Transmembrane TNFα-Expressed Macrophage Membrane-Coated Chitosan Nanoparticles as Cancer Therapeutics.

Transmembrane TNFα, a crucial signaling cytokine, holds anticell proliferative potential. Successful delivery of this intact transmembrane protein to the target site is quite intriguing. Amidst numerous nanocarriers, a novel class of new generation macrophage membrane-coated nanocarriers is endowed with innate tumor homing abilities and inherent capacity of escaping body's defense machinery. In this perspective, a novel therapeutic module has been fabricated by coating a nontoxic, biodegradable chitosan nanoparticle core with engineered macrophage membrane-tethered TNFα. Herein, the expression of membrane-bound TNFα was induced by challenging phorbol 12-myristate 13-acetate-differentiated THP-1 cells with bacterial lipopolysaccharide. Subsequently, the as-synthesized chitosan nanoparticle core was coated with a TNFα-expressed macrophage membrane through an extrusion process. While transmission electron microscopy imaging, sodium dodecyl sulphate polyacrylamide gel electrophoresis, and western blotting results demonstrated successful coating of the chitosan nanoparticles with the TNFα-induced membrane, the cell viability assays on several cancer cells such as-HeLa, MDA-MB-231, and MCF-7 revealed significant innate anticell proliferative potential of these membrane-coated nanoparticles. Additionally, evaluation of expression of several interleukins after treatment demonstrated excellent biocompatibility of the membrane-coated nanoparticles. The fabricated nanoparticles also demonstrated a dose-dependent cell death in tumor spheroids, which was further corroborated with calcein AM/propidium iodide dual staining results. Translation of the therapeutic efficacy of the synthesized nanoparticles from monolayers to tumor spheroids augments its potential in cancer therapy.

Introduction

The complex network of signaling cascades play a major role in orchestrating the delicate harmony between cell growth, division, and programmed death of cells.[1] Proteins, either in secreted or in membrane-bound forms, interact with their respective receptors, which modulates intricate signaling networks to maintain balance in the overall behavior of the cells. Highly regulated signaling pathways by the proteins replace dead cells with new healthy cells.[2]

In case of cell death-related pathways, death signals like TNF, CD95L, and TRAIL are important. Binding of these proteins on their corresponding receptors initiates the death-signaling cascade, which results in cell apoptosis.[3] Recently, the role of the transmembrane proteins in apoptosis has been widely studied. Among the signaling molecules, transmembrane tumor necrosis factor alpha has gained much interest in recent years because of its characteristic cell killing ability after binding to its receptor.[4] However, such recombinant proteins are unstable with short half-life and are susceptible to disintegration.[5] Therefore, in order to unleash the true potential of the biological macromolecule, there should be a judicious combination of the therapeutic moiety and a proper delivery vehicle.[6] This imperative need has motivated researchers to develop nano-based delivery systems.[7]

A diverse array of nanoparticles ranging from metallic, semimetallic to polymeric particles has emerged.[8] Amongst these, biodegradable polymeric nanoparticles are the most promising class for delivering biological molecules and drugs. These polymeric nanoparticles are armed with multitude of advantages such as, enhanced stability, higher drug payload, tunable physicochemical properties, homogeneous particle distribution, and controlled drug release.[9] Chitosan, consisting of α-(1–4)-2-amino-2-deoxy-β-d-glucan repeats, is one such FDA-approved biopolymer, which has been reported as a drug-delivery vehicle in several biological applications.[10−12] However, use of bare nanoparticles often leads to rapid clearance from blood stream because of opsonization.[13] Coating nanoparticles with layers of hydrophilic poly(ethylene glycol) (PEG) has been established to deceive the body immune system.[14] Yet, rapid clearance of the PEGylated nanoparticles has been reported when the animals were injected with the second dose of nanoparticles, owing to the formation of anti-PEG immunoglobulin M antibodies.[15] Hence, a new generation of novel biomimetics could be an alternative to cloak the synthetic nanocarriers by coating with natural membranes.[16−19] A wide array of natural membranes have been reported to coat nanoparticles. The natural membrane-coated nanoparticles could escape body’s defense machinery and retain prolonged circulation time in the body.[20] In this context, a variety of specialized cells such as, macrophages, dendritic cells, and T-helper cells are known to secrete cytokines. Among them, macrophages could easily produce TNFα upon induction with lipopolysaccharide (LPS). Furthermore, macrophage membranes deserve special mention because macrophages are the circulating sentinels of the body having innate characteristics of homing toward the inflammation-affected area.[21,22] Interestingly, the homing property of the whole macrophage cells has shown accumulation of the drug-carrying macrophage cells near cancer cells. However, the complete process of nanoparticle coating with the inert membrane is cumbersome and the therapeutic response would be exclusively dependent on the drug molecules loaded on the nanocarrier. Therefore, we have ventured to infuse the therapeutic potential in the membrane coating itself.

