Mesenchymal Stem Cells Engineering: Microcapsules-Assisted Gene

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Mesenchymal stem cells engineering: microcapsules assisted gene transfection and magnetic cell separation Albert Muslimov, Alexander S. Timin, Aleksandra Petrova, Olga Epifanovskaya, Alena Shakirova, Kirill Lepik, Andrey Gorshkov, Eugenia Il’inskaja, Andrey Vasin, Boris Afanasyev, Boris Fehse, and Gleb B. Sukhorukov ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00482 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 10, 2017

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ACS Biomaterials Science & Engineering

Mesenchymal stem cells engineering: microcapsules assisted gene transfection and magnetic cell separation Albert R. Muslimova,c,e, Alexander S. Timina,b*, Aleksandra V. Petrovac,d, Olga S. Epifanovskayaa, Alena I. Shakirovaa, Kirill V. Lepika,e, Andrey Gorshkovc, Eugenia V. Il’inskajac, Andrey V. Vasinc,d, Boris V. Afanasyeva, Boris Fehsef, Gleb B. Sukhorukovg

a

First I. P. Pavlov State Medical University of St. Petersburg, Lev Tolstoy str., 6/8, Saint-Petersburg,

Russian Federation b

c

RASA center in Tomsk, Tomsk Polytechnic University, pros. Lenina, 30, Tomsk, Russian Federation

Research Institute of Influenza, Popova str., 15/17, 197376, Saint-Petersburg, Russian Federation

d

Department of Molecular Biology, Peter The Great St. Petersburg Polytechnic University,

Polytechnicheskaya, 29, 195251, St. Petersburg, Russian Federation e

RASA center in St. Petersburg, Peter The Great St. Petersburg Polytechnic University,

Polytechnicheskaya, 29, 195251, St. Petersburg, Russian Federation f

Research Dept. Cell and Gene Therapy, Dept. of Stem Cell Transplantation, University Medical Center

Hamburg-Eppendorf, 20246, Martinistraße 52, 20251 Hamburg, Germany g

School of Engineering and Materials Science, Queen Mary University of London, Mile End Road,

London, E1 4NS, United Kingdom

*Corresponding author: Alexander S. Timin [email protected] KEYWORDS: magnetic capsules, internalization pathway, mesenchymal stem cells, In vitro cell separation, gene delivery, cell magnetization 1 ACS Paragon Plus Environment


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ABSTRACT Stem cell engineering, their manipulation and functionalization involving genetic modification, can significantly expand their applicability for cell therapy in humans. To this aim, reliable, standardized, and cost-effective methods for cell manipulation are required. Here we explored the potential of magnetic multilayer capsules to serve as universal platform for non-viral gene transfer, stem cell magnetization and magnetic cell separation to improve gene transfer efficiency. In particular, the following experiments were performed: (i) the study of internalization process of magnetic capsules into stem cells, including capsule co-localization with established markers of endo-lysosomal pathway,(ii) characterization and quantification of capsule uptake with confocal, electron microscopy and flow cytometry, (iii) intracellular delivery of messenger RNA (mRNA) and separation of gene-modified cells by magnetic cell sorting (MACS), (iv) analysis of the influence of capsules on cell proliferation potential. Importantly, based on the internalization of magnetic capsules transfected cells became susceptible to external magnetic fields, which facilitated to enrich gene-modified cells using MACS (purity ~95%), but also to influence their migration behavior. In summary our results underline the high potential of magnetic capsules in stem cell functionalization, namely (i) to increase gene-transfer efficiency, (ii) to facilitate enrichment and targeting of transfected cells. Finally, we did not observe a negative impact of the used capsules on the proliferative capacity of stem cells proving their high biocompatibility.

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1. INTRODUCTION Cell therapy is a promising treatment option in various areas of clinical medicine such as regenerative medicine or cancer therapy. Different cell types have been applied in therapeutic settings, e.g. lymphocytes

1 – 3

, dendritic cells

4, 5

, fibroblasts

6, 7

, hematopoietic stem cells

(HSCs)8, and mesenchymal stem cells (MSCs)9, 10. Among them, multipotent MSCs are one of the most commonly used cell types in clinical trials for cell therapy in humans 11. Fields of MSC application include regenerative medicine, oncology and oncohematology, infections, hereditary disease, cosmetology, and others12. The unique properties of MSCs such as significant immunomodulating activity and trophic effects, and, to a lesser extent, the abilities of engraftment and multilinear differentiation facilitate wide application of this cell type in cell therapy. MSC therapy has been a promising approach in the treatment of graft-versus-host disease after allogeneic hematopoietic stem cells transplantation, bone or cartilage injuries, inflammatory diseases such as Crohn's disease, and myocardial infarction.13 Clinical trials have shown the safety of MSC administration, but the effectiveness of the procedure remains to be proven. Consequently, there is a growing demand to enhance the required biological properties of MSCs in a given setting and to improve their tropism after administration. Another emerging field for MSCs is their application as delivery vehicles in cancertargeted therapy. This strategy exploits the inherent migratory ability of MSCs toward tumors 14, 15

