vein injected with Did solution, ANPs/Did, and RANPs/Did. After that, mice were subjected to anesthesia following i.p. injection of chloral hydrate, and ex vivo imaging was performed using IVIS® Spectrum (Caliper Life Sciences, PerkinElmer, USA) at 1 hr, 4 hrs, and 24 hrs post-injection of nanoparticles and Did solution. At given time points, mice were sacrificed, and their vital organs were excised for ex vivo fluorescence imaging.

In vivo Antitumor Efficacy

A total of 1 × 106 B16F10 cells were injected subcutaneously into the right flanks of female C57BL/6 mice. After one week, mice were randomly divided into four groups (n = 6), including saline, Taxol, ANPs/PTX, and RANPs/PTX. All PTX containing treatment groups were intravenously administered with the given formulation at an equivalent dose of 1 mg/kg PTX. Ten days after tumor inoculation, all treatments were administered via tail vein injection once every other day four consecutive times. The tumor diameters were measured every other day. Tumor volume (V) was then calculated using the following formula: volume = (L × W2)/2. Length (L) and width (W) represent the longest and shortest diameter, respectively. To evaluate proliferation, apoptosis, and neovascularization, immunohistochemical staining was performed on paraffin-embedded tumor sections.

Statistical Analysis

All data were presented as the means ± standard deviations (S.D.). For comparison among multiple groups, one-way ANOVA was performed, followed by a Tukey post hoc analysis. A p-value of less than 0.05 was considered statistically significant.

Results

Characterization of ANPs and RANPS

The hydrodynamic diameter of the membrane was less than 300 nm as measured by dynamic light scanning, and ANPs displayed an average size distribution of 138.7 ± 3.5 nm (Table 1). Upon fusion of the membrane vesicles with the albumin nanoparticles, the final average size of RANPs was 188.7 ± 4.8 nm (Figure 1C), which is between the size of ANPs and the membranes. For the surface charge, RANPs showed an average charge of −10.5 mV. Zeta potential measurements also suggested successful coating. Specifically, after being coated, the surface charge of the albumin cores increased to approximately the level of the membrane vesicles (Table 1). The membrane coating around the polymer nucleus was observed by transmission electron microscopy (TEM). RANPs were spherical and showed a core-shell structure using negative staining (Figure 1C). On the basis of some previous studies on membrane-coated nanoparticles,39 it was expected that the coating on RANPs would transmigrate faithfully to the surface of the nanoparticles as a membrane bilayer, resulting in the right outer conformation allowing the membrane to maintain its ability. Additionally, the encapsulation efficiencies of ANPs/PTX and RANPS/PTX were found to be 93.5 ± 2.1% and 83.4 ± 2.2%, respectively, demonstrating that the formulations can further be used in the antitumor study both in vitro and in vivo.

Figure 1 Size distribution and morphology of nanoparticles: Dynamic light scattering (DLS) size measurement and TEM image of (A) ANPs, (B) membranes, and (C) RANPs. Scale bar represents 100 nm.

Table 1 Characterization of ANPs, Membrane, and RANPs. Data Represent Mean ± SD (n = 3)

Cellular Uptake Efficiency, Internalization Pathways, and Intracellular Distribution

To investigate the cellular uptake efficiency of ANPs and RANPs, a near-infrared fluorescence probe (Did) was used instead of PTX. Did-loaded nanoparticles such as ANPs/Did and RANPs/Did were fabricated following the same method of PTX-loaded nanoparticles as described in the method section. To evaluate whether RANPs could be specifically taken up by tumor cells, we chose four kinds of tumor cells (4T1, A549, MCF-7, and B16F10 cells). According to the flow cytometry results, both ANPs/Did and RANPs/Did showed time-dependent increases in the fluorescence intensity in four tumor cells (Figure 2). At all given time points, RANPs/Did showed significantly higher uptake efficiencies (p < 0.05) compared to ANPs/Did in tumor cells. Additionally, in B16F10, the uptake efficiency of RANPS/Did was 3.41 times higher than that of cells treated with ANPs/Did at 0.5 hr, 4.3 times higher at 1 hr, and 5.88 times higher at 2 hrs, respectively (p < 0.05). Consistent with quantitative flow results, CLSM images showed a greatly enhanced intracellular distribution of Did fluorescence in the RANPs groups, and Did fluorescence intensity increased over time; in the ANPs/Did group, the fluorescence intensity remained much weaker (Figure 3A).

