Reversible Fusion Proteins as a Tool to Enhance Uptake of Virus

Jul 2, 2018 - For the efficient treatment of an increasing number of diseases the development of new therapeutics as well as novel drug delivery syste...
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Reversible Fusion Proteins as a Tool to Enhance Uptake of Virus-functionalized LbL Microcarriers Kira Scheffler, Claudia Claus, Megan L. Stanifer, Steeve Boulant, and Uta Reibetanz Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00360 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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Reversible Fusion Proteins as a Tool to Enhance Uptake of Virus-functionalized LbL Microcarriers Kira Scheffler1, Claudia Claus2, Megan L. Stanifer3, Steeve Boulant3,4, Uta Reibetanz1 1

Institute for Medical Physics and Biophysics, Faculty of Medicine, University of Leipzig, Leipzig, Germany 2

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Institute of Virology, Faculty of Medicine, University of Leipzig, Leipzig, Germany

Schaller research group at CellNetworks, Department of Infectious Diseases, Virology, Heidelberg University Hospital, Germany

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Research Group “Cellular polarity and viral infection” (F140), German Cancer Research Center (DKFZ), Heidelberg, Germany;

KEYWORDS: layer-by-layer microcarrier, vesicular stomatitis virus, surface assembly, enhanced cellular uptake, viability

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ABSTRACT

For the efficient treatment of an increasing number of diseases the development of new therapeutics as well as novel drug delivery systems is essential. Such drug delivery systems (DDS) must not only consider biodegradability and protective packaging but must also target and control the release of active substances, which is one of the most important points in DDS application. We highlight the improvement of these key aspects, the increased interaction rate of Layer-by-Layer (LbL) designed microcarriers as a promising DDS after functionalization with vesicular stomatitis virus (VSV). We make use of the unique conformational reversibility of the fusion protein of VSV as a surface functionalization of LbL microcarriers. This reversibility allows for VSV to be used both as a tool for assembly onto the DDS and as an initiator for an efficient cellular uptake. We could show that the evolutionary optimized viral fusion machinery can be successfully combined with a biophysical DDS for optimization of its cellular interaction.

INTRODUCTION Drug Delivery Systems (DDS), e.g. based on lipid1–3, solid4 or polymer

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designs, are under

intensive investigation with the aim to enhance their transport efficiency and specific release of active agents into the desired cell without side-effects. However, due to the most likely considered endocytotic uptake routes, an efficient uptake and intracellular processing to reach the cytoplasm as an intact carrier is still limited. In our investigations, we emphasize these problems and present a promising approach to conveniently functionalize the surface of a microcarrier system. As a drug delivery system, we used Layer-by-Layer constructed microcarriers8,9, as they are highly modular in design, and provide a wide functionality. In particular, LbL microcarriers present many options to independently integrate active, sensor and ACS Paragon Plus Environment

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functional agents, and components into the multilayer, either on their surface or entrapped inside the (hollow) core without internal interference.10–13 In our functional approach, we want to make use of the unique properties of (enveloped) viruses14–17 to efficiently gain access to the cell and cytoplasm. Viruses are especially advantageous, as they have evolutionary evolved a perfect machinery for the infiltration of their host cell to induce the subsequent release of their genetic information. Compared to other functionalizations to enhance carrier uptake, such as by antibody or cell penetrating peptides18,19, virus properties are not restricted to uptake improvement but can also be beneficial for the subsequent intracellular progress of attached drugs, genes or more complex structures20,21 towards the cytoplasm. With that, the question arose about the feasibility to transfer those properties onto the LbL microcarriers surface with preserved activity, while the underlying, prefunctionalized carrier system will not be affected aiming at a “building block” construction with variable functionalities. Previous studies have demonstrated that the general surface functionalization of LbL carriers with viruses can be achieved in a convenient way. Initially, they are equipped with a supported lipid bilayer (SLB) prior to virus assembly (SLB-LbL microcarrier). Then, fusion of SLB-LbL microcarriers with viruses or virus-like particles takes place through the exposure to low pH (virus-LbL microcarrier). This procedure was introduced by Fischlechner et al.14,15, with influenza viruses as well as rubella virus-like particles, and modified by Fleddermann et al.16, using immune-stimulating reconstituted influenza virosomes. However, both approaches have drawbacks regarding the surface functionalization of SLB-LbL microcarriers with viruses. The first drawback is that using intact viruses allows for the possibility of replication after transfer of its genetic material into the cell, which should be avoided or at least should have no impact on the health of the treated organism. Therefore, the virus type for an LbL microcarrier assembly has to be carefully considered, making human pathogenic viruses or virus-like particles (such as ACS Paragon Plus Environment

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the mentioned rubella and influenza-based particles) of limited use. A much better choice would be a non-human pathogenic virus, ideally equipped with an ubiquitous, non-species restricted receptor. Additionally, an alternative approach is to use virosomes as artificial virus-like particles which are created by the preparation of liposomes after mixing commercially available (phospho)lipids (representing virus membrane constituents) and defined viral fusion proteins, e.g., hemagglutinin (HA).22 This strategy makes them innocuous towards infectivity due to the lack of genetic material, but severely impairs the protein functionality found in natural viruses. Also the absence of the nucleocapsid can be disadvantageous, as it can be used as a nanocarrier itself or function as a stabilizing component of the virion. The second drawback is that viruses must first fuse with the SLB of the LbL microcarrier. Conformational changes of fusion proteins of most viruses are irreversible and are found in only two states: the initially folded protein structure which allows the virus binding to a specific receptor and the subsequent activated state which has undergone structural change, triggered through receptor binding or the trafficking to acidic intracellular compartments, induces membrane fusion. However, this natural course of infection (“direct virus application”) is contrary to the requirements as an LbL-based DDS constituent (“reverse virus application”). In the latter case, first the viral fusion protein is activated and used to fuse the viral membrane with the SLB during assembly on the SLB–LbL microcarrier, the virus then needs to be reset to an inactive state in order to have a second round of activation leading to cell adsorption and uptake of the entire microcarrier. Here, influenza viruses, rubella virus-like particles or virosomes are not able to fulfill these requirements as an effective surface functionality of an LbL microcarrier since the conformational changes of their fusion proteins are irreversible and lead to inactivation after the initial assembly on the SLB-LbL microcarrier.23,24 Additionally, their activity after being used as a functionalization on microcarrier surfaces has not been shown. Instead viruses with reversible, acid-induced activation of their viral fusion protein(s) are needed. However, ACS Paragon Plus Environment

