Specific Uptake of Lipid-Antibody-Functionalized LbL Microcarriers by

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Specific uptake of lipid-antibody-functionalized LbL-microcarriers by cells Martin Göse, Kira Scheffler, and Uta Reibetanz Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01159 • Publication Date (Web): 16 Oct 2016 Downloaded from http://pubs.acs.org on October 19, 2016

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Specific uptake of lipid-antibody-functionalized LbL-microcarriers by cells Martin Göse*, Kira Scheffler and Uta Reibetanz* Institute for Medical Physics and Biophysics, Faculty of Medicine, University of Leipzig, Germany

Layer-by-Layer, supported lipid bilayer, microcarrier, antibody functionalization, specific cell uptake

Abstract The modular construction of Layer-by-Layer biopolymer microcarriers facilitates a highly specific design of drug delivery systems. A supported lipid bilayer (SLB) contributes to biocompatibility and protection of sensitive active agents. The addition of a lipid anchor equipped with PEG (shielding from opsonins) and biotin (attachment of exchangeable outer functional molecules) enhances the microcarrier functionality even more. However, a homogeneously assembled supported lipid bilayer is a prerequisite for a specific binding of functional components. Our investigations show that a tightly packed SLB improves the efficiency of functional components attached to the microcarrier’s surface as illustrated with specific antibodies in cellular application. Only a low quantity of antibodies is needed to obtain improved cellular 1 ACS Paragon Plus Environment

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uptake rates independent from cell type as compared to a loosely packed lipid bilayer or directly assembled antibody onto the multilayer. A fast disassembly of the lipid bilayer within endolysosomes exposing the underlying drug delivering multilayer structure demonstrates the suitability of LbL-microcarriers as a multifunctional drug delivery system. Introduction The biomedical and pharmaceutical progress towards “personalized medicine” has led to extensive improvements in drug development as well as drug application.1–3 New drug delivery systems should now exhibit individual properties such as specific or low dose transport of active agents.4 Beside systems based on lipid structures (micelles or liposomes5,6), nano- and micrometer-sized modular polyelectrolyte-based delivery systems are very promising due to their versatility.7 Those systems can be designed by the assembly of different polyelectrolyte molecules onto a spherical template according to the Layer-byLayer (LbL) technique.8 Due to the large variety of possible driving forces to form the resulting polyelectrolyte multilayer (e.g. electrostatic interaction, coordination chemistry interaction, biologically specific interaction9) the LbL-technique offers the opportunity of creating drug delivery systems of variable composition with highly adaptable properties. Furthermore, specific functionalities (e.g. for stimuli responsive disassembly10,11) and active agents (e.g. α1-antitrypsin, insulin, siRNA12–15) can be integrated into the multilayer structure. In a final step, the microcarrier surface properties can be controlled by choosing the appropriate terminal polyelectrolyte layer or by immobilization/specific coupling of functional molecules like polyethylene glycol (PEG), zwitterionic peptides or antibodies.16–20 In addition, hybrid drug delivery systems can be created by the assembly of an artificial lipid membrane on the microcarrier surface.21–24 Such a supported lipid bilayer (SLB) shields the underlying polyelectrolyte multilayer, stabilizes integrated active agents,25 enhances the biocompatibility of the microcarrier system and reduces the interaction with serum 2 ACS Paragon Plus Environment

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components.26–28 Furthermore, functional lipid molecules can be integrated into the SLB. A firmly integrated functional lipid component provides many advantages, e.g. by variable equipment with a specifically required functionality without disturbing the inner structure of the core/shell/SLB design. For example, the addition of the functionalized lipid PE-PEGBiotin composed of a lipid anchor (phosphoethanolamine, PE), polyethylene glycol (PEG) and biotin, to the basis lipid mixture allows streptavidin mediated binding of a biotinylated functionality of choice, such as antibody or active agent.29 PEG as the middle part of the functionalized lipid serves as a spacer and facilitates a reduced microcarrier interaction with serum components.30 However, a prerequisite for the successful specific functionalization is a tightly arranged lipid bilayer. Only under this condition is it possible to efficiently reduce unwanted unspecific interaction of serum or functional components with the underlying LbLpolyelectrolyte multilayer. Nevertheless, the applied lipid composition drastically influences the formation of a SLB or the occurrence of irregularities exposing the underlying polyelectrolyte multilayer.31 In previous investigations29 we presented biopolyelectrolyte (protamine sulphate (PRM) and dextran sodium sulphate (DXS)) coated Silica (SiO2) microparticles which were further equipped with a 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) / 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (POPC) and PE-PEG-Biotin supported lipid bilayer as a hybrid LbL-microcarrier system. We could show that the primary lipid composition, the amount of the functional lipid component PE-PEG-Biotin, as well as the SLB assembly conditions exhibit a strong influence on the homogeneity and tightness of the resulting SLB. As a result, an optimal tightly packed SLB composed of POPS/POPC 1:1 and 0.5 mol% PEPEG-Biotin was prepared after spreading correspondent liposomes onto an LbL-microcarrier at a liposome to microcarrier surface ratio of 80:1. This construction leaves freely available

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outer biotin as a binding partner for biotinylated functional molecules via streptavidin coupling. We are now focused on extended investigations to assess the efficiency of such a multiple functionalized system in application in different culture cells. The emphasis is on the timedependent cell / microcarrier interaction under nearly physiological conditions (37°C, full medium, no blocking of unspecific cellular binding sites). We present the adaptability of such a microcarrier system regarding the variable outer functionality and the improvement of this system over other ways of LbL-microcarrier functionalization with antibodies. Our experiments have been carried out by the application of two distinct biotinylated antibodies addressing the respective cell types who carry the correspondent antigen, respectively. As cell lines 3T332 and Vero33 cells have been selected. Both cells lines are representatives of fibroblast like cells but express different interaction profiles with LbLmicrocarriers. Thus, this work addresses the time-dependent uptake rates of optimal antibody-functionalized

SLB-microcarriers

compared

to

non-functionalized

SLB-

microcarriers. The results were further analyzed regarding two different antibody application forms via LbL-microcarriers: On one hand an inhomogeneous SLB was used as a supporter for the specific antibody-functionalization and on the other hand the antibody was assembled directly onto the polyelectrolyte multilayer. Finally, the intracellular appearance of the SLB has been investigated to follow the fate of the lipid components after internalization and, to illustrate the transport and release capability of the overall multi-functionalized LbL-microcarriers. Materials and Methods (1) Materials Silica microparticles (SiO2, d = (5.53 ± 0.21) µm) were purchased from microparticles GmbH (Berlin, Germany). Dextran sodium sulphate (DXS) (MW~40,000 Da) was purchased from 4 ACS Paragon Plus Environment

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ICN Biochemicals (Irvine, California, USA). Protamine sulphate salt from herring (PRM), Poly(allylamine hydrochloride) (PAH) (MW~56,000 Da), Poly(sodium 4-styrenesulfonate) (PSS) (MW~70,000 Da), streptavidin and Hanks’ Balanced Salt solution without calcium and magnesium (HBSS-) were purchased from Sigma-Aldrich (Taufkirchen bei München, Germany).

