Coating a Self-Assembly Nanoconstruct with a Neutrophil Cell Membrane Enables High Specificity for Triple Negative Breast Cancer Treatment

Abstract

Breast cancer is one of the most commonly diagnosed cancers in American women. Triple negative breast cancer is among the most advanced and aggressive forms of breast cancer. Treatment options are limited for such cancers, making chemotherapy a convenient and effective treatment. Although these therapies can reduce morbidity and mortality, it is often followed by systemic side effects or relapse. Nanoparticles (NPs) have been considered for drug delivery approaches due to their ability to target various disease sites. Herein, we aim to develop a biomimetic NP construct (cell membrane-cloaked NPs) that exhibits specific affinity with triple negative breast cancer cells. In this regard, we designed biomimetic supramolecular nanoconstructs composed of a poly(vinyl pyrrolidone)-tannic acid (PVP-TA NPs/ PVT NPs) core and biofunctionalized with neutrophil cell membranes (PVT-NEU NPs). In this study, we have synthesized a PVT-NEU NP construct, characterized it, and evaluated it for improved targeting and therapeutic benefits in in vitro and in vivo models. Analysis of PVT-NEU NPs confirms the presence of the core of PVP-TA NPs coated with activated human neutrophil membranes. The study results confirmed that PVT-NEU NPs demonstrated an enhanced interaction and targeting with the tumor cells, thus improving the therapeutic activity of a model therapeutic agent (paclitaxel). Altogether, this study suggests the potential of biomimetic NPs as a promising therapeutic option for targeted drug delivery for advanced-stage breast cancer and other similar diseased conditions.

Graphical Abstract

1. INTRODUCTION

Breast cancer (BC) remains the second most commonly diagnosed cancer among women in the United States¹ with about 12% probability for women to develop BC in their lifetime. The American Cancer Society estimates for the year 2021 that there are 281,550 new BC cases diagnosed with 43,600 deaths predicted.² There are very few FDA-approved therapies for such aggressive forms of disease, leaving systemic administration of chemotherapeutic agents as the mainstay treatment for advanced BC, metastatic BC, and triple negative BC (TNBC). Chemotherapy with cytotoxic agents such as anthracyclines, taxanes, and platinum-based compounds³ significantly improved the survival rate of patients. However, chemotherapies can lead to adverse reactions such as induced neutropenia, peripheral neurotoxicity, myelosuppression, cardiotoxicity, etc. due to nonspecific distribution. The advancement of nanotechnology-based drug delivery systems has made significant improvements in overcoming conventional limitations.

Nanoparticle (NP)-based drug delivery systems have made a remarkable contribution to cancer treatment. NP-based drug delivery systems offer an enhanced permeability and retention (EPR) effect⁴ that renders them capable of penetrating the leaky tumoral neovasculature allowing NPs containing poorly soluble drugs to circulate longer, deliver site-specific chemotherapeutics,⁵ and improve the tolerability of cytotoxic agents.⁶ However, despite the therapeutic advantages, these exogenous materials suffer clearance by the reticular endothelial system (RES), which hinders tumor penetration by producing subtherapeutic concentrations in conjunction with the presence of a dense extracellular matrix.⁷

The biomimetic NP strategy has been emerging as a new type of nanoplatform that allows incorporation of bioinspired artificial cell membranes or naturally derived cell membranes into synthetic constructs or NPs. Such an interfacial combination can mimic the function of their source cell when interacting with surrounding biological components. The advantages are myriad, due to the utilization of naturally occurring cells which are intrinsically biocompatible, biodegradable, and nonimmunogenic.⁸ Additionally, the presence of proteins, glycans, and sialic acid moieties on the natural cell membrane serves as receptors/ligands for receptor-mediated endocytosis or for suppressing immune attack.⁹ The literature documents a number of cell membrane-coated NPs utilizing membranes derived from platelets and nucleated cells, such as macrophages, neutrophils, beta cells, and cancer cells. Literature reports so far suggest that cell membrane (MEM)-coated NPs inherently possess active self-marker expression that enables them to bind to tumor cells/sites.^10–13

Herein, we report the development of an ideal neutrophil-cloaked NP supramolecular construct for superior tumor-targeted delivery.¹⁴ We examined multiple cellular binding interactions, biological assays, and biodistribution/tumor targeting studies to identify a suitable membrane-cloaked NP construct. Finally, the superior antitumor activity of the neutrophil membrane-cloaked NP construct was verified in an ectopic xenograft tumor, the MDA-MB-231 breast cancer mouse model. Altogether, these NPs exhibited effective targeting and tumor delivery because of the inherent membrane properties, which cause increased circulation, self-binding capacity, and recognition/targeting functionalities of the source cells. These nanoconstructs due to cell-specific binding not only potentiate binding to the primary tumor site but can also target the metastatic tumor. This study’s findings can serve as the basis for design of experiments for targeted drug delivery to the tumor site for other disease models having similar characteristics.

2. MATERIALS AND METHODS

2.1. Chemicals and Solvents.

Poly(vinyl alcohol) (MW of 40,000), tannic acid, HEPES buffer, 96-well plates, and all other reagents and chemicals were procured from Sigma Aldrich (St. Louis, MO, USA). Tissue microarray (TMA) cores (catalogue no. BR243v) were procured from US Biomax, Inc. Tissue and cell culture supplies were obtained from Thermo Fisher Scientific (Grand Island, NY, USA). All chemicals, reagents, and solvents were used as received without further purification.

2.2. Cell Culture.

Human BC cell lines (MCF7, MDA-MB-231, and MDA-MB-468) and mammary epithelial cells (MCF10A) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Human neutrophils were procured from Astarte Biologics that are tested for bloodborne pathogens HIV-1 and 2, hepatitis B, hepatitis C, and HTLV-1 to ensure safety. Breast cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) that was supplemented with phenol red, 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin. MCF10A cells were cultured with MEGM mammary epithelial cell growth basal medium and MEGM SingleQuots supplements, purchased from Lonza/Clonetics Corporation. For all experiments, cell lines were incubated at 37 °C in a humidified 5% CO₂ and 95% O₂ atmosphere. At 80–90% confluency, these cells were trypsinized, counted using Trypan Blue (0.4%), and seeded as per specific experiments. Cells were allowed to adhere to the plate surface overnight before implementing any further treatment.