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such an approach has not yet been investigated, which lead us to pursue the use of vesicular stomatitis virus (VSV) as an LbL microcarrier constituent as it fulfills all above-mentioned parameters. This enveloped virus is nonpathogenic in humans.25 It is a negative-strand RNA virus, with the feature that its genome is never presented naked but always covered by its nucleocapsid proteins.26 It is widely known that VSV has a wide host range infecting both insects and vertebrates, leading to the conclusion that its receptor must be ubiquitous.27,28 Finkelshtein et al. proposed the low density lipoprotein receptor (LDLR) and its family members as the cell surface receptor for VSV.28 The widespread expression of those proteins could explain the broad tropism of the virus however the exact cellular receptor is highly debated. An additional advantage for LbL microcarrier assembly is that VSV can be produced at very high titers. The most important feature is the reversibility of the conformational changes of the glycoprotein (VSV-G), which is the main driving force for fusion of the viral membrane with cellular membranes and also serves as a cellular receptor.29 VSV fusion is accomplished by two fusion loops that face the viral membrane in the pre-fusion state at neutral pH, while at acidic pH those fusion loops are exposed to and able to interact with the target membrane.30 The VSV-G protein can switch from folded pre-fusion state (neutral pH) to unfolded fusion state (acidic pH) to folded post-fusion state (acidic pH) and from there back again to the folded pre-fusion state (neutral pH).31-33 Therefore we focused our investigation on the implementation of a functionalized LbL microcarrier with VSV as a surface modification of SLB-LbL microcarriers (VSV-LbL microcarrier). To establish this novel approach, several aspects were considered and addressed: Virus assembly: The optimal fusion conditions and the amount of virions necessary to interact with the SLB were identified. These conditions allowed for sufficient distribution and orientation of the VSV-G protein after fusion with the SLB.

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Functionality of VSV-G: Specific microcarrier-cell interactions were investigated to verify VSV-G as the moiety which induces specific cellular uptake. Uptake: LbL-microcarrier uptake by cells due to VSV modification was investigated and compared to SLB-LbL microcarriers. Toxicity: As DDS shall have no influence on cell viability, VSV-LbL microcarriers were investigated with regard to their cytotoxicity. We could show, that the advantages of VSV outlined above were beneficial as a surface functionalization of SLB-LbL microcarriers. An application ratio of 1:20 (SLB-LbL microcarrier:VSV virion) was best suited for cellular investigations. VSV was evenly distributed on the microcarrier surface as illustrated by labeled VSV-G and viral RNA. Neutralization assays with a monoclonal antibody against VSV verified that the VSV-G protein was the prominent driving force for uptake of the VSV-LbL microcarriers. The addition of VSV to the surface of SLB-LbL microcarriers did not lead to cytotoxicity on their target cells, highlighting their feasibility in cell culture or even in in vivo applications. Thus our novel and highly innovative approach introduces VSV-LbL microcarriers with a strong potential as a DDS.

MATERIALS AND METHODS Materials Silica-microparticles d=4.96 µm (SiO2) were purchased from microparticles GmbH. Dextran sodium sulphate (DXS) (MW~40,000Da) was purchased from ICN Biochemicals. Protamine sulphate salt from herring (PRM), Poly(allylamine hydrochloride) (PAH) (MW~56,000Da), Poly(sodium 4-styrenesulfonate) (PSS) (MW~70,000Da), Rhodamine B isothiocyanate (RITC), citric acid monohydrate (C6H8O7·H2O), sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) and Hanks’ Balanced Salt solution without calcium and magnesium (HBSS-) were purchased ACS Paragon Plus Environment

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from Sigma-Aldrich. Fluorescein isothiocyanate (FITC) was from Thermo Fisher Scientific, Germany. Sodium chloride (NaCl) was purchased from Carl Roth. 1-palmitoyl-2-oleoyl-snglycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were obtained from Avanti Polar Lipids. Polycarbonate membrane filter (d=50 nm) were obtained from Avestin. ZebaTM Spin Desalting Columns, Alexa Fluor TM 633 NHS Ester (Succinimidyl Ester), YoYoTM-1 Iodide, Hoechst 33342, trihydrochloride, trihydrate and Cholera Toxin Subunit B, Alexa FluorTM 555 Conjugate were purchased from Thermo Fisher Scientific, Germany and Rhodamine B Chloride (R18) from Biotium. Dulbecco’s Phosphate buffered Saline (DPBS), Dulbecco’s Modified Eagle Medium (DMEM) and Trypsin-EDTA (0.05 %) were from Thermo Fisher Scientific. 10x Minimum Essential Medium (MEM), PenicillinStreptomycin (Pen-Strep) and Fetal Bovine Serum (FBS) were purchased from Invitrogen Life Technologies, Germany. Sodium hydrogen carbonate (NaHCO3) was from Merck KGaA, Germany. The primary antibody against the fusion protein VSV-G (P5D4): sc-66180 and the isotype normal mouse IgG1:sc3877 were from Santa Cruz Biotechnologies. JC10, the enhanced derivative of 5,5’,6,6’-tetrachloro 1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide (JC1) was purchased from Enzo Life Science, Germany. Valinomycin and Triton X-100 were purchased from Sigma Aldrich, Germany. MultiTox-Fluor Multiplex Cytotoxicity Assay was purchased from Promega, Germany. Vero Cells (ATCC CCL-81) and Baby Hamster Kidney Cells (BHK) were purchased from ATCC (Manassas, VA, U.S.A.). Wild type Vesicular Stomatitis Virus (VSV) was kindly provided by Sean PJ Whelan, Harvard Medical School and cultured as previously described.48 Methods LbL Coating of SiO2 Microparticles 5µm SiO2 microparticles were coated according to the Layer-by-Layer (LbL) technique.54 The oppositely charged biopolymers were assembled by alternating adsorption of Protamine (PRM, positively charged) and Dextran Sulfate (DXS, ACS Paragon Plus Environment

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negatively charged). Both polymers were dissolved in 0.1 M NaCl with a final concentration of 2 mg/ml. 100 µl of the microparticle stock solution were incubated with 400 µl of the respective polymer solution for 10 min at room temperature and under constant shaking. After incubation the particles were centrifuged and washed three times with 200 µl 0.1M NaCl. The procedure was repeated until the desired number of nine layers polyelectrolytes was reached, following the schema: [PRM/DXS]4PRM. For interaction experiments two layers of PRM were substituted by fluorescence labeled PAHRITC as follows: PAH/PSS/[PAH-RITC/PSS]2PRM/DXS. SLB on LbL LbL microcarriers were surface coated with an additional lipid bilayer. Therefore liposomes were prepared as described according to GÖSE et al.15,44 POPS and POPC were mixed in a 1:1 molar ratio. The dried lipids were rehydrated with 522 µl PBS (without Calcium and Magnesium) to obtain a 10.2 mM lipid solution. Unilamellar vesicles were obtained with a 15 min ultrasonic treatment. Multilamellar vesicles with 50 nm diameter were produced by extrusion. Multilamellar vesicles were then incubated in PBS with LbL microcarriers equipped with [PRM/DXS]4PRM for 1 h at 37 °C and 1400 rpm based on a surface ratio (Liposomes:LbL microcarrier) of 80:1. Due to electrostatic attraction the liposomes adsorb and spread on the LbL microcarrier surface, leading to the formation of a Supported Lipid Bilayer (SLB). These carriers were referred to as SLB-LbL microcarriers. Pre-Evaluation with Liposomes Liposomes were prepared as described earlier15 and diluted in the respective buffer and pH. Fluorescence intensity of liposomes was measured with a plate reader (Perkin Elmer Victor X) and used as background intensity. Fluorescence intensity of the R18-labeled virus was measured at neutral pH, to prevent the virus from aggregating and fusing together. Subsequently, virus was added to the liposomes at respective pH and fluorescence intensity was measured immediately or after the indicated time period. Triton X-100 was added to fully release R-18 from the membrane which was set to 100% intensity. ACS Paragon Plus Environment