1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine

oleoyl-sn-glycero-3-phosphocholine

(POPC),

(POPS),

1-palmitoyl-2-

1,2-distearoyl-sn-glycero-3-

phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (PE-PEG-Biotin) and L-αPhosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (PE-RITC) were obtained from Avanti Polar Lipids (Alabaster, Alabama, USA). Biotinylated mouse monoclonal antibody [T21/8] against CCR5 (ABCCR5) has been purchased from Abcam (Cambridge, UK). Biotinylated mouse monoclonal antibody [12G5] against CXCR4 (ABCXCR4) was from eBioscience (Santa Clara, California, USA). Mouse monoclonal antibody [D171] against CD155 (Biotin) (ABCD155) as well as Alexa Fluor® 350 conjugated Wheat Germ Agglutinin (WGA-AF350) were from Life Technologies (Carlsbad, California, USA). LysoTracker® Green DND-26 (LysoTracker-green) and Lab-Tek II chamber slides (8 well) have been obtained from Thermo Fisher Scientific (Waltham, Massachusetts, USA). CELLSTAR® multiwell culture plates (48 well) were from Greiner Bio One (Kremsmünster, Austria). DMEM High Glucose w/ L-Glutamine w/o Sodium Pyruvate (DMEM), Trypsin-EDTA and Fetal Bovine Serum (FBS) were purchased from Biowest (Riverside, Missouri, USA). Phosphate buffered saline (PBS) was from PAA (Chalfont St Giles, UK) and sodium chloride was from Carl Roth (Karlsruhe, Germany). Polycarbonate membrane filter (d = 50 nm) were obtained from Avestin (Ottawa, Canada). NIH-3T3 cells expressing human CD4 and human CXCR4 (3T3) were obtained by Dr. Dann Littman and Vero (ATCC® CCL-81™) cells (Vero) were from ATCC (Manassas, Virginia, USA).


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(2) Methods LbL-coating of SiO2-microparticles SiO2-microparticles have been coated with polyelectrolytes according to the Layer-by-Layer (LbL) technique as described elsewhere.29 Briefly, 200 µl of microparticle stock solution was dispersed in 500 µl PRM solution (1 mg ml-1 in 0.1 M NaCl, positive charge) and incubated for 10 min under gentle shaking. Thereafter, microparticles have been centrifuged (2000 g, 1 min) and washed three times with 400 µl 0.1 M NaCl solution. The microparticles were then dispersed in DXS solution (1 mg ml-1 in 0.1 M NaCl, negative charge) and again incubated for 10 min. This procedure was repeated until the desired number of nine assembled polyelectrolyte layers had been reached: [PRM/DXS]4.5. For control experiments, fluorescence labelled PAH-RITC34 has been integrated into the coating scheme as follows: PAH/PSS/PAH-RITC/PSS/[PRM/DXS]2.5.

Polyelectrolyte

covered

microparticles

are

referred to as LbL-microcarriers. SLB assembly onto LbL-microcarriers LbL-microcarriers were additionally surface coated with a supported lipid bilayer (SLB) using the Liposome-Spreading technique.28,35,36 Liposomes (d = 50 nm) composed of POPS, POPC and PE-PEG-Biotin in a molar ratio of 1:1:0 (0 mol% PE-PEG-Biotin), 1:1:0.01 (0.5 mol% PE-PEG-Biotin) or 1:1:0.2 (10 mol% PE-PEG-Biotin) have been co-incubated with oppositely charged microcarriers for 1 h at 37°C in PBS under constant shaking. The lipid mixture has been additionally doped with 1 mol% L-α-Phosphatidylethanolamine-N(lissamine rhodamine B sulfonyl) (PE-RITC) for fluorescence based experiments. As previously described, an optimal SLB formation can be achieved at a liposome surface area to microcarrier surface area ratio (AL/AM) of 80:1,29 which has been exclusively used in this study. SLB covered LbL-microcarriers are referred to as SLB-microcarriers. 6 ACS Paragon Plus Environment

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Surface functionalization of LbL- and SLB-microcarriers LbL- as well as SLB-microcarriers were surface functionalized with biotinylated primary antibodies against CXCR4 (ABCXCR4), CCR5 (ABCCR5) and CD155 (ABCD155). LbLmicrocarriers with a terminal PRM layer (25 µl, 105 microparticles µl-1) were transferred to the correspondent antibody solution (100 µl, 20 µg ml-1) for 1 h under gentle shaking in PBS. SLB-microcarriers (100 µl, 105 microparticles µl-1) were at first incubated with streptavidin (0.15 µg µl-1 in PBS) for 1 h under gentle shaking. Thereafter, the microcarriers were centrifuged (2000 g, 1 min) and washed three times with PBS. Subsequently, the antibodyfunctionalization was applied according to the conditions mentioned previously. Cell / microcarrier co-incubation For flow cytometric cell /microcarrier co-incubation experiments, cells were seeded in a 48 well plate with 1×105 cells per well (500 µl) in DMEM + 10% FBS and allowed to settle 24 h prior to the application of microcarriers (37°C). Subsequently the cell medium was taken off and 5×105 microcarriers in DMEM + 2% FBS were added (500 µl). The samples were then incubated according to the indicated time period at 37°C, detached from the surface by Trypsin-EDTA incubation and subsequent DMEM + 10% FBS addition, centrifuged (400 g, 5 min, 4°C), washed with PBS and analyzed with a flow cytometer (BD FACS Calibur). For each measurement 1×104 cells were collected and analyzed according to standard procedures using the software Flowing Software. For confocal laser scanning microscopic analysis, 0.5×105 cells per well (500 µl) in DMEM + 10% FBS have been seeded in an 8 well Lab-Tek II chamber slide and allowed to settle for 24 h at 37°C. The medium was then replaced with 500 µl DMEM + 2% FBS containing 2.5×105 microcarriers and incubated at 37°C. 1 h before reaching the addressed time period, the medium was replaced with 100 mM LysoTracker-green solution (500 µl in 7 ACS Paragon Plus Environment

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DMEM + 2% FBS) for staining of acidic cell compartments. After reaching the addressed time period, the medium was replaced with WGA-AF350 solution (15 µg ml-1, 500 µl in HBSS-) and incubated for 10 min. Subsequently, the samples were washed three times with DMEM + 2% FBS solution and analyzed using a confocal laser scanning microscope Zeiss LSM 510 Meta equipped with a 63× objective. For observation of WGA-AF350 stained compartments an UV-laser (λEx = 351 nm) and a bandpassfilter λ = (385-470) nm were used. The detection of LysoTracker-green stained components was done using an Ar-laser (λEx = 488 nm) and a bandpassfilter λ = (505-550) nm. RITC-labelled components were visualized by excitation with a He-Ne-laser (λEx = 543 nm) and a bandpassfilter λ = (560615) nm. Results and Discussion As known from previous investigations,29 a stable, tight and homogeneously distributed SLB containing a functional lipid component can be built up on biopolyelectrolyte coated SiO2microcarriers. Such a terminal surface coverage inhibits the undesired attachment of serum components as proteins or even smaller peptides to the underlying multilayer. By using the biopolyelectrolyte pair protamine sulfate (PRM) and dextran sodium sulfate (DXS) as a multilayer and a POPS/POPC 1:1 mixture containing 0.5 mol% PE-PEG-Biotin a highquality SLB could be designed to provide a very specific binding of outer functionalities. The strategy of a sandwich-coupling of exchangeable outer biotinylized functional molecules via biotin-streptavidin-binding leaves the inner structure undisturbed. Nevertheless, it needs to be demonstrated by cell / microcarrier interaction under nearly physiological conditions (37°C, full medium, no blocking reagents) that the assembly of functionalities proceeds in the desired direction and is indeed preferable to other possible LbL-strategies. As a functionality, specific biotinylized antibody (AB) was used and attached to streptavidin.