2.3. Isolation of Cell Membranes.

Neutrophil membranes were activated using lipopolysaccharide (LPS, 100 ng/mL)-supplemented culture medium for 4 h to ensure activation followed by a wash in PBS. LPS treatment creates an inflammatory reaction that provides translocation of adhesion molecules on the cellular surface. To isolate the plasma membranes, cells were suspended in isolation buffer composed of 225 mM mannitol, 75 mM sucrose, 0.5% w/v essentially fatty acid-free BSA, 0.5 mM EDTA, 30 mM Tris–HCl, pH 7.4, and supplemented with protease inhibitor cocktail. Cells were homogenized in a Dounce homogenizer and centrifuged at 800g at 4 °C for 10 min, and the pellet containing unlysed cells and nuclei was discarded. The supernatant was centrifuged at 10,000g at 4 °C to pellet and remove the mitochondria. The remaining supernatant was ultracentrifuged using a Sorvall WX Ultra 80 (Thermo Scientific) at 100,000g at 4 °C for 1 h to pelletize the membranes. Finally, the plasma membrane pellet was suspended in 10 mM Tris–HCl, pH 7.4, with 0.5 mM EDTA and then stored at −80 °C for later use. To maintain the membrane integrity, freeze–thaw cycles were prevented by aliquoting membranes in smaller batches. Protein estimation was quantified using the BCA protein assay.

2.4. Preparation of Biomimetic Supramolecular Nanoconstructs.

The biomimetic supramolecular nanoconstructs were prepared in two steps beginning with production of PVP-TA (PVT) NPs by the solvent evaporation technique followed by cloaking/biofunctionalization of cellular membranes on PVP-TA NPs. Briefly, 2.5 mg amounts of PVP and TA were dissolved in 0.5 mL of aqueous medium separately and then mixed in a 1:1 ratio in an 8 mL glass vial under continuous stirring at 400 rpm on a stir plate. Then, 100 μL of PTX or coumarin-6 (C6) dissolved in acetone (1 mg/mL) was added dropwise, and the speed was increased to 900 rpm. After overnight stirring to ensure complete acetone evaporation, the final PVT NP solution was generated. To obtain PVT-NEU NPs, PVT NPs were mixed with the extracted cellular membranes at a concentration equivalent to 16:1 and subjected to solvent evaporation and simple stirring at 4 °C for 24 h. The next day, the mixture was sonicated at 100 W for 30 s with intermittent cooling on ice followed by centrifugation at 14,000 rpm at 4 °C for 10 min to remove the unbound membranes. The supernatant was collected bearing the final product, PVT-NEU NPs. All batches were freshly prepared to maintain the membrane integrity, and the experimental protocol was followed within 24 h from preparation.

2.5. Characterization of PVT-MEM NPs.

2.5.1. Particle Size and Zeta Potential.

The hydrodynamic mean particle size (nm), particle size distribution (PDI), and zeta potential of PVT NPs and PVT-NEU NPs were measured by a Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK) based on the dynamic light scattering principle. All measurements were performed at 25 °C with an angle of detection of 173°. Briefly, all of the PVT-NEU NPs were diluted in 1 mL of ultrapure water at a ratio of 1:10 and subjected to probe sonication for 30 s. Precaution was taken to prevent the samples from overheating during sonication. The zeta potentials were measured by a laser Doppler microelectrophoresis technique using the same dilution of 1:10 in ultrapure water with 1× PBS added in equal parts to PVT-NEU NPs. Each measurement was carried out for triplicate samples.

2.5.2. Short-Term Stability Study.

The stability of PVT NPs and PVT-NEU NPs was assessed at three different temperature conditions, namely, 4, 25, and 37 °C, using DLS particle size analysis. Briefly, samples were freshly prepared and incubated for 5 days, and the change in particle size with respect to time was assessed. Particle sizes of samples were measured in 1 mM HEPES buffer, pH 7.4, in ultrapure water supplemented with protease inhibitor cocktail at 4, 25, and 37 °C. A long-term stability study requires lyophilization with a suitable cryoprotectant. This can be implemented for future investigations.

2.5.3. Spectral Analysis.

Fourier transform infrared (FTIR) spectra were acquired using a universal ATR sampling accessory plate on a Spectrum 100 FTIR spectrophotometer (PerkinElmer, Waltham, MA) to confirm the structural composition of the cell membranes on the NPs. For this study, lyophilized powders (lyophilized using a Labconco freeze dry system, −48 °C and 133 × 10⁻³ mbar; Labconco, Kansas City, MO, USA) of PVT NPs and PVT-NEU NPs were placed on the tip of the ATR objective to obtain the spectra between 4000 and 650 cm⁻¹ at a scanning speed of 4 cm⁻¹ for 32 scans. The FTIR spectra are presented as %T vs cm⁻¹ and analyzed using Spectrum 100 software.

2.5.4. Transmission Electron Microscopy.

To confirm the size obtained from the DLS and to investigate the structural morphology of PVT NPs and PVT-NEU NPs, the NPs were imaged using a JEOL 2000EX transmission electron microscope (TEM) (JEOL Ltd., Tokyo, Japan) operating at 80 keV. For this study, freshly prepared samples were sonicated vigorously to prevent any agglomeration of NPs, 20 μL (5 μL over 4 × 10 min period) was slowly placed on the formvar carbon-coated side of a 150-mesh standard TEM grid (Electron Microscopy Sciences, PA, USA) and allowed to absorb at RT, and excess solution was wicked away. Then, NPs were stained using 1% w/v uranyl acetate solution, the excess amount of stain was wicked off, and the grid was allowed to air-dry followed by imaging using an AMT camera at a direct magnification of 100,000×.