. However, despite the promising results of preclinical studies, already completed randomized

clinical trials have found moderate or low therapeutic efficacy of the first generation of MSCbased cell products, only 16. According to the literature, but also based on our own experience, the therapeutic potential of MSCs is limited because of the low survival rate of these cells in the early post-transfusion period, their low ability to migrate into inflamed and / or ischemic tissue, and a non-optimal pattern of expression of therapeutically important enzymes and cytokines in 3 ACS Paragon Plus Environment


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MSCs17. We hypothesize that modifying or enhancing some of these properties will help to overcome the named limitations. This can be achieved via cell functionalization and/or genetic modification of MSCs 18, 19. Functionalization is currently a major strategy to improve MSCs for cell therapy19. It can be performed via the delivery of biologically active molecules (including genetic information) into the cells, which can, for example, lead to improvement of migration capacity or survival rate of MSCs. The main methods used for delivery may be divided to viral and non-viral20. Viral platforms are probably the most efficient method of getting genetic information (e.g., mRNA or plasmid DNA) into large numbers of cells, but viral gene transfer has some inherent limitations, associated, e.g., with low capacity, immunogenicity, and potential genotoxicity 21 – 24. Moreover, the production of viral vectors for clinical application remains cumbersome and expensive. In contrast, delivery of biologically active compounds using modern nanotechnology approaches offers a number of promises 25. Consequently, many nanostructured carriers have been developed for delivery of biologically active compounds, including mRNA and plasmid DNA26. However, the engineering of a universal platform for MSC functionalization and simultaneously for genetic modification of target cells is still a very challenging purpose. Multifunctional multilayer microcapsules fabricated via so-called layer-by-layer (LbL) technique can serve as universal instrument for intracellular delivery of biologically active compounds and for construction of MSC-based multifunctional stem cell platform 27 – 30. The main advantages of these microcapsules include precise controlling over size and shape of capsules, fine-tuning of wall thickness, and variable functional wall compositions allowing impregnation of designed parameters such as responsiveness, permeability, biodegradability and surface modification 36

31 –

. Based on the possibility of coating capsule shell with magnetic nanoparticles and

simultaneous immobilization of mRNA or plasmid DNA, it allows to generate genetically 4 ACS Paragon Plus Environment

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modified cells with required standardized, efficient and minimal-step procedures for transfection, isolation and manipulation. Here we report on the application of magnetic microcapsules for the functionalization of human MSCs (hMSCs). Exemplified for mRNA we studied whether these magnetic microcapsules can mediate efficient delivery of genetic material in the target cells with low toxicity. Also, we investigated how these capsules can be applied for magnetic cell separation in order to obtain a high purity of transfected hMSCs in vitro using magnetic cell sorting (MACS). Together, in our work we demonstrate the conceptual possibility for the functionalization of hMSCs with magnetic capsules.

2. EXPERIMENTAL SECTION 2.1 Materials Poly-L-arginine hydrochloride (PARG, MW > 70 000), Dextran sulfate (DEXS, MW > 500 000), Bovine serum albumin-fluorescein isothiocyanate conjugate (FITC-BSA), Bovine serum albumin-tetramethylrhodamine isothiocyanate (TRITC-BSA), calcium chloride dehydrate, anhydrous sodium carbonate, ethylenediaminetetraacetic acid trisodium salt (EDTA) were obtained from Sigma-Aldrich and used without further purification. Deionized (DI) water with specific resistivity higher than 18.2 MΩ/cm from a three-stage Milli-Q Plus 185 purification system was used. 2.3 Preparation of magnetic capsules and immobilization of eGFP-mRNA PARG and DEXS were applied to fabricate capsules on 2 – 3 micron sized calcium carbonate cores used as template. The cores were formed during precipitation across the reaction between calcium chloride (CaCl2 × 2H2O) and sodium carbonate (Na2CO3)

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. After layer-by-layer

assembly of PARG/DEXS, the calcium carbonate cores were removed by EDTA to form hollow 5 ACS Paragon Plus Environment


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microcapsules with following shell architecture: (PARG/DEXS)3. For visualization of capsules using confocal microscope, they were labeled by TRITC-BSA and FITC-BSA. The TRITC-BSA or FITC-BSA had been co-precipitated within porous calcium carbonate microparticles before the multilayer assembly of PARG/DEXS

