A Study of The Cellular Uptake of Magnetic Branched Amphiphilic Peptide Capsules

Abstract

Understanding cellular uptake mechanisms of nanoparticles with therapeutic potential has become critical in the field of drug delivery. Elucidation of cellular entry routes can aid in the dissection of the complex intracellular trafficking and potentially allow for the manipulation of nanoparticle fate after cellular delivery (i.e. avoid lysosomal degradation). Branched amphiphilic peptide capsules (BAPCs) are peptide nanoparticles that have been and are being explored as delivery systems for nucleic acids and other therapeutic molecules in vitro and in vivo. In the present study, we determined the cellular uptake routes of BAPCs with and without a magnetic nanobead core (BAPc-MNBs) in two cell lines: macrophages and intestinal epithelial cells. We also studied the influence of size and growth media composition in this cellular process. Substituting the water filled core with magnetic nanobeads might provide the peptide bilayer nanocapsules with added functionalities, facilitating their use in bio/immunoassays, magnetic field guided drug delivery and magnetofection among others. Results suggest that BAPc-MNBs are internalized into the cytosol using more than one endocytic pathway. Flow cytometry and analysis of reactive oxygen and nitrogen species (ROS/RNS) demonstrated that cell viability was minimally impacted by BAPc-MNBs. Cellular uptake pathways of peptide vesicles remain poorly understood, particularly with respect to endocytosis and intracellular trafficking. Outcomes from these studies provide a fundamental understanding of the cellular uptake of this peptide-based delivery system which will allow for strengthening of their delivery capabilities and expanding their applications both in vitro and in vivo.

Graphical Abstract

1. INTRODUCTION

Self-assembled peptide nanostructures have gained increasing importance as delivery systems since they are highly biocompatible, biodegradable, and tunable. Their structure and function can be easily modified by changing the amino acid sequence and/or environment of assembly (temperature, solvent, pH).^1–3

For the past decade, our team has focused on studying the properties and applications of two branched amphiphilic peptides that self-assemble to form nano-capsules. They assemble into bilayer delimited vesicles called branched amphiphilic peptide capsules (BAPCs) whose molecular architecture is similar to liposomes.^{4, 5} BAPCs are formed by mixing equimolar concentrations of bis-(Ac-FLIVI)-KKKKK-CONH₂ and bis-(Ac-FLIVIGSII)-KKKKK-CONH₂. They can also be prepared using either sequence individually which varies the thickness of the assembled bilayer.⁶ The size of BAPCs is controlled by temperature changes that induce a shift in the secondary structure of the peptide bilayer.⁷ Thus, BAPCs of varying size can be generated as per the requirement of a specific application.

BAPCs have been successfully used to deliver nucleic acids in different animal models. In mice, intramuscular injections of BAPCs loaded with a DNA vaccine enhanced immune responses and controlled tumors induced by type 16 human papilloma virus (HPV-16).⁸ In insects, oral administration of BAPCs bound to dsRNA enhanced the silencing of vital genes, leading to the premature death of two species from two different orders: Tribolium castaneum and Acyrthosiphon pisum.⁹ We also reported that BAPCs are easily degraded by a common soil fungus thereby suggesting that their use would not result in any accumulation in the environment.¹⁰

Recently, we developed Branched Amphiphilic Peptide coated (BAPc) magnetic nanobeads (MNBs).¹¹ The peptide bilayer was assembled in a controlled manner i.e. one layer at a time on the magnetic bead surface. We refer to these as BAPc-MNBs. Substituting the water filled core of BAPCs with magnetic nanobeads might provide the peptide bilayer nanocapsules with added functionalities, thereby permitting their use in a multitude of applications such as bio/immunoassays, magnetic field guided delivery in vitro and in vivo, magnetofection, magnetic resonance imaging (MRI) and hyperthermia in cancer treatment.^{1, 12–14} Nonetheless, fundamental aspects of BAPc-MNBs such as cellular uptake routes have yet to be fully elucidated.

In this study, the cellular internalization pathways of the newly developed BAPc-MNBs were explored along with their effect on cell viability. Uptake was evaluated in two cell types: mouse macrophages (J774A.1) and rat intestinal epithelial cells (IEC-18). Ileum epithelial cells are known to be a major barrier for oral drug delivery animals. Thus, we consider that in vitro studies using the intestinal epithelial cells can serve as proof of concept of BAPCs uptake by gut cells.^15,16,17 Foreign particles when administered in vivo commonly encounter phagocytes and therefore, we chose to study BAPc-MNBs uptake by macrophage as well.

The magnetic core allowed for easy quantification using a colorimetric assay¹⁸, offering a numerical analysis of the uptake process with a relatively simple benchtop processing. We also analyzed cellular uptake of fluorescent labeled water filled BAPCs, to establish a comparison between the two nanoparticles with differing cores. The fluorescent dye Rhodamine B was incorporated in the N-terminal lysine of the BAPCs forming the peptide bis-(Ac-FLIVI)-KKKKK-CONH₂. We named these modified branched amphiphilic peptide capsules as Rh-BAPCs. By using this approach, cellular internalization was monitored qualitatively using confocal microscopy and quantitatively by flow cytometry.

In general, interaction between nanomaterials and the exterior of the plasma membrane results in cell entry via endocytosis. Depending on the proteins involved in the internalization process, endocytosis can be classified as clathrin-dependent and independent endocytosis¹⁹. The clathrin-independent routes are further classified as 1) caveolae-mediated endocytosis, 2) clathrin- and caveolae-independent endocytosis and 3) macropinocytosis. To probe the dependency of these nanoparticles on these pathways, we used selective inhibitors (individually or in combination) to disrupt these endocytic routes²⁰.

Our results indicated that BAPc-MNBs and Rh-BAPCs enter epithelial cells via multiple endocytic pathways- clathrin and caveolae mediated endocytosis as well as macropinocytosis. Some differences were observed between BAPc-MNBs and Rh-BAPCs, most likely due to the different nanoparticle core (magnetic versus water filled). We also included reactive oxygen and nitrogen species analysis (ROS/RNS) to determine the downstream effects following BAPc-MNBs internalization. Cell viability and ROS/RNS analysis suggest that BAPc-MNBs did not induce significant toxicity in epithelial cells and macrophages.

Few peptide vesicles have been reported in the literature compared to the numerous synthetic polymers capable of self-assembling into vesicles. The studies presented in this report, provided fundamental knowledge on peptide nanocapsules-based delivery systems and can lay a foundation for novel therapeutic applications.