2.5.5. Identification of Membrane-Associated Proteins.

The presence of proteins on the NPs was verified by Coomassie blue staining. For the SDS-PAGE study, the PVT NPs and PVT-NEU NPs were processed as mentioned in Kang et al.¹¹ Briefly, samples were lysed and centrifuged at 13,000g for 5 min at 4 °C. The protein concentration was quantified using a standard BCA assay. Subsequently, the supernatant was mixed with SDS loading buffer and heated to denature the proteins, and 20 μg of protein (equivalent to the cellular membranes of NEU and cellular membranes of NEU in PVT-NEU NPs) per sample was loaded in a 10–20% SDS-PAGE gradient gel and electrophoresed until the dye front left the gel. The protein gels were stained using Coomassie blue fast staining solution and imaged using the ChemiDoc MP System (Bio-Rad, Hercules, CA, USA).

2.6. In Vitro Cellular Uptake.

To determine the preferential targeting of membrane-cloaked NPs, cellular uptake of PVT NPs and PVT-NEU NPs was conducted in BC cells (MCF7, MDA-MB-231, and MDA-MB-468) and breast epithelial cells (MCF10A, serves as an experimental control). For this experiment, C6 was labeled in PVT NPs and PVT-NEU NPs for visualization or tracking of NPs. In brief, cells were seeded at 0.5 × 10⁶ cells in a 12-well plate and allowed to adhere to the plate surface overnight. The following day, the cells were treated with a 5 μg dye equivalent amount of C6-labeled PVT and C6-labeled PVT-NEU NPs for 3 h. This time was optimal to ensure the stability of C6 but to not allow for leaching out from the NPs and to offer sufficient time for the cells to bind/uptake. The cells were then washed thrice with 1× PBS to remove the C6 bound to the outer cell surface. The DMEM phenol red-free medium was used in this study to reduce phenol red-induced autofluorescence. For qualitative images, cells were imaged using an EVOS FL imaging system (AMF4300, Life Technologies, Carlsbad, CA, USA), and quantitative analysis was done after injecting samples into an Accuri C6 flow cytometer (Accuri Cytometer, Inc., Ann Arbor, MI, USA). Quantitative uptake of C6 was acquired using the fluorescence levels in the FL1 channel (488 excitation, blue laser, 530 ± 15 nm, FITC/GFP). The average reading and the standard error of the mean were calculated from 3 runs. Data were reported as normalized mean fluorescence values (after normalizing all values with respect to control values for the respective cell lines).

2.7. Evaluation of In Situ Tumor Cell Targeting.

For this study, tumor and normal breast adjacent epithelial tissue sections were analyzed for cellular binding of NPs. Briefly, slides were deparaffinized and rehydrated by different concentrations of alcohol content followed by heat-induced antigen retrieval (HIAR) at pH 6.0. On these TMAs, 100 μg/mL C6 equivalent C6-labeled PVT NPs and C6-labeled PVT-NEU NPs were used to stain the tissues overnight followed by three consecutive washes for 5 min in PBS. Tissues were then imaged using a Zeiss 710 laser scanning confocal microscope. All images were captured using the same laser power and gain settings to ensure comparability for intensity analysis. Mean fluorescence intensities (MFI) were quantified using ImageJ, and the corrected total cell fluorescence (CTCF) was measured using eq 1. This was done to normalize the actual fluorescence inside the cells, independent of the size and structure of each cell.¹⁵

$$\text{CTCF}=\text{integrated density}-(\text{area of the selected cell}\times \text{MFI of background readings})$$ (1)

2.8. In Vitro Biological Assays.

2.8.1. Cell Viability.

Cell viability was assessed using MTT assay on MDA-MB-231. Briefly, 5 × 10³ cells were dispersed in 100 μL of media/well and seeded in 96-well culture plates and allowed to stand overnight. The next day, these cells were treated with 0–20 nM PTX or PTX equivalent PTX-loaded PVT NPs/PVT-NEU NPs for 48 h. PVT NPs, also referred to as blank NPs, were not loaded with PTX and served as treatment group. Cell lines with no treatment served as experimental controls. After the required incubation time, 20 μL of MTT reagent solution was added and incubated for 3 h at 37 °C. After formazan crystals formed, the media was aspirated and 100 μL of DMSO was added to dissolve the salt formation. Absorbance readings were recorded at 490 nm using a microplate reader (BioTeK Cytation 3, Winooski, VT, USA). Cell proliferation with respect to control cells was calculated. All experiments were performed in triplicates at a minimum. Cell morphology changes with respect to treatment were further confirmed by imaging using an EVOS FL imaging system at a 20× magnification.

2.8.2. Cell Migration.

Boyden’s chamber cell migration assay was used to confirm the migration of cancer cells upon treatment with the PTX-loaded PVT NPs and PTX-loaded PVT-NEU NPs. Briefly, 0.5 × 10⁵ cells were suspended in serum-free media along with the 5 nM PTX or 5 nM PTX equivalent PTX-loaded PVT NPs/PVT-NEU NPs and added to the upper chamber of the 96-transwell inserts (Corning, NY, USA). Cells receiving no treatment and blank NPs were used as an experimental controls. These inserts were then placed inside the 96-well plates in 10% FBS media. After 24 h, the migrated cells at the lower inside of the transwell inserts were fixed using 4% paraformaldehyde and then further stained using crystal violet. The cells on the upper side of the insert were removed using a cotton swab. Inserts were then imaged using the EVOS FL imaging system.

2.8.3. Clonogenic Assay.

A colony-forming assay was performed to investigate the proliferation ability of BC cells. For this study, BC cells were seeded (250 cells/well) in a 12-well plate and treated with 5 nM PTX or 5 nM PTX equivalent PTX-loaded PVT NPs/PVT-NEU NPs and set in the incubator for 15 days with intermittent media changing. Visible colonies were fixed, stained with hematoxylin, and imaged.¹⁶

2.9. In Vivo Evaluation.

2.9.1. Tumor Xenograft Generation.

To grow the ectopic xenografts in mice, we purchased six-week-old female athymic nude mice (nu/nu) from Jackson Laboratory (Bar Harbor, ME USA) and maintained in a pathogen-free environment. All animals were housed and maintained according to the recommendation of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) guidelines. All studies were conducted using UTHSC Institutional Animal Care and Use Committee (UTHSC-IACUC) protocol ID 18-046.0. To establish xenograft tumors in mice, MDA-MB-231 cells (2 × 10⁶ cells) were suspended in DMEM medium and Matrigel (BD Biosciences) solution in a 1:1 ratio and then injected subcutaneously on the hind flanks of each mouse. Tumor volumes in mice were monitored using a digital Vernier caliper and calculated using eq 2.

$$\text{ellipsoid formula for the tumor volume}(m{m}^3)=\pi /6^{\ast }\left(L^{\ast }{W}^{\ast }H\right)$$ (2)

Mice were euthanized either when the tumor volume reached ~1200 mm³ or the mouse lost weight of more than 10% body weight during any studies. Mice were euthanized at the end of experiments by CO₂ asphyxiation followed by cervical dislocation.