In the current study, we have prepared innate therapeutic module using engineered macrophages. Reports suggest that the macrophages, when challenged with endotoxins like LPS, start secreting several cytokines such as TNFα and interleukins within a short span of 4 to 5 h.[23] In view of the above rationale, we speculated that the membrane-bound form of TNFα could be obtained by modulation of the LPS induction time.[24,25] Time-bound induction of TNFα expressed in the macrophage membrane showed antiproliferative action against several types of cancer cells—HeLa, MCF7, and MDA-MB-231 cells. Following this, the membrane vesicles were prepared from the LPS-induced macrophage cells by extrusion methods. To ensure a stable framework for these therapeutic vesicles, nontoxic biodegradable polymeric FDA-approved chitosan nanoparticles were synthesized and coated with the transmembrane TNFα-expressed macrophage membrane. The fabricated membrane-coated nanoparticles were characterized for biocompatibility, hemocompatibility, and stability. Maintenance of functional integrity of the engineered macrophage membrane after coating over the chitosan nanoparticle core was validated by cytotoxicity studies on several cancer cell lines. The cell viability studies demonstrated a dose-dependent decrease in cell viability. Furthermore, assessment of the mode of cell death revealed that the cells were undergoing apoptosis, a regulated and programmed manner of cell death. The therapeutic efficiency of the designed therapeutic nanoparticles was further validated on tumor spheroids, which mimic the complexity and heterogeneity of in vivo conditions.[26] Succinctly, in the present study, fabrication and therapeutic application of a novel biocompatible transmembrane TNFα membrane-coated nanocarrier has been illustrated.

Results and Discussion

The preparation of transmembrane TNFα-expressed macrophage membrane coated-chitosan nanoparticles is a three-step procedure as discussed in the Materials section. Initially, THP-1 monocytes were differentiated into macrophages using varying concentrations of phorbol 12-myristate 13-acetate (PMA). Schematic representation in Figure A illustrated differentiation of monocytes to macrophages in presence of PMA. Microscopic visualization demonstrated (Figure B) that the monocytes became larger, granular, and less refractive, in a concentration-dependent manner accompanied with a gradual conversion of floating monocytes into adherent macrophages with increasing concentrations of PMA (0–100 μM). In addition, flow cytometry-based studies also depicted an increase in the granularity of the cells with higher concentration of PMA, essentially indicating successful conversion of monocytes into granular macrophages (Figure C).

Figure 1

(A) Schematic representation of differentiation of monocytes to macrophages upon addition of PMA. The cells become larger and granular. (B) Microscopic assessment of monocytes differentiating into macrophages with increasing concentrations of PMA, (i) 0, (ii) 40, (iii) 80, and (iv) 100 μM [scale bar: 50 μm]. (C) Flow cytometric assessment of monocytes differentiating into macrophages with increasing concentrations of PMA, (i) 0, (ii) 40, (iii) 80, and (iv) 100 μM, respectively.