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For specific uptake, NIH-3T3.CD4.CXCR4 (3T3), a murine fibroblast cell line and Vero ATCC CCL-81 (Vero), a fibroblast-like cell line from the African green monkey (kidney) were selected. The expression of respective surface antigens was determined by flow cytometry investigations of primary antibody as well as Cy5-labelled secondary antibody (ABsec-Cy5) binding. Figure S1 (supplement) illustrates the highly specific binding of antibody against CXCR4 (ABCXCR4) to 3T3 cells and antibody against CD155 (ABCD155) to Vero cells. As an isotype control for both cell lines, antibody against CCR5 (ABCCR5) was identified. Interaction time frame To analyze the interaction of antibody-functionalized SLB-microcarriers with cells, at first the relevant time frame of cell /microcarrier interaction was determined with flow cytometry using pre-functionalized SLB-microcarriers (0 mol% and 0.5 mol% PE-PEG-Biotin). Therefore, the SLB was equipped with a fluorescence label in terms of 1 mol% PE-RITC. This label serves a double purpose by not only allowing the determination of interaction rate but also localization and processing of the lipid components within a cell. After interacting with cells (microcarrier adsorption or uptake), cell / microcarrier region (high forward scatter intensity (FSC): cells, high fluorescence intensity: microcarriers) can be evaluated with respect to total cell number (for more details regarding data evaluation see Figure S2, supplement). Since it can be expected that microcarriers, lacking the presence of a specific AB, follow a rather time-delayed interaction with cells compared to antibody-functionalized microcarriers, Figure 1 shows flow cytometry investigations focusing on control SLBmicrocarriers after interaction with 3T3 and Vero cells.


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Figure 1. Flow cytometrically detected time-dependent cell / SLB-microcarrier interaction rate. As a fluorescence label, 1 mol% PE-RITC has been inserted into the lipid mixture. A) illustrates a very fast interaction of POPS/POPC as well as POPS/POPC + 0.5 mol% PE-PEGBiotin coated SLB-microcarriers with 3T3 cells. A1), a2), a3) and a4) exemplarily show confocal laser scanning microscopy (CLSM) images of cell / microcarrier co-incubation after 1 h (a1 and a2) and 24 h (a3 and a4). B) reveals that Vero cells exhibit a slower and lower interaction with POPS/POPC as well as POPS/POPC + 0.5 mol% PE-PEG-Biotin coated SLB-microcarriers. Statistical analysis was carried out using a two-tailored student’s t test: * p ≤ 0.05, n.s. non-significant (for detailed statistical analysis regarding the influence of PEPEG-Biotin see Table T1, supplement). B1),b2), b3) and b4) again present CLSM images of cell / microcarrier co-incubation after 1 h (b1 and b2) and 24 h (b3 and b4). The CLSM images have a size of 133 µm × 133 µm. In Figure 1a (3T3 cells), cell / microcarrier interaction proceeds very fast and after 3 h at the latest a plateau is reached. Although basically the interaction rate is very high (1 h: 10 ACS Paragon Plus Environment

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(29.9 ± 2.5) % for 0 mol% PE-PEG-Biotin and (34.7 ± 2.7) % for 0.5 mol% PE-PEG-Biotin), microcarriers equipped with an SLB containing 0.5 mol% PE-PEG-Biotin show even a significant higher interaction rate with increasing incubation time compared to an nonfunctionalized SLB (0 mol% PE-PEG-Biotin) (3 h: (29.9 ± 6.9) % for 0 mol% PE-PEG-Biotin and (52.7 ± 7.9) % for 0.5 mol% PE-PEG-Biotin). Confocal images in Figures 1a1 and a2 illustrate the microcarrier fluorescence intensity distribution (red) of the two designs (0 mol% and 0.5 mol% PE-PEG-Biotin) after 1 h co-incubation with 3T3 cells, whereas the cell membrane is visualized by wheat germ agglutinin staining (WGA-AF350, blue) and acidic cell compartments are stained with LysoTracker® Green DND-26 (LysoTracker-green, green). It can be seen that the fluorescence label PE-RITC is homogeneously distributed all over the microcarrier surface in circular appearance and the microcarriers are located inside the cells or close to the cell membrane in both images. Furthermore, no distinct difference in the overall microcarrier distribution in dependence of the PE-PEG-Biotin concentration can be visualized as predicted by the flow cytometry data. In Figure 1b (Vero cells), a different interaction behavior regarding time dependence as well as absolute interaction rate can be observed. The time frame of increasing cell / microcarrier interaction for both designs is prolonged, and a plateau was reached not before 5 h of coincubation. Starting with a lower interaction rate compared to 3T3 cells (1 h: (18.0 ± 3.1) % for 0 mol% PE-PEG-Biotin and (9.1 ± 2.6) % for 0.5 mol% PE-PEG-Biotin), a final rate of ~30 % is not exceeded ((28.2 ± 9.7) % for 0 mol% PE-PEG-Biotin after 5 h, (27.3 ± 6.2) % for 0.5 mol% PE-PEG-Biotin after 24 h). Furthermore microcarriers equipped with a functionalized SLB (0.5 mol% PE-PEG-Biotin) show a reduced interaction behavior compared to non-functionalized SLB-microcarriers, which is in contrast to 3T3 cells. This is supported by confocal laser scanning microscopy (CLSM) images after 1 h co-incubation. In Figure 1b1 the localization of microcarriers with 0 mol% PE-PEG-Biotin appears to be 11 ACS Paragon Plus Environment

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similar to the one observed with 3T3 cells (Figure 1a1). In contrast Figure 1b2 reveals that microcarriers with 0.5 mol% PE-PEG-Biotin as functional lipid component can mainly be found on the outer cell membrane, as visualized by the image obtained in the focal plane of the microcarriers. Again, the fluorescence label PE-RITC is clearly visible and homogeneously distributed all over the microcarrier surface in all cases. These results illustrate, that without a specific antibody-functionalization the absence or presence of the functional lipid component inside the SLB, that is 0 mol% or 0.5 mol% PEPEG-Biotin, results in an inconsistent effect on both the cell / microcarrier interaction rate and kinetics depending on the examined cell line. The effect could either be caused by structural differences of the microcarriers or different cell properties. To rule out the influence of the microcarrier design, several observations were made. Zeta potential measurements as well as investigations of unspecific microcarrier / protein interaction do not show noticeable changes of the microcarrier’s surface characteristics in dependence on the absence or presence of PE-PEG-Biotin.29 Thus, a surface charge effect does not occur. Another potential cause would be an undesired interaction of parts of the functional lipid component, particularly of biotin, with components of the cell membrane. Some cells express or overexpress a biotin receptor.37 3T3 and Vero cells have been thus investigated more closely towards a possible specific biotin interaction. As reporters, fluorescence labeled (1 mol% PE-RITC) liposomes (d = 50 nm) composed of POPS/POPC 1:1 either unaffected or equipped with 0.5 mol% PE-PEG-Biotin or 0.5 mol% PE-PEG were used (Figure S3, supplement). However, no distinct relation between the lipid composition and the interaction rate could be observed as all lipid mixtures show an equal interaction rate with the cells. This result also eliminates biotin as a variable for increased interaction with 3T3 cells, the cell line reacting differently to the SLB-microcarrier design than expected.