2.9.2. Biodistribution and Tumor Targeting.

The biodistribution and tumor accumulation/targeting behavior of NPs were evaluated by in vivo imaging. PVT NPs and PVT-NEU NPs were loaded with ICG (NIR fluorescence dye) for in vivo imaging (3 mice per group), similarly to NPs mentioned in Section 2.4. Briefly, upon reaching tumors of ∼400 mm³, mice were randomly assigned into three treatment groups: plain ICG solution, ICG-loaded PVT NPs, or ICG-loaded PVT-NEU NPs. The NPs were intraperitoneally administered once with 100 μg equivalent of ICG solution or ICG-loaded NPs. Then, mice were imaged at 3, 6, 24, 48, and 72 h using the XRMS imaging system (Caliper Life Sciences, Waltham, MA) equipped with a coupled device camera to find the tissue localization fate and retention of the NPs. These animals were fed with a special diet of Teklad Global Rodent Diets (Envigo, Huntingdon, UK) to avoid background autofluorescence from regular diet.¹⁷ Mice were anesthetized with 2% isoflurane and analyzed for fluorescence intensity (integration: 30 s; binning: 4). The peak fluorescence signal intensity was calculated by the sum of all detected photon counts within the region of interest (ROI) after subtraction of background fluorescence, in photon/s/cm²/sr. After the necessary time, mice were euthanized, and organs were excised. Biodistribution of ICG was examined by assessing the ROI of the excised heart, lungs, liver, kidney, and spleen along with tumor retention.

2.9.3. Anticancer Efficacy.

Once the tumor volumes reached ∼150–200 mm³, the mice were randomly divided into four groups (5 mice per group). Mice were treated with either control solution (PBS-treated), PTX (10 mg/kg), PTX-loaded PVT NPs, or PTX-loaded PVT-NEU NPs (PTX equivalent to 10 mg/kg) by intraperitoneal injections twice a week for 4 weeks. Tumor volumes were measured once a week. The body weight of animals was also supervised during the experimental periods. The mice were euthanized at the end of the experiment, and whole blood as well as organs were collected for immunohistochemistry (IHC) and blood/toxicity profiling. Tumors were excised and processed for tissue sections, histopathology, and hemotoxicity images. Blood was collected after postmortem cardiac puncture and submitted to the UTHSC Research Histology Core Laboratory for histopathology and blood chemistry analysis (IDEXX Bioresearch). Also, hepatotoxicity was assessed by measuring serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) enzymes, and total bilirubin (TBIL), and kidney functions were marked by blood urea nitrogen (BUN) levels.

2.9.4. Immunohistochemistry.

The effect of PTX-loaded PVT NPs and PTX-loaded PVT-NEU NPs on the expression of BAK, BCL2, β-tubulin, cleaved caspase 3, and PCNA was observed. The standard IHC protocol was followed.¹⁸ Briefly, tumor tissues were deparaffinized, rehydrated, soaked with 0.3% hydrogen peroxide, and processed for HIAR. After blocking with a background sniper, the samples were subjected to staining for expression of respective proteins in the tissues. The slides were counterstained with hematoxylin, dehydrated, and mounted in Permount mounting medium (Fisher Scienctific). The histological differences in organ samples were analyzed by H&E staining to study organ toxicity.

2.10. Statistical Analysis.

All data were generated and analyzed using GraphPad Prism 5.03 software (GraphPad Software, San Diego, CA), and results are presented as the mean ± standard error of the mean (SEM). Statistical significance was performed using a Student’s t-test analysis when comparing between two groups. A p-value of ≤0.05 was considered significant.

3. RESULTS

Several studies suggest a multitude of binding or interaction(s) occurring between cellular membranes and the NPs, thus forming a simple yet effective cellular membrane-cloaked nanoconstruct.^19,20 To investigate if membrane-associated proteins would have maximal interactions generating the best delivery platform, we tested human neutrophil cellular membranes. We herein report human neutrophils, activated by lipopolysaccharide (LPS), which induces an inflammatory response allowing for maximal adhesion moieties to translocate to the cell surface. The enhanced receptor expression on the surface allows better targeting and binding capability to the neutrophil cell membranes (NEU). We utilized a series of differential centrifugation and Dounce homogenization techniques for extracting the cell membranes followed by cloaking the surface of PVT NP cores using solvent evaporation techniques. The self-functionalization on the surface of the membranes imparts cell-like properties to the nanoconstruct for effective tumor targeting. The PVP layer serves as a stabilizing agent²¹ to the PVP-TA NP core, thus stabilizing the core of PVT NPs, which can extend binding ability to cellular membranes. A schematic representation of preparation of PVT-NEU NPs is presented in Figure 1A.

Figure 1.