Synthesis of soluble TNF by the macrophages is known to be driven by the interaction of LPS with TLR on the macrophage surface as early as 4 h post stimulation.[25] Therefore, in order to obtain membrane-expressed TNFα, the time duration of LPS stimulation was initially optimized (data not shown). It was found that 2.5 h of LPS stimulation was most suitable for maximum transmembrane TNFα expression. Amount of LPS required for maximum induction of transmembrane TNFα was also optimized by using varying concentrations of LPS (upto 500 ng/mL). While Figure A illustrates the induction of TNFα on the macrophage membrane, western blot analysis (Figure B) with anti-TNFα antibody on the isolated membrane showed a gradual rise in the transmembrane TNFα concentration up to 100 ng/mL, followed by a steady level of expression up to 500 ng/mL (Figure S1). Therefore, in order to induce expression of transmembrane TNFα in THP-1 monocytes, the above stated concentrations of PMA and LPS were used for all the subsequent experiments. The expression on TNFα by the differentiated macrophages upon LPS stimulation was also confirmed by semiquantitative polymerase chain reaction (PCR) using TNFα specific primers. The gel image (Figure C) revealed a distinct band corresponding to transmembrane TNFα around 750 bp in 100 ng/mL LPS-treated macrophages, while there was no amplification in the untreated ones. The LPS-induced macrophage membranes were isolated by hypotonic lysis buffer (details in Materials) and the homogenized suspension was centrifuged to remove cell debris. Subsequently, the supernatant was centrifuged to obtain the plasma membrane fractions of the macrophages.

Figure 2

(A) Schematic: formation of membrane-expressed TNFα upon LPS addition. (B) Western blot of THP-1 membrane induced at increasing concentrations (ng) of LPS using anti-TNFα antibody. (C) Semi-quantitative PCR using TNFα-specific primers—lane 1: untreated, lane 2: 100 ng/mL LPS treated. Lane 3, 4: β actin controls for the samples.

After successful isolation of the TNFα expressed-macrophage membrane fraction, we embarked on the study of its therapeutic potential. HeLa, MCF7, and MDA-MB-231 cells were treated with varying concentrations (protein) of the membrane fractions for 48 h following which cell viability was assessed using MTT assay by recording the absorbance at 590 nm and keeping 630 nm as the reference. Cell viability percentage was calculated and the results (Figure S2A) demonstrated a dose-dependent decrease in proliferation of the cells treated with membrane fractions expressing transmembrane TNFα, whereas no substantial decrease in cell viability was observed for the uninduced membrane-treated control group (Figure S2B). Thus, the inherent anticell proliferative potential of the transmembrane TNFα-expressed membrane fraction was validated, which paved the way for the subsequent experiments.

Capitalizing on the antiproliferative potential of the macrophage membrane, we proceeded to fabricate this engineered membrane into stable nanocarrier having inherent therapeutic potential. In this regard, it was imperative to design a steady framework for the therapeutic membrane. Therefore, the polymeric chitosan nanoparticle core was synthesized using a well-established ionic gelation method. The as-prepared chitosan nanoparticles were negatively charged (−0.7 mV) as determined by zeta potential analysis (Figure S3) and the hydrodynamic diameter of the nanoparticles was recorded as 237.5 nm (Figure C). Characterization by field-emission scanning electron microscopy (FESEM) (Figure A) and transmission electron microscopy (TEM) (Figure B) established the successful synthesis of uniform spherical chitosan nanoparticles having roughly 200 nm diameter. Once the chitosan nanoparticles were synthesized, the subsequent step was to evaluate cytotoxicity of the bare nanoparticles on cancer cell lines. Results of MTT assay revealed no apparent cytotoxicity of chitosan nanoparticle alone (Figure S4).

Figure 3

(A) FESEM image of the synthesized chitosan nanoparticles, (B) TEM analysis of the nanoparticles, and (C) hydrodynamic diameter of the chitosan nanoparticles as evident from DLS measurement.