Figure 2 Time-dependent cellular uptake of ANPs/Did and RANPs/Did after incubation with B16F10 (A), A549 (B), 4T1 (C)and MCF-7 cells (D) for 0.5 hr, 1 hr, and 2 hrs as determined by flow cytometry analysis. Data represent means ± SD (n = 3). *p < 0.05 vs control. #p < 0.05 vs ANPs/Did.

Figure 3 (A) Representative confocal laser scanning microscopy images of B16F10 cells after incubation with ANPs/Did and RANPS/Did for 0.5 hr, 1 hr, and 2 hrs. DAPI stains for cell nuclei (blue); Did represents NPs or RNPS (red). Scale bar represents 100 µm. (B, C) Internalization pathways of ANPs and RANPs in B16F10 cells. B16F10 cells were preincubated under various conditions, including 4°C, chlorpromazine, verapamil, amiloride, and colchicine. Data represent means ± SD (n = 3). *p < 0.05 vs 37°C.

To elucidate the internalization pathways of RANPs/Did in B16F10 cells, classic endocytosis inhibitors were selected.40 The uptake efficiencies of ANPs/Did and RANPs/Did at 4°C were much lower than at 37°C (p < 0.05), indicating that the internalization processes of both ANPs and RANPs were energy-driven. Additionally, both chlorpromazine and verapamil treated groups exhibited significant uptake reduction compared to the control (p < 0.05), while other endocytosis inhibitors such as amiloride did not affect the uptake efficiency, which suggested that the endocytosis of both nanoparticles was mainly mediated by clathrin-dependent, caveolae-independent and macropinocytosis-independent pathways (Figure 3B and C).

Next, the intracellular distribution profiles of ANPs and RANPs in B16F10 cells were studied using CLSM. In the following study, coumarin 6 (C6) was selected as the model compound to demonstrate the intracellular distribution pattern of nanoparticles. LysoTracker Red was used to label lysosomes in B16F10 cells. At given time points, both ANPs and RANPS showed internalization in B16F10 cells (Figure 4). Notably, RANPs showed more enhanced fluorescence intensity than ANPs at 2 hrs (Figure 4B), which is consistent with the flow cytometry results. At 24 hrs, both ANPs and RANPS showed extensive intracellular distributions in the cytoplasm (Figure 4); the green and red fluorescence overlapped significantly, suggesting a lysosomal pathway after internalization. Regarding the internalization behavior and intracellular distribution pattern, no obvious differences were observed between ANPs and RANPs.

Figure 4 Representative confocal images of B16F10 cells incubated with coumarin 6-loaded nanoparticles (ANPs/C6 (A) and RANPs/C6 (B)) and LysoTracker Red for 1 hr, 2 hrs, and 24 hrs. Scale bar represents 20 µm.

Immune Cell Uptake Efficiency in Tumor Microenvironment

It is now widely believed that the immune system is also capable of constantly checking and removing precancerous cells to prevent the development of melanoma.41 DC cells and RAW264.7 cells, as two important immune cell types in the tumor microenvironment, were chosen to investigate the uptake efficiency of RANPs. According to flow cytometry results, both ANPs/Did and RANPs/Did showed time-dependent increases in the fluorescence intensity in the two types of immune cells. At all given time points, RANPs/Did showed significantly higher uptake efficiencies (p < 0.05) compared to ANPs/Did in tumor cells (Figure 5). The results showed that the preparation could be specifically ingested by immune cells, thereby may affect tumor growth at the immune level.

Figure 5 Time-dependent cellular uptake of ANPs/Did and RANPs/Did after incubation with DCs (A) and RAW 264.7 cells (B) for 0.5 hr, 1 hr, and 2 hrs as determined by flow cytometry analysis. Data represent means ± SD (n = 3). *p < 0.05 vs control. #p < 0.05 vs ANPs/Dids.

Cytotoxicity

First, cell viability of blank ANPs and blank RANPS against B16F10 cells was evaluated by standard MTT assay. Both blank NPs and RANPS displayed no obvious cytotoxicity against B16F10 cells at all concentrations under investigation (Figure 6), indicating nanoparticle vehicles were of minimum cytotoxicity against B16F10 cells. At each concentration under investigation, both ANPs/PTX and ARNPs/PTX showed much higher inhibitory effect compared to PTX solution in B16F10 cells after 24 hrs of treatment. Moreover, RANPs/PTX showed greater cytotoxicity as compared to ANPs/PTX (Figure 6).

Figure 6 Cell viability of B16F10 after 24 hrs of treatment of blank ANPs, blank RANPs, Taxol, ANPs/PTX, and RANPs/PTX with varying concentrations. Data represent mean ± SD (n = 3). *p < 0.05 vs control. #p < 0.05 vs ANPs/Did.