Characterization of PVT and PVT-NEU NPs. (A) Schematic representation of PVT-NEU NP preparation. (B) Dynamic light scattering measurement for PVT and PVT-NEU NPs in water using 50 μL of freshly prepared samples added to 1 mL of ultrapure water and probe-sonicated for 30 s and measured using a Zetasizer (Nano ZS, Malvern Instruments, Malvern, UK) at 25 °C. PVT NPs exhibited a particle size of 220.53 + 12.35 nm. There was a slight increase in size after addition of the cell membrane bilayer with 243.3 + 4.65 nm. Data are presented as the mean ± standard error of the mean (n = 3). (C) Zeta potential was measured in 1× PBS and the average of 3 readings (each reading = 30 runs). PVT NPs had a charge of −2.89 ± 0.28 mV, causing electrostatic repulsion between the NPs and the negatively charged bilayers, resulting in a zeta potential of −4.26 ± 0.24 mV. (D) Fourier transform infrared (FTIR) spectral analysis of PVT and PVT-NEU NPs were acquired on a universal attenuated total reflectance (UATR) accessory plate by a Spectrum 100 FTIR spectrophotometer (Waltham, MA), between 4000 and 650 cm⁻¹ at a scanning speed of 4 cm⁻¹ for 32 scans. Characteristic N–H stretching at 3258 cm⁻¹ and C═O stretching at 1715 cm⁻¹ confirm the presence of PTX consistently in PVT and PVT-MEM NPs. (E) PVT and the representative PVT-NEU NP transmission electron microscopic (TEM) images. A UranyLess EM stain was used for negatively staining the NPs. Particles are shown by arrows on TEM images. The image was acquired by using a JEOL 200EX TEM at a direct magnification of 100,000×. (F) Presence of membrane proteins in NEU and PVT-NEU membrane proteins as seen from SDS-PAGE Coomassie blue staining. Membrane proteins marked at equivalent molecular weight (kDa).

3.1. Preparation and Characterization of PVT-NEU NPs.

The hydrodynamic mean particle sizes of PVT NPs and PVT-NEU NPs were determined by DLS (Figure 1B). There was a slight increase in the size after the coating with cellular membranes. PVT NPs (NPs without any membrane coating) exhibited an average size of 220.53 ± 12.35 nm, which slightly increased to 243.3 ± 4.65 nm after coating with NEU cellular membranes. The zeta potential of PVT NPs was −2.89 ± 0.28 mV, and the addition of the negatively charged cellular membrane causes the zeta potential to decrease resulting in −4.26 ± 0.24 mV for PVT-NEU NPs (Figure 1C), suggesting the successful cloaking of the cell membranes on the surface of PVT NPs. A short-term stability assay (Figure S1) confirmed the stability of the formulation in HEPES buffer. HEPES is the buffer of choice as it is a physiological buffer that mimics the body’s buffering capacity. This provides insights about the stability of the membrane-cloaked NPs, especially in the presence of cell membrane proteins, during in vitro and in vivo studies. No significant changes in size of PVT-NEU NPs were observed for 5 days after storing the PVT-NEU NPs at 4, 25, and 37 °C. However, a long-term stability study is warranted. FTIR spectra of PVT NPs demonstrate distinct peaks of N–H stretching at 3258 cm⁻¹ and C═O stretching at 1715 cm⁻¹,²² which are also consistently present in PVT-NEU NPs, demonstrating that the functional integrity of PVT NPs was maintained after the cloaking with membrane. The presence of characteristic peaks at 1638 cm⁻¹ due to stretching of conjugated C═O, 1292 cm⁻¹ due to CN, and at 1198 cm⁻¹ due to C–O^23,24 confirms the presence of TA and PVP in the NPs. The FTIR spectra (Figure 1D) of PVT-NEU NPs corroborated the large peak intensities of amide groups at 1638 due to −CO–NH₂²⁵ proteins and lipids present in the cellular lipid bilayer. The infusion of the membranes on the surface of the PVT NPs was further confirmed by TEM (Figure 1E), which displays the core–shell structure of the PVT-NEU NPs that encapsulates the PVT NPs in the core and the surrounding NEU layer on the shell. The presence of a darker core–shell structure surrounding a lipid bilayer was seen in PVT-NEU NPs. To further ensure that the membrane proteins were successfully translocated to the surface of the PVT NPs, Coomassie blue-SDS-PAGE analysis was conducted (Figure 1F).

3.2. PVT-NEU NPs Exhibit Improved Cancer Cell Targeting.

Biologically derived cell membrane nanoconstructs are capable of bypassing the subject’s immune system while also having enhanced tumor cell targeting. The targeting capacity of PVT-NEU NPs to cancer cells was tested in BC cells and breast epithelial cells (Figure 2A). PVT-NEU NPs showed pronounced binding to MDA-MB-231 and MDA-MB-468 cells compared to PVT NPs, whereas breast epithelial cells, MCF10A, show very minimal internalization, demonstrating the preferential uptake in cancer cells vs noncancer cells. The quantitative analysis of flow cytometry was reported (Figure 2B). In all tested cell lines, the fold change with PVT-NEU is the highest in comparison to PVT NPs. To further confirm PVT-NEU NPs’ preferential targeting on BC tumor cells, an in situ cellular targeting experiment was performed on fixed tissue slides. The visual fluorescence imaging and ImageJ quantification of normal adjacent tissue vs tumor tissues are presented in Figure 3A. A targeted efficacy is observed for the cancerous section vs the normal adjacent tissue section with PVT-NEU NPs exhibiting significantly higher intensity as well as the highest statistical increase in uptake in comparison to PVT NPs (Figure 3B), elaborating the preferential targeting ability of the PVT-NEU NPs. It is important to note that PVT-NEU NPs are not very effective in targeting MCF7 cells (Figure S2). This implies its suitability for TNBC.

Figure 2.

Intracellular uptake of PVT and PVT-NEU NPs. (A) Intracellular uptake of dye (C6)-loaded NPs, instead of drugs, to view the fluorescence uptake of the NPs, after allowing 3 h of incubation. The excess dye adhered to the surface of the cells was washed with PBS, 3 times for 5 min each. Cells were qualitatively imaged under the EVOS FL imaging system (AMF4300, Life Technologies, Carlsbad, CA, USA). (B) Quantitative analysis of the cellular uptake was conducted using an Accuri C6 flow cytometer (Accuri Cytometer, Inc., Ann Arbor, MI, USA). FL1 channel (488 excitation, blue laser, 530 ± 15 nm, FITC/GFP). The average reading and the standard error of the mean were calculated from 3 runs.

Figure 3.