2. MATERIALS AND METHODS

Chemical reagents and cell lines

Ethanol (99% pure, Sigma, ChromSolv, Denatured ethanol), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Acros Organics, ThermoFisher), Magnetic nanobeads (Ocean Nanotech, San diego, CA), Trifluoroethanol (TFE) (Tokyo Chemical Industry Ltd.), Rhodamine B (Sigma-Aldrich Corp., St. Louis, MO), 6-well plate (TPP® tissue culture plates, Sigma-Aldrich), 2.0 mL Low binding tubes (VWR, North America), Dulbecco’s modified Eagle’s medium (Gibco®, Sigma-Aldrich), OptiMEM-I® (Gibco®, Sigma-Aldrich), L-Glutamine (GlutaMAX^™, Sigma-Aldrich), Insulin (Bovine Pancreas, Millipore Sigma), 3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5’, 5”-disulfuric acid disodium salt (Ferene-s, Sigma Ultra), IEC-18 (ATCC® CRL-1589^™), J774A.1 (ATCC® TIB-67^™), pH 7.4 Phosphate buffered Saline (PBS) with Ca⁺² and Mg⁺², L-ascorbic acid (Sigma-Aldrich), Acetate Buffer (Glacial Acetic acid, Fischer), methyl-β-cyclodextrin (Millipore Sigma), Chlorpromazine, Dynasore and Nystatin(Sigma-Aldrich), Cytochalasin D (Tocris Biosciences), 7AAD (Tonbo, San Diego, CA), Sodium azide (Sigma-Aldrich), Dichlorofluorescein-diacetate (DCF-DA) (Sigma – Aldrich), potassium hexacyanoferrate (II) trihydrate 98.5–102% (Sigma-Aldrich), Paraformaldehyde (37%w/v, Fisher Scientific), Hema III stain (Solution 1) (Fischer Scientific).

Synthesis of Branched Amphiphilic Peptide-Magnetic Nanobeads (BAPc-MNBs)

A. Peptide layer on MNBs

The strategy employed to synthesize the modified branched amphiphilic peptide coated-magnetic nanobeads (BAPc-MNBs) bears some similarity with a method previously reported by Natarajan et al.¹¹ A cysteine residue was added on the oligo-lysine segment, C-terminus of bis-(Ac-FLIVIGSII)-KKKKK-C-CONH₂ and was covalently linked to maleimide groups embedded on the magnetic nanobeads surface, in 75% Ethanol: HEPES solvent. Thus, the peptide hydrophobic chains orient themselves towards the organic solvent which promotes the peptides to adopt a helical secondary structure thereby keeping them monodispersed. Assembly of peptides into capsules only occurs when they adopt a beta structure in an aqueous solvent.

To determine whether the peptides were bound to the MNBs and assemble into a tightly packed monolayer, we substituted Phe of the peptide with its fluorescent counterpart, cyanophenylalanine (F_CN) i.e. bis-(Ac-F_CNLIVIGSII)-KKKKK-C-CONH₂, as described previously.¹¹ Varying amounts of this peptide was added to MNBs in 75% ethanol to form the inner peptide monolayer. The pH of the solution was adjusted to 7.5 using 1N NaOH and the mixture was placed on a shaker overnight at RT. The tube containing the reaction mixture was placed in a magnetic separation rack (OzBiosciences, San Diego, CA) for a minimum of 2 h and maximum MNBs were recovered after separation from excess, unbound peptides. The peptide monolayer coated MNBs were washed twice in ethanol and resuspended in 100% Trifluoroethanol (TFE). The peptide binding was measured as a function of cyanophenylalanine fluorescence at ƛ_emission = 290 nm when excited at ƛ_excitation at 240 nm, on a Cary eclipse fluorescence spectrophotometer (Varian). Fluorescence saturation or curve flattening was observed at 600 nmoles of peptide per 0.5 mg of MNBs, which was the point at which the MNBs surface was saturated with peptides.

B. Synthesis of BAPc-MNBs

Peptide monolayer coated MNBs was synthesized using bis-(Ac-FLIVIGSII)-KKKKK-C-CONH₂ peptide (as the chloride salt) as described above and redispersed in 100% TFE. The second peptide bis-(Ac-FLIVIGSII)-KKKKK-CONH₂ was added to it at twice the concentration used to form the bilayer (1200 nmoles per 0.5 mg of MNBs). Water was added to dilute the TFE (final water to TFE ratio of 9:1) and promote beta structure formation thereby allowing the branched sequences to interact and form a bilayer on the MNBs. After sitting for 20–30 minutes on the magnetic rack, the BAPc-MNBs were carefully collected and was concentrated on a rotavapor with a 40 °C water bath. Thus, the TFE was completely removed during this process and the final product i.e. BAPc-MNBs were re-dispersed in water alone. After the overnight incubation at 4 °C the BAPc-MNBs were extruded through a sterile 0.22 μM syringe filter (Millex-GS, Millipore-Sigma). This sterilizes the BAPc-MNBs and excludes any large aggregated BAPc-MNBs. The typical yield after the separation of aggregates is between 25–40%.

Synthesis of Water-filled Rhodamine labeled branched amphiphilic peptide capsules (Rh-BAPCs)

Rhodamine B labeled bis(FLIVI)-K-K₄ peptide was synthesized as described in Sukthankar et al.²¹ Rh-BAPCs were prepared as indicated in Avila et al.⁸, with 30% Rhodamine B labeled bis(FLIVI)-K-K_4. Briefly, water was added dropwise to the dried peptide mixture containing equimolar concentrations of bis-(Ac-FLIVIGSII)-KKKKK-CONH₂ and bis(FLIVI)-K-K₄ (30% Rh-labeled and 20% unlabeled) and allowed to stand for 30 min at 25 °C to form the water-filled nanocapsules. Subsequently, the solution was cooled to 4 °C, and incubated for 1 h prior to returning them to room temperature for an additional 30 min. This protocol yields the conformationally constrained Rh-BAPCs.

Transmission electron microscopy (TEM)

Five μL of undiluted samples (50nm and 200nm BAPc-MNBs) were spotted on Parafilm paper Individual grids (Lacey F/C 200 mesh Au, Cat No. 01882G, TedPella) were carefully placed on the surface of each sample and allowed to stay in contact for ~ 5 min. Grids were then sequentially washed with 20 μL deionized water on the parafilm. Excess sample or water were removed by gently putting the side edge of grids in contact with Kim wipes. Grids were allowed to dry overnight at ~ 50°C in petri dishes. For imaging, grids were mounted in specimen holders specific for TEM. Conditions for imaging were set to 25 KV on a SEM Model S-4800 (Hitachi) or they were adjusted occasionally according to quality of images.