Following successful synthesis of the core, TNFα-expressed macrophage membrane-coated chitosan nanoparticles were developed by the process of serial extrusion of the membranes through 0.8 and 0.4 μm pore-sized membrane followed by combined extrusion of both the membrane and the nanoparticles with 0.2 μm pore-sized membrane as illustrated in Figure A. Successful membrane coating over the chitosan nanoparticles was validated by TEM imaging, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and western blotting with anti-TNFα antibody. TEM image (Figure B) showed distinct halos around the inner chitosan nanoparticle cores denoting successful membrane coating around the chitosan nanoparticles. Furthermore, SDS PAGE was also performed (Figure C), which revealed similar protein profiles of the induced macrophage membrane and the membrane-coated nanoparticles confirming membrane coating over the chitosan nanoparticles. Henceforth, transmembrane TNFα-expressed membrane-coated nanoparticles are termed as “nanoassembly” for our subsequent studies. To confirm the presence of TNFα in the nanoassembly, western blotting on nanoassembly was performed with the anti-TNFα antibody. The results in Figure D revealed a distinct band (lane 3) corresponding to TNFα in the nanoassembly. The dynamic light scattering (DLS) data in Figure E also depicted an increase in size of the nanoassembly as compared to the bare chitosan nanoparticles because of the membrane coating around the nanoparticles.

Figure 4

(A) Schematic: extrusion process. (B) TEM image demonstrating successful membrane coating over chitosan nanoparticles. (C) SDS PAGE image confirming successful membrane coating. Lane 1: uninduced cell lysate, lane 2: LPS-induced cell membrane, lane 3: induced macrophage membrane-coated nanoparticles. (D) Western blotting of the membrane-coated chitosan nanoparticles. Lane 1: uninduced cell lysate, lane 2: LPS-induced cell membrane, lane 3: induced macrophage membrane-coated nanoparticles. (E) DLS of the nanoassembly.

Before evaluating therapeutic potential of the system on cancer cells, it was crucial to ensure the safety of the designed nanoassembly for further biological application. Therefore, the biocompatibility of the THP-1 membrane was examined. For this, the expression of several interleukins that play a role in inflammation and in generation of immunogenic response were assessed after treatment.[27] Because macrophages are the key mediators of immunity, human monocytes (THP-1 cells) were differentiated into macrophages and the cells were treated with the nanoassembly. Subsequently, the expression of several interleukins, IL6 and IL1β were studied with quantitative PCR keeping LPS-treated macrophages as a positive control. The fold changes in expression of the genes were calculated and as evident from Figure A, the membrane-coated nanoparticles did not elicit any immune response because the expression of IL6 and IL1β was quite low in comparison to LPS (0.5 μg/mL)-treated positive control samples. Simultaneously, the hemocompatibility of the designed nanoassembly was also evaluated by incubating them with intact human RBCs for 3 h on ice.[27] Samples treated with 1% Triton-X 100 were considered to have undergone 100% hemolysis, while phosphate buffer saline (PBS)-treated RBCs were kept as the negative control. From Figure B, it was evident that the designed nanoassembly did not induce any hemolysis, whereas a visible hemolysis in the Triton X 100-treated positive control was seen. In order to accomplish appropriate biomedical potential, stability is crucial for any nanocarrier. Hence, the stability of the nanoassembly was assessed over a period of 7 days by recording DLS measurements. Interestingly, there was (Figure C) negligible difference in size of the nanoassembly over a period of 7 days, which offers an additional advantage to the designed therapeutic module.

Figure 5

(A) Assessment of biocompatibility after treatment with the nanoassembly. (B) Assessment of hemocompatibility. Courtesy of Srirupa Bhattacharyya, Copyright 2019, (C) evaluation of hydrodynamic diameters of the nanoassembly by DLS for consecutive 7 days.