In situ tissue dye binding by PVT NPs and PVT-NEU NPs. (A) In situ tumor targeting abilities of PVT and PVT-NEU NPs were assessed using tissue microarray slides. Slides were incubated with C6 equivalents on each slide for overnight, after processing them followed by imaging. (B) Breast carcinoma tissues show significant fluorescence by PVT-NEU NPs when compared to PVT NPs (n = 3). ImageJ was used to quantify the fluorescence intensity. Data were measured using the distance in pixel and the same pixel aspect ratio. There is no change in adjacent normal tissue fluorescence levels by both formulations.

3.3. PVT-NEU NPs Promote Anticancer Activity of Therapeutic Agents.

The in vitro cytotoxicity of PTX in solution and PTX-loaded in the PVT NPs (PVT-PTX NPs) and PTX-loaded PVT-NEU NPs (PVT-NEU-PTX NPs or PVT-PTX-NEU NPs) was evaluated. Cell proliferation was conducted by MTT assay with PTX concentrations ranging from 0 to 20 nM when treated on MDA-MB-231. The IC₅₀ values were significantly lowered by PTX-loaded PVT NP treatment in comparison to free PTX. Meanwhile, PTX-loaded PVT-NEU NPs showed a significant change of 2.95-fold reduction when compared to PTX alone in MDA-MB-231 (Figure 4A). We observed the changes in the cell structure after treatment with 5 nM (images are displayed in Figure S3) showing distinct morphological changes on treatment with PTX solution and PTX-loaded PVT NPs at similar concentrations. To further confirm the superior anticancer efficacy of PTX-loaded PVT-NEU NPs over free PTX, we conducted Boyden’s migration assay and the colony-forming ability on MDA-MB-231. As shown in Figure 4B,C, after 5 nM equivalent treatment with PTX solution or NP treatment with PTX-loaded PVT/PVT-NEU NPs, migration and colony formation assays show statistically significant differences with the plain drug, PTX. Representative pictures of cell proliferation, migration, and colony assays are presented in Figure S3. These results altogether suggest that the NPs significantly improved the therapeutic effect over the free drug.

Figure 4.

In vitro activity of PTX-loaded PVT and PTX-loaded PVT-NEU NPs in MDA-MB-231 cells. (A) Proliferation profiles of cancer cells upon treatment with PTX and PTX-containing PVT and PVT-NEU NPs. (B,C) Migration and colony formation assays: PVT-MEM NPs after exposure to a 5 nM equivalent of PTX show higher inhibition in comparison to PVT NPs and significantly more than PTX solution at an equivalent concentration. Treatment group: blank PVT NPs (with no PTX) serve as negative a control. Significant changes in MDA-MB-231 cells suggest superior in vitro efficacy by PVT-NEU NPs.

3.4. PVT-NEU NPs Exhibit Superior Tumor Targeting and Antitumor Potential of Therapeutic Agent.

To verify the tumor targeting ability and biodistribution of PVT-NEU NPs, a noninvasive NIR fluorescence imaging study was conducted in MDA-MB-231 ectopic xenograft tumor-bearing mice. We conducted kinetic tracking of ICG-labeled PVT NPs and ICG-labeled PVT-NEU NPs for 3, 6, 24, 48, and 72 h. As shown in Figure 5A, the ICG solution is cleared out most rapidly from the system compared to all the other treated groups, suggesting prolonged retention and longer circulation. In comparison, PVT-NEU has more selective accumulation at the tumor site, indicating a high affinity toward tumor cells. After this experiment, we investigated the accumulation of NPs in other organs, such as the heart, lungs, liver, kidney, and spleen, which are shown in Figure 5B where little to no retention of ICG was found indicating the rapid clearance of the dye due to the absence of a delivery system. Tumor retention of the ICG dye after 72 h showed a considerably higher retention observed in PVT-NEU NPs when compared to PVT NPs. An ex vivo analysis (Figure 5B,C) of PVT NPs shows the highest retention in the liver in comparison to any other organ. There was a significantly lower retention of the dye in all the organs, except the liver (heart, lung, kidney, and spleen), which can likely be traced to the inescapable macrophagic uptake by the liver.

Figure 5.

Biodistribution tumor targeting and ex vivo analysis of excised tumors and organs in ectopic xenograft tumor-bearing mice. (A) Kinetic immunofluorescence imaging with ICG-loaded PVT and PVT-NEU NPs. Ectopic tumors of MDA-MB-231 cells were grown, mice were treated with an ICG equivalent of 100 μg/mL of PVT or PVT-NEU NPs, and plain ICG solution was used as an experimental control. Mice were subjected to imaging after 3, 6, 24, 48, and 72 h to determine the retention of the fluorescent dye using an IVIS Spectrum system (Caliper Life Sciences). ICG was cleared out within 24 h, whereas PVT-NEU NPs exhibited a prolonged retention over PVT NPs. (B) Ex vivo biodistribution of the immunofluorescent dye ICG, ICG-loaded PVT, and PVT-NEU NPs. (C) Quantitative analysis of the fluorescence intensity of excised organs; the heart, lungs, liver, kidney, spleen, and tumor were collected, and ROI values were collected and assessed using an IVIS Spectrum system (Caliper Life Sciences) at the UTHSC core facility.

Altogether, based on less biodistribution to other organs, prolonged circulation, and higher tumor retention, PVT-NEU was selected for evaluating therapeutic efficacy in the in vivo xenograft model. For this, we used MDA-MB-231 tumor-bearing mice (Figure 6A). Control mice received PBS treatment. PTX solution showed ∼52% tumor growth inhibition in comparison to control mice. In contrast, tumor growth was well-inhibited by PTX-loaded PVT NPs (67%) and PTX-loaded PVT-NEU (∼72%), in comparison to the control group (Figure 6B,C). All tumors excised from mice are presented in Figure S4 for representation.

Figure 6.

In vivo anticancer efficiency. (A) Representative of various treatment groups’ influence on tumor growth in mice bearing tumors. (B) Average tumor volumes of the control, PTX, PVT-PTX NPs, and PVT-NEU-PTX NPs over the 4-week treatment. (C) Net tumor growth of MDA-MB-231 tumors in the control and PTX versus PVT-PTX NPs and PVT-NEU NP groups. Data in the bar graph represent the mean ± standard error of the mean (n = 5).