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Therefore, the difference between the global interaction rate as well as interaction kinetic of the two different cell lines (Figure 1) suggest that general differences in cell morphology or cell metabolism might have an important impact on the interaction rate with the investigated microcarriers and have to be taken into consideration when choosing the LbL-microcarrier design and characteristics. One intriguing observation is the unexpected drop of the cell / microcarrier interaction rate after 24 h of co-incubation with both 3T3 and Vero cells. Corresponding confocal images (Figures 1a3, a4, b3 and b4) reveal that the previously detected circular microcarrier

fluorescence intensity distribution (red) can now only be partially observed in all cases. This finding indicates a time-dependent loss of the fluorescence label. The subsequent intensity decrease then suggests misleadingly obtained drop of the cell / microcarrier interaction rate. However, the origin of this behavior was investigated in more detail and will be separately addressed in a later section (see Figure 5).

Antibody-functionalized SLB-microcarriers Within the early time frame (1 h to 3 h of co-incubation), detailed investigations have now been made according to the interaction of cells with differently surface functionalized microcarriers.

The

optimal

SLB

assembly

on

LbL-microcarriers,

that

is

POPS/POPC + 0.5 mol% PE-PEG-Biotin, was now equipped with antibody against CXCR4 or CD155, specifically addressing 3T3 or Vero cells (Figure 2). Furthermore, a nonexpressed receptor (CCR5) was addressed, resp., serving as an isotype control for both cell lines. For good comparability, pre-functionalized SLB-microcarriers (see Figure 1, POPS/POPC + 0.5 mol% PE-PEG-Biotin, dark grey dots) were again presented in this image (dark grey bars).


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Figure 2. Flow cytrometric investigations of antibody-functionalized microcarriers previously equipped with a POPS/POPC + 0.5 mol% PE-PEG-Biotin lipid bilayer (striped bars) interacting with a) 3T3 (ABCXCR4) and b) Vero (ABCD155) cells. As control, a nonspecific antibody (ABCCR5) was used compared to basis SLB with and without streptavidin (filled bars). Statistical analysis was carried out using a two-tailored student’s t test: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05. For more detailed statistical analysis see Table T2, supplement. As previously shown in Figure 1, cells show a time-dependent increasing interaction rate with functionalized SLB-microcarriers within 3 h, whereas 3T3 (Figure 2a) are more accessible than Vero cells (Figure 2b). Observing 3T3 cells interacting with SLBmicrocarriers, the assembly of streptavidin (+Streptavidin) as well as the addressing of the non-expressed CCR5-receptor (+Streptavidin+ABCCR5) resulted in similar interaction rates compared to only pre-functionalized SLB-microcarriers (0.5 mol% PE-PEG-Biotin). In comparison, the interaction rate of those microcarriers with Vero cells shows a slight increase towards the isotype control (+Streptavidin+ABCCR5). This behavior can be explained by the low unspecific interaction of the antibody against CCR5, as observed in control experiments determining the expression of different surface receptors for both cell lines (Figure S1, supplement). 14 ACS Paragon Plus Environment

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However, the addition of specific antibody, that is ABCXCR4 for 3T3 and ABCD155 for Vero cells, leads in all cases to a significant increase in interaction, up to factor 1.7 for 3T3 cells and factor 3.2 for Vero cells. But while all cell types show a gradual increase of their interaction with non-targeted microcarriers (0.5 mol% PE-PEG-Biotin, +Streptavidin, +Streptavidin+ABCCR5) over 3 h, the application of specific antibody accelerates the interaction significantly already reaching a plateau after only 1 h of co-incubation. Differently antibody-functionalized LbL- and SLB-microcarriers To illustrate the efficacy of the here presented antibody-functionalized SLB-microcarriers, our design (Figure 3I) was compared with two different approaches known from literature: (a) the direct assembly of antibody onto the LbL-microcarrier surface (Figure 3II) and (b) antibody assembled to a random SLB on LbL-microcarriers (Figure 3III).

Figure 3. Schematic representation of different types of antibody-functionalized LbL- and SLB-microcarrier systems: I) represents an optimal SLB-microcarrier system with terminal antibody-functionalization. Due to the incorporated functional lipid, a specific antibody binding can be realized exclusively. II) shows the direct assembly of antibody onto the charged polyelectrolyte surface of a LbL-microcarrier, resulting in a high amount of unspecifically bound antibody. III) illustrates an SLB-microcarrier, which exhibits inhomogeneities inside the SLB due to a high concentration of the functional lipid. However, the high concentration leads to the binding of larger amounts of antibody compared to the microcarrier system I. Thus, those systems have been under additional consideration: 15 ACS Paragon Plus Environment

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(a)

As one alternative approach, a direct AB assembly onto the biopolyelectrolyte

multilayer was investigated (Figure 3II). Hereby, PRM and DXS were coated onto the SiO2microparticles as already described. To facilitate a microcarrier localization within the cell, RITC labelled PAH was used as an additional inner multilayer constituent. As outermost layer, negatively charged antibody was then assembled onto the outer PRM layer using electrostatic interaction as a driving force (PRM+AB). This procedure was previously described by Cortez et al.38 presenting a fast and easy method of functionalizing LbLmicrocarriers with antibodies, whereas an efficient microcarrier adsorption to the addressed cell membrane was described after only 1 h co-incubation at 4°C and BSA blocking of unspecific binding sites. Using this assembly strategy, our previous investigations29 have shown a much higher amount of antibody assembled onto the polyelectrolyte multilayer (by a factor of 7) compared to specific antibody-binding to a functional lipid component via biotin/streptavidin coupling. This is not surprising due to overall accessible charged polyelectrolytes vs a considerably lower number of specific streptavidin binding sites in the second case.