Upon completion of evaluation of safety and stability of the nanoassembly, the subsequent step was to determine its functional integrity. Hence, MTT cell survival assay was performed on several cell lines, viz—HeLa, MCF7, and MDA-MB-231 to determine the therapeutic potential of nanoassembly. For this, the cells were incubated with increasing concentrations of the nanoassembly and MTT assay was performed after 48 h. Results of MTT assay (Figure ) depicted a dose-dependent decrease in cell viability with increase in concentration of the nanoassembly, which implied that the TNF-expressing membrane retained its functionality even after serial extrusion. It should be mentioned here that there was negligible cell death in the samples treated with chitosan nanoparticles alone (Figure S4) essentially indicating the superior biocompatibility of chitosan and thereby re-establishing the innate therapeutic potential of TNFα-expressed membrane coating over the chitosan nanoparticle core. From Figure it is observed that the amount of nanoassembly required to bring about IC50 in the HeLa cells (IC50: 1.317 μg/mL) was much lower than that in MDA-MB-231 (IC50: 7.525 μg/mL) and MCF7 (IC50: 5.9 μg/mL) cells. This can be attributed to the differential expression of TNFα receptors on the surface of these cell lines. The anticell proliferative effect of transmembrane TNFα was reduced when the nanoassembly was preincubated for 1 h in presence of the anti-TNFα antibody (Figure S5). This confirmed the specific role of the transmembrane TNFα component of the nanoassembly in growth inhibition, and the other proteins present in the membrane after PMA induction did not show any additive effect in the present study. Retention of the activity was assessed by measuring the anticell proliferative potential of the nanoassembly on HeLa cells following 7 days storage. MTT-based cytotoxicity assay was performed, which demonstrated a dose-dependent decrease in cell viability (Figure S6), essentially indicating that the nanoassembly retained activity even after storage for 7 days. Although, it should be mentioned here that there was around twofold reduction in efficiency after storage as compared to the freshly prepared nanoassembly.

Figure 6

Assessment of cell viability upon treatment with increasing concentrations of nanoassembly.

Cell death occurs by two mechanisms—either apoptosis or necrosis. While necrosis is a sudden unprogrammed event, apoptosis is fundamentally a plan of programmed cell death involving a series of regulated events.[28] Therefore, to investigate the mode of cell death induced by the designed nanoassembly, dual staining of treated HeLa cells with calcein AM and propidium iodide (PI) was performed. Calcein AM is a vital dye, which is converted to a membrane impermeable fluorescent analogue by the cellular esterases. Hence, calcein AM only stains live cells and the fluorescence leaks out in case of completely membrane compromised cells.[29] Whereas, PI is a DNA intercalating agent, which is selectively permeable to the membrane compromised cells. Exploiting the differential behavior of the two dyes, both treated and untreated HeLa cells were stained for 30 min and visualized under a confocal microscope. Confocal microscopy images (Figure ) illustrated the presence of bright green or yellowish bodies, which essentially denoted the cells that were undergoing early apoptosis; whereas, cells with red nuclei were indicative of late apoptotic cells.

Figure 7

Assessment of the mode of cell death using calcein AM and PI dual staining of HeLa cells. (A) Untreated and (B) treated with nanoassembly. E.A. and L.A. indicate early apoptotic and late apoptotic cells, respectively.

During apoptosis, the initiator caspase cleaves and activates downstream executioner caspases, which in turn cleaves subsequent proteins.[30] Therefore, activation of executioner caspases is considered as a signature of apoptotic cells. Therefore, the expression of executioner caspases—caspase 3 and 7 were studied using an executioner caspase detection kit. The cells were treated with the nanoassembly for 12 h and incubated with the florigenic substrate for 30 min followed by visualization under a confocal microscope. The confocal microscopic images (Figure ) displayed appearance of intense bright green fluorescence in treated cells indicative of the activation of the executioner caspases.

Figure 8

Assessment of activation of executioner caspases 3/7 in HeLa cells after treatment with nanoassembly, demonstrating activated executioner caspases. (i) Untreated cells and (ii) treated cells illustrating activation of executioner caspases (green) denoted with arrows.

To further validate the effect of therapeutic efficacy of the engineered system, tumor spheroids of HeLa cells were generated by a facile forced floatation method, as discussed in the Materials section.[31] The spheroid growth was monitored after every 24 h and 3 day old compact spheroids were used for experimental purposes. For quantitative evaluation of the therapeutic effect of the fabricated nanoassembly on the spheroids, 3 day old spheroids were treated with increasing concentrations of the nanoassembly for 48 h. The results (Figure ) demonstrated a dose-dependent decrease in cell viability of the spheroids with increase in concentration of the nanoassembly. However, it should be mentioned that the amount of the nanoassembly required for the attainment of IC50 in HeLa spheroids was higher than IC50 of the corresponding monolayer culture.