Cell Cycle and Cell Apoptosis

PTX has been proven to impact the cell cycle by arresting cells in the G2/M phase.42 To estimate the in vitro antimitotic effect of ANPs and RANPs at the cellular level, cell cycle analysis was performed, and the results are presented in Figure 7. Compared to Taxol, ANPs/PTX and RANPs/PTX arrested fewer cell populations in the G0/G1 phase (p < 0.05) and more cells in the G2/M phase (p < 0.05). Furthermore, ANPs/PTX-treated groups showed significantly more cell populations in the sub-G1 phase than that in the Taxol-treated group (p < 0.05). Because the sub-G1 phase is a characteristic hypodiploid DNA content peak representing apoptotic and necrotic cell populations, more cells in the sub-G1 phase in the RANPs/PTX-treated group may indicate more cells likely undergo apoptosis. Further, RANPs/PTX could induce more cell apoptosis, which is likely due to the coating of the macrophage cell membrane.

Figure 7 (A) Cell cycle distributions of B16F10 cells after various treatments with equivalent doses of PTX. (B) The apoptotic cell percentages of B16F10 cells after various treatments of blank ANPs, blank RANPs, Taxol, ANPs/PTX, and RANPs/PTX. An equivalent dose of 1 μg/mL PTX was used, and the concentrations of blank nanoparticles were maintained the same as the PTX-loaded nanoparticles. Data represent means ± SD (n = 3). *p < 0.05 vs Taxol, #p < 0.05 vs ANPs/PTX.

Biodistribution of RANPs/Did in Melanoma Xenograft Model

To gain insight into biodistribution of RANPs, ex vivo fluorescence imaging was performed in B16F10 bearing C57BL/6 mice at 1 hr, 4 hrs, and 24 hrs after systemic administration of ANPs/Did and RANPs/Did. The biodistribution of the nanoparticles in major organs and tumors at the given time points is shown in Figure 8. Among two treatment groups, dramatic accumulations of fluorescence signals in the liver and kidney were observed (Figure 8A). Regarding Did distribution in the tumor tissues, RANPs/Did group displayed remarkably higher accumulation at the tumor site than ANPs/Did group after 24 hrs, indicating RANPs have greater tumor-specific distribution (Figure 8C). Meanwhile, RANPs/Did also displayed the highest fluorescence intensity in blood after 24 hrs (Figure 8B), suggesting a prolonged circulation of RANPs as compared to ANPs.

Figure 8 Biodistribution profiles of Did-loaded nanoparticles in melanoma xenograft mice. Did-loaded nanoparticles were administered via tail vein injection at an equivalent dose of 0.015 mg/kg. (A) Ex vivo fluorescence imaging of major organs at 1 hr, 2 hrs, and 24 hrs after injection. (B) Ex vivo imaging of blood at given time points. (C) Ex vivo imaging of excised tumors at the given time points.

In vivo Antitumor Efficacy and Systemic Toxicity

To explore the antitumor efficacy of RANPs/PTX in vivo, melanoma xenograft models were established in C57BL/6 mice. As shown in Figure 9, the RANPs/PTX-treated group showed remarkably better treatment effects than the saline control, Taxol, and ANPs/PTX-treated group, presenting the smallest tumor volumes among treatment groups at all given time points (p < 0.05) (Figure 9A and B). Although the tumor inhibition rate of ANPs/PTX and RANPs/PTX was distinctly higher than that of the Taxol-treated group, the RANPs/PTX-treated group showed the highest tumor inhibition rate among all treatment groups (Figure 9D). On the other hand, the weight changes and morphology of the different treatment groups were not significant (Figure 9C and E), revealing reduced systemic toxicity of the nanoparticles.

Figure 9 The antitumor efficacy of PTX-loaded nanoparticles in C57BL/6 mice bearing B16F10 melanoma xenografts. (A) Images of excised B16F10 tumors collected from each treatment. (B) Average tumor volumes after treatment over the investigative period (n = 5). *p < 0.05 vs Taxol, #p < 0.05 vs ANPs/PTX. (C) Bodyweight variations of mice bearing B16F10 melanoma xenografts after each treatment over time. (D) Tumor inhibition rate after treatment. (E) Morphology of RANPs/PTX-treated groups. Sections were isolated and stained with hematoxylin and eosin (H&E) for histopathological analysis. Scale bar represents 100 μm. Data represent means ± SD (n = 5).