Dynamic light scattering (DLS) and Zeta potential analysis

50 nm and 200 nm BAPc-MNBs and control beads were resuspended in sterile DDI H₂O to have a final concentration of 10⁹ particles/mL. Dynamic light scattering (DLS) and zeta potential (ZP) analyses were performed for nanoparticles in a 10 mm path length cuvettes (Sarstedt® Standard Cuvettes) on a Zetasizer Nano ZSP (Malvern Instruments Ltd., Westborough, MA).

Uptake of BAPc-MNBs by IEC-18 and J774A.1

IEC-18

Rat Intestinal Epithelial Cells (IEC-18) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 2% FBS (DMEM₂), 10% OptiMEM-I®, 3% FBS, 5% NuSerum, 2mM L-Glutamine and 180 μM Insulin. IEC-18 were seeded in a 6-well plate at 5×10⁵ cells/mL and incubated overnight. After overnight incubation, the media in the wells was replaced with fresh media and 10⁴ magnetic nanobeads per cell or 17 pg /cell were added to the wells directly. Therefore, the total number of magnetic nanobeads added to each well was 5×10⁹ or 8.4 μg total. Cells were then incubated at 37 °C for 4 h. Three treatments (Table 1) were performed in a plate i.e. 2 wells per treatment. The experiments were repeated three times to account for any experimental variations/errors and to test the reproducibility of data. Thus, we obtained up to 6 readings per treatment. After 4 h, the cells were briefly washed with PBS and trypsinized and pelleted.

Table 1.

Summary of treatments to determine uptake of BAPc-MNBs by IEC-18 and J774A.1 cells in DMEM 10% Serum Media and OptiMEM® 5% Serum Media.

Treatments	Cell Type Tested
Only Cells	IEC-18 and J774A.1
Control beads (Only MNBs)	IEC-18 and J774A.1
BAPc-MNBs in DMEM 10% Serum Media	IEC-18 and J774A.1
BAPc-MNBs in OptiMEM® 4% Serum Media	IEC-18 only
BAPc-MNBs in DMEM 10% Serum Media with Lipopolysaccharide (LPS)	J774A.1 only

The supernatant was stored at −80 °C for reactive nitrate species analysis as discussed later, and the pellet resuspended in 1 mL PBS by gently mixing using pipet. The number of cells in each tube were counted on a Moxi-Z^™ cell counter (ORFLO technologies). The magnetic separator (Permagen® Labware) was placed in an ice bath to chill prior to sorting the cells. The tubes containing cells were placed in the separation rack on ice for 30 min. After incubating on ice, the PBS was gently aspirated without disturbing the cells adhering to the side of the tube facing the magnet.

The PBS with residual cells was transferred to another tube and the adhered cells in the tube on the magnetic rack were resuspended in 1 mL of PBS. The number of successfully separated cells containing the magnetic nanobeads were counted on the cell counter as stated before. Using this approach, we could determine the percentage of cells that took up the magnetic nanobeads. The cells were then gently spun down and the supernatant discarded. The pellets were then stored at −80 °C to determine the number of magnetic nanobeads in the cells using ferene-s assay. The same procedure was used to determine uptake of magnetic beads by IEC-18 in OptiMEM® as well as for different temperatures and times.

J774A.1

Mouse macrophages were cultured in media with same media composition as for IEC-18 with the exception of insulin, which was not required for macrophage growth. The macrophages adhere strongly to any surface and hence to ensure consistent counts of the cells, they were kept in suspension for the 20 min incubation period and in low binding tubes, to ensure consistent cell counting. 5×10⁹ magnetic nanobeads were added to 5×10⁵ cells in 1 mL of media, and the tube was inverted a couple of times to ensure maximal dispersion. The tubes were then placed in a 37 °C incubator on a shaker (Labquake,Barnstead Thermolyne rotator) that rotates the tubes 360°, for 20min.

Macrophages were then spun down as stated above at 4 °C. Cells were then resuspended in PBS and separated using a magnetic separation rack. For macrophage activation, the adherent cells were stimulated with 1μg/mL of lipopolysaccharide from E.coli O55:BS (List Biological Lab Inc.) in media for 4 h at 37 °C. The media was then replaced with fresh media without LPS to determining uptake of magnetic nanobeads by cells as previously described.

Quantification of BAPc-MNBs in cells by Ferene-s assay

The Ferene-s chromophoric assay used was adapted from Hedayati et al.¹⁸ The 3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5’, 5”-disulfuric acid disodium salt working solution was prepared by mixing 10 mL of 5 times working buffer 2 g L-ascorbic acid in 11 mL 2 M Acetate Buffer and 500 μL of 0.5 M Ferene-s in DDIH₂O (0.5 g in 2 mL water). The volume was subsequently brought up to 50 mL with DIH₂O. One mL of the ferene-s working solution was added to the cell pellets after bringing them to RT. The solution was vortexed and then incubated overnight in the dark at RT. This allows for the cells to break open and release the magnetic beads. The iron beads released, reacted with ferene-s leading to a measurable color change. After incubating overnight, the cells were vortexed again and the cell debris (including some magnetic beads remained bound to the cell membrane.) spun down at 16,000 rcf (Centrifuge 5415D, Eppendorf) for 3 min. The supernatant was then transferred to a 1 mL disposable cuvette and the absorbance of the solution read at 595 nm using a Varian Cary 300 UV-Vis spectrophotometer against a blank containing only the ferene-s working solution. The magnetic nanobeads in the cell supernatants were sorted on the magnetic separator overnight and quantified using ferene-s. Thus, we could determine the quantity of magnetic beads recovered from the cell supernatant after incubation with cells.

The standard curve for this assay was generated using freshly prepared 0.1 mg/mL FeCl₃ (Fischer Scientific) solution in DIH₂O. Fe standards containing 0.1 μg, 0.2 μg, 0.5 μg, 1 μg, 2 μg, 4 μg and 5 μg were used. 1mL of Ferene-s solution was added to each tube with mixing and allowed to stand at RT for 30 min. In order to calculate the number of magnetic nanobeads in cell extract solution, a control tube containing a known amount of magnetic nanobeads was used along with the cell extract samples. According to the manufacturer, 1 mg of 50 nm MNB contains 6×10¹¹ beads. Using this relationship, the number of magnetic nanobeads in solution was calculated (i.e. 0.5 mg beads/mL contains 3 ×10¹¹ beads).