Figure 4. Flow cytometric investigations of antibody-functionalized as well as control microcarriers in co-incubation with a) 3T3 and b) Vero cells. The antibodies have been assembled directly onto biopolyelectrolyte multilayer (left pair of bars, dark grey), onto a 16 ACS Paragon Plus Environment

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homogeneous POPS/POPC + 0.5 mol% PE-PEG-Biotin lipid bilayer (middle pair of bars, grey) and onto an inhomogeneous POPS/POPC + 10 mol% PE-PEG-Biotin lipid bilayer (right pair of bars, light grey). Statistical analysis was carried out using a two-tailored student’s t test: *** p ≤ 0.001, ** p ≤ 0.01. For more detailed statistical analysis see Table T3, supplement.

However, applying those microcarriers to 3T3 (Figure 4a, left pair of bars, dark grey) and Vero cells (Figure 4b, left pair of bars, dark grey), two observations can be made. On one hand, the cellular interaction with antibody-functionalized polyelectrolyte microcarriers (PRM+AB) results in a very different behavior as compared to unfunctionalized polyelectrolyte microcarriers (PRM). Interacting with 3T3 cells, the electrostatically attached ABCXCR4 exhibits a much lesser interaction rate of (32.6 ± 3.9) % after 1 h compared to only PRM coated control microcarriers ((46.2 ± 4.1) %). In the contrary, interaction with Vero cells reveals an inverted pattern: A surface functionalization with ABCD155 increases the interaction rate after 1 h co-incubation to (27.5 ± 3.3) % compared to only (16.5 ± 0.7) % for PRM coated control microcarriers. This behavior can be explained by the generally different unspecific interaction rate of the two investigated cell lines with unfunctionalized LbL-microcarriers. After coating ABCXCR4 or ABCD155, cell /microcarrier interaction is now dominated by only specific interaction. 3T3 cells, highly accessible to unspecific interaction with LbL-microcarriers, represent a relative decrease of interaction rate with specific antibody introduction. Vero cells in contrast are less accessible to unspecific interaction, leading to an increase in the interaction rate after the antibody assembly. On the other hand, the absolute interaction rate of antibody-functionalized LbL-microcarriers as compared to the optimal SLB based antibody-functionalization (Figure 4, middle pair of bars, grey) varies according to the investigated cell line. Comparing ABCXCR4-covered 17 ACS Paragon Plus Environment

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microcarriers (PRM+ABCXCR4 and 0.5 mol% PE-PEG-Biotin + ABCXCR4) after 1 h coincubation with 3T3 cells, PRM+ABCXCR4 exhibit only about half of the respective interaction rate of antibody-functionalized SLB-microcarriers. Furthermore, this ratio can be observed up to 3 h of co-incubation, indicating that both surface functionalizations have reached their maximum interaction rate within the first hour. Analyzing ABCD155 functionalized microcarriers in co-incubation with Vero cells after 1 h, a comparable interaction rate in both cases (PRM+ABCD155 and 0.5 mol% PE-PEG-Biotin+ABCD155) can be observed (~30 %). In contrast to 3T3 cells the maximum interaction rate is not reached within this short time frame which is demonstrated by the gradually increasing interaction rate over time in both cases. These findings demonstrate that the electrostatically driven irregular functionalization of LbL-microcarriers with antibody may results in a diffuse and unexpected cellular interaction behavior with the respective microcarrier system. In dependence of the investigated cell line, a decrease (3T3 cells) or increase (Vero cells) of the overall interaction rate due to the antibody surface functionalization was observed. Furthermore, an effective improvement of the cell / microcarrier interaction rate that is comparable to the optimal SLB-microcarrier system (0.5 mol% PE-PEG-Biotin) was only achieved in the case of Vero cells. Thus it can be concluded that the direct assembly of antibody onto LbL-microcarriers may result in an irregular behavior and a constant or comparable efficacy of this antibody-functionalization method is not achievable. Despite much less assembled antibody, the previously described antibody-functionalization of SLB-microcarriers (0.5 mol% PE-PEG-Biotin+AB, Figure 3I) show a consistent increase as well as an acceleration of the interaction rate independent of the investigated cell line. Thus, the presented microcarrier design appears to be highly promising in providing an adaptable general platform for targeted drug delivery systems.


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(b)

In a second step, specific antibody-binding to a functional lipid component integrated

into an inhomogeneous SLB was investigated (Figure 3III). It has been shown that inhomogeneities in the SLB are generated e.g. by high concentration of functional lipid.29 In our investigations, a lipid composition of POPS/POPC + 10 mol% PE-PEG-Biotin was used, obtaining an increase in specific antibody-binding by a factor of 4 compared to a PE-PEGBiotin concentration of 0.5 mol%. As can be seen (Figure 4, right pair of bars, light grey), SLB-microcarriers containing a high concentration of PE-PEG-Biotin exhibit a decrease of the interaction rate for both cell lines compared to a low PE-PEG-Biotin concentration of 0.5 mol%. This result suggests, that the blocking of unspecific interaction due to the high concentration of PEG20,30 is more dominant than the accessibility of the underlying polyelectrolyte multilayer due to inhomogeneities inside the SLB, resulting in an reduced interaction rate. Furthermore, this behavior is independent of the investigated cell line as well as the co-incubation time, as both 3T3 and Vero cells show a slight reduction of the interaction rate at all observed time points. Thus, it can be concluded that a certain concentration of PEG is needed in order to effectively inhibit unspecific cell / microcarrier interaction, which is not fully accomplishable using only 0.5 mol% PE-PEG-Biotin. The antibody-functionalization of such microcarriers results in a course of interaction that is comparable to that of a homogeneous, antibody-functionalized lipid bilayer (Figure 4, middle pair of bars, grey), but not in absolute values. The presence of ABCXCR4 and ABCD155 increases the interaction rate with 3T3 and Vero cells, resp., compared to non-functionalized microcarriers. After 1 h, an improvement by a factor of 1.6 (3T3 cells) and 2.2 (Vero cells) can be realized. But despite the high binding efficiency of antibody onto the functional SLB containing 10 mol% PE-PEG-Biotin, the overall interaction rate is much lower compared to antibody-functionalized SLB-microcarriers containing 0.5 mol% PE-PEG-Biotin. These findings appear to be contrary to the expectation that a higher amount of antibody should


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result in a higher interaction rate. But in case of 10 mol% PE-PEG-Biotin the specific interaction due to the antibody-functionalization is overlaid by the previously observed reduced interaction rate due to the high amount of PEG. Furthermore, the high concentration of PE-PEG-Biotin inside the SLB results in a specific state of the PEG-molecules, the so called “brush state” (dPE-PEG-Biotin = 2 nm).39,40 It is known that PEG-molecules which are arranged in such a state are less flexible compared to the “mushroom state” at lower PEGconcentrations (e.g. 0.5 mol%). As the antibody is directly coupled to the PEG moiety via a biotin-streptavidin binding, the flexibility and availability of the antibody might as well be reduced, resulting in a restrained interaction with the corresponding receptor on the cell membrane. This explanation is further supported by the observation regarding a SLB with a low PEPEG-Biotin-concentration of 0.5 mol%. Here the blocking characteristics of PEG are not dominant, as demonstrated by the comparable interaction rates of 0 mol% and 0.5 mol% SLB-microcarriers (Figure 1). Long-time cell / microcarrier interaction An interesting phenomenon of SLB-microcarriers carrying a SLB-integrated PE-RITC label in co-incubation with both cell lines is the already mentioned apparent reduction of cell / microcarrier interaction rate after 24 h co-incubation (Figure 1). Since there is a significant contrast to the stable interaction plateau reached after several hours, this reduction was investigated in more detail. Figure 5 exemplarily shows flow cytometric as well as CLSM investigations after 1 h and 24 h co-incubation, both for 3T3 (Figure 5a) and Vero cells (Figure 5b). In a1, a4, b1, and b4, dot plots of FSC (size) vs microcarrier PE-RITC fluorescence intensity are plotted, whereas a2, a5, b2 and b5 show the correspondent fluorescence intensity histograms. After


