Protein A Functionalized Polyelectrolyte ... - ACS Publications

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Protein A Functionalized Polyelectrolyte Microcapsules as a Universal Platform for Enhanced Targeting of Cell Surface Receptors Tatiana A. Kolesnikova,* Gayane Kiragosyan, Trang H. N. Le, Sebastian Springer,‡ and Mathias Winterhalter‡ Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany S Supporting Information *

ABSTRACT: Targeted delivery systems recognizing specific receptors are a key element in personalized medicine. Such systems allow the delivery of therapeutics to desired sites of the body, increasing their local concentration and thus reducing the side effects. In this study, we fabricate chemically cross-linked (PAH/PAA)2 microcapsules coated with specific cell-targeting antibodies in random (via direct covalent coupling to the surface) or optimized (via supporting layer of protein A) orientation. We use these antibody-functionalized capsules to target major histocompatibility complex (MHC) class I receptors in living cells and quantify the efficiency of targeting by flow cytometry. We show for the first time the selective binding of polyelectrolyte microcapsules to MHC class I receptors, and confirm that targeting is allotype-specific. Remarkably, protein A assisted immobilization of antibodies enhances targeting efficiency by 40−50% over capsules with randomly attached antibodies. Moreover, biofunctionalized capsules reveal low levels of cytotoxicity and nonspecific binding, excluding the need of additional modification with poly(ethylene glycol). Thus, protein A coated (PAH/PAA)2 microcapsules represent a unique example of universal targeting tools providing high potential for selective binding to a broad range of cell surface receptors. KEYWORDS: antibody−antigen interaction, layer-by-layer, MHC class I protein, polyelectrolyte microcapsules, protein A, selective targeting

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INTRODUCTION Recent developments in genomics and molecular biology have made it possible to introduce a large variety of proteins, peptides, enzymes, and nucleic acids as therapeutic agents in molecular medicine and cancer therapy.1,2 However, most of these molecules are prone to hydrolysis, and small conformational changes may reduce their activity; therefore, their stabilization is required. Polyelectrolyte microcapsules (PEMs) nicely fulfill this requirement, since they are able to load a sufficient amount of therapeutic cargo.3−5 The cargo stays protected from hydrolyzing enzymes by a separating polymeric shell, and thus, it can be delivered to the cell in its active form.6,7 PEMs of different sizes and charges can be easily produced by a well-established layer-by-layer (LbL) technique.8,9 Adjustability of physicochemical properties is a substantial advantage of PEMs over other nano- and microcontainers, since PEMs can be chemically engineered to meet the needs of individual experimental design.10−13 For instance, © XXXX American Chemical Society

the cargo can be released into the cytosol in response to a welldefined stimulus, e.g., light or ultrasound if PEMs are sensitized with nanoparticles,14−18 pH and temperature if PEMs are functionalized with star polyelectrolytes,19,20 or in the course of biodegradation if PEMs are made of biodegradable materials.21 Functionalized capsules can be introduced into living cells via electroporation and passive uptake;14,22,23 however, the more sophisticated active uptake mechanism is driven by the molecular recognition system of cells. PEMs equipped with targeting ligands, such as antibodies, receptor peptides or aptamers, have the potential to selectively bind to a certain receptor at the plasma membrane, and thus to target a specific cell type.24−27 Using this concept, delivery of therapeutics to a particular site of the body could be greatly improved, thus Received: January 25, 2017 Accepted: March 14, 2017 Published: March 14, 2017 A

DOI: 10.1021/acsami.7b01313 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. (PAH/PAA)2 microcapsule fabrication and functionalization with targeting antibodies. A. Structural formula of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) sodium salt (PAA). B. Scanning electron microscopy. SEM image of a 6-μm (PAH/PAA)2 microcapsule after shell cross-linking and CaCO3 core dissolution. C. Scheme: Random vs optimized antibody immobilization. Random immobilization by direct covalent coupling of primary amino groups of the antibody to carboxyl groups on the capsule surface using EDC/sulfoNHS activation chemistry (top). Optimized immobilization via protein A (bottom): protein A is covalently attached to the capsule surface first, then the Fc domain of the antibody is coupled to protein A. The schematic is not to scale. D. Flow cytometry study of random vs optimized antibody immobilization. Murine Y3 mAb is coupled to the capsule surface in random (orange curve) and oriented (dark green curve) configuration. The antibodies are detected by Alexa Fluor 488-labeled secondary goat antimouse antibody (GαM-AF488) by flow cytometry. Two secondary antibody controls (blue and light green curves) reveal no nonspecific binding to the capsules, giving the same signal as nonfunctionalized capsules (red curve). MFI values are given for each sample.

coreceptors for binding to class I antigens on target β cells, leading to β cell selective destruction and a deficiency of insulin secretion. Because the autoimmune process is undetected until the point of diagnosis, the trigger of T1D remains unknown. We believe that selective targeting of MHC class I molecules in β cells with PEMs loaded with genetic material aiming to backregulate the expression level of class I to normal, could inhibit autoimmune responses against β cells, thereby suppressing the disease. This would allow to keep at least some β cells alive and functional, which could be the first step toward the cure of type 1 diabetes. The two important questions that need to be answered first are the possibility of targeting class I receptors with PEMs, and the selectivity of targeting, which is one of the major current challenges. The strategy of surface functionalization of PEMs to achieve the best targeting efficiency has been insufficiently explored; still there are many open problems to solve, for example the improvement of signal-to-background ratios,24,27 the simplification of the construction of the shell,25,34 the development of the quantitative high-throughput screening techniques to monitor the interaction of capsules with cells,26 etc. In this work, we determine the degree of capsule association with cells by quantitative flow cytometry and perform dedicated experiments using nonfunctionalized (PAH/PAA)2 capsules, and capsules coated with targeting antibodies or control proteins to ensure the selectivity of binding. By testing different