Endocytosis inhibition study

IEC-18 cell (growth as previously described) cells were used to study the effect of chemical inhibitors on the uptake of BAPc-MNBs and Rh-BAPCs. The inhibitors tested were- methyl-β-cyclodextrin (5 mM), chlorpromazine (10 μM), Nystatin (50 μM), Cytochalasin D (4 μM) and dynasore (80 μM). We also tested the combined effect of inhibitors on uptake – methyl-β-cyclodextrin + chlorpromazine, Cytochalasin D + Nystatin, Cytochalasin D + Chlorpromazine.

Prior to incubation of IEC-18 with BAPc-MNBs and Rh-BAPCs, cells were incubated with 1 mL media containing the respective inhibitor (or combinations of inhibitors) for 30 min at 37 °C. The BAPc-MNBs and Rh-BAPCs were then added to the media at 5×10⁹ and 60 μM respectively. After 1 h or 4 h of incubation time, plates were washed with PBS twice. . The analysis of BAPc-MNBs uptake was performed as previously described and Rh-BAPCs uptake was evaluated using confocal microscopy and flow cytometry. For confocal analysis, cells were fixed with paraformaldehyde (Sigma-Aldrich) for 30 min and washed twice with PBS. Subsequently, coverslips were mounted to microscope slides and imaging was carried out with a Nikon AR-1 confocal microscope. BAPCs labeled with rhodamine B were prepared as previously described by Gudlur et al.⁴

For flow cytometry analysis, cells were detached using acutase, the well contents were loaded into a micro-centrifuge tube, centrifuged at 1500 rpm for 5 min, and were then washed twice with PBS. Flow cytometry was carried out in a MACSquant (Miltenyi Biotec, Germany) at 488 Excitation/585–640 Emission channel.

BAPc-MNBs toxicity in IEC-18 cells

IEC-18 cells were seeded in a 12 well plate at 100,000 cells/mL and were allowed to adhere overnight prior to nanoparticle treatment. Further, cells were washed once with sterile PBS and treated with 10⁴ magnetic nanobeads / cell of 50 nm MNBs and 50 nm and 200 nm of BAPc-MNBs in OptiMEM® for 4 h. Upon treatment, supernatant was removed and adherent cells were detached using accutase enzyme. The cells were collected in 1.5 mL tubes and were then spun at 1500 rpm. Next, the pellet was washed twice with PBS containing 2mM EDTA. Prior to flow cytometry analysis, 0.05 μg of 7AAD dye was added to samples and incubated for 5–10 min. Percentage of dead cells was determined by measuring 7AAD fluorescence using flow cytometry MACSquant (Miltenyi Biotec, Germany) at 488 Excitation/655–730 Emission channel.

BAPc-MNBs toxicity in J774A.1 cells

To test cell viability in J774A.1, we used an Abcam® cell viability kit (catalog no. ab112118) and the recommended protocol was followed. Briefly, J774A.1 cells were seeded in 96 well, clear bottom, black walled plates at 10,000 cells/well. The cells were treated with 50 nm and 200 nm BAPc-MNBs and control beads at 10000:1 bead to cell ratio as stated previously for 4 h in DMEM without phenol red. Sodium azide was used as the positive control while the negative control consisted of untreated cells. 20 μL of the reagent compound provided in the kit was added to all wells, except one set of wells containing untreated cells to account for background due to just cells. The cells were incubated at 37 °C for 1.5 h. The absorbance was measured at 570 nm and 605 nm for each well using a Bio-rad microplate reader (Model 680). The percent cell viability was calculated as percent cell viability= 100 × (R_sample-R₀)/(R_ctrl-R₀), where R₀= ratio of OD₅₇₀/OD₆₀₅ for negative control cells without the reagent, Rsample= ratio of OD₅₇₀/OD605 in the presence of the test compound and R_ctrl = ratio of OD₅₇₀/OD₆₀₅ in the absence of the test compound.

Determination of Reactive Nitrogen Species (RNS)- Nitric oxide (NO) detection using Griess reagent

IEC-18 and J774A.1 cells supernatant was collected after treatment with BAPc-MNBs for varying time periods and were used for RNS detection using Griess agent. Control beads, untreated and lipopolysaccharide treated cells served as controls for the study. 25 μM of sodium nitrite was serially diluted in the appropriate cell media to generate a standard curve in the 12.5 μM to 0.195 μM range. Cell supernatants (150 μL) were placed in duplicates in the 96 well plate. A volume of 150μL of the Griess reagent (1% sulfanilamide+0.1%naphthylene diamine dihydrochloride+2.5%H₃PO₄) was added to each well and incubated at RT for 10 min. The absorbance was read using a plate reader (Bio-Rad Model680 microplate reader) at 550 nm. The data was analyzed using Microplate Manager 5.2 software.

Determination of Reactive oxygen species (ROS) using DCF-DA

IEC-18 and J774A.1 cells were seeded at 10⁵ cells per well in a clear bottom, black walled, 96 well plate. The cells were allowed to adhere overnight and washed with Hanks buffered salt solution (HBSS). 20mM stock (1000X) Dichlorofluorescein-diacetate (DCF-DA) was prepared in neat DMSO. Fresh solution of 1X DCF-DA was prepared in HBSS and added to the cells for a final concentration of 20 μM. The cells were incubated for 30min in the 37 °C incubator and the supernatant was discarded. DMEM without phenol red was added to the cells followed by addition of 50 nm BAPc-MNBs and control beads at 10000:1 beads to cell ratio. Negative control included untreated cells. Positive control cells were treated with a final concentration of 10 μM hydrogen peroxide. The cells were treated with the magnetic nanobeads for 4 h and 24 h at 37 °C. The fluorescence was scanned from 500 nm – 600 nm with excitation wavelength of 495 nm using Cary eclipse fluorescence spectrophotometer (Varian) with plate reader accessory. The normalized fluorescence was calculated as the ratio of the fluorescence intensity of the treated cells to negative control cells minus the autofluorescence of cells not treated with DCF-DA and magnetic nanobeads. A ratio of > 1 suggests increased fluorescence in comparison to the negative control and indicates release of reactive oxygen species.

Prussian Blue staining of cells for visualization of magnetic nanobeads within cells.