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Preparation of Giant Unilamellar Vesicles (GUVs) Giant Unilamellar Vesicles were prepared from the same lipids as the described SUVs with electroformation. The lipid solution was adjusted to 1.3 mM in chloroform, deposited on two platinum wires and GUVs were obtained in 250 mM sucrose solution with a 2V AC electric field (10 Hz for 1.5 hours and 2 Hz for 0.5 hours respectively).55,56 UV inactivated, R-18 labeled VSV was added to the GUVs in the respective buffer and visualized with a Confocal Laser Scanning Microscope (CLSM). VSV isolation To obtain high titer virus stocks, BHK cells were seeded 24 h prior to infection. When they had grown to 80% confluence they were infected with VSV with an MOI < 1. 16 h post infection supernatant was collected. VSV was purified by ultracentrifugation with a 10% sucrose cushion and resuspended overnight in PBS at 4 °C. Plaque Assay The titer was determined with a plaque assay as already described elsewhere.42,56 In brief, Vero cells were seeded 24 h prior to infection. The virus was then diluted in DMEM supplemented with 2 % Fetal Bovine Serum (FBS) and cells were infected with the respective dilution for 1 h at 37 °C, 5% CO2. The infected cells were overlaid with a mixture of 60 % Agar, 36% DMEM, 1 % FBS, 3% NaHCO3 (5%) and 1% Penecillin-Streptomycin (Pen-Strep). At 48 h post-infection the plaques were manually counted. Fusion Procedure VSV was incubated with the SLB equipped LbL microcarriers with 0.2 mM phosphate/0.25 mM citrate buffer with the respective pH and temperature for 10 min with gentle shaking, followed by three washings with PBS. For interaction experiments the fusion procedure was conducted at 4 °C in 0.2 mM phosphate/0.25 mM citrate buffer with pH 4.0 for 10 min with gentle shaking. Staining of Viral Membrane, RNA and Fusion Protein The virus stock was incubated with the respective dye for 90 min at room temperature with gentle shaking, followed by purification with Zeba Spin desalting columns. Viral membrane VSV was stained with 100 µM Octadecyl

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Rhodamine B chloride (R-18) in a quenching state. Viral RNA was stained with 1µM YoYoTM-1 Iodide and the VSV-G protein with 25 µM Alexa Fluor TM 633 NHS Ester. Co-Incubation of Cells with Microcarriers For flow cytometric (FC, BD AccuriTM C6) experiments cells were either seeded in a 48- or 96 well plate with 5×104 / 1.25×104 cells per well in 500 µl / 200 µl DMEM 10 % FBS, respectively. Cells were allowed to settle at 37 °C and 5 % CO2 for 24 h before microcarrier application. Media was replaced by DMEM+2% FBS containing 2.5×105 or 6.25×105 microcarriers per well. All experiments based on cellular interactions were conducted with virus that was UV-inactivated with 750,000 µJ/cm2 (UVStratalinker 2000, Stratagene). Samples were incubated for the indicated time periods at 37°C and 5% CO2, treated with 0.5 % Trypsin EDTA to detach and centrifuged at 500×g for 5 min. For non-UV-inactivated virus cells were fixed with 2% PFA for 20 min at room temperature and washed with PBS afterwards. Subsequently cells were analyzed with a FC. For each measurement between 5×103 and 1×104 cells were measured and analyzed according to standard procedures. Blocking of Cellular Interaction. SLB-LbL microcarriers were incubated with virions. To reduce unspecific virus binding the SLB-LbL microcarriers were pre-incubated with 3 % BSA for 30 min at 37 °C 900 rpm and washed with PBS three times. Anti-VSV-G and isotype control antibodies (VSV-G (P5D4) and normal mouse IgG 1, both from Santa Cruz Biotechnology) were diluted in PBS and incubated with microcarriers for 1 h at 37 °C and 900 rpm. Microcarriers were washed three times with PBS and incubated with cells for 1 h in DMEM + 2% FBS. After one hour co- incubation cells were analyzed by means of FC. Mitochondrial Membrane Potential and Cytotoxicity Assays The viability of cells was measured as described elsewhere.58 In brief, cells were incubated with LbL microcarriers for the respective time periods. Changes in mitochondrial membrane potential, which is an indicator for apoptosis, can be detected by applying the lipophilic dye JC-10. JC-10 aggregates emitting at ACS Paragon Plus Environment

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590 nm can be exclusively detected in vital cells, while the JC-10 monomer emitting at 527 nm can be detected in apoptotic cells. JC-10 was diluted in HBSS- to a final concentration of 10 µM. Staining solution was added to cells in medium and incubated at 37 °C for 15 min. To obtain a positive control, untreated cells were incubated with valinomycin. For FC measurements, cells were resuspended in HBSS-. To assess the ratio between living and dead cells the fluorescent MultiTox-Fluor Multiplex Cytotoxicity Assay (Promega) was used according to the manufacturer’s instructions. Briefly cells were seeded 24 h prior to incubation with LbL microcarriers. After the respective time period assay reagents were added to cells and incubated at 37 °C, 5% CO2 for 60 min. Fluorescence intensity was measured with a multiple plate Reader (Perkin Elmer Victor X). Confocal Laser Scanning Microscopy studies. For confocal laser scanning microscopy analysis, Vero cells were seeded in a 8-well chamber slide in a concentration of 5 x 104 cells per well. After 24 hours microcarriers were added to cells at a ratio of 5:1 (microcarrier:cell) in DMEM supplemented with 2 % FBS. After the respective time points nuclei were stained with 16.2 µM Hoechst 33342 for 10 minutes at 37 °C. After three washing steps cells were fixed with 2 % paraformaldehyde and cellular membranes were labeled with 10µg/ml Cholera Toxin-B for 20 minutes at 37°C. After three subsequent washing steps cells were left in PBS and analyzed using a Leica SP 8 Confocal Microscope.

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RESULTS AND DISCUSSION A unique characteristic of the VSV-G protein is its conformational reversibility, its property allowing it to restore its pre-fusion state after induction of fusion between the viral and any other present lipid membrane. This unique property of the VSV-G protein was now investigated in its applicability for the assembly of native VSV virions on SLB-LbL microcarriers. This was followed by the analysis of their retained fusion capacity in subsequent cellular applications. Figure 1 illustrates our approach to assemble (Figure 1a) and make use of VSV as an LbL microcarrier constituent with full cell-interaction capacity (Figure 1b).