To analyze if PTX induces any adverse toxic effects, we evaluated the serum level of liver enzymes (ALT, AST, and TBI) and a marker of kidney function (BUN) (Figure 7A). The normal ranges for these enzymes/markers are as follows: ALT, 17–77 U/L; AST, 54–298 U/L; TBili, 0.0–0.9 mg/dL; and BUN, 8–33 mg/dL. There was slight elevation of AST and TBili in PTX-loaded PVT NPs and PTX-loaded PVT-NEU NPs, signifying potential liver damage caused by PTX,^26,27 which was also observed from the liver retention in the biodistribution studies. All other tested enzymes were normal, exhibiting the regular kidney function. On the contrary, PTX reduced the number of WBC, neutrophils, and lymphocytes with respect to the control, as seen in Figure 7B. Conversely, no myelosuppression was noticed in PTX-loaded PVT-NEU NPs with counts similar to the control, although slight decreases in WBC, neutrophils, and lymphocytes in PVT NPs with respect to the control were observed. Although there was no statistically significant difference between PTX and PTX-loaded PVT NPs or PTX-loaded PVT-NEU NPs, there was an increasing trend in neutrophil counts by 43.33% in contrast to PTX, showing lesser chances of PTX-loaded PVT-NEU NPs to cause neutropenia similarly to PTX solution.²⁸ To assess the hemocompatibility, fresh blood was withdrawn by cardiac puncture during the termination of the in vivo anticancer efficacy study. As indicated in Figure 7C, the mouse group treated with PTX shows the distorted morphology of the RBC (indication of systemic toxicity), whereas PTX-loaded PVT NPs and PTX-loaded PVT-NEU NPs do not exhibit any morphological changes, thus suggesting that PTX-loaded PVT NPs and PTX-loaded PVT-NEU NPs are nontoxic to healthy cells.

Figure 7.

Evaluation of potential toxicity of PVT-PTX NPs and PVT-NEU-PTX NPs. (A) Serum chemistry levels of liver enzymes (ALT, AST, and TBI) and a kidney function marker (BUN) were determined in all the groups; the dashed black line indicates the normal range for each of the enzyme. (B) Blood count analysis determined levels of WBC, RBC, neutrophils, and lymphocytes. The WBC, neutrophil, and lymphocyte count was lower in the PTX-treated group, indicating changes of myelosuppression, in contrast to PVT-NEU-PTX NPs that had a WBC, RBC, neutrophil, and lymphocyte count equivalent to the control group. (C) Hemocompatibility was assessed from red blood cell morphological changes, after blood was collected at the end point of the study by cardiac puncture. Data were presented as the mean ± standard error of the mean (n = 3).

Tumors were excised and collected for IHC and H&E histological analysis, as shown in Figure 8. As observed from the images, there was an increase in apoptotic markers BAK and cleaved caspase-3 and a decrease in cell survival protein BCL-2 expression in tumors treated with PTX-loaded PVT-NEU NPs comparatively more than PTX-loaded PVT NPs and PTX solution. The control shows upregulation of PCNA, a cell proliferation marker, whereas with treatment groups, there is a decrease in PCNA expression. IHC analysis for β-tubulin shows a higher expression of protein in tumor treatment with treatment groups as compared to the control, indicating that the treatment causes mitotic cell cycle arrest of the tumor cells. The β-tubulin expression is enhanced with PTX-loaded PVT-NEU NPs over PTX, suggesting more inhibition of microtubules causing cell cycle arrest during mitosis. H&E staining shows apoptosis, evident from the clusters of nuclei that are consistent with tumor inhibition, whereas the PBS-treated control group displayed less apoptosis observable from the intact nuclei. Taken together, we report that PTX-loaded PVT NPs and PTX-loaded PVT-NEU NPs significantly improved therapeutic efficacy. Furthermore, PTX-loaded PVT-NEU NPs demonstrated a comparatively safer profile in terms of biocompatibility, biodistribution to healthy organs, and analysis of various enzymes and blood work. Taken together, the potential of biomimetic PTX-loaded PVT-NEU NPs, as a personalized tool for cancer therapeutics is significantly better due to the enhanced biocompatibility and other therapeutic virtues.

Figure 8.

Immunohistochemistry analysis and histological evaluation of tumors treated with PVT-PTX NPs and PVT-NEU-PTX NPs. Representative immunohistochemical images of excised xenograft tumors for expression of apoptotic markers (BAK and cleaved aspase-3), cell survival protein (Bcl-2), a proliferation marker (PCNA), β-tubulin suggesting mitotic cell cycle arrest, and H&E staining for distinguishing morphological differences.

4. DISCUSSION

A major goal in engineering of biomimetic nanoconstructs is to locally retain a potent therapeutic agent at the tumor sites. Various cellular membranes have been explored to camouflage NPs with red and white blood cells, platelets, viruses, and exosomes, extending potential therapeutic response and mimicking properties.^29–33 Herein, we report neutrophil membrane-cloaked NPs for selective tumor targeting and reducing systemic side effects. Towards the construction of these biomimetic NPs, we initially developed a PVP-TA-based NP construct of which both base components (poly(vinyl pyrrolidone) and tannic acid) have their associated advantages. It was reported by Le et al. that PTX molecules aggregate with tannic acid by a hydrogen bond. However, on addition of PVP to this tannic acid-PTX core, a compact and stable core could be achieved due to stronger hydrogen bond interactions.²¹ This would be an ideal stabilized core for neutrophil membrane-cloaked NPs, as the membrane can form supramolecular assembly on the top of the NPs. For this reason, we choose PVT (PVP-TA self-assembled) NPs as our core for the delivery system. At first, we developed an NP core (PVT NPs) based on our previous findings^16,34 that shows therapeutic efficacy over commonly administered chemotherapeutic agent, PTX, in TNBC and prevents P-glycoprotein-mediated drug efflux, one of the major hindrances resulting in the reduced efficacy of PTX.^35–37