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In order to engineer capsules with magnetic

properties, Fe3O4 nanoparticles with a mean diameter of 30 nm were layered between polyelectrolytes as negatively charged layer 38. Immobilization of eGFP-mRNA into capsules was performed in the presence of RNase inhibitor as previously described39. RNA solution contained a 1:100 volume part of the RNase inhibitor. The in-situ immobilization of eGFP-mRNA was performed using previously described methods

39, 40

. After immobilization process, the actual amount (µg) of immobilized genetic

materials (mRNA) was quantified via measurements by absorbance using the Nanodrop 2000 Spectrophotometer. 2.4 Characterization of magnetic capsules The morphology of capsules was studied using a JEOL JEM 1011 (Japan) transmission electron microscope (TEM). The capsules were also examined using the LSM 880 Confocal Laser Scanning Microscope (Carl Zeiss, Germany). 2.5 Cell culture Human mesenchymal stem cells (hMSCs) were derived from bone marrow of 3 healthy donors who had signed informed consent. Cells were isolated using direct plating procedure. Briefly, 1 ml of whole bone marrow, heparinized, were re-suspended in alpha-MEM (Lonza, Switzerland); supplemented with 100 IU/ml penicillin, 0.1 mg/ml streptomycin (Biolot, Russia), and 10% FBS (Hyclone, USA) and 2mM Ultraglutamin I (Lonza, Switzerland). After attachment of the cells to the culture plastic, the medium containing non-adherent cells was replaced by fresh culture medium. Culture medium was changed every 3 days until reaching confluence (80%). For 6 ACS Paragon Plus Environment

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passaging, MSCs were detached with trypsin/EDTA solution (Invitrogen, USA) for 5 min and replated at a density of 5.0×103 cells/cm2. hMSCs were characterized by flow cytometry to confirm the expression of CD73, CD90, and CD105 surface molecules and absence of CD34, CD45, CD14, and CD20. Cultures of the 2nd passage were used for further experiments. 2.6 Evaluation of uptake efficiency of magnetic capsules To monitor the cellular uptake efficiency, the magnetic capsules were labeled with FITCBSA. Confocal Laser Scanning Microscope (CLSM, Carl Zeiss, Germany) was used to visualize the cells with internalized capsules. Z-stack series were performed to build 3D reconstruction of cells with internalized capsules. Percentage of cells with internalized capsules was evaluated using flow cytometry (FACS Aria, BD, USA). The cells were stained with anti-CD90-PE. Additionally, TEM was used to analyze capsule uptake. To do so, hMSCs in 6-well plates (5x105 cells per plate) were incubated in alpha-MEM, containing magnetic capsules (10 capsules per cell). After 24 hours incubation, the culture medium with residual non-captured capsules were removed and cells were rinsed twice with PBS. Then, the cells were detached with trypsin/EDTA solution, centrifuged at 1000g for 10 min and fixed with 2.5% PBS-buffered glutaraldehyde solution (Sigma Inc.) for 45 min. The fixed cells were washed by 2 changes of PBS and postfixed in 1% OsO4 solution for 1h. Then, the cells were dehydrated in series of ethanol solutions of gradually increasing concentration, infiltrated with acetone and embedded in the Epon epoxy resine. The ultrathin sections (50-70 nm) of the cells under study were obtained using ultramicrotome Leica UC7 (Germany). The sections were collected on the copper grids for the electron microscopy and contrasted with uranyl-acetate and lead citrate. TEM analysis of the sections was performed using the electron microscope JEOL JEM 1011 (Japan), equipped with a high-resolution digital camera Morada (Olympus, Japan).



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2.7 In-vitro gene transfection To determine transfection efficiencies of the capsules for mRNA, we made use of the enhanced green fluorescence protein (eGFP) reporter gene. EGFP mRNA was generated by in-vitro transcription as described previously 34. Transfection efficiencies were evaluated in hMSCs using flow cytometry. Briefly, cell suspensions were mixed with capsules loaded with eGFP-mRNA at cell-to-capsule ratio from 1:1 to 1:10 (Supporting Information, Table S1) and seeded into 12well plates (5×105 cells/cm2). Transfected cells were further incubated at 37°C for 24 h. After 24 h of cultivation, cells were gently washed with phosphate buffer solution (PBS), and images of eGFP-positive cells were analyzed using CLSM. Then, the cells were detached from flasks by trypsinization, and the transfection efficiency was quantified by flow cytometry (FACS Aria, Becton Dickinson). The same experiments were performed for adherent cells to compare the effect of adherent state of cells on the transfection efficiency. 2.8 Cell viability assay Magnetic capsules (containing from 0 to 2 Fe3O4 layers) at different cell:capsule ratios were added into the wells of 96-well plates with confluent monolayer of hMSCs (6 000 cells) in 8 repeats. Cells were incubated for 24 h at 37˚C in a 5% CO2 atmosphere. For evaluation of cytotoxicity of microcapsules AlamarBlue cell viability assay (Thermo Fisher Scientific) was performed: cells were washed twice with PBS, 0.01% alamarBlue solution in PBS was added, and cells were incubated for 1 h at 37˚C. Absorbance was measured at 535 nm/600 nm wavelengths using a multifunctional reader CLARIOstar (LABTECH, Germany). All data were normalized on background control reading measurement (600 nm). 2.9 Immunofluorescence