In order to visualize the magnetic beads within cells, potassium hexacyanoferrate (II) trihydrate 98.5–100% was used to stain the magnetic iron nanobeads within cells. For the purpose of staining, IEC-18 and J774A.1 cells were cultured as described previously. After incubation with magnetic nanobeads for the different times at 37 °C, the supernatant was collected in 2 mL tubes and the cells were washed with PBS while adhering to the plate. The cells were then fixed with 3% paraformaldehyde in PBS by adding 1mL of 3% Paraformaldehyde (PFA) in each well and incubating for 30 min at RT. After the 30 min incubation, the PFA was removed by aspiration and the cells were washed with PBS.

Fresh stain was prepared by mixing equal volumes of 10% Potassium Ferrocyanide solution (5 g in 50 mL DDI H₂O) and 20% HCl (Add 10 mL HCl to 15 mL DDI H₂O and bringing the volume to 50 mL with DDI H₂O). One mL of the fresh acidified potassium Ferrocyanide solution was added to the wells and allowed to incubate at 37 °C for 30 min. The solution was then aspirated and the cells washed with water to remove excess stain. Cells were counterstained for 30 sec using Hema III stain (Solution 1) which stains the cytoplasm pink. The excess of stain was washed out with water. The cells were imaged using a light microscope (Olympus KX31) and a Digital sight DS-5M Nikon lens placed on the eyepiece and connected to Nikon DS-L1 screen, for capturing the images.

3. RESULTS AND DISCUSSION

Biophysical Characterization of BAPc-MNBs

Two sizes of magnetic iron nanobeads (MNBs) (50 and 200 nm diameter), were used in this study. The branched amphiphilic peptide bilayer on the MNBs was formed as described before.¹¹ We determined the concentration of peptide required to form a monolayer i.e. to saturate the surface by titrating the MNBs with varying concentrations of the inner leaflet peptide, containing the fluorescent amino acid residue, cyanophenylalanine (F_CN), in 75% ethanol:HEPES as described in methods. Six hundred nmoles of peptide per 0.5 mg of 50 nm MNBs was required to form the monolayer (Fig. 1A).

Figure 1. Biophysical characterization of BAPc-MNBs.

(A) Representative graph showing titration of MNBs with bis-(Ac-F_CNLIVIGSII)-KKKKK-C-CONH₂ that demonstrates the saturation of MNB surface with the fluorescent peptide at 600 nmoles of peptide per 0.5 mg of magnetic nanobeads. The cyanophenylalanine residue of the modified peptide sequence emits fluorescence close to 290 nm when excited by light of wavelength 240 nm. Transmission Electron Microscopy (TEM) images of (B) 50 nm and (C) 200 nm BAPc-MNBs on Lacey TEM grids, shows well dispersed magnetic Nanobeads.

The formation of a second peptide layer on magnetic nanobeads in water was confirmed using Förster resonance energy transfer (FRET) analysis as demonstrated in Natarajan et al.¹¹ The peptide forming the inner layer i.e. binding directly to the MNBs surface were modified to have the fluorescent amino acid - cyanophenylalanine in place of the N-terminal phenylalanine. The phenylalanine of peptides in the outer layer were substituted with the fluorescent aromatic amino acid, tryptophan. Cyanophenylalanine when excited with light of wavelength 240 nm, emits light at ~290 nm which is close to the excitation wavelength of tryptophan.²² Thus, the formation of the bilayer was determined using the inherently fluorescent amino acids which do not cause change in the secondary structure of the peptide. Further, the BAPc-MNBs were purified (i.e. separated from the aggregates) by placing them on a magnetic separation rack for a specific amount of time, as shown in Fig S1.

TEM images of the 50 nm and 200 nm BAPc-MNBs showed dispersed magnetic nanobeads lining the edges of the lacey TEM grid (Fig. 1 B, C). Few, if any, BAPc-MNB clusters were observed as a result of the drying process in the grid. Dynamic light scattering (DLS) size analysis indicated that the hydrodynamic size of BAPc-MNBs synthesized using the 50 nm and 200 nm MNBs were 208 nm and 331 nm, respectively (Table 2), while the commercially supplied 50 nm MNBs displayed a hydrodynamic radius of ~80 nm (Fig. S2 A). The polydispersity index of BAPc-MNBs was < 0.2 which is indicative of monodispersed nanoparticles.²³ The intrinsic property of large nanoparticles such as irregularity in shape and the modified surface coating changes the solvation sphere around the nanoparticles increasing the apparent hydrodynamic size of the nanoparticles and thus contributes to the apparent disparity in size between TEM and DLS.²⁴ Zeta potential analysis further confirmed the presence of the BAP-bilayer on MNBs. Zeta potential of MNBs prior to peptide bilayer coating is −30.9 mV, indicating that their surface charge is negative. (Fig. S2 B) BAPc-MNBs with 50 nm and 200 nm cores by contrast have a zeta potential of +23.1 mV and +37.8 mV, respectively (Table 2). This suggests that the BAPc-MNBs are positively charged due to the oligo-lysine tails of the peptides present on the outer, solvent-exposed layer of the nanoparticles. For the sake of simplicity, we refer to the TEM size (50 nm and 200 nm) of BAPc-MNBs in further experiments. In Fig. 2, we compare the structure of BAPc-MNBs and fluorescent rhodamine labeled water filled Rh-BAPCs.

Table 2-.

Size and zeta potential data for BAPc-MNBs determined by Dynamic light scattering and transmission electron microscopy.

Nanoparticle	Size by TEM	Hydrodynamic size by DLS	Polydispersity Index (PdI)	Zeta Potential
50nm BAPc-MNBs	50nm	208nm	<0.2	+23.1mV
200nm BAPc-MNBs	200nm	331nm	<0.2	+37.8mV

Figure 2. Schematic representation of water-filled BAPCs and BAPc-MNBs.

Left panel: water filled BAPCs and the amino acid sequence of the capsules forming peptides. The lysine tagged with rhodamine has been represented in red. Right panel: BAPc-MNBs and the sequence of the bilayer forming peptides. Cysteine (in red) is covalently bound to the maleimide groups embedded in the dextran coating, on the surface of the magnetic nanobeads. The poly-lysine tail has been indicated in green and the hydrophobic tail in blue.