increasing their local concentration and reducing undesirable toxic effect on other cells and tissues. Recently, we have demonstrated that PEMs made of synthetic polyelectrolytes PAH (polyallyllamine hydrochloride) and PAA (poly(acrylic acid) sodium salt) can be chemically cross-linked and functionalized with biomolecules, allowing for ultrasensitive detection of soluble analytes in biological fluids.28 In the current study, we produce cross-linked (PAH/PAA)2 microcapsules, and develop a protocol of their biofunctionalization with antibodies aiming to target specific receptors at the plasma membrane of living cells very selectively. In particular, we target peptide receptors of the immune system called major histocompatibility complex (MHC) class I molecules. Briefly, these are transmembrane proteins, which are able to bind intracellular peptide fragments derived from endogenous antigens, and transport them to the cell surface for inspection by cytotoxic T lymphocytes (CTLs);29 if peptides are pathogenic, CTLs will immediately react and kill the infected cell. MHC-associated diseases include almost all autoimmune diseases, because of the very high gene diversity in the MHC region.30 In some cases, like autoimmune disorder type 1 diabetes (T1D),31 class I molecules are overexpressed on the surface of the insulin-secreting β cells of the islets of Langerhans in the pancreas.32,33 The T1D scenario is the following: class I proteins present fragments of pancreatic β cell peptides to cytotoxic T cells; the CD8 molecules on CTLs act as B


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ACS Applied Materials & Interfaces Table 1. Overview of Cell Lines, MHC Class I Proteins and Antibodies Used in the Current Studya cell type

cell line

origin

adherent suspension

STF1 RMA

human mouse

H-2Kb-GFP H-2Kb

MHC class I

suspension

T1

human

HLA-A2, HLA-B

antibody used

specificity

binding

Y3 (murine) Y3 (murine) 5D3 (hamster) W6/32 (murine) Y3 (murine) 5D3 (hamster)

α-H-2Kb α-H-2Kb α-tapasin α-HLA α-H-2Kb α-tapasin

+ + − + − −

a Includes cell type and abbreviation, origin of cells, allotypes of MHC class I molecules expressed, origin and specificity of the antibodies used for cell staining experiments or capsule surface functionalization, and expected binding of the Ab-functionalized capsules to the cells.

Figure 2. Targeting MHC class I receptors in human fibroblasts STF1 cells with the antibody-functionalized (PAH/PAA)2 microcapsules. A. Schematics. Microcapsules are coated with protein A and postfunctionalized with murine Y3 mAb that recognizes specifically the H-2Kb allotype of class I molecules at the plasma membrane of STF1 cells. Kb molecules are expressed as a GFP fusion, allowing direct fluorescent imaging by microscopy. Interaction between the antibody and the antigen allows for selective binding of the capsules to the cell surface with accumulation of the Kb-GFP at the point of contact (green asterisk). Schematic is drawn not to scale. B. Confocal microscopy study. Accumulation of Kb-GFP at the points of contact between the cell surface and the Y3-functionalized capsules (bright green dots marked with the white arrows) after 3 h of capsule incubation with STF1/Kb-GFP cells at 37 °C. The 6-μm sized microcapsules are filled with BSA-AF647 (red). Several confocal planes are merged together in order to obtain maximum contrast. Scale bars are 10 μm.

cores, and subsequently cross-linked with 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC)39,40 to form mechanically stable polyelectrolyte shells (Figure 1B).28,41,42 Cross-linking allows us to limit the number of fabrication steps to only four polyelectrolyte layers without compromising the integrity and the stability of the shell even at the extreme conditions (see the Supporting Information, Figure S1). After cross-linking, the particle surface was functionalized with targeting antibodies using two approaches: direct covalent coupling of their lysines to the carboxylated surface via EDC and N-hydroxysulfosuccinimide (sulfo-NHS), resulting in their random orientation on the surface,43 or immobilization via adaptor protein A that was covalently attached to the surface and served as a supporting layer for optimized antibody orientation.44 Random orientation of the antibodies in “headon” and “sideways-on” spatial configurations, as well as their optimized orientation in the “end-on” configuration are represented schematically in Figure 1C. In this study, several monoclonal antibodies (mAb) were used for immobilization, namely Y3,45 5D3,46 and W6/3247 (see Table 1 for specification). Each fabrication and functionalization step was characterized by zeta potential measurements (see the Supporting Information, Figure S2). To detect the presence of the antibodies, PEMs were stained with fluorescently labeled secondary antibodies and measured by flow cytometry (Figure S3) and confocal microscopy (Figure S4). As observed, protein A-coated PEMs with immobilized mAb show stronger fluorescence than PEMs with directly attached mAb (Figure 1D; dark green vs orange curves, respectively). Since the EDC/sulfo-NHS-induced reaction might occur with

cell lines expressing different allotypes of class I molecules, we demonstrate for the first time that targeting is allotype-specific. Remarkably, by introducing Staphylococcus aureus protein A as a supporting layer for optimized antibody immobilization on the capsule surface, we are able to increase the targeting efficiency by 40−50% over randomly attached antibodies, with signal-tobackground ratio enhanced by a factor of 3. Generally, our capsules reveal low cytotoxicity and low level of nonspecific binding. The developed protocol of biofunctionalization is universal and can be potentially applied to other targets.

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RESULTS AND DISCUSSION For delivery applications, the size of the vehicles is a very important parameter, since it can affect their biodistribution and functionality. The size can range from 10 nm to several micrometers in diameter; however what size is best remains an ongoing debate, suggesting that each particular application requires particular experimental design.35 It is known that particles similar to red blood cells in size (6−8 μm) demonstrate longer circulation times compared to the smaller ones.36,37 We therefore produced 3 and 6 μm capsules, as these sizes could offer good circulation times and high payload, while still enabling cellular uptake.21,38 Poly(allylamine hydrochloride) and poly(acrylic acid) were chosen as shell components due to their well-studied physicochemical properties and availability of functional amino- and carboxyl-groups that allow for further cross-linking and functionalization with biomolecules (Figure 1A). (PAH/PAA)2 microcapsules were produced by a sequential adsorption of polyelectrolytes on 3- or 6-μm calcium carbonate (CaCO3) C