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hMSCs were seeded on microscope slides (50 000 cells). After 24 hours FITC-labeled magnetic microcapsules were added to cells at cell:capsule ratio of 1:10. To evaluate endosomal capsule co-localization, the cells with capsules were washed twice with 1xPBS and then were fixed (4% paraformaldehyde) and permeabilized (0.1 % Triton) after different time periods (5 min - 24 h) After that, the obtained samples were incubated with primary antibodies: anti-EEA1 antibody (rabbit) – early endosome marker Abcam ab2900; anti-RAB7 antibody – late endosome marker Abcam ab50533; anti-LAMP1 antibody (rabbit) – lysosome marker Abcam ab24170 and subsequently secondary antibodies [α-IgG_R-AlexaFluor 568; α-IgG_M-Alexa Fluor 528; αIgG_R-AlexaFluor 568]. Cell nuclei were stained with DAPI (6-diamidino-2-phenylindole; 10 nM). The capsule co-localization was visualized using a CLS microscope Leica TCS SP8 in multiple cells across 3 replicates. 2.10 Magnetic cell sorting of hMSCs that internalized magnetic capsules To address the possibility of magnetic-field directed cell delivery and sorting, magnetic separation of hMSCs after microcapsule uptake was performed. Cells were trypsinized, washed for three times in PBS (pH 7.4), re-suspended at 1x106 cells/ml and then separated using miniMACS cell separation system (Miltenyi Biotec) as proposed by manufacturer. Efficiency of magnetic cell sorting (MACS) was evaluated by counting eGFP-positive cells using a hemocytometer under fluorescent microscope. 2.11 The influence of capsules with and without mRNA on proliferation of hMSCs To estimate cell proliferation, hMSCs were first incubated with empty and mRNA-loaded capsules at different cell:capsule ratios (1:1, 1:3, 1:5, 1:10) for 1 day. Cells were cultured in their growth medium for 1, 4 and 8 days, respectively. At each time point cell nuclei were stained with DAPI, and CyTell imaging system (GE Healthcare Life Sciences) was used to make images. Obtained images with cells nuclei were examined by FiJi cell counter java application 9 ACS Paragon Plus Environment


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(http://imagej.net/Cell_Counter) to count the number of cell nuclei. To examine long-term effects of magnetic capsules on cell proliferation, hMSCs treated with capsules at capsule-to-cell ratio of 10:1 were subcultured for 3 serial passages (around 80 – 90 % monolayer confluence) with full media exchange every 3 days.

3. RESULTS AND DISCUSSION Magnetic capsules are highly interesting means for cell functionalization and genetic modification of cells followed by magnetic cell sorting. Therefore, detailed studies of interactions between magnetic capsules and target cells is of great interest. We here addressed this question focusing on a cell type used in various therapeutic settings, namely hMSCs. Better understanding of the main principles of hMSCs interactions with magnetic capsules will help to develop a kind of biomimetic platform consisting of live cells with internalized drug carriers. Potentially, the magnetic capsules can also be applied for obtaining genetically engineered cells for therapy in humans. 3.1 Microscopic characterization of capsule uptake and intracellular localization of capsules To understand the interactions of magnetic capsules with hMSCs, several important factors should be considered, including the uptake mechanism of capsules, internalization capability of hMSCs, and toxicity of capsules in hMSCs. First, we have established hMSC cultures following commonly used protocols. Confirmation of cell type was performed using FACS with multicolor antibody panel in accordance with the consensus criteria of the International Society of Cellular Therapy

41

(Figure S1). The analysis show that cell population expressed low levels ( 95%) of typical hMSC markers: CD105, CD90, CD73