PVT NPs were cloaked with neutrophil membranes. These cells, when activated with LPS, endow their plasma membranes with membrane proteins such as lymphocyte function-associated antigen-1 (LFA-1), macrophage-1 antigen (MAC-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), and P-selectin glycoprotein ligand-1 (PSGL-1) that have high affinity for receptor sites specific on BC/TNBC.³⁸ The SDS-PAGE Coomassie stain results show the possible presence of LFA-1, MAC-1, PSGL-1, and PECAM-1 (molecular weights of ~140, ~47, ~43, and ~130 kDa, respectively). The profile of the membrane proteins on both NEU and PVT-NEU NPs can be found around those molecular weights. Although the presence of the proteins in PVT-NEU NPs seems to be lesser than NEU, the presence of the membrane proteins suggests proper translocation of these proteins on the surface of PVT NPs. We would suggest a further verification by Western blotting and bottom-up proteomics analysis.³⁹ The presence of these proteins explains the enhanced PVT-NEU NP accumulation at the tumor site due to the overexpression of ICAM-1, VCAM-1, and E-selectin on TNBC that alters tight junctions between endothelial cells, resulting in increased vessel permeability.^40–43

We chose neutrophil cells to extract the cell membranes for cell-specific binding of NPs to the specific tumor site and the metastatic region in BC/TNBC. After extraction of cell membranes, we cloaked the NPs with these lipid bilayer membranes, which can explain the slight increase in particle size also seen from the TEM images of PVT-NEU NPs over PVT-NPs. The prime reason is that tannic acid has a tendency to bind strongly to lipids and proteins; thus, we anticipate that this may be the key mechanisms for the formation of the NP core with a cell membrane-cloaking ability.^44–47 The binding of the NPs with the cancer cells can be confirmed by various types of cellular targeting and binding assays.^14,16,34,48 To evaluate the interaction of activated neutrophil membranes, we cloaked NPs with these membranes and tested them on an aggressive, metastatic TNBC cell line (MDA-MB-231). The intracellular and in situ uptake shows higher uptake of PVT-NEU NPs as compared to PVT NPs when the NPs are loaded with the fluorescent dye C6. In vitro cytotoxicity assays as seen from cell proliferation, migration, and colony-forming assays suggest the efficient anticancer ability of PTX-loaded PVT-NEU over PTX-loaded PVT NPs in TNBC cells. These studies suggest that the in vitro activity of the NPs was intact upon the membrane coating and did not have any impact on the anticancer potential. A plausible reason would be the presence of self-markers on the surface of the PTX-loaded PVT-NEU NPs that provides cell-specific binding to the potential targets on TNBC cells. This can explain the enhanced uptake on MDA-MB-231 versus other cell lines, especially the breast normal epithelial cells, MCF10A.

The in vivo circulation time of plain ICG shows clearance of the dye completely, probably due to clearance by the RES, which would also be expected to produce subtherapeutic tumor concentrations⁴⁹ over NPs. Nannoparticles, through prolonged circulation over plain solution and due to the EPR effect, circulate longer but not as long as PVT-NEU NPs. The presence of membrane coating over NPs prolongs circulation as they are not opsonized by macrophagic uptake¹⁹ as seen throughout all the time points observed in the bioimaging study. However, ex vivo analysis shows that higher accumulation of the dye by PVT and PVT-NEU NPs occurs in the liver, which is probably due to it being a highly perfused organ causing inescapable macrophage uptake.⁵⁰ A xenograft mouse model for TNBC was established using MDA-MB-231 to generate an ectopic xenograft model of TNBC (MDA-MB-231 cells) through subcutaneous injections, and this is considered a suitable model for evaluation of therapeutics and anticancer drug candidates for TNBC.^51–53 The accumulation of membrane-coated NPs over plain NPs causes higher tumor inhibition as seen from PTX-loaded PVT-NEU NPs than PTX-loaded PVT NPs in the in vivo model. After evaluation of the therapeutic potential of PTX-loaded PVT-NEU NPs and its improved efficacy over PTX solution and PVT NPs, the safety perspective was evaluated from serum enzymes and blood counts. PTX is known to cause leukopenia and neutropenia and severe peripheral neurotoxicity, which often ranges from 30 to 40% of patients receiving PTX chemotherapy.⁵⁴ Although no statistical significance was observed, a lessened trend to cause myelosuppression or neutropenia could be suggested for PTX-loaded PVT-NEU NPs over PTX. Additionally, no hemotoxicities were observed to regular red blood cells except for PTX, which also correlates to the toxicities observed from serum enzymes. Furthermore, histological analysis shows enhanced apoptosis with PTX-loaded PVT-NEU NPs over other treatment groups, and the morphology of the distorted tumor elucidates the enhanced therapeutic activity. Taken together, we demonstrate that the cell membrane-coated NPs could be utilized for efficient delivery of anticancer drugs to the TNBC cells due to the enhanced biocompatibility and other therapeutic virtues.

5. CONCLUSIONS

In summary, we report the development, optimization, and characterization of a biomimetic nanoconstruct that combines cell membrane characteristics and imparts a biological identity to the NPs. Neutrophil membrane-coated nanoconstructs (PVT-NEU NPs) exhibit improved cellular targeting, tumor retention, and comparatively less biodistribution in healthy organs as seen in a xenograft mouse model. Furthermore, drug-loaded nanoconstructs (PTX-loaded PVT-NEU NPs) demonstrate an improved antiproliferative, antimigration, and anticolonogenic activity. Additionally, this nanoconstruct demonstrates an improved in vivo therapeutic effect and lowered systemic toxicity and good hemocompatibility when compared to uncoated NPs and the native drug paclitaxel. In conclusion, this strategy of biomimetic engineered nanoconstructs shows promise as a drug delivery system with potential for active tumor targeting, improved therapeutic effects, and reduced adverse effects as encountered from standard chemotherapy.

Supplementary Material

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsabm.2c00614.

Additional data on stability of biomimetic NPs; effect of biomimetic NPs on proliferation, migration, and colony formation against breast cancer cells (images); excised tumor tissue images of treatment groups (PDF)