Figure 9

Cell viability study with the nanoassembly on 3 day old HeLa spheroids.

For visual evaluation of live and dead cells, the spheroids treated with IC50 (7.1 μg/mL) and IC75 (10 μg/mL) concentrations of the nanoassembly were further subjected to calcein AM/PI dual staining procedure. The dual stained spheroids imaged using a confocal microscope (Zeiss LSM 880), illustrated an increase in dead (PI stained) cells after treatment as compared to the untreated spheroids (Figure ). Dual staining results on treated spheroids corroborated the findings of the cell viability assay by alamar blue, as evident in Figure .

Figure 10

Calcein-AM/EtBr dual staining study. (A) (i) phase contrast, (ii) Calcein AM stained, (iii) PI stained, and (iv) merged images of untreated HeLa spheroids, (B) (i) phase contrast, (ii) calcein AM stained, (iii) PI stained, and (iv) merged images HeLa spheroids incubated with IC50 concentration of the nanoassembly, (C) (i) phase contrast, (ii) calcein AM stained, (iii) PI stained, and (iv) merged images of HeLa spheroid incubated with IC75 concentration of the nanoassembly, and (D) Z-stack projection of HeLa spheroid incubated with IC75 concentration of the nanoassembly [scale bar: 200 μm].

Conclusions

The present work amalgamates therapeutically relevant transmembrane-TNFα expressed-macrophage membranes with nontoxic chitosan nanoparticles to formulate a nanoassembly. Without interference of other cytokines, increased cell death signal TNFα imparted therapeutic efficiency to the otherwise noncytotoxic macrophage membrane upon LPS induction. Excellent stability, hemocompatibility, and biocompatibility of the fabricated nanoassembly paved the way to explore its functional integrity. The membrane-coated nanoparticles retained dose-dependent anticell proliferative properties when examined on three different cancer cells. Evaluation of the mode of cell death unveiled triggering of apoptosis in the cells after treatment with the nanoassembly. Successful regressive effect of the nanoassembly on spheroids boosts the biological significance of our study in the prospective new regime in cancer therapy, as the spheroids closely mimic the in vivo tumor microenvironment. The translation of cell retardation activity confirms the immense potential of this novel therapeutic nanoassembly as a “nanodrug”.

Materials

Cell Culture

MCF7 (breast cancer), HeLa (human cervical cancer), MDA-MB-231 (triple negative breast cancer), and THP-1 (human monocytes) cells were obtained from National Center for Cell Science, Pune, India. MCF7, HeLa, and MDA-MB-231 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% penicillin–streptomycin. THP-1 cell lines were maintained in RPMI media containing 10% heat-inactivated FBS supplemented with 1% antibiotic solution. All the cells were maintained in a humidified condition at 37 °C, with 5% CO2.

Monocyte Differentiation

THP-1 cells (monocytes) were differentiated into macrophages by treating them with 100 nM PMA for 24 h. The PMA concentration used for differentiation was optimized and differentiation into macrophages was confirmed by microscopy and flow cytometry.

Optimization of LPS Concentration

The macrophages were challenged with bacterial LPS to produce TNFα. The literature suggests that the time of secretion of soluble TNFα by the macrophages is 4–5 h. Our aim was to obtain transmembrane TNFα, so we reduced the time to 2.5 to 3 h and optimized the LPS concentration for induction of TNFα.

Isolation of the TNFα-Expressed Macrophage Membrane

The macrophages were harvested by scraping and were kept overnight in a hypotonic lysis buffer containing sodium bicarbonate, ethylenediaminetetraacetic acid, phenylmethylsulfonyl fluoride. After overnight incubation, the cell suspension was loaded into hand held Dounce Homogeniser and disrupted with 30 passes each pestle. The suspension was centrifuged at 3500g to remove the larger debris, the supernatant was again centrifuged at 15 000g for 40 min, and the pellet was obtained containing the plasma membrane of the macrophages. The pellet was washed 4–5 times with PBS by repeated centrifugation to remove any other contaminants. To evaluate the expression of TNFα in the isolated membrane of the macrophages, western blotting with the anti-TNFα antibody was performed. The expression on TNFα after adding 100 ng/mL LPS was also confirmed by semiquantitative PCR using TNFα-specific primers. β-Actin was used as an internal positive control.