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ACS Applied Materials & Interfaces any and all lysine residues all over the protein, the protein molecule will be attached to the surface in random configuration.43 When the antibody is randomly attached to the particle surface, the antigen binding sites might be blocked by modification of their lysine residues, which may compromise the functionality and prevent the binding of the secondary antibody. However, when protein A is attached to the surface in random orientation, it can still bind antibodies very efficiently, since protein A has several IgG binding sites per molecule.48 As in both approaches the amounts of mAb used were the same, we hypothesized that the presence of protein A preserves functionality and probably optimizes the antiboy orientation, which may result in a better efficiency of targeting.49,50 Previously, we have quantified the number of mAb that can be captured by protein A-coated capsules by UV−vis spectroscopy and SDS-PAGE analysis, which was about 4 × 105 and 1 × 105 molecules per 6- and 3-μm capsule, respectively.28 Importantly, due to the porosity of the polyelectrolyte network, small and medium-sized biomolecules can diffuse through the shell if the core is removed and become trapped inside the capsules.51 Recently, we have demonstrated that if particles are maintained in core−shell structure during all functionalization steps, such undesired internal accumulation of biomolecules can be avoided.28 Herein, we remove the cores just before addition of capsules to the cells (see the Materials and Methods section). To support our hypothesis and to study the targeting capability of the antibody-functionalized microcapsules, we tested a number of conditions using various cell lines (human fibroblasts STF1, murine lymphoma RMA, and human lymphoma T1 cells). In particular, RMA and T1 cells expressing high levels of class I were used to model type 1 diabetes conditions in pancreatic β cells. Selection of cell lines was based on the allotype of MHC class I receptors expressed, and their reactivity with the antibodies (see Table 1). The murine Y3 mAb is highly specific for the H-2Kb (Kb) allotype of class I proteins,45 which were expressed as a green fluorescent protein (GFP) fusion at the plasma membrane of STF1 cells (abbreviated as STF1/Kb-GFP), or in their native nonfluorescent form in RMA cells. The 5D3 mAb from Armenian hamster was used as a negative control for Kb in RMA cells, since it recognizes the irrelevant cell-internal protein tapasin.46 The murine W6/32 mAb specifically recognizes human HLA-A2 and HLA-B class I proteins, which are expressed in human T1 cells,47 while Y3 and 5D3 mAb serve as negative controls for T1. As a proof-of-concept, we first tested the functionality of the antibodies coupled to the PEM surface by performing a series of experiments on adherent STF1 cells (Figure 2A). Cells were incubated with 6 μm-sized (PAH/PAA)2 capsules functionalized with Y3 mAb in optimized orientation. After 3 h of incubation, cells were analyzed by confocal laser scanning microscopy (CLSM). A series of z-stack images were taken and merged to obtain maximum contrast (Figure 2B). Remarkably, the CLSM revealed an accumulation of the GFP signal at the point of contact between the cell surface (green) and the capsules (red), suggesting that the Kb-GFP antigen, which is diffusible in the plane of the membrane, was being bound strongly and trapped by the antibody. In the control sample, incubation with nonfunctionalized PEMs did not reveal any binding (see the Supporting Information, Figure S5). To improve the quantification, we next used flow cytometry for measuring association of capsules with suspension cells. First, to ensure the specificity of the antibody−antigen interaction,

Figure 3. Characterization of surface expression of MHC class I receptors in suspension cells. A. Staining RMA cells with antibodies at RT. Flow cytometry histograms represent fluorescence of RMA cells incubated with the monoclonal antibodies (Y3 or 5D3, 1 μg each) for 1.5 h at RT, followed by staining with the polyclonal AF488-labeled goat-αmouse secondary antibody (GaM-AF488, 1 μg). Y3 mAb specifically binds to Kb class I proteins, whereas 5D3 mAb is nonspecific for Kb. Legend: RMA cells (red curve) are used as a background control; negative controls (blue and orange) do not reveal any fluorescence; staining with Y3 mAb (light green) provides more than 280-fold signal increase above the background; importantly, post-treatment of Y3-stained RMA cells with trypsin for 15 min (dark green curve) do not reveal any loss of fluorescence. B. Staining T1 cells with antibodies at RT. Flow cytometry histograms of T1 cells incubated with the monoclonal antibodies (Y3, 5D3 or W6/32, 1 μg each) for 1.5 h at RT, followed by staining with the GaM-AF488 secondary antibody (1 μg). The W6/32 mAb specifically recognizes human HLA-A2 and HLA-B class I proteins, whereas Y3 or 5D3 mAb serve as negative controls.

we performed a series of the antibody stains for the suspension cells at different conditions (Figure 3). For RMA cells, the Y3 stain was clearly visible due to recognition of Kb molecules, whereas the 5D3 stain was negative (Figure 3A). Importantly, the level of Kb did not change in RMA cells additionally treated with trypsin after the staining, confirming that class I molecules cannot be cleaved by trypsinization. For T1 cells, the W6/32 stain was clearly specific for HLA-A2 and HLA-B class I, whereas Y3 and 5D3 stains were both negative (Figure 3B). Next, we tested the interaction of RMA cells with 3 μm-sized (PAH/PAA)2 capsules functionalized with Y3 mAb in random or optimized orientation at 4 °C vs room temperature (RT), using nonfunctionalized PEMs as control (Figure 4A). When measured by flow cytometry, gating was performed on cells (see the Supporting Information, Figure S6), and the fluorescence of the capsules bound to the cell was plotted on the x-axis. As observed, the antibody-functionalized PEMs bound specifically to their antigenic proteins at the cell surface at both temperatures, whereas in the control no association of PEMs with RMA cells was observed (Figure 4B). The level of association was stronger for capsules with Y3 mAb attached in optimized orientation than for capsules with randomly attached Y3. Remarkably, the signal-to-background ratio increased approximately by a factor of 3 due to optimization of mAb orientation: from 10 to 35 at 4 °C and from 35 to almost 100 at RT. The efficiency of binding was lower at 4 °C (blue curves) than at RT (orange curves). This is expected, since at low D