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1 h (upper row, (I)), both cell lines represent clearly distinguishable regions of unaffected cells (low auto-fluorescence intensity, R1) and of cells interacting with one or more microcarriers (high microcarrier fluorescence intensity, R2). As already shown in Figure 1, in Figure 5a2 3T3 cells express a higher interaction rate even with several microcarriers which is illustrated by a high number of distinguishable peaks in region R2. Vero cells interact with less amount of microcarriers per cell (Figure 5b2). CLSM images in a3 and b3 illustrate the homogeneous circular fluorescence intensity distribution of the lipid bilayer on top of the microcarrier surface after 1 h.


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Figure 5. Flow cytrometric and CLSM investigations of SLB-microcarriers previously equipped with a POPS/POPC + 0.5 mol% PE-PEG-Biotin lipid bilayer (including 1 mol% PE-RITC) with a) 3T3 and b) Vero cells after 1 h (I) and 24 h (II) co-incubation. A1), a4), b1) and b4) show FSC (size) vs microcarrier PE-RITC fluorescence intensity dot plots. In a2),


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a5), b2) and b5) PE-RITC intensity histogram represents the different intensity distribution in more detail. In a3), a6), b3) and b6) CLSM images (overlay of brightfield signal in grey and PE-RITC signal in red) illustrate the microcarriers appearance after cell interaction. The images have a size of 133 µm × 133 µm. After 24 h co-incubation, no distinct discrimination between cell and cell / microcarrier fractions can be observed on the fluorescence intensity scale (lower row, (II)). While cellular auto-fluorescence seems to be slightly increased (observable only on log-scale), the cell / microcarrier regions shift towards lower intensities. This process is particularly pronounced in case of 3T3 cells (Figure 5a5). CLSM images confirm (Figure 5a6 and b6), that the microcarrier fluorescence intensity is significantly reduced. Furthermore, the distribution of the fluorescence label PE-RITC is no longer limited to the microcarrier surface, but can be visualized in a spot like pattern all over the cells. This observation points out that the cell / microcarrier interaction rate cannot be reliably determined due to the loss of microcarrier fluorescence intensity within a 24 h time period, leading to an inaccurate interpretation of the obtained data. Nevertheless, the question arises about the nature of the disappearance of the fluorescence label. On one hand, a mere instability or degradation of the label could occur. On the other hand, the whole lipid bilayer could be affected by the uptake process or intracellular processing. To rule out a possible influence of the environment onto the RITC fluorescence intensity, at first PAH-RITC coated microcarriers were investigated using the same CLSM intensity amplification (Figure 6). For both cell lines, 3T3 (Figure 6a) and Vero (Figure 6b), a stable microcarrier fluorescence intensity with circular appearance could be observed after 1 h, 5 h as well as 24 h coincubation. Therefore, PAH-RITC represents a stable fluorescent probe and is not affected by the intracellular processing as well as lysosomal degradation. 23 ACS Paragon Plus Environment

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Figure 6. CLSM investigation of cell / microcarrier interaction after 1 h, 5 h and 24 h coincubation for a) 3T3 and b) Vero cells. RITC labelled PAH as multilayer constituent (red) marks the LbL-microcarriers, LysoTracker-green (green) represents lysosomes and endolysosomes and WGA-AF350 (blue) was used for staining cell membranes. The individual images have a size of 52 µm × 52 µm. However, differences could be observed regarding cellular uptake behavior and intracellular processing. After 1 h, 3T3 cells have already incorporated a certain number of microcarriers (Figure 6a1), whereas an uptake by Vero cells could not be observed (illustrated by out-offocus confocal cell layer compared to in-focus confocal microcarrier layer, Figure 6b1). Furthermore, Vero cells do not exhibit microcarriers in endolysosomes, whereas 3T3 cells already show a few microcarriers in an acidic environment, as illustrated by co-localization of the microcarriers with the fluorescence signal of LysoTracker-green (green). After 5 h, both cell lines present incorporated and acidified microcarriers. However, while nearly all endocytosed microcarriers are located in endolysosomes, some microcarriers were still visualized in the extracellular space yet, both for 3T3 and Vero cells. After 24 h, the majority of microcarriers is located in endolysosomes independent of the cell line. Nevertheless, Vero cells still show some microcarriers which have not yet been acidified. This observation 24 ACS Paragon Plus Environment

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clearly supports the results of cell / microcarrier interaction shown in Figure 4, illustrating a reduced and delayed interaction behavior of Vero cells compared to 3T3 cells. Furthermore it could be seen that the fluorescence label RITC exhibits a stable fluorescence intensity independent of the surrounding pH-value (e.g. neutral in extracellular space and acidic in endolysosomes). Thus it can be concluded, that the previously observed drop in the RITCintensity in the case of SLB-microcarriers cannot be attributed to the stability of the RITCmolecule but to the consistency of the SLB. Focusing on the critical time points 1 h and 24 h of cell / microcarrier co-incubation (Figure 7), microcarriers equipped with either non-functionalized (POPS/POPC + 0 mol% PE-PEGBiotin

(I),

POPS/POPC + 0.5 mol%

PE-PEG-Biotin

(II))

or

functionalized

(POPS/POPC + 0.5 mol% PE-PEG-Biotin + AB (III)) SLB demonstrate again a different appearance. After 1 h, 3T3 cells have already interacted with non-functionalized as well as functionalized SLB-microcarriers (Figure 7a1) as demonstrated by the proportion of endocytosed microcarriers. However, in case of functionalized microcarriers, the majority of the microcarriers can be detected inside the cells or attached to the cell membrane. Similar to PAH-RITC-microcarriers, microcarriers can be partially localized in endolysosomes (colocalized LysoTracker-green staining) independent of the surface coating. The lipid bilayer, visualized by the PE-RITC label, appears to be not affected by the interaction process and is still represented by a circular fluorescence distribution on top of the microcarrier surface. Vero cells in comparison show a delay in interaction, represented by microcarriers that are mainly localized in the extracellular space, as shown in off-focus images (Figure 7b1). Only in the case of surface functionalized microcarriers (Figure 7b1 (III)), microcarriers can be visualized inside the cell. Again, microcarriers in interaction with Vero cells still exhibit a detectable PE-RITC fluorescence signal that is only localized on the microcarrier surface.