Preliminary Cell Viability Assay with the Membrane Fractions

In order to study the therapeutic potential of the TNFα-expressing macrophage membrane, HeLa, MCF7, and MDA-MB-231 were seeded in 96-well plates and treated with increasing concentration of the LPS-induced macrophage membrane. After 48 h, cell viability was evaluated using MTT assay by recording the absorbance at 590 and 630 nm as background.

Synthesis of Chitosan Nanoparticles

Chitosan nanoparticles were synthesized using the well-established protocol of ionic gelation method using 0.5 mg/mL chitosan and 0.75 mg/mL TPP. The solution was stirred for 24 h at 600 rpm. After synthesis, the solution was centrifuged at 15 000 rpm for 30 min to pellet down the nanoparticles and washed 4–5 times with water by repeated centrifugation to remove any unreacted components. Zeta potential and hydrodynamic diameter were measured by a PerkinElmer Lambda 25 spectrophotometer.

Imaging of the Chitosan Nanoparticles

The chitosan nanoparticles were characterized by FESEM and TEM. For FESEM, the nanoparticles were drop-cast on an aluminum-coated piece of glass coverslip and allowed to dry overnight followed by imaging. For TEM imaging, 10 μL nanoparticle suspension was drop-cast on a carbon-coated copper grid, air-dried, and observed using TEM (JEOL, MA).

Macrophage Membrane Coating around the Chitosan Nanoparticles

Macrophage membranes were serially extruded through 0.8 and 0.4 μm pore-sized membranes in the extruder (Avanti Polar Lipids). To prepare macrophage membrane-coated chitosan nanoparticles (nanoassembly), chitosan nanoparticles and macrophage membrane vesicles were combined and extruded through a 0.2 μm pore-sized membrane.

Confirmation of Membrane Coating

Membrane coating over the chitosan nanoparticle core was confirmed by TEM imaging, SDS PAGE, and western blotting with the anti-TNFα antibody. For TEM analysis, 10 μL of the as-synthesized nanoparticles were drop-cast on a carbon-coated copper grid, air dried overnight, and observed under TEM (JEOL, MA). In order to ensure successful coating of the macrophage membranes over the chitosan nanoparticle, SDS PAGE was carried out. Proteins from uninduced macrophages, LPS-induced cell membranes, and induced macrophage membrane-coated nanoparticles were electrophoresed in 12% SDS PAGE. Silver staining was performed to visualize and compare the proteins in the gel. Furthermore, western blotting was also performed to validate successful membrane coating over the chitosan nanoparticles. For this, equal amount of proteins from uninduced macrophages, LPS-induced cell membranes, and induced macrophage membrane-coated nanoparticles were electrophoresed in 12% SDS PAGE for separation according to molecular weight. Subsequently, the proteins were transferred from the gel to the poly(vinylidene difluoride) membrane followed by blocking for 2 h with 4% bovine serum albumin. The protein-containing membrane was then incubated with the anti-TNFα primary antibody overnight followed by incubation with the horseradish peroxidase-tagged secondary antibody. The blots were developed using a chemiluminescent peroxidase substrate (Sigma) and visualized under a ChemiDoc transilluminator (Bio-Rad). The hydrodynamic diameter of the membrane-coated nanoparticles was recorded by a PerkinElmer Lambda 25 spectrophotometer.

Biocompatibility Study

Biocompatibility of the THP-1 membrane-coated nanoparticle was studied before evaluating therapeutic potential of the system on cancer cells. For this, the expression of several interleukins was studied by real time PCR. For this purpose, human monocytes (THP-1 cells) were differentiated into macrophages using PMA (100 ng/mL) for 24 h. Following differentiation the cells were treated with membrane-coated nanoparticles. Bacterial LPS (500 ng/mL) was used as a positive control for the experiment. Following treatment, RNA was isolated using a TRI Reagent (Sigma) and cDNA was synthesized using a Bio-Rad cDNA synthesis kit. Real time PCR was performed (Rotor, GeneQ, Qiagen) using glyceraldehyde 3-phosphate dehydrogenase as an internal control and the expression of interleukins-IL6 and IL1β was studied with quantitative PCR.