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Figure 4. Targeting MHC class I receptors in RMA cells with the antibody-functionalized (PAH/PAA)2 microcapsules. A. Schematics. Microcapsules are functionalized with the murine Y3 mAb in optimized orientation (via protein A) or in random orientation (via EDC/sulfo-NHS reaction). Y3 recognizes specifically the H-2Kb class I expressed on the surface of RMA cells. Nonfunctionalized capsules are used as a negative control, where no recognition/binding to RMA cells occurs. Schematic is drawn not to scale. B. Flow cytometry study. Flow cytometry histograms of the interaction of RMA cells with three sets of capsules: nonfunctionalized (left), Y3-functionalized capsules in optimized (middle) and random (right) orientation. The 3-μm sized capsules filled with BSA-AF647 are incubated with RMA cells for 1.5 h at 4 °C (blue curves) or RT (orange curves). Ratio of capsules to cells is 30:1. Numbers on each histogram represent the corresponding mean fluorescence intensity (MFI) values for better comparison. RMA cells alone are used to define a background signal (red curves). C. Imaging flow cytometry. Interaction of RMA cells with the corresponding three sets of capsules (see Figure 4B). Ratio of capsules to cells is 30:1. The data confirm that optimized orientation results in a higher targeting efficiency than the random orientation of the antibody on the capsule surface. Ch01: phase-contrast; Ch02/Ch11: merged fluorescent channels.

ized PEMs, a few cells had one capsule bound (Figure 4C, left), whereas most of the cells showed no binding at all. This explains the slight increase of fluorescence above the background in Figure 4B (left). In the case of the antibody-functionalized PEMs, the average numbers of associated capsules per cell were more than 15 for optimized (Figure 4C, middle) and less than 10 for random orientation of Y3 on the PEM surface (Figure 4C, right). This explains the difference in fluorescence in the corresponding histograms in Figure 4B (middle and right). We conclude that the orientation of antibodies on the PEM surface is important, since it improves their targeting capabilities. Clearly, after 1.5 h of incubation, PEMs were only bound to the cell surface and not taken up by cells (see the Supporting Information, Figure S8).

temperatures the plasma membrane undergoes a phase transition from liquid to gel state, when membrane proteins are restricted in their lateral movement due to low fluidity but still retain their physiological functions;52,53 thus, at 4 °C class I molecules cannot diffuse in the membrane and accumulate at the point of contact with the capsules. This explanation is supported by the results of the RMA stain with Y3 mAb at 4 °C and RT, revealing similar surface levels of Kb in both conditions (see the Supporting Information, Figure S7). The optimal ratio of capsules to cells was determined as 30:1 and kept constant throughout all experiments. In addition, RMA cells were characterized by imaging flow cytometry, revealing similar results for binding the antibodyfunctionalized capsules at RT. As observed for nonfunctionalE


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Figure 5. Summary of the flow cytometry study of interaction between the antibody-functionalized (PAH/PAA)2 microcapsules and the suspension RMA cells at different conditions. A. Incubation with capsules at 4 °C vs RT. RMA cells are incubated with the capsules at 4 °C or RT, and the difference in association is measured by flow cytometry (n = 15). B. Post-treatment of cells with trypsin after incubation with capsules. RMA cells are incubated with capsules at RT. Half of each sample is then treated with trypsin for 15 min at RT, followed by 3× washing with PBS (pH 7.2). The difference in association before/after the trypsin treatment is then measured by flow cytometry (n = 3). C. Incubation with the mixture of specific/ nonspecific capsules at RT. RMA cells are incubated with the mixture of red and green capsules at RT. The Y3-functionalized capsules of one color are added together with the 5D3-fuctionalized capsules of another color. Ratio of capsules to cells is 30:30:1. The selectivity of targeting is measured by flow cytometry (n = 3). D. Variation of surface level of class I molecules in RMA cells. RMA cells are cultured overnight at 25 °C vs 37 °C, followed by incubation with capsules at RT. The difference in association is measured by flow cytometry (n = 3). Set of capsules used: nonfunctionalized (A); coated with protein A (B); functionalized with Y3 (C) and 5D3 (D) antibodies in optimized orientation; or Y3 (E) and 5D3 (F) antibodies in random orientation; and coated with BSA (G). The Y3-functionalized capsules (C, E) are used for selective targeting of H-2Kb molecules in RMA cells. Other capsules (A, B, D, F, G) serve as negative controls. Ratio of capsules to cells in each sample is 30:1, unless stated otherwise. Cells are incubated with the capsules for 1.5 h, gently mixing, followed by 6× washing with PBS (pH 7.2). Mean fluorescence intensity (MFI) values are normalized by the maximum value. Error bars indicate the standard error of the mean (SEM); n is the number of independent experiments.

capsules next to the cell surface during the incubation time,54 and treatment with trypsin disrupts weak interactions with those receptors, releasing the noninternalized PEMs from the cell surface. Importantly, RMA cells that were incubated with Y3-functionalized PEMs (both in optimized and random orientation) showed the strongest fluorescence, even after exposure to trypsin, thus additionally confirming the specificity of targeting Kb surface receptors. Next, the specificity of cellular binding was investigated by incubating RMA cells with a mixture of specific green and nonspecific red (and vice versa) PEMs functionalized with antibodies. As observed, the interaction was very specific (Figure 5C): from the corresponding mixtures, the Y3-functionalized PEMs of one color selectively bound to RMA cells, while the 5D3fuctionalized PEMs of another color did not. In controls, neither nonfunctionalized nor protein A- or BSA-coated PEMs of any color bound to the cells. It is worth mentioning that the obtained results open up avenues for multiplexing, when colorcoded capsules with an attached library of targeting antibodies would recognize different antigens on the cell surface simultaneously. Additionally, we tried to model different physiological conditions by varying the Kb expression level in RMA. It is known that the surface level of Kb can be increased at low temperature (25 °C) due to the reduced endocytic destruction.55,56 To test whether binding of capsules to the cells depends on the expression level of class I molecules, RMA cells were cultured at 37 and 25 °C overnight, followed by incubation with the

To ensure the reliability and reproducibility of our targeting approach, we performed a number of additional controls using RMA cells and the following set of PEMs: nonfunctionalized; coated with protein A; functionalized with specific (Y3) or nonspecific (5D3) antibodies in optimized or random orientation; and capsules coated with BSA. We tested protein A for its ability to induce the nonspecific binding of capsules to the cells, while BSA was used as a model irrelevant protein. We expected that only Y3-functionalized PEMs would bind to RMA cells, and not the controls. Indeed, the association of capsules with cells was very selective for incubation at both 4 °C and RT (Figure 5A), although the effect was more pronounced at RT, resulting in ∼80% association increase. The difference in binding capsules with optimized and randomly oriented Y3 was about 40−50%, which proves that orientation of antibodies on the capsule surface is important for improved targeting. We observed an increase in the background signal from 1% to 25% while conducting the experiment at RT; however, the total signal-to-background ratio remained constant for both conditions. Based on this observation, we decided to perform all further experiments at RT. To test whether capsules were just attached to the surface and not internalized by cells, cells were treated with trypsin after incubation with capsules and again measured in the flow cytometer (Figure 5B). Exposure to trypsin resulted in a 60% decrease of fluorescence in all samples; the background signal dropped to almost 10% in comparison to initial 25%. Possibly, integrin-type or other receptors become involved in holding the F