Figure 1. Schematic representation of the optimal virus assembly on an SLB-LbL microcarrier using the unique conformational reversibility of the VSV-G protein. (a) Assembly strategy is

shown making use of the unfolded state of the protein at pH 4 to initiate fusion with the SLB. (b) After neutralization, VSV-G regains its pre-fusion state allowing the protein to be available for specific cell interaction via receptor binding. ACS Paragon Plus Environment

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To determine the optimal conditions for the assembly of VSV as a surface functionalization of SLB-LbL microcarriers, we first mimicked virus fusion at low pH by assessing different pH conditions. Additionally, to increase the specificity of virus binding before the pH shift, different temperature conditions and different time frames were investigated. To rule out virus interaction with potentially accessible charged polymers of the SLB-underlying multilayer, investigations were initiated with liposomes as a model for an impenetrable lipid bilayer structure. Therefore, small unilamellar vesicles (SUVs, 50 nm) were built under the same conditions as liposomes which are used to form a SLB on LbL microcarriers. Octadecyl Rhodamine B chloride (R-18) was utilized as a marker to monitor membrane interactions. R-18 fluorescence is quenched when incorporated into a lipid membrane when applied in concentrations up to 9 mol%.34 Upon fusion with another lipid membrane, R-18 disperses resulting in fluorescence dequenching and subsequent intensity increase thereby making it a good tool to monitor fusion events. VSV virions were labeled with R-18 and incubated with liposomes in the respective fusion buffer, pH 7.4, 5.2, or 4.0 and fluorescence intensity was measured spectroscopically. As a reference for full fusion liposomes were permeabilized with Triton X-100 to obtain 100 % fluorescence intensity. Figure 2a shows the detected fluorescence intensity as an indicator for VSV liposome fusion events as a function of time, temperature, and pH value. Virus–liposome fusion experiments were conducted based on conventional virus-cell fusion assays, such that virions incubated with cells at 4 °C delays endocytosis and equalizes receptormediated attachment thus reducing unspecific interactions between virions and cells. Fusion is then initiated at 37 °C.

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Figure 2. Interaction of liposomes with VSV virions. (a) R-18 dequenching assay was used to assess the fusion of the viral membrane with liposomes. R-18 labeled VSV virions were incubated with liposomes in citrate/phosphate buffer with different pH, at different time intervals

and at different temperatures. The resulting fluorescence intensity was measured by fluorescence spectroscopy. As a positive control, lipid membranes were permeabilized with Triton X-100 and this total fluorescence intensity was used as a reference value. (b) Corresponding Confocal Laser Scanning Microscopy (CLSM) images show lateral distribution of R-18 after virion fusion with

giant unilamellar vesicles (GUVs). In (b1), fusion was induced at pH 7.4 (scale bar: 100 µm), in (b2) fusion was induced at pH 4 (scale bar: 100 µm) illustrating the different distribution patterns.

Thus we can assume that unspecific interactions between VSV virions and liposomes can also be reduced at 4 °C which could explain the up to two times lower fluorescence intensity between neutral and acidic pH both at 4 °C and 37 °C (Figure 2a). This leads to the possibility, that at 4 °C as well as 37 °C also electrostatic interactions between the VSV virions and liposomes take place, but at 37 °C this interaction rate is accelerated. Similar results could be found at 25 °C. ACS Paragon Plus Environment

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The electrostatic nature could facilitate stable interaction with SLB-LbL microcarriers in the absence of a low pH shift, explaining the fusion events that are observed even at neutral pH. The amount of pre-incubation time also slightly changes the rate of VSV virion fusion with the liposomes, however this is independent of temperature and pH. Fusion starts very fast, as can be seen with the high intensity values at the starting time point (0 min), but the increase over the next 10 min is minimal (Figure 2a). However, the most prominent effect can be observed within the pH spectrum. Independent of incubation time and temperature, pH 7.4 produces the lowest fluorescence intensity values indicating the lowest fusion rate, pH 5.2 displays medium values, and pH 4.0 has the highest intensity values and highest fusion rate (67.29 ± 9.2 , 73.54 ± 4.3, 69.83 ± 2.25 for 4 °C, 25 °C and 37 °C, respectively) (Figure 2a). It was previously shown that irrespective of temperature and pH, the presence of any anionic lipid is sufficient to promote VSV virion fusion. This occurs in the absence of acid-induced conformational changes within the G fusion protein and is probably based on the strong electrostatic nature.35,36 Fusion activity of VSV virions with liposomes at neutral pH could be explained by the presence of phosphatidylserine (PS) in the investigated liposomes. Although PS is not the binding site for the VSV-G protein as proposed by Schlegel et al.37, VSV generally shows a high affinity for anionic lipids.38 This further emphasizes the electrostatic nature of the interaction between VSV virions and membranes. Since the investigated liposomes are made of 50 % 1-palmitoyl-2-oleolyl-sn-glycero-3-phosphoL-serine (POPS), the role of PS-dependent interactions of VSV with the synthetic membranes cannot be neglected.39 Our results lead to the conclusion that the highest rate of fusion interaction can be achieved by activating the VSV-G protein at pH 4.

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Since SUVs cannot be visualized by Confocal Laser Scanning Microscopy (CLSM), giant unilamellar vesicles (GUVs), prepared from the same lipid mixture, were incubated with R-18 labeled virions to visualize virus-lipid fusion. Confocal images (Figure 2b1 and 2b2) show the distribution of dequenched R-18 within the GUVs after fusion with the R-18 labeled VSV virions at pH 7.4 and pH 4, respectively. Both images show that fusion occurs at both neutral and acidic pH. To confirm the virus assembly on SLB-LbL microcarriers, a different staining protocol was applied specifically labeling VSV-G protein with Alexa Fluor 633 (AF 633). These labeled virons were incubated with SLB-LbL microcarriers at selected conditions based on previous VSV virion-liposome experiments: co-incubation took place at 4 °C for 10 min and acidic pH was compared to neutral pH. As can be seen in Figure 3, co-incubation again led to a high assembly ratio at pH 4, while the assembly ratio at pH 7.4 is significantly reduced by a

Figure 3. VSV virion assembly and stability on SLB-LbL microcarriers at acidic and neutral pH. VSV-G was labeled with AF 633 and incubated with SLB-LbL microcarriers at 4 °C and the respective pH value for 10 minutes. VSV-LbL microcarriers were then stored at 4 °C in PBS and fluorescence intensities were measured by means of FC after the respective time points.