41

. Further, we studied the internalization process of 10

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magnetic capsules by hMSCs. In our study we applied PE capsules consisting of PARG and DEXS. To facilitate visualization of capsules by confocal microscopy we made use of FITClabeled BSA. In order to engineer PE capsules with magnetic properties, Fe3O4 nanoparticles were layered between polyelectrolytes as negatively charged layer. The morphology of magnetic capsules was determined by transmission electron microscopy (TEM). The TEM images with corresponding CLSM images can be found in Supporting Information (Figure S2). To examine the internalization of magnetic capsules by hMSCs, the capsules were incubated with hMSCs for 24 h. Nuclei were stained with DAPI (blue), cell membranes with anti-CD90-PE (red), while the green fluorescent signals correspond to the fluorescence of capsules labeled by FITC-BSA. (Supporting Information, Figure S3). Figure 1A depicts threedimensional confocal image of hMSC from the glass slide to the top of the cell at a series of depths along the z-axis. The main panel shows the fluorescent image in the x-y cross section at a given z-location. The two smaller panels reveal the structure along the x-z (top panel) and y-z cross sections (right side panel). It can be seen that the magnetic capsules (green) are located between two red layers of cell membrane. We additionally built a 3D reconstruction of an hMSC (Figure 1B). As evident, magnetic capsules (green) were inside the cell in places, covered by red membrane, confirming the successful internalization of capsules into hMSCs. TEM images also confirm cellular internalization of magnetic capsules (Figure 1C).



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Figure 1. Efficient uptake of magnetic microcapsules. (A) Z-stack CLSM image confirming the internalization of magnetic microcapsules labelled with FITC-BSA. (B) 3D reconstruction image of hMSC with internalized microcapsules. Nuclei: stained violet with DAPI; Membrane: stained red with anti-CD90-PE; Green fluorescence: magnetic microcapsules labeled with FITC-BSA (C) TEM images of hMSC in the absence of capsules (control) and incubated with capsules.

Next, we aimed at establishing the intracellular pathway of capsules in hMSCs, which provides us information about the uptake mechanism of these capsules. So far, internalization pathways and trafficking of microcapsules with analysis of the influence of their physical and chemical properties on their intracellular fate were studied only on tumor cell lines. In contrast, we here investigated these pathways on primary human cells. In order to determine the intracellular fate of microparticles, we used early endosome antigen 1 (EEA-1), Ras-related protein Rab-7 (RAB7) and the lysosomal-associated membrane protein 1 (LAMP-1). EEA-1 is 12 ACS Paragon Plus Environment

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required for fusion of early and late endosomes and for sorting at the early endosome level thus being a marker of early endosomes

42

. RAB7 functions as a key regulator in endo-lysosomal

trafficking, governs endosomal maturation, endosomal migration and positions, and endosomelysosome transport and considered as late endosomes marker 43. Finally, LAMP-1 is a marker of lysosomes 44. After 15 min we observed co-localization of the capsules with the EEA-1 marker, indicating that microparticles were located inside the early endosomal compartment (Figure 2, first row). Concomitantly with vesicular trafficking, EEA-1 co-localization was no longer seen at 30 min after internalization. Instead, microcapsules now co-localized with late endosome marker RAB7, and this process was apparently increasing during the 4-hour observation period (Figure 2, middle row). After 6 hours, we observed co-localization of capsules with structures containing LAMP1, which is a marker for lysosomes, showing that lysosomes are the final localization of the capsules (Figure 2, third row). Thus, we have characterized the intracellular trafficking of the capsules from early endosomes, to late endosomes and lysosomes. These results indicate that capsules undergo fast (within 15 min) internalization to the endosomal membrane compartment. Based on the size of the magnetic capsules (2-4 µm), we suppose that they are internalized via phagocytosis or macropinocytosis. The important question that should be addressed is further cargo release from lysosomes to cytosol. For gene transfection, the cargo molecules (e.g., plasmid DNA, protein, mRNA and siRNA) should be localized in the cytosol. Respectively, the microcapsules can facilitate the cytosolic delivery of cargo molecules as demonstrated by W. Parak, underlying the potential of this platform for non-viral gene delivery 45, 46.



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Figure 2. Internalization of capsules by hMSCs can be studied based on their co-localization with endo-lysosomal markers. CLSM images after definite time internals show co-localization of fluorescently labeled magnetic capsules (green) with EEA1 (early endosomes), RAB7 (late endosomes) and LAMP1 (lysosomes). Eventually, capsules accumulated in the lysosomes. Blue, nuclei (DAPI); red, early, late endosomes (EEA1, RAB7) and lysosomes (LAMP1); green, magnetic capsules.