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ACS Applied Materials & Interfaces capsules. In the case of 25 °C, approximately 40% increase in capsule association with cells was observed (Figure 5D), which is in a good agreement with the antibody staining experiments, revealing about 40% signal increase due to accumulation of Kb molecules at 25 °C in comparison to 37 °C (see the Supporting Information, Figure S9). Thus, our observations confirm that the mAb-functionalized capsules are able to differentiate between variations in the surface level of class I molecules, resulting in higher association with those cells, which express more antigens. Again, PEMs functionalized with Y3 mAb in optimized orientation bound best to RMA cells at both conditions. To be effective as a targeting system, mAb-functionalized PEMs must be able to bind exclusively to the antigenexpressing cells, while showing minimal association with cells that do not express the antigen. To confirm this requirement, we tested the versatility of our approach using a different cell line. We demonstrated that suspension T1 cells are able to bind W6/32-functionalized capsules very selectively, revealing no binding of Y3- or 5D3-functionalized capsules (Figure 6).

difference in total cell viability. Therefore, the low level of cytotoxicity suggests that (PAH/PAA)2 capsules can be potentially used in both in vitro and in vivo studies.

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CONCLUSION In summary, we have shown here that (PAH/PAA)2 microcapsules functionalized with targeting antibodies can selectively recognize and bind to their corresponding antigenic receptors at the plasma membrane, in particular to MHC class I proteins. Remarkably, recognition properties of the antibodies are fully preserved after being attached to the capsules, especially in the case of their protein A-assisted immobilization. As observed, it gives 40−50% increase of targeting efficiency as compared to the randomly attached antibodies, revealing no more than 25% of nonspecific binding in all controls. If one type of capsules can target a certain cell line or receptor on the cell surface successfully, it often fails in targeting another cell line/receptor. Here, the versatility of our approach has been demonstrated in vitro for different cell lines expressing different allotypes of class I molecules, and the results were reproducible under various experimental conditions, confirming that targeting is selective and allotype-specific. Moreover, by incubating cells with the capsules of two colors, we have shown the possibility of multiplexing, which would enable simultaneous targeting of several antigens at the plasma membrane with color-coded capsules functionalized with their respective antibodies. Importantly, six washing steps performed after incubation of capsules with cells in every set of experiments did not decrease the signal-to-background ratio, assuring the specificity of interaction. In our study, we use nondegradable micrometer-sized (PAH/ PAA)2 capsules for several reasons: (i) The shell can be produced in a robust form by covalent cross-linking, yielding containers suitable for long-term investigation under broad range of conditions, which is an important prerequisite for the optimization of targeting both in vitro and in vivo. (ii) Carboxyl groups in the terminating layer are available for covalent attachment of a broad spectrum of biomolecules to the capsule surface to provide them with unique properties and functions. (iii) Negative surface charge reduces the chances of nonspecific binding of capsules to the cells due to electrostatic attraction and thus ensures the specificity of the antibody−antigen interaction. (iv) Due to their size and ability to encapsulate fluorescent cargo, capsules are ideally suited for detection by optical techniques, such as microscopy and flow cytometry, providing the possibility for multiplexing. (v) Capsules with sized in the micrometer range could offer high payload and good circulation times in vivo, while still enabling cellular uptake. Indeed, selective targeting of cell surface receptors is just the first step toward microcapsule internalization by cells. After targeting is being achieved, the actual uptake mechanism allowing for encapsulated cargo to be delivered to the cell, as well as the uptake specificity must be investigated in detail.38,58 It is known that particle size, shape, and mechanical properties could influence the interaction with cells and uptake substantially.59,60 If the capsules are taken up by cells via the endocytic pathway, which is the major uptake mechanism of cells for larger entities, their endosomal escape can be facilitated by several viral and bacterial proteins, synthetic biomimetic peptides, polymers, etc.61 Different cell-penetrating peptides have been developed recently that can go directly into the cytosol of cells without being endocytosed and trapped in the

Figure 6. Summary of flow cytometry study of interaction between the antibody-functionalized (PAH/PAA)2 microcapsules and the suspension T1 cells. Set of capsules used: nonfunctionalized (A); coated with protein A (B); functionalized with Y3 (C), 5D3 (D), and W6/32 (E) antibodies in optimized orientation; or Y3 (F), 5D3 (G), and W6/32 (H) in random orientation; and coated with BSA (I). The W6/32-functionalized capsules (E, H) are used for selective targeting of human HLA-A2 and HLA-B class I proteins expressed on the surface of T1 cells. Other capsules serve as negative controls. Ratio of capsules to cells is 30:1. Cells are incubated with the capsules for 1.5 h at RT, gently mixing, followed by 6× washing with PBS (pH 7.2). The association is measured by flow cytometry. Mean fluorescence intensity (MFI) values are normalized by the maximum value; error bars indicate the standard error of the mean (SEM) (n = 5); n is the number of independent experiments.