Uptake of BAPc-MNBs by rat ileum intestinal epithelial cells (IEC-18) and mouse macrophages (J774A.1) in vitro

Cellular uptake of BAPc-MNBs was quantified in IEC-18 and J774A.1. Different parameters were taken into consideration for this analysis, such as size of BAPc-MNBs and the percentage of serum present in the cell culture media (Fig. 3A–C). IEC-18 cells exposed to ~50 nm and ~200 nm BAPc-MNBs for 4 h at 37 °C showed increased uptake (~ 2.5-fold) in media containing only 4% of serum (OptiMEM®) compared with DMEM supplemented media containing up to 10% of serum. The composition of OptiMEM® probably improved uptake by IEC-18, in part, due to the reduced number of serum proteins adsorbed to the surface of BAPc-MNBs. This behavior is analogous to other cationic liposomes and nanoparticles, which have been shown to have better transfection efficiency and/or uptake in reduced-serum media.^25–27 With respect to the nanoparticle size, quantitative analysis shows IEC-18 cells internalize significantly more ~50 nm BAPc-MNBs than ~200 nm BAPc-MNBs in both cell culture media. (Fig. 3 A–B). Similar results have been reported by Foged et al.²⁸ with polystyrene nanoparticles in dendritic cells as well as by Oh et al.²⁹ with positively charged gold nanoparticles of varying sizes, in which smaller nanoparticles are internalized by cells more efficiently than larger nanoparticles.³⁰

Figure 3. Uptake of BAPc-MNBs and magnetic nanobeads by IEC-18 and J774A.1 cell.

IEC-18 and J774A.1 cells were incubated with BAPc-MNBs of different sizes and control beads for 4 h in DMEM 10% serum or OptiMEM® 4% serum. The untreated cells were the negative controls. (A) 50 nm BAPc-MNBs internalized by IEC-18 and J774A.1, (B) 200 nm BAPc-MNBs internalized by IEC-18 and J774A.1 and (C) 50 nm BAPc-MNBs internalized by J774A.1 in the presence or absence of Lipopolysaccharide (LPS). sJ774A.1 treated with OptiMEM® could not be analyzed for uptake ANOVA followed by Bonferroni’s posttest was applied for statistical analysis. n=5 (ns=not significant, *p-value<0.05, **p-value<0.01, ***p-value<0.001).

Mouse macrophages (J774A.1) treated under similar experimental conditions used for IEC-18, showed that nanoparticle size did not affect the number of nanoparticles internalized by this cell type, in DMEM 10% Serum (Fig. 3 A–B). Addition of lipopolysaccharides (LPS) to this media yielded similar results (Fig. 3C). LPS induces production of pro-inflammatory cytokines such as IL-1ß and IL-6, activating the macrophages and upregulating phagocytosis of the foreign particles.³¹ Since LPS stimulation did not increase uptake of the beads, we concluded that the uptake of BAPc-MNBs and bare MNBs (control beads) was not TLR-4 mediated or low levels of LPS were present which led to endotoxin tolerance. For both cell types, critical controls (i.e. control beads, untreated cells) were included in all the uptake studies.

Uptake of BAPc-MNBs in IEC-18 and J774A.1 cells was quantified using the ferene-s based spectrophotometric assay.¹⁸ Ferene-s is a triazine compound that binds to iron, allowing the colorimetric detection of iron by measuring the absorbance at 595 nm. The iron content from magnetic beads internalized by treated cells was determined by subtracting the background iron from negative control. Since the iron content of each magnetic nanoparticle is known the number of magnetic nanoparticles internalized can be ascertained. Loss of beads was observed for some treatments, possibly due to a percentage of the beads binding to the cell membrane (or organelle membranes). However, quantification of BAPc-MNB in the cell membrane would lead to inclusion of not just the internalized beads but also the cell membrane bound nanoparticles. This would not be an accurate representation of internalized BAPc-MNBs in the cytosol and therefore, insoluble membranes or cell debris that reacted with ferene-s were spun down prior to spectrophotometric measurements and hence not detected in the assay. However, we can estimate the number of cell membrane-bound nanoparticles by subtracting the nanobeads quantified in the cytosol from the nanobeads recovered i.e in media. (Fig. S3A)

Mechanism of uptake of BAPc-MNBs and Rh-BAPCs by IEC-18.

The effect of different endocytic inhibitors on BAPc-MNBs and Rh-BAPCs uptake was tested in IEC-18 cells. Elucidation of cellular internalization mechanisms can help to determine the sub-cellular processing of the nanoparticles and their potential downstream effects. To inhibit clathrin mediated endocytosis we used chlorpromazine (Cpz) and dynasore. Methyl-b-cyclodextrin (M-β-CD) and nystatin were used to inhibit caveolae mediated endocytosis. M-β-CD is commonly used as an inhibitor for caveolae mediated endocytosis by sequestering cholesterol which in turn perturbs plasma membrane fluidity in lipid rafts. However, there is growing evidence that suggests M-b-CD can also inhibit clathrin mediated endocytosis and macropinocytosis, making it a non-specific inhibitor.^{32, 33} Thus, we also used Nystatin, which is more specific towards caveolae mediated endocytosis. Dynasore is a dynamin inhibitor known to be less specific towards clathrin mediated endocytosis³⁴, therefore, it was used in combination with chlorpromazine (Cpz). To prevent macropinocytosis, we treated cells with Cytochalasin D (Cyt D), which is specific towards macropinocytosis and phagocytosis because it induces depolymerization of actin filaments which are essential for coating the macropinosomes.³⁵ A viability assay was performed to ensure that the selected concentrations of all the inhibitors were nontoxic for cells. (Fig. S4A).

IEC-18 cells were incubated at 1 h and 4 h in the presence of inhibitors and BAPc-MNBs or Rh-BAPCs. Cellular uptake was monitored using microscopy, flow cytometry and UV/Vis spectroscopy. We selected the 50 nm BAPc-MNBs since uptake appeared to be more efficient, as discussed in previous section. Quantitative analysis by UV/Vis spectroscopy suggested that at 1 h, BAPc-MNBs uptake was reduced in the presence of M- β-CD + Cpz as well as Cytochalasin D + Cpz (Fig. 4A–B). This significant reduction in uptake only in the presence of combinatorial treatments indicates that BAPc-MNBs enter cells via multiple endocytic pathways simultaneously and, disrupting one pathway might cause the upregulation of other active pathways. However, excluding M-β-CD, other inhibitors had no significant effect on the uptake of the 50 nm BAPc-MNBs when incubated with cells for 4 h (Fig. 4C–D). We believe that the combinatorial treatments did not elicit a stronger effect because Cpz (clathrin inhibitor) did not have a significant impact on the uptake of BAPc-MNBs. It is also possible that BAPc-MNBs may be endocytosed via a clathrin and caveolae independent pathway at this incubation time. This pathway is independent of dynamin function and caveolae dependent microdomains but majorly dependent on the cell membrane cholesterol which affects membrane fluidity.^{19,36, 37} Cells may also be using an energy-dependent pathway which is unaffected by the chemical inhibitors used in this study, over 1 h of incubation with BAPc-MNBs.