The amount of incorporated magnetic capsules was further quantified using flow cytometry (FC). hMSCs were stained with CD90-PE. Cellular uptake of capsules was evaluated for different cell:capsule ratios (1:1, 1:3, 1:5 and 1:10) after 24 h of incubation. Figure 3A shows 14 ACS Paragon Plus Environment

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representative FC data plots reflecting cellular uptake of capsules labeled with FITC-BSA. It should be noted that these experiments were performed in cell suspension. It can be clearly observed that the increase in amount of capsules per cell leads to proportional increase in capsule uptake. The average fraction of cells with internalized capsules was ~ 34% (cell-to-capsule ratio = 1:1), ~ 60% (cell-to-capsule ratio = 1:3), ~ 77% (cell-to-capsule ratio = 1:5) and ~ 93% (cellto-capsule ratio 1:10), respectively. It is important that the uptake efficiency depends on adherence state of hMSCs. As shown in Figure 3B, the uptake of capsules incubated with adherent cells was significantly lower compared with capsules incubated with cell suspensions. It was shown previously that the cell culturing conditions have influence on uptake efficiency of particles47. Higher uptake rates were also reported for several other cell types cultured in suspension48. The higher efficiency of capsule internalization in cell suspension can be explained by uniform cell capsule dispersion, higher cell membrane area exposed for cell-particle contact, and retention of cytoskeleton potential while cells are non-adherent. The cell viability assay demonstrated that capsules made of PARG/DEXS exert very mild toxicity (Figure 3C). Among microcapsule components, iron nanoparticles toxicity seems to be most pronounced. In fact, magnetic iron nanoparticles (Fe3O4) were previously demonstrated to be associated with significant toxic effects such as inflammation, the formation of apoptotic bodies, impaired mitochondrial function, membrane leakage of lactate dehydrogenase, and generation of reactive oxygen species (ROS). Therefore, the influence of the number of Fe3O4 layers on the capsule toxicity was also examined. As seen from Figure 3C, the cell viability decreased with increasing numbers of Fe3O4 layers. Thus, using one-layer coating with Fe3O4 seems to be the most promising for further application, because of very limited toxicity.



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Figure 3. Uptake efficiencies of capsules by hMSCs at different cell:capsule ratios. (A) Experiments were performed in cell suspension. hMSCs were stained with anti-CD90-PE. (B) Comparison of uptake efficiencies of capsules incubated with hMSCs in adhesion and suspension cultures. (C) Cell viability of hMSCs incubated with magnetic capsules at different cell:capsule ratios after 24 h incubation. Bars represent mean ± S. D.

3.2 Magnetic-capsules mediated delivery of eGFP-mRNA As shown previously, magnetic capsules were internalized and engulfed into endosomes. The latter eventually become lysosomes, where enzymatic decay processes occur. This physiological process can be related to the degradation of biologically active compounds contained in capsules. In case of gene-delivery techniques, it is crucial that the capsules form stable complexes with plasmid DNA or RNA to prevent their premature degradation. Several approaches have been proposed to protect plasmid DNA or RNA from premature degradation using endosomal escape agents, e.g., PEI, lipofectamine, or glycoprotein H. However, these agents can be toxic at high 16 ACS Paragon Plus Environment

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concentrations, which would be detrimental for clinical application. Therefore, PE microcapsules can be more attractive given their low toxicity and strong complex ability with mRNA/ plasmid DNA. The main problem is that capsules accumulated in lysosomes, which results in mRNA degradation by RNases.39 The usage of RNase inhibitors can help to protect the mRNA from its degradation in lysosomes. To assess the ability of capsules to deliver mRNA into the hMSCs, we immobilized eGFP-mRNA in the capsule shell with and without RNase inhibitors at different cell:capsule ratios. The amount of immobilized eGFP-mRNA for each cell:capsule ratio is presented in Supporting Information (Table S1). The transfection efficiency was determined as proportion of eGFP-positive cells, measured by flow cytometry. As shown in Figure 4A, B, the eGFP-mRNA was successfully delivered via magnetic capsules containing RNase inhibitor into the cells leading to eGFP expression in transfected hMSCs. We observed dose-dependent effectiveness in eGFP-mRNA delivery – with an increase in the capsule-to-cell ratio the estimated proportion of transfected GFP-positive cells also increased (Figure 4A, B). The maximum eGFP transfection efficiency of ~ 62 % was obtained for a capsule-to-cell ratio of 10:1. It was interesting to note that there was an apparent decrease in the relative rates of eGFP mRNA transfer as related to capsule-uptake efficiencies (compare Figure 3). Indeed, only at the lowest capsule-to-cell ratio (1:1) the capsule-uptake rate (~ 34 %) was in good agreement with the resulting percentage of eGFP-positive cells (~30 %). In contrast, at the highest ratio (10:1), only ~ 62 % of hMSCs became eGFP-positive, whereas the capsule uptake efficiency reached app. 93%. This finding may potentially be explained by high number of capsules that were associated with hMSC membranes, but were not internalized. Alternatively, there might be some differences in the capacity of cells to express foreign genes, e.g. depending on the cell cycle state. Finally, higher amounts of mRNA might more efficiently stimulate intracellular immune mechanisms resulting 17 ACS Paragon Plus Environment