Remarkably, the nonspecific binding background for T1 cells in control samples was very low, corresponding to less than 10% of the maximum signal. The difference in binding PEMs with optimized and randomly oriented W6/32 was about 40−50%, which is the same as association of the Y3-functionalized PEMs with RMA cells observed earlier (Figure 5). Finally, we evaluated the influence of PEMs on cell viability for both cell lines using the WST-1 (4-[3-(4-iodophenyl)-2-(4nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) cytotoxicity assay (see the Supporting Information, Figure S10).57 As observed, the viability decreased no more than 30% for all samples incubated with capsules (A-I), whereas centrifugation/ washing steps themselves resulted in 20% viability decrease (C2) as compared to the untreated cells in the control sample (C1). T1 cells were slightly more sensitive to the experimental conditions than RMA, but altogether there was no significant G


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ACS Applied Materials & Interfaces endocytic compartments.62 Consequently, the capsule surface could be decorated with those peptides to be able to pass through the plasma membrane. In the future, to study the uptake mechanism of the antibody-functionalized microcapsules, we are going to extend our work by reducing the capsule size, selecting biodegradable polymers, such as polysaccharides and polyamino acids,63,64 or proteins as building blocks for production of biodegradable capsules,65 which will be then functionalized with biomolecules using the protocol developed in the current study. In this work, we demonstrated the ability of targeting MHC class I receptors with polyelectrolyte microcapsules, which are only one possible subject of our targeting approach. They are a significant subject because they are currently the focus of attention in antitumor and antiviral vaccination, and tumor immunotherapy. Moreover, they are responsible for almost all autoimmune diseases, including type 1 diabetes. However, rather than being restricted to class I proteins, our system can be universally adjusted for targeting any receptor at the cell surface, for instance tumor-associated receptors such as insulinlike growth factor receptor 1 (IGFR-1),66 epidermal growth factor receptor (EGFR/HER2),67 estrogen (ER) and folate (FR) receptors,68,69 etc. By selecting cognate antibody−antigen pairs, our capsules have the potential to target any receptor at the plasma membrane that is specific for a certain disease, and thus to serve as a “smart” delivery tool that could improve the efficiency of the drug in the future. We expect that researchers in academia and industry will adapt our system to their individual purpose, using their own antibodies and ligands. Our manuscript provides clear and comprehensive guidelines and criteria for the translation of the principle into practical use.

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adjusted to pH 8.3 with 0.2 M sodium bicarbonate buffer (NaHCO3, pH 9) to increase the number of unprotonated amino groups in the protein and to achieve high conjugation yield. BSA solution was free of any amine-containing substances (TRIS, free amino acids or ammonium ions). The reaction mixture was kept for 1 h at RT under gentle agitation and protection from light. The reaction was quenched by 200 mM Tris-Cl buffer (pH 8). The excess dye was separated from the labeled BSA using Vivaspin 2 centrifugal concentrator by triplicate washing with PBS (pH 7.2). The labeled BSA is referred to as BSA-AF488 and BSA-AF647, respectively. For long-term storage, the conjugate solution (10 mg/mL) was divided into small aliquots and kept at −20 °C protected from light. Preparation of Core−Shell Particles. CaCO3 particles (0.05 g) were suspended in 2 mL of Milli-Q water (pH 7.0) and sonicated for 5 min. The particles were then centrifuged at 3000 rpm for 2 min, and the supernatant was discarded. To prepare fluorescent capsules, CaCO3 particles were incubated with 0.5 mL of fluorescently labeled BSA (1 mg/mL in Milli-Q water, pH 7.0) for 30 min at RT upon continuous shaking under light-shielded conditions. The washing/ centrifugation steps were repeated three times, and the particles were used for the adsorption of the first polyelectrolyte layer. A total of 2 mL of PAH (2 mg/mL in 0.5 M NaCl, pH 7.0) were added and incubated for 10 min, continuously shaking at 1200 rpm using TS1 ThermoShaker (Biometra, Germany). The particles were then centrifuged/washed three times with Milli-Q water (pH 7.0), and 2 mL of PAA (2 mg/mL in 0.5 M NaCl, pH 7.0) were added and incubated for 10 min to adsorb the second layer of polyelectrolyte. Four layers of PAH and PAA were adsorbed in total, resulting in the (PAH/PAA)2 shell structure. Cross-Linking, Surface Activation, and Functionalization with Proteins. The polymer layers were covalently cross-linked with 10 mg/mL EDC in MES buffer (0.1 M MES in 0.5 M NaCl, pH 6.0) overnight while shaking at 1200 rpm at room temperature (RT). The unreacted EDC was removed by triple washing of the core−shell particles in ice-cold Milli-Q water. Free carboxyl groups remaining on the surface after cross-linking were activated by incubating the particles with 500 μL of freshly prepared 0.4 M EDC/0.1 M sulfo-NHS mixture in MES buffer (0.1 M MES in 0.5 M NaCl, pH 6.0) while shaking at 1200 rpm for 1 h at RT. After activation, particles were washed/ centrifuged three times with PBS (pH 8.2). To functionalize with protein A, surface-activated particles were incubated with 50 μg of protein A (0.1 mg/mL in 500 μL of PBS, pH 8.2) for 2 h at RT, shaking at 1200 rpm, followed by triple washing of the particles with PBS (pH 8.2) to remove weakly bound protein molecules that were not covalently immobilized on the surface. Remaining active NHS-esters were quenched by addition of 500 μL of 50 mM TrisCl (pH 8.8) for 30 min at RT. To attach the antibodies in optimized orientation, protein A-functionalized particles were incubated with 20 μg of monoclonal antibody (0.04 mg/mL in 500 μL PBS, pH 8.2) for 2 h at RT, shaking at 1200 rpm (depending on the experiment, monoclonal Y3, 5D3, or W6/32 antibodies were used). To attach the antibodies to the particle surface in random orientation, EDC/sulfoNHS surface-activated particles were incubated directly with 20 μg of monoclonal antibody (0.04 mg/mL in 500 μL PBS, pH 8.2) for 2 h at RT, shaking at 1200 rpm. Particles were then washed/centrifuged three times with PBS (pH 7.2) and quenched with 50 mM Tris-Cl (pH 8.8) for 30 min at RT. To prepare additional sample of BSA-coated capsules, EDC/sulfo-NHS surface-activated particles were incubated with BSA (2 mg/mL in 500 μL PBS, pH 8.2) for 2 h at RT, shaking at 1200 rpm, then washed and quenched with 50 mM Tris-Cl. Finally, all samples were blocked with 5% IgG-free BSA solution in PBS (pH 7.2) for 2 h at RT, shaking at 1200 rpm. Then the core−shell particles were resuspended in 0.2 M EDTA (pH 7.2) to dissolve the CaCO3 core, thoroughly washed three times with PBS (pH 7.2), and collected using Vivaclear spin filters at 800 rpm. For further use, capsules were redispersed in 1 mL of PBS (pH 7.2) and kept at 4 °C protected from light. Immunostaining. To detect the efficiency of the antibody conjugation, core−shell particles with preattached monoclonal antibodies (Y3, 5D3 or W6/32) were incubated with 1 μg of polyclonal