Figure 4. Endocytosis inhibition assay to determine uptake mechanism of ~50 nm BAPc-MNBs by IEC-18.

(A) and (B) IEC-18 incubated for 1 h with BAPc-MNBs + single and combinatorial inhibitor treatments respectively.(C) and (D) IEC-18 incubated for 4 h with BAPc-MNBs + single and combinatorial inhibitor treatments respectively. ANOVA followed by Bonferroni’s posttest was applied for statistical analysis. (ns=not significant, *p-value<0.05, **p-value<0.01, ***p-value<0.001).

Some individual treatments with inhibitors such as Nystatin revealed higher uptake (120%) compared with the “No inhibitor” group. It has been reported in the literature that inhibition of a single endocytic pathway can up-regulate another pathway.³⁸ Thus, inhibition of caveolae mediated endocytosis by Nystatin may have increased internalization via macropinocytosis. Moreover, one cell type can sometimes endocytose the same nanoparticle using multiple pathways, as nanoparticle formulations are often made up of a group of heterogeneous particles, which makes the process more complicated to be analyzed.³⁹

Rh-BAPCs (~ 50 nm) were subjected to similar experimental conditions used for BAPc-MNBs. Confocal imaging showed that water-filled Rh-BAPCs internalization in IEC-18 was inhibited by Nystatin, Cpz, CytD, Cpz + Cytochalasin D, M-β-CD + Cpz and to some degree with M-β-CD after 1 h of incubation (Fig. 5). These results confirm that at 1 h internalization occurs via multiple pathways. After 4 h of incubation, uptake was inhibited only by Cytochalasin D + Cpz and to some degree by Cpz, showing that at longer incubation times clathrin mediated endocytosis and macropinocytosis are the major uptake pathways for Rh-BAPCs (Fig. 5). Flow cytometry was used as well to verify results obtained by confocal microscopy. This analysis also showed a significant decrease in the uptake of fluorescent BAPCs in the presence of Cpz and Cpz + Cytochalasin D, which agreed with confocal analysis (Fig. S5 A–B). Nevertheless, a limitation of this analytical technique is that Rh-BAPCs trapped or bound to the cell membrane can result in the detection of a false positive for cells that did not actually uptake the nanoparticles. Therefore, results from this analysis should be always complemented with a secondary technique.

Figure 5. Endocytosis inhibition assay to determine mechanism of rhodamine labelled BAPCs uptake by IEC-18.

IEC-18 cells incubated with rhodamine BAPCs for 1 h and 4 h after preincubation with respective inhibitors of endocytosis Inhibitors used are indicated in the bottom right corner of each micrograph. The pathway inhibited has been indicated above the micropgraphs for individual inhibitor treatments. For combinatorial treatments, the micrographs are placed directly below one pathway inhibited and to the right of the other pathway inhibited. The time of incubation is indicated to the left of the micrographs. Control group were cells treated exclusively with saline solution (PBS) and served as a positive control. BAPCs only group was not exposed to inhibitors. Bright field images showing cell boundaries are shown in Fig. S6.

Endocytosis of nanoparticles is a complex process involving several proteins that play a role in the identification of the cargo and subsequent internalization via vesicle formation.^{39, 40} In the case of BAPCs, different cores (magnetic vs water) and sizes influenced the uptake route. These discrepancies were noticeable at both incubation times and Table. S1 summarizes the effect of endocytic inhibitors on BAPc-MNBs and Rh-BAPCs. Recent discoveries also indicated that incubation time with inhibitors may be cell type and nanoparticle size dependent, therefore a careful optimization would be required for each system.^{41, 42} Review of literature suggest that endocytosis of nanomaterials can take around 20–30 min or the whole process can take up to 4 h,¹¹ besides different uptake routes can be activated depending on the exposure time to nanoparticles.³¹

Co-localization of magnetic nanobeads inside cells and their influence on cell viability and free radical generation.

Toxicity of BAPc-MNBs in IEC-18 cells was evaluated by flow cytometry. This is a rapid and reliable method to quantify cell viability.⁴³ Dead cells are identified by the fluorochrome 7-AAD, which binds to the DNA of damaged cells. As a result, those cells emit fluorescence at 647 nm and are identified as non-viable cells. As seen in Fig. 6A, viability of IEC-18 treated with 50 nm BAPc-MNBs was minimally affected (<5% cell mortality). However, the number of non-viable cells increased at bead to cell ratios of 10000:1 for the 200 nm BAPc-MNBs. Due to some degree of cellular stickiness observed in macrophages, we did not use the 7AAD dye-based flow cytometry assay used to determine cell viability. Instead we used an MTT-like assay kit available commercially, which is claimed to be more sensitive than the traditional MTT assay for determining the macrophages viability.⁴⁴ The viability of macrophages treated with 50 nm and 200 nm BAPc-MNBs as well as the control MNBs was not significantly affected. Sodium azide (0.04 mM) was used to generate a positive control group, as it is cytotoxic and results in significant cell death. Thus, 50 nm BAPc-MNBs do not have an adverse effect on the viability of IEC-18 and J774A.1 cells (Fig. 6B).

Figure 6. Cell viability assay and reactive oxygen species (ROS) assay to determine toxicity of BAPc-MNBs to cells.

Flow cytometry analysis using 7AAD was used to determine cell viability of (A) IEC-18, treated with 50nm and 200nm BAPc-MNBs and control MNBs at 10000:1, beads to cell ratio. Negative control=No treatment. (B) Cell viability of J774A.1 cells were determined using an MTT like assay. The cells were treated with 50nm and 200nm BAPC-MNBs and control MNBs at 10000:1, beads to cell ratio. Reactive oxygen species generated by (C) IEC-18 and (D) J774A.1 cells after 4h and 24h incubation with 10000:1 beads to cells, as determined by DCF-DA fluorescence-based assay. A normalized fluorescence greater than 1 signifies higher ROS generated in comparison to untreated cells. Negative control=No treatment, Positive control = treated with hydrogen peroxide. (* p-value < 0.05, ** p-value < 0.01, n=4)