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in more rapid degradation. Altogether, our observation requires further exploration and indicates a potential point of action for future improvement of the delivery platform. When the transfection was performed without RNase inhibitor, we observed a strong three-tofive-fold decrease in gene-transfer efficiencies reaching 4 – 15 %, only (Figure 4C). This data indicates that mRNA protection is essential to mediate efficient gene expression. As we demonstrated above by CLSM, at 24 h the capsules are co-localized with established endosomal marker LAMP1. This indicates that the lysosomal compartment represents the final point of intracellular trafficking of capsules. It is well-known that the lysosomes act as the waste disposal system of the cell by digesting unwanted materials in the cytoplasm, both from outside of the cell and obsolete components inside the cell.49 The lysosomal compartment contains more than 60 different enzymes enabling the cell to degrade various biomolecules it engulfs, including peptides, nucleic acids, carbohydrates, and lipids.

50

Lysosomal acid ribonuclease provides digestion of

ingested RNA, as well as turnover of cytoplasmic RNA.

51

This aspect of capsule trafficking and

localization should be taken into account, and premature cargo RNA degradation should be prevented. The RNase inhibitor used in our study is a recombinant murine enzyme that specifically inhibits a wide spectrum of nucleases. 52 In the presence of RNase inhibitor we observed a significant increase in the percentage of eGFP-positive cells (Figure 4C). We also observed that hMSCs transfected with magnetic capsules containing mRNA/RNase inhibitor showed a strong expression of eGFP just 6 h after transfection and maintained this high eGFP expression for more than 72 h posttransfection (Supporting Information, Figure S6).


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Figure 4. Influence of cell:capsule ratio and RNase inhibitor on the transfection efficiency. hMSCs were incubated with magnetic capsules containing eGFP mRNA. (A) Percentages of GFP-positive hMSCs were determined using flow cytometry 24 hours after transfection. (B) CLSM images demonstrate eGFP-expressing hMSCs after transfection. (C) The histogram shows the influence of cell:capsule ratios and the presence of RNase inhibitor in capsules on the transfection efficiency of hMSCs. Bars represent mean ± S. D.



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3.3 Magnetic cell sorting to enrich genetically modified cells In many settings, (almost) pure populations of genetically modified cells are desirable or even required. On the other hand, high transfection rates are often associated with unwanted side effects such as high cytotoxicity, e.g. after plasmid electroporation

53

, or unnecessary high

transgene copy numbers. Enrichment of gene-modified cells represents an interesting approach to address these limitations. The magnetic capsules used here to transfect hMSCs should offer the opportunity to separate the population of transfected from non-transfected cells under the influence of a strong magnetic field in vitro. To verify this assumption, we established a protocol for the magnetic separation of cells with engulfed capsules using commercially available devices (miniMACS by Miltenyi Biotec) for magnetic cell sorting (MACS). A flow chart of the experimental procedure and the results of the experiment are depicted in Figure 5A. As shown in Figure 5B, we observed high sensitivity of hMSCs with internalized magnetic capsules towards an external magnetic field. The magnetic sorting process allowed straight sorting of an initially mixed suspension of cells containing app. 60% hMSCs with internalized capsules (capsule-to-cell ratio = 3:1) into two, very pure populations of cells – hMSCs containing magnetic capsules (purity app. 99%) and hMSCs without magnetic capsules (impurity app. 1%). This result clearly demonstrates the potential to control microcapsules-containing hMSCs by applying an external magnetic field. Since the MACS procedure was very efficient for capsule-transfected cells, we went on to apply the same strategy for hMSC transfected with eGFP-mRNA containing capsules. We used the cell suspension transfected at a capsule-to-cell ratio of 1:1, because for this sample the percentage of eGFP-positive cells was similar to the observed capsule-uptake value. Figure 5 C,D shows CLSM images of transfected hMSCs with internalized magnetic capsules before (Figure 5C) and after (Figure 5D) MACS. Again, we observed strong enrichment of eGFP-positive cells to ~ 20 ACS Paragon Plus Environment

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95 % after the magnetic separation procedure (Figure 5D). Based thereon we suppose that our strategy can be used to overcome poor gene transfer efficiencies and/or high cytotoxicity associated with high transfection rates. Moreover, cell separation by MACS has been widely used in clinical cell therapy, and the application of magnetic capsules can be expected to be much cheaper in comparison with antibodies used for MACS. Finally, magnetization of celltherapy products such as hMSCs may be effectively used to increase targeted delivery to desired tissues/organs, e.g. in tissue engineering, or pathological sites. In view of low toxicity and biocompatibility of microcapsules, we propose that our protocol can be further used for the development of efficient and safe gene therapy applications, including ex-vivo gene transfer.