MATERIALS AND METHODS

Materials. Calcium carbonate (CaCO3) particles (ca. 6 and 3 μm diameter) were purchased from PlasmaChem GmbH. Polyacrylic acid sodium salt (PAA, MW ∼ 60 kDa), 35% solution in water was purchased from Polysciences Inc. Poly(allylamine hydrochloride) (PAH, MW ≈ 70 kDa), sodium chloride (NaCl), sodium bicarbonate (NaHCO3), bovine serum albumin (BSA), ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA), 1-(3-(dimethylamino)propyl)3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS) were purchased from SigmaAldrich GmbH. Tris(hydroxymethyl)aminomethane (Tris) ultrapure, 2-(N-morpholino)ethanesulfonic acid (MES) monohydrate buffer grade were obtained from PanReac AppliChem. All materials were used as received without further purification. Protein A was obtained from Thermo Scientific. Alexa Fluor 488 labeled polyclonal goat antimouse antibody, Alexa Fluor 488 (AF488), and Alexa Fluor 647 (AF647) carboxylic acid succinimidyl esters were purchased from Invitrogen. WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) cell proliferation assay was obtained from Roche Diagnostics GmbH. Monoclonal Y3,45 5D3,46 and W6/3247 antibodies were purified from the corresponding hybridoma supernatants with standard methods using protein A agarose beads. Vivaclear mini high-flux poly(ether sulfone) (PES) membrane filers with 0.8 μm pore size were purchased from Sartorius Stedim Biotech GmbH. Vivaspin 2 centrifugal concentrators with 10 kDa cutoff were obtained from Vivaproducts, Inc. High purity deionized water with a resistivity higher than 18.2 MΩ·cm was prepared in a three-stage Milipore Milli-Q Plus 185 purification system (Milli-Q water). All buffers were made up in Milli-Q water and pH adjusted with sodium hydroxide (0.1 M) or hydrochloric acid (0.1 M) accordingly. Fluorescent Labeling of BSA. BSA solution (10 mg/mL in Milli-Q water) was mixed with AF488 or AF647 NHS-esters (5 mg/mL stock solutions in anhydrous DMSO) in equimolar concentration. Prior to mixing BSA with the labeling reagents, pH of the protein solution was H


Research Article


AExperimental is the absorbance of the sample, ABlank is the absorbance of the blank, and A100% control is the absorbance of the control sample with 100% cell viability. Characterization Techniques. Microcapsules were characterized by confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), ζ-potential measurements, and flow cytometry. Confocal micrographs (1.0 Airy units) of microcapsules in solution were recorded with a Zeiss LSM 510 ConfoCor 2 scanning system (Carl Zeiss, Germany) using a Plan-Apochromat 63× oil immersion objective with numerical aperture (NA) of 1.4. SEM images of microcapsules were obtained on a Zeiss Supra 40 VP scanning electron microscope (Carl Zeiss, Germany) at an acceleration voltage of 5 keV. For SEM measurements, a drop of the capsule suspension was placed on a glass slide and allowed to dry at RT; samples were sputtered with gold and subsequently analyzed using a 10 keV electron beam. Zeta potential analysis was performed on a Zetasizer nanoseries (Malvern Instruments Ltd., UK) using aqueous suspension of calcium carbonate cores (mean diameter 3 μm) in Milli-Q water. Recordings were made after each polyelectrolyte/protein deposition step on the surface of CaCO3 particles. Concentrations of polymers/proteins used were identical to the concentrations used in the aforementioned microcapsule fabrication section. Measurements were performed in triplicate; each result represents an average of ten subsequent runs. Fluorescence data for microcapsules and cells were acquired on a CyFlow Space flow cytometer (Partec) and FloMax 3.0 was used in the data analysis. Threshold forward and side scattering were adjusted to maximize the signal-to-noise ratio. Fluorescence was detected in the respective channels using green (488 nm) and red (638 nm) lasers and analyzed using FlowJo V.10.1 software (FlowJo Enterprise). Over 50 000 counts were analyzed for each data point and the peak intensity was used as the value. Experiments were performed at least in triplicate. Imaging flow cytometry was done on ImageStream Mark II device (Amnis Corporation) using IDEAS 6.1 analytical software for data analysis. Over 10 000 cells were acquired for each sample, and the fluorescence from cells/capsules was detected simultaneously in two channels (Ch2/Ch11). Confocal imaging of cells was performed with a laser scanning microscope LSM 700 (Carl Zeiss) equipped with 488 nm (10 mW) and 639 nm (5 mW) solid-state excitation lasers and the integrated Variable Secondary Dichroic (VSD) beamsplitter. Images were merged and pseudocolored in Carl Zeiss confocal software ZEN 2012 (blue edition). All microscopy experiments were performed in triplicate; in each experiment, at least ten cells were analyzed to get a sustained statistics of interaction with capsules. Data Analysis. All our repeats were performed at different time points (different days or weeks); therefore, the data was normalized to an internal control that was taken the same day as the original data set. That allows accounting for different outside conditions, thus ruling out external influences that are different for each time point. Since capsules with optimized Y3 antibody always show the strongest binding to the cells (i.e., the highest MFI values), we used optimized-Y3 entries as the internal control, so-called “normalization by the maximum value”. Although the MFI values describing the interaction of cells with capsules having the same surface coating may vary significantly from one day to another, based on this calculation in every data set there will be always a 100% control sample with zero error bar. All other error bars represent variation in the binding capacity based on the difference in the surface coating of the capsules.