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factor of 2.5 but still not at a negligible level (white bars). These experiments support the previous finding using R-18 staining labeled virions and liposomes. However, the difference between pH 4 and pH 7.4 is even more pronounced in AF 633 labeling compared to R-18 labeling. Therefore, we investigated in a next step the stability of the VSV-G protein on the SLB-LbL microcarrier surface over time. The fluorescence intensity was measured at additional five time points by means of Flow Cytometry (FC) after VSV-LbL microcarrier storage in PBS at 4°C. Figure 3 shows that the fluorescence intensity does not significantly change over one week for both pH incubation conditions. No significant loss or increase in fluorescence intensity can be observed both conditions tested, suggesting that all interactions take place in a short time frame and the labeled VSV-G protein can be detected at the same intensity on the SLB-LbL microcarrier surface for one week when stored in PBS at 4°C. Both experiments demonstrate that acidic pH is most efficient in triggering most fusion events and that events can take place efficiently at low temperature. Therefore, we used low pH conditions and 4 °C incubations for all subsequent investigations of assembly of VSV virions on the SLB-LbL microcarriers.

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Figure 4. Labeling scheme and detection of viral RNA and the VSV-G protein on the SLBLbL microcarrier surface. (a) Viral RNA as well as the VSV-G protein were stained with YoYo-1 Iodide and Alexa Fluor 633 prior to fusion with the SLB-LbL microcarrier. VSVLbL microcarriers were then analyzed by means of CLSM (b1,b3,c1,c3) and FC (b2 and c2)

(upper panel for viral RNA and lower panel for protein). In (b2 and c2), RNA as well as VSV-G staining show narrow peaks, and fluorescence intensity increases with increasing assembly ratio. CLSM images in contrast illustrate, that both parameters, RNA/VSV-G and

assembly ratio 1:20 / 1:2000 result in a patchy corresponds to 10 µm.

lateral fluorescence distribution. Scale

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These conditions were used to address two essential questions. 1) which incubation ratio between SLB-LbL microcarriers and VSV virions is best and if this results in altering the virus nucleocapsid and 2) whether VSV-G proteins are accessible on the microcarrier surface after fusion to promote cellular interaction. Virus distribution on the SLB-LbL microcarrier surface was then investigated with FC and CLSM through the use of specific fluorescent labels applied to VSV virions prior to acid-induced fusion with SLB-LbL microcarriers at 4°C. The VSV-G protein was again labeled with AF 633 (presented in red) and viral RNA with YoYo-1 Iodide (YoYo, presented in green) (Figure 4) using a SLB-LbL microcarrier:VSV virion ratio of 1:2000 and 1:20. The first ratio symbolizes a calculated total coverage of 100%, and 1:20 is a medium ratio allowing for sufficient coverage but providing an economical aspect as well. SLB-LbL microcarriers assembled with labeled virions can be visualized with either fluorescence dye and clearly distinguished from unlabeled control (Figure 4b2: YoYo-labeled viral RNA, Figure 4c2: AF 633-labeled VSV-G protein). The fluorescence intensity distributions are quite narrow, indicating a rather consistent and homogenous distribution of the viral RNA and VSV-G protein on the surface of the SLB-LbL microcarrier. The respective confocal images (Figure 4b1/b3 and 4c1/c3) illustrate that the fluorescence signal covers almost the entire microcarrier surface, with having a smaller assembly ratio of 1:20 (left panel) and a higher coverage with 1:2000 (right panel). Nevertheless, fluorescence patches can be detected in either case, particularly in the case of YoYo. As introduced earlier, the RNA can be strongly associated with the virus nucleocapsid, since the RNA is always encapsulated by nucleocapsid proteins,27 thus a YoYo fluorescence signal is an indicator for the nucleocapsid distribution on the microcarrier surface. Considering the rigidity of the nucleocapsid, a patchy appearance instead of a homogenous fluorescence distribution is not unlikely. In contrast, the fusion protein-related fluorescence distribution appears to be more homogenous. The fluidic nature of the lipid

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membrane allows homogenous and rapid mixing of the viral, VSV-G containing lipid membrane with the SLB.40-42 Nevertheless, it has to be mentioned that those investigations do not allow to draw conclusions about the exact position of the nucleocapsid within the lipid membrane. An assembly underneath the fused lipid membrane (Figure 1) therefore represents only one, although the most likely

Figure 5. Labeling scheme and detection of VSV-G protein on the SLB-LbL microcarrier surface. (a) AF 633 is added to SLB-LbL microcarrier after fusion with VSV. In (b) AF 633 staining of VSV-G protein after virion fusion with SLB-LbL microcarriers is presented demonstrating a high unspecific interaction of AF 633 with the SLB of the micocarrier. In (c) VSV-G mediated interaction of VSV-LbL microcarriers ( in a 1:20 ratio ) compared to SLB-LbL microcarriers (CTR) with Vero cells after one hour is shown, illustrating a significant influence of the protein on cell interaction in its function as a receptor binding moiety.

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option considering the spreading process of the enveloped virions onto the lipid bilayer preassembled LbL microcarrier according to FISCHLECHNER et. al.14 Even (Scanning Electron Microscopy (SEM) images are not sufficient for distinct conclusions about the capsid position (data not shown). However, for our application those informations are of secondary interest since the main aspect is the orientation, accessibility and functionality of the fusion proteins on the surface. Our results show, that virion assembly on the SLB-LbL microcarrier surface is achievable and can be easily recapitulated. RNA as well as the fusion proteins are detectable on the SLB-LbL microcarrier and even a moderate SLB-LbL microcarrier:VSV virion ratio is sufficient for assembly. Therefore we next investigated the external accessibility and functionality of the VSVG protein (Figure 5). To make use of the reversibility of the fusion conformation of the VSV-G protein as a SLBLbL microcarrier constituent, the VSV-G protein has to retain its fusion function and thus its prefusion conformation has to be restored on the microcarrier surface. Instead of pre-labeling VSV virions, AF 633 staining was applied after fusion of VSV virions with the SLB-LbL microcarrier. In Figure 5b, fluorescence intensities of SLB-LbL microcarriers and VSV-LbL microcarriers are shown after incubation with AF 633 as determined by FC. As it can be seen SLB-LbL microcarriers are unspecifically labeled by AF 633 (CTR+AF 633) since the fluorescence intensity is 20000 times higher compared to SLB-LbL microcarriers without AF 633 (CTR). Fluorescence intensities for VSV-LbL microcarriers (1.20 and 1:2000) are a bit higher compared to SLB-LbL microcarriers but due to the high unspecific binding it is not possible to use this marker for VSV-LbL microcarriers. Therefore we confirmed that VSV-G was still functional by applying VSV-LbL microcarrier to Vero cells, a commonly used cell line for VSV propagation.38,43 Since VSV is known to induce apoptosis in cells, the following cell experiments were conducted with UV inactivated VSV virions. Rhodamine B isothiocyanate (RITC) labeled ACS Paragon Plus Environment

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poly(allylamin hydrochloride) (PAH-RITC) was used as an underlying layer on the microcarriers, facilitating the distinction between cells that did not interact with SLB-LbL microcarriers and cells that did by means of FC. In Figure 5c, SLB-LbL microcarriers without VSV and with UV-inactivated VSV assembly (SLB-LbL microcarrier:VSV virion ratio 1:20) have been explored. Compared to control (SLB-LbL microcarrier), after 1h of carrier coincubation with cells a significant higher interaction rate can be observed. These experiments demonstrate, that the VSV-G protein was not only located on the surface of the SLB-LbL microcarrier but also retained its fusion capacity. Nevertheless, with increasing cell coincubation time the advantage of VSV as a surface functionalization vanishes and interaction rate