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Figure 5. Principle and application of magnetic cell sorting after transfection with magnetic capsules. (A) Schematic representation of the principle of magnetic cell sorting (MACS). (B) Efficient enrichment of hMSCs with internalized magnetic capsules labeled by FITC-BSA (green) by MACS. hMSCs with magnetic capsules (capsule-to-cell ratio = 3:1) (1) were sorted using the miniMACS cell separation device facilitating efficient separation of capsule-negative (2)

and positive (3) cells (C). MACS-based enrichment of 5 × 105 eGFP-transfected (1)

(capsule-to-cell ratio of 1:1) hMSCs cells (2). (D) The histogram shows a significant increase in percentage of eGFP-positive cells after magnetic sorting. Bars represent mean ± S. D. 22 ACS Paragon Plus Environment

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3.4 Proliferation potential of hMSCs with internalized magnetic capsules After establishing efficient cellular uptake and gene delivery using our biocompatible capsules, we next wanted to address a potential impact on proliferative capacity of hMSCs. Their high proliferation potential is one of the defining features of hMSC, and impaired clonogenic activity of hMSCs plays a crucial role in a number of human diseases. 54, 55 We therefore examined the influence of magnetic capsules (empty vs. loaded with mRNA) on proliferation of hMSCs using Cytell Cell Imaging. At defined time points, hMSCs were stained with DAPI and cell quantities were assessed via cell nuclei counting on images taken from Cytell Cell Imaging (Figure 6A). Cell counting showed that there were significant differences in the numbers of cells cultured with capsules (~1000 cells/cm2 for capsule-to-cell ratio of 1:1 and ~ 600 cells/cm2 for capsule-to-cell ratio of 10:1) compared to control cells (~3000 per cm2) after one day of incubation (Figure 6B). Besides mild influence of capsules on the cell viability, we suggest that the capsule uptake can decrease adhesion capability of hMSCs due to interaction of capsules with the cytoskeleton during internalization process as discussed above. After four days of cultivation, the differences in cell densities between control group and cells incubated with capsules decreased. Still there was an inverse correlation between the capsule-to-cell ratio and the observed hMSCs density, i.e. proliferative activity (e.g., ~ 5000/cm2 for capsule-to-cell ratio of 1:1, and ~ 3000/cm2 for capsule-to-cell ratio of 10:1). On day 8 all groups achieved almost 8090% confluence and differences between the four capsule-transfected groups were minimal. However, quantitative analyses at this time point were not reliable anymore, since hMSC proliferation was already suppressed by contact inhibition.56 In order to assess a potential impact of the mRNA cargo of the capsules on cell growth, e.g. due to the induction of an intracellular immune response, we also evaluated hMSC proliferation after



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transfection with mRNA (Figure 6B). As shown, there was no additional effect of the mRNA on the proliferation of hMSCs in comparison with hMSCs incubated with empty capsules. Next, we examined the long-term impact of magnetic microcapsules on hMSC proliferation. Cells were maintained for 3 sequential passages. As shown in Figure S10, there was no significant difference in cell growth rate between control cell group and hMSCs treated with capsules. It is obvious that the cells treated with capsules conserve their proliferation potential throughout the long-term cultivation warranting the safe use of magnetic microcapsules for transfection and cell sorting.

Figure 6. Proliferation of hMSCs cultured with magnetic capsules at different cell:capsule ratios. (A) Images of cell nuclei staining with DAPI (blue). (B) Influence of the amounts of capsules 24 ACS Paragon Plus Environment

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and of the mRNA cargo on growth of hMSCs. Growth was evaluated by counting the number of cells at definite time points (1, 4 and 8 days) using Cytell cell imaging. Bars represent means ± S. D.

4. CONCLUSIONS In this study, we investigated the applicability of magnetic capsules for genetic modification and magnetic separation of hMSCs. We demonstrated that LbL magnetic capsules can be successfully internalized by hMSCs, where they were co-localized with endo-lysosomal pathway markers. Lysosomal localization of capsules may lead to enzymatic decay process and the degradation of mRNA contained in capsules. However, co-immobilization of mRNA with RNase inhibitors during capsule

synthesis

effectively protects mRNA

from

premature

degradation with only mild cytotoxicity, resulting in the high transfection efficiency (~ 62 % of hMSCs expressing eGFP) at cell:capsules ratio of 1:10. We observed that capsule uptake was

associated

internalized

with

magnetic

a transient capsules

suppression

of

hMSCs

proliferation.

hMSCs with

became sensitive to magnetic fields, which facilitated

enrichment of transfected cells using magnetic cell sorting. Based thereon, almost pure populations of transfected hMSCs could be generated by MACS even with relatively low (

Mesenchymal Stem Cells Engineering: Microcapsules-Assisted Gene

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