AF488-labeled goat antimouse (GaM-AF488) antibody solution (0.003 mg/mL in 300 μL PBS, pH 7.2) for 30 min at RT, shaking at 1200 rpm, followed by triple washing with PBS (pH 7.2). Finally, particles were resuspended in 0.2 M EDTA (pH 7.2) to dissolve the core, and capsules were then measured in the flow cytometer. Cell Culture. Mouse lymphoma RMA cells (kindly provided by Peter Cresswell, Yale School of Medicine) and human lymphoma T1 cells (kindly provided by Alain Townsend, University of Oxford) were cultured in RPMI 1640 medium (GE Healthcare Europe) supplemented with 10% fetal calf serum (FCS) (Biochrom), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/mL streptomycin. TAPdeficient human fibroblasts STF1 (kindly provided by Henri de la Salle, Etablissement de Transfusion Sanguine de Strasbourg) were grown in low-glucose (1 g/L) DMEM medium (GE Healthcare Europe) supplemented as above. STF1 cells were stably transduced with Kb-GFP; lentiviruses were produced and used for gene delivery as described previously.70 Cell cultures were maintained at 37 °C in a 5% CO2 humidified incubator. Staining Cells with Antibodies. RMA cells were stained with monoclonal Y3 antibody (positive control, specific for H-2Kb class I) and 5D3 antibody (negative control, irrelevant for H-2Kb). T1 cells were stained with monoclonal W6/32 antibody (specific for HLA-A2 and HLA-B class I), and Y3 and 5D3 antibodies (negative controls, irrelevant for HLA-A2 and HLA-B). In each case, 106 cells/tube were centrifuged (300 g, 3 min) and washed twice with PBS (pH 7.2). Cells were then incubated with 1 μg of monoclonal antibody (0.002 mg/mL in 500 μL PBS, pH 7.2) for 1.5 h at 4 °C or RT (depending on the experiment), gently mixed, followed by 3× washing with PBS (pH 7.2). For staining, polyclonal AF488-labeled goat-anti mouse (GαM-AF488) secondary antibody was used. Cells were incubated with 1 μg of GaM-AF488 antibody solution (0.003 mg/mL in 300 μL PBS, pH 7.2) for 15 min at RT, followed by triple washing with PBS (pH 7.2). Finally, cells were resuspended in PBS (pH 7.2) and counted in the flow cytometer. Incubation of Cells with Capsules. To study the interaction with cells, capsules were preloaded with fluorescently labeled BSA (BSAAF488 or BSA-AF647) as described above. The following capsules including several controls were prepared: nonfunctionalized; coated with protein A; functionalized with specific and nonspecific monoclonal antibodies in both optimized and random orientations; and coated with BSA. 106 cells/tube were centrifuged (300 g, 3 min) and washed twice with PBS (pH 7.2). Cells were then incubated with capsules (in 500 μL PBS, pH 7.2) for 1.5 h at 4 °C or RT (depending on the experiment), gently mixing, followed by 6× washing with PBS (pH 7.2). The ratio of capsules to cells in each sample was 30:1, unless stated otherwise. Finally, cells were resuspended in 2 mL of PBS and measured by flow cytometry. To accumulate H-2Kb at the plasma membrane, RMA cells were cultured at 25 °C overnight, then washed twice with PBS (pH 7.2), followed by incubation with capsules for 1.5 h at RT as described above. WST-1-Based Cytotoxicity Assay. Cytotoxicity study was performed using the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)2H-5-tetrazolio]-1,3-benzene disulfonate) cell proliferation assay (Roche) following manufacturer’s instructions.57 In brief, 106 cells (RMA or T1) were washed/centrifuged twice with PBS (pH 7.2) and incubated with microcapsules for 1.5 h at RT as described above. A set of unlabeled capsules was used in this study in order to avoid interference of fluorophore with the assay performance. Ratio of capsules to cells was 30:1. Cells were washed 6× with PBS (pH 7.2) and transferred to 96-well plate. Finally, 100 μL/well of WST-1 reagent diluted with RPMI 1640 media 1:9 was added to cells and incubated for 2 h at 37 °C with 5% CO2 to reduce the reagent into the dye form. Wells with WST-1 containing RPMI medium were treated as a blank. Cells incubated only with WST-1 containing RPMI medium were treated as a control (100% cell viability). Cells that were handled the same way as other samples but without addition of microcapsules were used as an additional control. All experiments were performed in triplicate. The absorbance of the wells was measured in a Spark 20 M microplate reader (Tecan) at 450 nm. The percent cell viability was calculated as (AExperimental − ABlank)/(A100% control − ABlank) × 100, where

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01313. Stability of (PAH/PAA)2 microcapsules after covalent cross-linking; zeta potential study of microcapsule shell assembly and functionalization with biomolecules; flow cytometry study of binding the antibodies to the microcapsule surface; confocal microscopy study of binding 3-μm sized capsules to the adherent STF1 cells; imaging I


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flow cytometry study of binding 3-μm sized capsules to the suspension RMA cells; gating on RMA cells and capsules in a flow cytometry study; staining RMA cells with antibodies at 4 °C vs RT; staining RMA cells with antibodies after overnight culture at 25 vs 37 °C; viability of RMA and T1 cells after incubation with (PAH/PAA)2 microcapsules determined by WST-1 cytotoxicity assay. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; t.kolesnikova@ jacobs-university.de. Phone: +49 421 200 3588. Fax: +49 421 200 3249. ORCID

Tatiana A. Kolesnikova: 0000-0002-3409-0812 Author Contributions ‡

S.S. and M.W. contributed equally.

Author Contributions

T.K., G.K., and T.L. conducted experiments with capsules and cells. S.S. and M.W. provided laboratories and equipment and gave general guidance and advice. T.K. devised research, supervised students, and wrote the draft; all authors revised the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank their colleagues for support; Dr. Mihaela Delcea, Dr. Raghavendra Palankar, and Dr. Mykola Medvidov (Nanostructure group, ZIK-HIKE, University of Greifswald) for an opportunity to use SEM and imaging flow cytometer; Prof. Helmuth Möhwald (Max-Planck-Institut für Kolloid- und Grenzflächenforschung), Prof. Detlef Gabel (Jacobs University Bremen), Prof. Gerd Klöck (Hochschule Bremen), and Dr. Malgorzata Garstka (Netherlands Cancer Institute) for fruitful scientific discussions; and Ursula Wellbrock for technical assistance. Support by Bundesministerium für Bildung und Forschung (BMBF) (Kooperationsprojekt 031A153A-B “Prozessüberwachung in vivo und in vitro mit PolyelektrolytNanokapseln”) is gratefully acknowledged.

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ABBREVIATIONS EDC, 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide LbL, layer-by-layer MFI, mean fluorescence intensity MHC I, major histocompatibility complex class I PAA, poly(acrylic acid) PAH, polyallyllamine hydrochloride PEM, polyelectrolyte microcapsule SD, standard deviation SEM, standard error of the mean

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