Figure 6. Assembly of different ratios of virions on SLB-LbL microcarriers. Viral RNA has been pre-labeled with YoYo (grey bars) and the fusion protein VSV-G with AF 633 (dark grey bars)

before incubation with LbL microcarriers. Microcarriers were then analyzed with FC, and fluorescence detected in FL1 (YoYo) and FL4 (AF 633). With a rising microcarrier:VSV virion

ratio the microcarrier fluorescence intensity becomes higher indicating a steady increase in virion assembly or adsorption. ACS Paragon Plus Environment

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of both VSV-LbL and SLB-LbL microcarriers reach the same level of interaction rate, as Figure S1 illustrates. However, the question arises to which extent the amount of assembled VSV virions influences the cellular interaction rate. To assess this influence, at first the SLB-LbL microcarrier:VSV virion ratio was investigated towards highest assembly rate (Figure 6). While previous investigations concentrated on selected examples of LbL microcarrier:VSV virion ratios (1:20 and 1:2000, Figure 4 and 5) a more comprehensive approach was now applied. As described before, both the viral RNA (YoYo) and the VSV-G protein (AF 633) were stained prior to acid-induced fusion with SLB-LbL microcarriers and four different ratios (1:2; 1:20; 1:200; 1:2000) were tested. Microcarriers were then analyzed by means of FC. An increasing SLB-LbL microcarrier:VSV virion ratio correlates with an increasing fluorescence intensity of either dye. No differences between labeling of RNA and VSV-G were detected. But even at the highest ratio of 1:2000 (100 % theoretical coverage) the fluorescence intensity of both dyes was still increasing. From such as strong increase it can be assumed that with higher ratios a notable rate of unspecific binding of virions to each other occurs which then assemble onto the SLB-LbL microcarrier possibly even without membrane fusion. We therefore investigated the influence of the amount of VSV virions assembled on SLB-LbL microcarriers on the interaction rate with Vero cells. To prevent virions from affecting cell viability, VSV was UV-inactivated for all following experiments. To rule out the possibility of reduction in fluorescence intensity due to VSV dis-assembly from the surface of the microcarrier during cellular uptake, the fluorescence label was applied within the polymer multilayer of the VSV-LbL microcarrier by using PAH-RITC (Figure 7). From previous investigations it has been shown that a significant interaction between LbL microcarriers and Vero cells does not occur prior to 3 h post co-incubation.44 However, cellular internalization of lipid-enveloped viruses via clathrin-mediated endocytosis occurs within a few minutes.45

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Figure 7. a) Interaction rate of Vero cells with VSV-LbL microcarriers as a function of time and SLB-LbL microcarrier:VSV virion ratio. Microcarriers equipped with PAH-RITC were incubated

with VSV virions in ratios of 1:2; 1:20; 1:200 and 1:2000 for 30 min and 60 min and analyzed by means of FC afterwards. The red label in the microcarrier shell allows the discrimination between cells that interacted with microcarriers and cells that did not. CLSM analysis was conducted with Vero cells after incubation with b) VSV-LbL and c) SLB-LbL microcarriers equipped with a PAH-FITC Layer (green) for 4 h. The nuclei were stained with Hoechst 33342 (blue) and the cell membrane with Cholera Toxin-B (red). The scale bars correspond to 25 µm.

Therefore, the interaction of the VSV-LbL microcarriers was monitored by means of FC over relatively short time frames of 30 and 60 minutes (comparing red fluorescent cells and nonfluorescent cells). Figure 7 shows that within these selected time points LbL microcarriers have already intensively interacted with Vero cells. All applied VSV-LbL microcarriers show a significant higher interaction rate with cells compared to the control (SLB-LbL microcarriers).

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The analysis of the different SLB-LbL microcarrier:VSV virion ratios revealed a maximum in cell interaction rate at 1:20, but only at a ratio of 1:2000 a significant decrease in interaction was detected compared to the maximum value. Based on this, an interaction rate of 1:20 ratio was chosen for further investigations. While up to a ratio of 1:20 virions seem to assemble without restrictions, the subsequent reduction with increasing virions can be explained by two phenomena. First, steric hindrance can impede fusion of virions with SLB-LbL microcarriers, and several intact single virions may adhere on the SLB-LbL microcarrier surface restricting the availability of directly attached VSVG protein. Secondly, high concentrations can provoke aggregation of virions themselves before assembly on SLB-LbL microcarriers. Virions become hydrophobic after exposure to acidic pH, leading to fusion events between themselves if there is no membrane in close proximity.46 These large aggregates will be unlikely to fuse with the SLB-LbL microcarrier, since they are much bigger and more rigid than single virions. It is rather likely that they do not fuse but only adsorb on the surface, which would be even more restricting for the activity of the fusion protein.29 That indeed a higher interaction leads to a higher uptake was demonstrated by CLSM analysis. Vero cells were incubated for 4 h with microcarriers that were additionally equipped with Fluorescein isothiocyanat (FITC, green) labeled PAH as a deeper multilayer. This time frame was chosen since interaction rates for both kinds of microcarriers with cells have then reached a plateau (see Supplement S1). To distinguish intracellular microcarriers from external microcarriers, cell membranes and nuclei were stained with Cholera Toxin subunit-B (red) and Hoechst 33342 (blue), respectively. Figure 7 shows that a higher uptake into cells takes place for VSV-LbL microcarriers (b) compared to LbL microcarriers solely equipped with a SLB (c). Viral neutralization was then used to investigate that indeed the VSV-G protein as surface functionalization of the SLB-LbL microcarrier is solely responsible for the enhanced microcarrier-cell interaction. In these experiments, the pre-determined optimized virion assembly ACS Paragon Plus Environment