Discussion

With increasing understanding of the biomimetic science, nanoscale delivery systems coated with plasma membranes have attracted extensive attention in the past few years. Thus far, nanoparticles coated with cancer cell membranes, bacterial membranes, red blood cell membranes, leukocyte membranes, and macrophage membranes have been reported for the delivery of small molecule drugs, antibodies, and vaccines. Various technologies have been developed to fabricate plasma membrane-coated nanoparticles. For example, the self-assembly of leukocyte membrane-coated nanoparticles was driven by electrostatic and hydrophobic interactions between the negatively charged proteo-lipid patches and the positively charged nanoparticle surface under continuous rotation, while emtansine liposomes coated with macrophage membranes were fabricated by a direct extrusion method. These “biomimetic camouflage” strategies utilize the function of plasma membranes to help nanoparticles escape from the immune system and cross the biological barriers. Moreover, nanoparticles coated with plasma membranes possess cell-like properties and behaviors, thus increasing the affinity toward target cells and cell membrane interactions.

In this study, we successfully prepared PTX-loaded albumin nanoparticles coated with macrophage membranes to achieve targeted therapy of melanoma in vivo. PTX has demonstrated potent tumor inhibitory effects against a variety of tumor cells. For example, PTX could successfully suppress A549 cancer growth.43 Additionally, PTX can inhibit cancer cell invasion via suppression of NF-ĸB-mediated matrix metalloproteinase-9 expression.44,45

In our study, compared to ANPs/PTX without a macrophage membrane coating, RANPs/PTX showed improved antitumor efficacy both in vitro and in vivo (Figure 6). First, the cell uptake efficiency of RANPs in B16F10 cells and three other tumor cells was significantly increased when coated with macrophage membranes compared to that of ANPs (p < 0.05). However, coating nanoparticles with macrophage membranes did not change the overall internalization pathway and the intracellular distribution pattern of the nanoparticles (Figure 4). This result indicates that the presence of macrophage membranes effectively increased the internalization efficiency of nanoparticles in B16F10 cells without changing its internalization pathway. The increased cell uptake efficiency in tumor cells might be due to the enhanced affinity between RANPs and the tumor cell membrane. The results of uptake of DC and RAW cells also suggest that RANPs may also be involved in tumor immunotherapy due to the specific proteins on the surface of RANPs. Albumin-based cancer drug delivery systems have shown prolonged blood circulation time and high drug accumulation in tumors.46,47 However, albumin-based nanoscale delivery systems have low stability in blood circulation, resulting in premature nanoparticle dissociation and early drug release before reaching the tumor target.48 The biodistribution study using tumor-bearing mice demonstrated that RANPs had relatively longer retention in circulation and selective accumulation in the tumor sites (Figure 8). Nanoparticles coated with plasma membranes can be more easily taken up by tumor cells and showed enhanced internalization efficiency in tumor cells compared to nanoparticles without plasma membrane coatings, which were similar to RBC-membrane-coated nanoparticles.49 These results were also consistent with in vitro cytotoxicity and cellular uptake results, demonstrating that nanoparticles coated with macrophage membranes showed enhanced efficacy both in vitro and in vivo.

In systemic circulation, macrophages recognize nanoparticles coated with macrophage membranes and consider them as a part of the body, thereby escaping clearance by the immune system. Then, the granulocyte colony-stimulating factor in the inflammatory microenvironment of the tumor releases a signal to gather nanoparticles coated with macrophage membranes because of the receptor–ligand interactions.50 Pharmacodynamic studies demonstrated the antitumor efficacy of RANPs/PTX in vivo. These results revealed that RANPs could be targeted to cancer cells. The targeting ability of nanoparticles coated with macrophage membranes is extremely likely because the macrophage membrane coated on albumin nanoparticles may retain inflammation-associated proteins, which may facilitate the accumulation of nanoparticles in the tumor inflammatory microenvironment.51,52

Conclusion

Overall, albumin-based nanoscale delivery systems coated with macrophage plasma membranes remain a highly promising delivery system with a prolonged circulation profile and without premature drug release; further, these systems offer improved tumor targetability that can be applied to various drug compounds with varying physicochemical properties. Specifically, the presence of macrophage membranes on the nanoparticle surface significantly enhanced the cellular uptake efficiency in B16F10 cells without changing their major internalization pathways. Additionally, the presence of macrophage membranes induced more specific accumulation at the tumor site and stronger antitumor efficacy. Taken together, albumin nanoparticles coated with macrophage membranes present a neat and efficient strategy to achieve targeted melanoma chemotherapy.

Acknowledgment

Authors are very grateful for the financial support from Anhui Province Natural Science Foundation (No.1808085MH235).

Disclosure

The authors report no conflicts of interest in this work.

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