Reactive oxygen species generated by cells in response to nanoparticles is also a potent early marker for nanoparticle toxicity.^{45, 46} Transition metals such as iron (Fe⁺²) in iron oxide nanoparticles can generate ROS by reacting with hydrogen peroxide (H₂O₂) to form hydroxyl free radical (OH•) which is called the Fenton reaction.^{45, 47} These can disrupt the mitochondrial activity, cause damage to DNA and cause lipid peroxidation. This in turn destabilizes the cell membrane making it more susceptible to oxidation.^{47, 48} Phagocytes generate reactive nitrogen species (RNS) owing to their inducible nitric oxide synthase (iNOS) activity in response to activation by foreign molecules.⁴⁷ Therefore, measuring ROS/RNS generated by epithelial cells and macrophages will help us determine the downstream effects of BAPc-MNBs from a toxicity perspective. Reactive oxygen species (ROS) were detected using Dichlorofluorescein-diacetate (DCF-DA) fluorescence assay. The release of reactive oxygen species causes increase in fluorescence of membrane permeable DCF-DA. BAPc-MNBs do not cause a significant increase in the ROS (Fig. 6 C, D) in either epithelial cells or macrophages. Similarly, no reactive nitrogen species (RNS) were detected in IEC-18 and J774A.1 cells treated with BAPc-MNBs. (Table S2)

To verify that BAPc-MNBs gain access to IEC-18 and J774A.1 cytosolic space, cells were stained by incubation with Prussian blue (Fig. 7 A–B). This staining method uses an inorganic compound, potassium ferrocyanide, along with hydrochloric acid to permeate the fixed cells and bind to iron, developing a Prussian blue colored pigment. Thus, from these observations we confirmed that BAPc-MNBs and the control MNBs co-localized within IEC-18 and J774A.1 without affecting their morphology, growth or survival rate.

Figure 7. Prussian blue staining for visualization of magnetic nanobeads in J774A.1. and IEC-18 cells.

The cells incubated with magnetic nanobeads at 10000:1, bead to cell ratio were imaged using an Optical light microscope (45X magnification for J774A.1 & 20X for IEC-18), to visualize the magnetic nanobeads internalized by cells as blue aggregates. (Panel 1) J774A.1 macrophage with (A.1) BAPc-MNBs, (B.1) MNBs and (C.1) no nanobeads; counter stained with Hema Stain® solution I (stains cytoplasm pink). (Panel 2) IEC-18 epithelial cells with (A.2) BAPc-MNBs, (B.2) MNBs and (C.2) no beads.

4. CONCLUSIONS

In summary, we studied the endocytic uptake routes of BAPc-MNBs in epithelial cells. The magnetic beads facilitated the separation and sorting of the cells that have internalized BAPCs and allowed us to quantify nanoparticles per cell using the ferene-s method. Results indicated that after exposing cells to BAPc-MNBs for 1 h, the preferable route of entrance is clathrin and caveolae mediated endocytosis and macropinocytosis. Most likely these routes act synergistically. At 4 h, caveolae mediated endocytosis appeared to be the predominant pathway. However, future studies will elucidate and confirm if BAPc-MNBs may be endocytosed via a clathrin and caveolae independent pathway at this incubation time. This pathway is independent of dynamin function and caveolae dependent microdomains, but it is highly dependent on the cell membrane cholesterol which affects membrane fluidity.

We compared uptake results of BAPc-MNBs with water-filled Rh-BAPCs using the same incubation periods. Confocal imaging showed that at 1 h internalization is similar to BAPc-MNBs. However, after 4 h of incubation, uptake was inhibited only by Cytochalasin D + Cpz and to some extent by Cpz, showing that at longer incubation times clathrin mediated endocytosis and macropinocytosis are the major uptake pathways for Rh-BAPCs. These slight variances observed mostly at longer incubation times can be attributed to the type of nanoparticle used (BAPCs with different cores). Similar discrepancies have been reported for other nanoparticles with different cores⁴⁵.

The study also demonstrated that while epithelial cells preferentially internalize BAPc-MNBs over unmodified control beads, macrophages indiscriminately phagocytose the magnetic beads with different surface compositions. Uptake also appeared to be more efficient for 50 nm BAPc-MNBs than 200 nm BAPc-MNBs in epithelial cells and hence 50 nm BAPc-MNBs were used to determine the endocytic uptake mechanism. Cell viability was carefully assayed and we confirmed that it is minimally affected by BAPc-MNBs as < 5% of non-viable cells were detected using flow cytometry and MTT analysis in IEC-18 and macrophages, respectively. In addition, BAPc-MNBs did not significantly induce ROS generation in the cell lines we tested. i.e. induce oxidative stress, that could cause damage to the DNA and mitochondria.

We have also explored different techniques in this study and have therefore been able to weigh their advantages and disadvantages. Peptide bilayer coated magnetic beads are quantitative tools, however, a limitation of this technique is the exclusion of BAPc-MNBs bound to the cell membrane. Confocal imaging allows for visual analysis with the limitation that it is not quantitative. Flow cytometry on the other hand is not very suitable for the kind of study conducted here given that interference was observed from membrane embedded fluorescent particles. Overall, applying different methods helps to compensate for each other’s limitation.

Biophysical characterization of BAPc-MNBs confirmed the presence of the peptide bilayer coating on MNBs and the homogeneity/ monodispersity of the nanoparticles. Thus, we have described herein, a relatively easy and reliable method to synthesize cationic peptide bilayer coated magnetic nanoparticles. We explored the similarities and differences between the magnetic and water filled core BAPCs in cell culture, for their use in other applications in vitro and in vivo. This study brings us a step closer to understanding BAPCs and their interactions with cells, that will allow us to further investigate the potential of magnetic and water filled core BAPCs in therapeutic applications.

ABBREVIATIONS

BAP

Branched amphiphilic peptides

BAPCs

Branched amphiphilic peptide capsules

MNBs

magnetic nanobeads

BAPc-MNBs

Branched amphiphilic peptide coated magnetic nanobeads

CPP

cell penetrating peptides

MPS

mononuclear phagocytic system

DLS

Dynamic light scattering

TEM

Transmission electron microscopy

LPS

lipopolysaccharide

TLR4

Toll-Like receptor 4

IEC

Intestinal epithelial cells

M-b-CD

methyl-b-cyclodextrin

Cpz

Chlorpromazine

Nys

Nystatin

CytD

cytochalasin D

Dyn

dynasore

CME

Clathrin mediated endocytosis

DIH₂O

Deionised water

RT

room temperature

TFE

Trifluoroethanol

ROS

Reactive Oxygen Species

RNS

Reactive Nitrogen Species