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conditions were used along with the 1:20 ratio. Again, PAH-RITC was used as a multilayer constituent to follow cellular interaction via FC. SLB-LbL microcarriers were used as a negative control as they represent the minimal interaction rate (43 ± 14 %, data not shown) that can be achieved with microcarriers. VSV-LbL microcarriers without antibody (CTR+VSV) represent the positive control and were set as 100 % (data not shown). VSV-LbL microcarriers were then incubated with different VSV-G antibody concentrations to saturate the fusion protein before incubation with Vero cells. As can be seen in Figure 8, the application of high antibody dilution factors did not affect the interaction rate of antibody saturated VSV-LbL microcarriers (grey bars, VSV-LbL+AB) with Vero cells (105 ± 8 % (1:500) and 96 ± 33 % (1:100), respectively). At dilution factors of 1:50 and 1:10 the interaction rates decreased significantly (51 ± 14 % (1:50) and 52 ± 12 % (1:10)) and were reduced to the level of the negative control (SLB-LbL microcarriers). To confirm specificity of the applied antibody, VSV-LbL as well as SLB-LbL microcarriers were incubated with IgG1 isotype control antibody in a dilution range similar to the specific antiVSV-G antibody (Figure 8, red circles: VSV-LbL+IgG1 and black squares: SLB-LbL+ IgG1). As expected, no effect on cellular interaction rate was detected for either LbL microcarrier type in the presence of isotype control antibody (VSV-LbL+IgG1 and SLB-LbL+ IgG1). In conclusion, the functionality conferred to LbL by VSV was completely neutralized by the VSVG specific antibody, but not by an isotype control. While neutralization experiments clearly show a VSV-G mediated uptake of the VSV-LbL microcarriers through reduction of internalizing rate to 60 %, an unspecific uptake of SLB-LbL microcarriers still took place. Different endocytotic routes of SLB-LbL microcarriers and VSV virions may explain the higher cellular interaction rate of VSV-LbL microcarriers compared to SLB-LbL microcarriers. Microcarriers can be generally taken up by different endocytotic routes, but is has been previously shown that macropinocytosis is the most likely mechanism.47 VSV ACS Paragon Plus Environment

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infection is dependent on clathrin-mediated endocytosis, as it has been previously described by Cureton et al.45,48 An additional conclusion can be drawn: Since the higher interaction rate of VSV-LbL microcarriers compared to SLB-LbL microcarriers can be reduced by saturation of the VSV-G protein, the VSV-G protein seems to be able to act as a cellular receptor on the VSV-LbL microcarrier surface.

Figure 8. Neutralization of VSV-LbL microcarrier-cell interaction was carried out with an antibody against VSV-G assembled in different concentrations (grey bars). Interaction with cells was measured by FC using fluorescently labeled microcarriers. VSV-LbL microcarriers without antibody served as a positive control (100%) and SLB-LbL microcarriers as a negative control. Specificity for the VSV-G glycoprotein was proven by conducting isotype control experiments for VSV-LbL microcarriers (red circles) and SLB-LbL microcarriers (black squares). Cellular interaction was determined by the discrimination between fluorescent cells and non-fluorescent cells.

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Thus, those investigations comprehensively demonstrate the feasibility not only to assemble a virus with a fusion protein that bears reversible conformational changes on top of an SLB-LbL microcarrier but that the protein is able to regain its pre-fusion conformation and acts as a cellular receptor. The biocompatibility and lack of cytotoxicity of a drug delivery systems is one of the most important criteria to be fulfilled. Therefore, the cytotoxicity of the VSV-LbL microcarriers was investigated. While LbL biopolymer microcarriers are typically non-cytotoxic49, surface functionalization with VSV may have a negative impact on cell viability.50,51 Vero cells were used to study the influence of VSV-LbL microcarriers on cell viability. The impact on cell viability was analyzed through examining the loss of the mitochondrial membrane potential (as stained with the dye JC-10), which indicates early apoptosis. Furthermore the loss of membrane integrity was detected by measuring live and dead cell proteases (Multitox Promega). Figure 9 shows the amount of living cells after time-dependent microcarrier application. SLB-LbL microcarriers were equipped with UV-inactivated virions and incubated with Vero cells for the respective time intervals. Solely SLB-LbL microcarriers serve as a control for carrier influence and all results are referred to untreated cells. In Figure 9a no significant impact of VSV-LbL microcarriers on cell viability was detected (98.9 ± 4.2 %, 93.1± 17.2 % and 97.98 ± 6.8 % amount of living cells after 1, 6 and 24 h co-incubation, respectively). Also membrane integrity was not altered (Figure 9b), as no change in dead as well as living cell protease activity could be detected. While dead cell protease activity (Figure 9b1) remains constantly low independent on co-incubation time, live cell protease activity (Figure 9b2) decreases slightly but is in accordance to the control (SLB-LbL microcarriers). Regarding the most effective uptake time frame of 1 h-6 h in Figure S1, a non-toxic behavior of the VSV-LbL microcarriers within 24 h covers the full range of the application time frame.

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Figure 9. JC-10 assay (early apoptosis) and Multitox assay (live and dead cell proteases) as indicators for Vero cell toxicity induced by VSV-LbL microcarriers. In (a), no early apoptosis was induced. VSV-LbL as well as SLB-LbL microcarriers show no induction of viability loss with increasing time as determined by the JC-10 assay. Measurement of dead cell (b1) and living cell (b2) proteases show no significant influence of the SLB-LbL microcarriers over time as

determined by fluorescence intensity measurements with a plate reader. Live cell protease activity was measured at 400 nmex / 505 nmem while dead cell protease activity was measured at 485 nmex / 520nmem. For live cell protease activity untreated cells served as a control, while for dead cell protease activity cells were incubated with valinomycin prior to incubation with the substrates.

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CONCLUSION In our work we investigated VSV as an LbL microcarrier surface modification. Additionally, we characterized its properties on the microcarrier and its ability to bind and enter cells. The fusion reversibility of VSV-G protein was a key feature allowing for both the construction of the microcarrier and cell entry using the same protein. All other classes of fusion proteins are irreversible, thereby limiting their use to a single step, either LbL microcarrier assembly or cell entry. VSV was able to be fused on the SLB-LbL microcarrier using low pH conditions. These particles are then “re-set” by bringing them back to neutral pH. This allows them to be available for cell binding and subsequent entry into the cell is achieved by a second activation in the low pH endosomal environment. While preliminary investigations14–17 exclusively used virions with irreversible fusion proteins to investigate and establish their assembly routine onto the LbL microcarrier after pre-assembly of a supported lipid bilayer (SLB), we now focused on a strategy to make use of the fusion protein again to enhance the cellular interaction by the application of vesicular stomatitis virus (VSV) expressing the VSV-G protein with reversible conformational changes. In our study we determined the specific assembly characteristics such as pH, temperature and assembly time. Interestingly, we found that the amount of applied virions has a significant impact on the resulting functionalization; increasing RNA (virus nucleocapsid) and VSV-G protein (virus membrane) amount on the SLB-LbL microcarrier surface, Figure 6, does not necessarily result in more efficient protein accessibility and VSV-SLB microcarrier uptake. Only a moderate ratio (SLB-LbL microcarrier:VSV virion

ratio of 1:20) is required for the

functionality of the VSV-LbL after pH-mediated re-folding. Additionally, we confirmed that the cellular uptake was virus specific as interaction could be blocked when neutralizing antibodies were applied prior to incubation with VSV-LbL microcarriers. Finally, we could show that VSVLbL microcarriers do not induce toxicity after VSV-G mediated uptake. ACS Paragon Plus Environment

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In conclusion we have shown that virions with reversible protein folding capacity as a LbL microcarrier constituent are strongly capable to enhance the cellular uptake of the entire microcarrier. Wild type VSV was used as a proof of concept in our studies. For in vivo applications as a drug delivery system, wild type VSV can be substituted by attenuated recombinant VSV vectors. Such vectors are currently in a phase III clinical trial as an Ebola virus vaccine candidate.52,53

Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Interaction experiments with Vero cells over a 6 h time frame for SLB-LbL microcarriers and VSV-LbL microcarriers which can be seen in Figure S1. The fluorescence intensities of R-18 and AF 633 were tested with regard to pH values and the resulting spectra are depicted in Figure S2. AUTHOR INFORMATION Corresponding Author PD. Dr. U. Reibetanz, Institute for Medical Physics and Biophysics, Faculty of Medicine, University of Leipzig, Härtelstr. 16-18, 04107 Leipzig, Germany E-mail: [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.