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Figure 7. CLSM investigations of differently functionalized SLB-microcarriers after interaction with (a) 3T3 and (b) Vero cells. Microcarriers are either non-functionalized (POPS/POPC + 0 mol% PE-PEG-Biotin (I) and POPS/POPC + 0.5 mol% PE-PEG-Biotin (II)) or are functionalized with a specific antibody against the correspondent cell line (POPS/POPC + 0.5 mol% PE-PEG-Biotin + antibody (ABCXCR4 for 3T3 and ABCD155 for 26 ACS Paragon Plus Environment

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Vero) (III)). Microcarriers are labelled with PE-RITC as lipid bilayer constituent (red), lysosomes and endolysosomes are visualized by LysoTracker-green (green) and cell membranes are stained with WGA-AF350 (blue). The individual images have a size of 52 µm × 52 µm. Expanding the co-incubation time to 24 h (Figure 7a2 and b2) an increased interaction with all differently surface functionalized SLB-microcarriers can be detected. Independent of the cell line or final microcarrier surface modification, almost all microcarriers can be localized inside the cells. Furthermore a distinct acidification, that is localization in endolysosomes, can be visualized, as illustrated by a co-localized LysoTracker-green staining. Focusing on the PE-RITC distribution, a dramatic difference in comparison with 1 h co-incubation time can be observed. The lipid bilayer label is no longer homogeneously distributed on the microcarrier surface. Regarding both cell lines, microcarriers with an apparently intact lipid bilayer and microcarriers undergoing different stages of lipid bilayer disassembly can now be observed in parallel. Again, this process appears to be time-delayed for microcarriers incorporated by Vero compared to 3T3 cells. However, the disassembled lipid bilayer will not be totally degraded and removed, fragments scattered all over the cell are still visible. Due to a co-localization with LysoTracker-green, it can be concluded that those fragments are located in acidic cell compartments. In contrast to the PAH-RITC microcarriers (Figure 6), the remaining microcarriers do express only a partial co-localization with LysoTracker-green. These findings illustrate an endocytotic processing of the microcarriers, whereas the assembled lipid bilayer is contemporary disassembled after acidification of the endo(lyso)some. Observing microcarriers lacking PE-RITC as well as LysoTracker-green staining, one can conclude that after successful lipid bilayer disassembly, the microcarriers are released into the cytoplasm.


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These results show that independent of the nature of the SLB-microcarriers, that is equipped either with a plain POPS/POPC SLB, a SLB containing a functional component or a finally antibody-functionalized lipid bilayer, such microcarriers will intracellularly lose their lipid coating followed by the expected exposure of the underlying polyelectrolyte multilayer. The LbL-microcarrier as a drug delivery system may than follow a desired delivery route within the cell. Our investigations show, that the process of stripping of the lipid bilayer does not take place during the endosomal uptake, since immediately after incorporation the lipid bilayer appears to be intact. But although microcarriers are located in endolysosomes after 24 h, the original lipid bilayer coating will be gradually disassembled and removed into separate endolysosomes distributed all over the cell.

Conclusion This study emphasizes antibody-functionalized SLB-microcarriers and their application as a potential drug delivery system. As delivery targets, two fibroblast-like cell lines (NIH3T3.CD4.CXCR4 and Vero ATCC CCL-81) have been investigated in detail regarding interaction with functionalized microcarriers. It was shown that an optimal SLB-microcarrier system can be easily adapted to the addressed cell line and a large increase of the interaction rate as well as an acceleration of the interaction kinetics can be realized. Furthermore,

two

widely explored

microcarrier systems with

surface

antibody-

functionalization, that is using the mere polyelectrolyte multilayer as well as a random (porous) SLB assembly as a platform for antibody-functionalization, were investigated in comparison. However, none of these systems exhibits the same consistent properties as the above mentioned, that is a tightly packed, SLB based system, whereas either an inconsistent


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effect of the antibody-functionalization on interaction (decrease (3T3) or increase (Vero)) or a lower interaction rate have been obtained. In addition, a first insight into the intracellular processing of an SLB-microcarrier was achieved. CLSM images show that the microcarriers are successfully endocytosed, possessing a still intact SLB on the microcarrier surface. Subsequently, an acidification of the SLB-microcarriers occurs, which can be correlated with an endolysosomal localization. Here, the SLB is disassembled exposing the underlying polyelectrolyte structures. Finally, microcarriers which no longer carry an SLB can be visualized inside the cytoplasm. This intracellular processing further promotes the application of the investigated microcarrier system as a drug delivery system as the SLB can be used to target specific cells, but may not influence intracellular disassembly of the biodegradable multilayer and therefore the release of transported active agents. It can be concluded that the presented microcarrier system will have future impact in biomedical applications as a targeted drug delivery system.


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ASSOCIATED CONTENT Supporting Information The Supporting Information (SI) comprises further detailed investigations of specific cell membrane receptor expression (Figure S1) as well as description about analysis of cell / microcarrier co-incubation raw data (Figure S2). Furthermore, the interaction of 3T3 as well as Vero cells with liposomes of different lipid concentrations is illustrated in order to investigate specific cell membrane biotin receptors (Figure S3). Detailed statistically analysis of Figure 1, Figure 2 and Figure 4 can be found in Table1, Table2 and Table3, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Martin Göse, Universität Leipzig, Institute for Medical Physics and Biophysics, Härtelstr. 16-18, 04107 Leipzig, Germany [email protected] * PD Dr. Uta Reibetanz, Universität Leipzig, Institute for Medical PhysicsandBiophysics, Härtelstr. 16-18, 04107 Leipzig, Germany [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT


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The work presented in this paper was made possible by funding from the German Research Foundation (DFG, RE 2681/2-2), the European Union, and the Free State of Saxony and was supported by the DFG graduate school 185 “Leipzig School of Natural Sciences Building with Molecules and Nano-objects” (BuildMoNa). The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: NIH-3T3 CD4+CXCR4+ Cells from Dr. Dan R. Littman.