Hemocompatibility Studies

For hemocompatibility testing, 1 mL blood was centrifuged at 1000 RCF for 5 min and the supernatant was discarded. To the pellet, 1 mL PBS was added and centrifuged again. Finally, the freshly isolated RBCs were incubated on ice for 3 h with the macrophage membrane-coated nanoparticle, 0.1% Triton (positive control), and PBS (negative control). Percentage hemolysis was measured by recording the absorbance at 550 nm, considering 100% hemolysis with the Triton X sample.

Stability Study of the Nanoassembly

The stability of the nanoassembly was studied, by recording DLS by a PerkinElmer Lambda 25 spectrophotometer over a period of consecutive 7 days.

Cytotoxicity Studies

In order to study the therapeutic potential of the fabricated module, HeLa, MCF7, and MDA-MB-231 cells were seeded in 96-well plates and treated with increasing concentration of macrophage membrane-coated nanoparticles. After 48 h, cell viability was assessed using colorimetric MTT assay. For this, the treated cells were incubated with 0.5 mg/mL MTT in DMEM for 2 h. Subsequently, MTT was removed and 150 μL of organic solvent dimethyl sulfoxide was added followed by 10 min of incubation. Absorbance was recorded using a multiplate reader (Tecan) at 590 and 630 nm as background. Cell viability percentage was calculated. To understand whether the nanoassembly retained its activity, the synthesized the membrane-coated nanoparticles were stored for 7 days in 4 °C. After 7 days, cytotoxicity assay was performed on HeLa cells. To ascertain specific role of transmembrane TNFα, the nanoassembly was also preincubated for 1 h in rocking condition at room temperature in presence of the anti-TNFα antibody. Thereafter, HeLa cells, seeded at a density of 5 × 103 cells/well, were treated with only the nanoassembly and with the antibody-treated nanoassembly for a period of 48 h. Subsequently, MTT assay was performed for both of the treatment groups and absorbance was recorded at 590 nm.

Calcein AM/PI Dual Staining

To qualitatively evaluate the mode of cell death differential staining was performed. HeLa cells were treated with the nanoassembly for 48 h. Following treatment, the media was removed and the cells were gently washed with 100 μL of PBS. Thereafter, calcein AM/PI solution was added in the final concentrations of 2 and 4 μm followed by a 30 min incubation in the dark. The cells were the visualized by a confocal microscope (Zeiss LSM 880).

Caspase 3/Caspase 7 Assay

Activation of effector caspases after treatment was studied with a CellEvent Caspase-3/7 Green Detection Reagent (Thermo Fisher Scientific). For this, HeLa cells were seeded at a density of 5 × 103 cells/well in a 96-well plate. Following 24 h of treatment, 100 μL of diluted reagent solution (4 μM) was added and incubated for 30 min. Subsequently, the cells were imaged using a confocal microscope (Zeiss LSM 880).

Generation of 3D Tumor Spheroids of HeLa Cells

To create three-dimensional spheroids, 96-well plates were precoated with serum-free media containing 1.5% (w/v) agarose. HeLa cells were seeded at a density of 2 × 104 cells/well in an agarose precoated plate with a final media volume of 200 μL. Subsequently, the plates were centrifuged at 1500g for 10 min to form aggregates and incubated at 37° with 5% CO2 in a humidified atmosphere.

Assessment of Viability of the Spheroids

Three days old spheroids were treated with increasing concentrations of the nanoassembly. Following 72 h of treatment, resazurin disodium salt was added to each well and incubated for 4 h in a CO2 incubator at 37 °C. Thereafter, absorbance at 570 nm was measured using a multiplate reader. Live/dead cell imaging of the treated spheroids were carried out with 2 μM calcein-AM and 4 μM PI, respectively. Following 30 min of incubation, the spheroids were washed with PBS and imaged using a confocal microscope (Zeiss LSM 880).