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ACKNOWLEDGMENT The work presented in this paper was made possible by funding from German Research Foundation (DFG, RE 2681/2-2).

ABBREVIATIONS

DDS, Drug delivery system; LbL, Layer-by-Layer; SLB, Supported lipid bilayer; VSV, Vesicular Stomatitis Virus; LDLR, low density lipoprotein receptor; VSV-G, VSV glycoprotein; SUV, Small unilamellar vesicle; R-18, Octadecyl Rhodamine B chloride; CLSM, Confocal Laser Scanning Microscope; GUV, Giant unilamellar vesicle; PS, Phosphatidylserine; FC, Flow Cytometry; RITC, Rhodamine B isothiocyanate; FITC, Fluorescein Isothiocyanate

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Schematic representation of the optimal virus assembly on an SLB-LbL microcarrier using the unique conformational reversibility of the VSV-G protein. (a) Assembly strategy is shown making use of the unfolded state of the protein at pH 4 to initiate fusion with the SLB. (b) After neutralization, VSV-G regains its pre-fusion state allowing the protein to be available for specific cell interaction via receptor binding.

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Interaction of liposomes with VSV virions. (a) R-18 dequenching assay was used to assess the fusion of the viral membrane with liposomes. R-18 labeled VSV virions were incubated with liposomes in citrate/phosphate buffer with different pH, at different time intervals and at different temperatures. The resulting fluorescence intensity was measured by fluorescence spectroscopy. As a positive control, lipid membranes were permeabilized with Triton X-100 and this total fluorescence intensity was used as a reference value. (b) Corresponding Confocal Laser Scanning Microscopy (CLSM) images show lateral distribution of R-18 after virion fusion with giant unilamellar vesicles (GUVs). In (b1), fusion was induced at pH 7.4 (scale bar: 100 µm), in (b2) fusion was induced at pH 4 (scale bar: 100 µm) illustrating the different distribution patterns.

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VSV virion assembly and stability on SLB-LbL microcarriers at acidic and neutral pH. VSV-G was labeled with AF 633 and incubated with SLB-LbL microcarriers at 4 °C and the respective pH value for 10 minutes. VSVLbL microcarriers were then stored at 4 °C in PBS and fluorescence intensities were measured by means of FC after the respective time points. 68x56mm (300 x 300 DPI)

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Labeling scheme and detection of viral RNA and the VSV-G protein on the SLB-LbL microcarrier surface. (a) Viral RNA as well as the VSV-G protein were stained with YoYo-1 Iodide and Alexa Fluor 633 prior to fusion with the SLB-LbL microcarrier. VSV-LbL microcarriers were then analyzed by means of CLSM (b1,b3,c1,c3) and FC (b2 and c2) (upper panel for viral RNA and lower panel for protein). In (b2 and c2), RNA as well as VSV-G staining show narrow peaks, and fluorescence intensity increases with increasing assembly ratio. CLSM images in contrast illustrate, that both parameters, RNA/VSV-G and assembly ratio 1:20 / 1:2000 result in a patchy lateral fluorescence distribution. Scale corresponds to 10 µm. 146x157mm (300 x 300 DPI)

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Labeling scheme and detection of VSV-G protein on the SLB-LbL microcarrier surface. (a) AF 633 is added to SLB-LbL microcarrier after fusion with VSV. In (b) AF 633 staining of VSV-G protein after virion fusion with SLB-LbL microcarriers is presented demonstrating a high unspecific interaction of AF 633 with the SLB of the micocarrier. In (c) VSV-G mediated interaction of VSV-LbL microcarriers ( in a 1:20 ratio ) compared to SLB-LbL microcarriers (CTR) with Vero cells after one hour is shown, illustrating a significant influence of the protein on cell interaction in its function as a receptor binding moiety.

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Assembly of different ratios of virions on SLB-LbL microcarriers. Viral RNA has been pre-labeled with YoYo (grey bars) and the fusion protein VSV-G with AF 633 (dark grey bars) before incubation with LbL microcarriers. Microcarriers were then analyzed with FC, and fluorescence detected in FL1 (YoYo) and FL4 (AF 633). With a rising microcarrier:VSV virion ratio the microcarrier fluorescence intensity becomes higher indicating a steady increase in virion assembly or adsorption.

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a) Interaction rate of Vero cells with VSV-LbL microcarriers as a function of time and SLB-LbL microcarrier:VSV virion ratio. Microcarriers equipped with PAH-RITC were incubated with VSV virions in ratios of 1:2; 1:20; 1:200 and 1:2000 for 30 min and 60 min and analyzed by means of FC afterwards. The red label in the microcarrier shell allows the discrimination between cells that interacted with microcarriers and cells that did not. CLSM analysis was conducted with Vero cells after incubation with b) VSV-LbL and c) SLB-LbL microcarriers equipped with a PAH-FITC Layer (green) for 4 h. The nuclei were stained with Hoechst 33342 (blue) and the cell membrane with Cholera Toxin-B (red). The scale bars correspond to 25 µm. 100x59mm (600 x 600 DPI)

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Neutralization of VSV-LbL microcarrier-cell interaction was carried out with an antibody against VSV-G assembled in different concentrations (grey bars). Interaction with cells was measured by FC using fluorescently labeled microcarriers. VSV-LbL microcarriers without antibody served as a positive control (100%) and SLB-LbL microcarriers as a negative control. Specificity for the VSV-G glycoprotein was proven by conducting isotype control experiments for VSV-LbL microcarriers (red circles) and SLB-LbL microcarriers (black squares). Cellular interaction was determined by the discrimination between fluorescent cells and non-fluorescent cells. 68x56mm (600 x 600 DPI)

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JC-10 assay (early apoptosis) and Multitox assay (live and dead cell proteases) as indicators for Vero cell toxicity induced by VSV-LbL microcarriers. In (a), no early apoptosis was induced. VSV-LbL as well as SLBLbL microcarriers show no induction of viability loss with increasing time as determined by the JC-10 assay. Measurement of dead cell (b1) and living cell (b2) proteases show no significant influence of the SLB-LbL microcarriers over time as determined by fluorescence intensity measurements with a plate reader. Live cell protease activity was measured at 400 nmex / 505 nmem while dead cell protease activity was measured at 485 nmex / 520 nmem. For live cell protease activity untreated cells served as a control, while for dead cell protease activity cells were incubated with valinomycin prior to incubation with the substrates.  144x122mm (300 x 300 DPI)

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