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(14) Reibetanz, U.; Schoenberg, M.; Rathmann, S.; Strehlow, V.; Goese, M.; Lessig, J. ACS Nano 2012, 6 (7), 6325–6336 (15) Strehlow, V.; Lessig, J.; Goese, M.; Reibetanz, U. J. Mater. Chem. B 2013, 1 (30), 3633–3643 (16) Cortez, C.; Tomaskovic-Crook, E.; Johnston, A. P. R.; Scott, A. M.; Nice, E. C.; Heath, J. K.; Caruso, F. ACS Nano 2007, 1 (2), 93–102 (17) Reibetanz, U.; Lessig, J.; Hoyer, J.; Neundorf, I. Adv. Eng. Mater. 2010, 12 (9), B488B495 (18) Wattendorf, U.; Merkle, H. P. J. Pharm. Sci. 2008, 97 (11), 4655–4669 (19) Heuberger, R.; Sukhorukov, G.; Vörös, J.; Textor, M.; Möhwald, H. Adv. Funct. Mater. 2005, 15 (3), 357–366 (20) Khopade, A. J.; Caruso, F. Langmuir 2003, 19 (15), 6219–6225 (21) Moya, S.; Donath, E.; Sukhorukov, G. B.; Auch, M.; Baumler, H.; Lichtenfeld, H.; Mohwald, H. Macromolecules 2000, 33 (12), 4538–4544 (22) Tanaka, M.; Sackmann, E. Nature 2005, 437 (7059), 656–663 (23) Angelatos, A. S.; Katagiri, K.; Caruso, F. Soft Matter 2006, 2 (1), 18–23 (24) Katagiri, K.; Caruso, F. Adv. Mater. 2005, 17 (6), 738–743 (25) Becker, A. L.; Johnston, A. P. R.; Caruso, F. Small 2010, 6 (17), 1836–1852 (26) Alessandrini, A.; Facci, P. J. Mol. Recognit. 2011, 24 (3), 387–396 (27) Qi, W.; Wang, A.; Yang, Y.; Du, M.; Bouchu, M. N.; Boullanger, P.; Li, J. J. Mater. Chem. 2010, 20 (11), 2121–2127 (28) McConnell, H. M.; Watts, T. H.; Weis, R. M.; Brian, A. A. Biochim. Biophys.Acta 1986, 864 (1), 95–106 (29) Goese, M.; Pescador, P.; Reibetanz, U. Biomacromolecules 2015, 16 (3), 757–768 (30) Wattendorf, U.; Kreft, O.; Textor, M.; Sukhorukov, G. B.; Merkle, H. P. Biomacromolecules 2008, 9 (1), 100–108 (31) Fischlechner, M.; Zaulig, M.; Meyer, S.; Estrela-Lopis, I.; Cuéllar, L.; Irigoyen, J.; Pescador, P.; Brumen, M.; Messner, P.; Moya, S.; Donath, E. Soft Matter 2008, 4 (11), 2245


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Biomacromolecules

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Flow cytometrically detected time-dependent cell / SLB-microcarrier interaction rate. As a fluorescence label, 1 mol% PE-RITC has been inserted into the lipid mixture. A) illustrates a very fast interaction of POPS/POPC as well as POPS/POPC + 0.5 mol% PE-PEG-Biotin coated SLB-microcarriers with 3T3 cells. A1), a2), a3) and a4) exemplarily show confocal laser scanning microscopy (CLSM) images of cell / microcarrier co-incubation after 1 h (a1 and a2) and 24 h (a3 and a4). B) reveals that Vero cells exhibit a slower and lower interaction with POPS/POPC as well as POPS/POPC + 0.5 mol% PE-PEG-Biotin coated SLB-microcarriers. Statistical analysis was carried out using a two-tailored student’s t test: * p ≤ 0.05, n.s. non-significant (for detailed statistical analysis regarding the influence of PE-PEG-Biotin see Table T1, supplement). B1),b2), b3) and b4) again present CLSM images of cell / microcarrier co-incubation after 1 h (b1 and b2) and 24 h (b3 and b4). The CLSM images have a size of 133 µm × 133 µm. 113x73mm (300 x 300 DPI)

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Biomacromolecules

Flow cytrometric investigations of antibody-functionalized microcarriers previously equipped with a POPS/POPC + 0.5 mol% PE-PEG-Biotin lipid bilayer (striped bars) interacting with a) 3T3 (ABCXCR4) and b) Vero (ABCD155) cells. As control, a non-specific antibody (ABCCR5) was used compared to basis SLB with and without streptavidin (filled bars). Statistical analysis was carried out using a two-tailored student’s t test: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05. For more detailed statistical analysis see Table T2, supplement. 177x68mm (300 x 300 DPI)


Biomacromolecules

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Schematic representation of different types of antibody-functionalized LbL- and SLB-microcarrier systems: I) represents an optimal SLB-microcarrier system with terminal antibody-functionalization. Due to the incorporated functional lipid, a specific antibody binding can be realized exclusively. II) shows the direct assembly of antibody onto the charged polyelectrolyte surface of a LbL-microcarrier, resulting in a high amount of unspecifically bound antibody. III) illustrates an SLB-microcarrier, which exhibits inhomogeneities inside the SLB due to a high concentration of the functional lipid. However, the high concentration leads to the binding of larger amounts of antibody compared to the microcarrier system I. 26x8mm (300 x 300 DPI)


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Biomacromolecules

Flow cytometric investigations of antibody-functionalized as well as control microcarriers in co-incubation with a) 3T3 and b) Vero cells. The antibodies have been assembled directly onto biopolyelectrolyte multilayer (left pair of bars, dark grey), onto a homogeneous POPS/POPC + 0.5 mol% PE-PEG-Biotin lipid bilayer (middle pair of bars, grey) and onto an inhomogeneous POPS/POPC + 10 mol% PE-PEG-Biotin lipid bilayer (right pair of bars, light grey). Statistical analysis was carried out using a two-tailored student’s t test: *** p ≤ 0.001, ** p ≤ 0.01. For more detailed statistical analysis see Table T3, supplement. 177x69mm (300 x 300 DPI)


Biomacromolecules

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Flow cytrometric and CLSM investigations of SLB-microcarriers previously equipped with a POPS/POPC + 0.5 mol% PE-PEG-Biotin lipid bilayer (including 1 mol% PE-RITC) with a) 3T3 and b) Vero cells after 1 h (I) and 24 h (II) co-incubation. A1), a4), b1) and b4) show FSC (size) vs microcarrier PE-RITC fluorescence intensity dot plots. In a2), a5), b2) and b5) PE-RITC intensity histogram represents the different intensity distribution in more detail. In a3), a6), b3) and b6) CLSM images (overlay of brightfield signal in grey and PE-RITC signal in red) illustrate the microcarriers appearance after cell interaction. The images have a size of 133 µm × 133 µm. 232x304mm (300 x 300 DPI)


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Biomacromolecules

CLSM investigation of cell / microcarrier interaction after 1 h, 5 h and 24 h co-incubation for a) 3T3 and b) Vero cells. RITC labelled PAH as multilayer constituent (red) marks the LbL-microcarriers, LysoTracker-green (green) represents lysosomes and endolysosomes and WGA-AF350 (blue) was used for staining cell membranes. The individual images have a size of 52 µm × 52 µm. 86x41mm (300 x 300 DPI)


Biomacromolecules

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CLSM investigations of differently functionalized SLB-microcarriers after interaction with (a) 3T3 and (b) Vero cells. Microcarriers are either non-functionalized (POPS/POPC + 0 mol% PE-PEG-Biotin (I) and POPS/POPC + 0.5 mol% PE-PEG-Biotin (II)) or are functionalized with a specific antibody against the correspondent cell line (POPS/POPC + 0.5 mol% PE-PEG-Biotin + antibody (ABCXCR4 for 3T3 and ABCD155 for Vero) (III)). Microcarriers are labelled with PE-RITC as lipid bilayer constituent (red), lysosomes and endolysosomes are visualized by LysoTracker-green (green) and cell membranes are stained with WGAAF350 (blue). The individual images have a size of 52 µm × 52 µm. 222x278mm (300 x 300 DPI)


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Biomacromolecules

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Specific Uptake of Lipid-Antibody-Functionalized LbL Microcarriers by

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