Safe and Effective Delivery of Antitumor Drug Using Mesenchymal

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Biological and Medical Applications of Materials and Interfaces

Safe and effective delivery of antitumor drug using mesenchymal stem cells impregnated with submicron carriers Alexander S. Timin, Oleksii O Peltek, Mikhail Valeryevich Zyuzin, Albert R Muslimov, Timofey E Karpov, Olga S Epifanovskaya, Alena I Shakirova, Mikhail V Zhukov, Yana V Tarakanchikova, Kirill V Lepik, Vladislav S Sergeev, Gleb Sukhorukov, and Boris V Afanasyev ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b22685 • Publication Date (Web): 18 Mar 2019 Downloaded from http://pubs.acs.org on March 19, 2019

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ACS Applied Materials & Interfaces

Safe and Effective Delivery of Antitumor Drug using Mesenchymal Stem Cells Impregnated with Submicron Carriers Alexander S. Timin1,2,*, Oleksii O. Peltek3, Mikhail V. Zyuzin4, Albert R. Muslimov2,5, Timofey E. Karpov3, Olga S. Epifanovskaya2, Alena I. Shakirova2, Mikhail V. Zhukov4, Yana V. Tarakanchikova3,5, Kirill V. Lepik2, Vladislav S. Sergeev2, Gleb B. Sukhorukov6, Boris V. Afanasyev2 1Research

School of Chemical and Biomedical Engineering, National Research Tomsk

Polytechnic University, Lenin Avenue 30, 634050 Tomsk, Russia 2First

I. P. Pavlov State Medical University of St. Petersburg, Lev Tolstoy str., 6/8, 197022 Saint-

Petersburg, Russian Federation 3RASA

Center, Peter the Great St. Petersburg Polytechnic University, Polytechnicheskaya, 29,

195251 Saint Petersburg, Russian Federation 4Department

of Nanophotonics and Metamaterials, Saint Petersburg National Research

University of Information Technologies, ITMO University, 197101 Saint Petersburg, Russia 5Nanobiotechnology

Laboratory, St. Petersburg Academic University, 194021 Saint Petersburg,

Russia 6School

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

London, E1 4NS, United Kingdom *Corresponding

author:

Dr. Alexander S. Timin. [email protected] , [email protected]. Address: National Research Tomsk Polytechnic University, Lenin Avenue 30, 634050 Tomsk, Russia

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Keywords: human mesenchymal stem cells, silica capsules, spontaneous and directed migration, vincristine, malignant tumor, tumor therapy, tumor spheroid.

ABSTRACT An important area in modern malignant tumor therapy is the optimization of the antitumor drugs pharmacokinetics. The use of some antitumor drugs is limited in clinical practice due to their high toxicity. Therefore, the strategy for optimizing the drug pharmacokinetics is focusing on the generation of high local concentrations of these drugs in the tumor area with minimal systemic and tissue-specific toxicity. This can be achieved by encapsulation of highly toxic antitumor drug (vincristine/VCR that is 20-50 times more toxic than widely used in antitumor therapy doxorubicin) into nano- and microcarriers with their further association into therapeutically relevant cells that possess ability to migrate to sites of tumor. Here we fundamentally examine the effect of drug carrier size on behavior of human mesenchymal stem cells (hMSCs), incl. internalization efficiency, cytotoxicity, cell movement, to optimize the conditions for the development of carrier-hMSCs drug delivery platform. Using the malignant tumors derived from patients, we evaluated the capability of hMSCs associated with VCR loaded carriers to target tumors using a 3D spheroid model in collagen gel. Compared to free VCR, developed hMSCsbased drug delivery platform showed enhanced antitumor activity regarding to those tumors that express CXCL12 (SDF-1) gene, inducing directed migration of hMSCs via CXCL12 (SDF1)/CXCR4 pathway. These results show that the combination of encapsulated antitumor drugs and hMSCs, which possess the properties of active migration into tumors, is therapeutically beneficial and demonstrated high efficiency and low systematic toxicity, revealing novel strategies for chemotherapy in the future.


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Abbreviations: Layer-by-Layer – LbL Polyelectrolytes poly(sodium 4-styrenesulfonate) – PSS Poly(allylamine hydrochloride) – PAH Ethylene glycol – EG Tetraethyl orthosilicate – TEOS Ethylenediaminetetraacetic acid trisodium salt – EDTA Human mesenchymal stem cells – hMSCs Phosphate buffer solution – PBS Bovine serum albumin conjugated with tetramethylrhodamine – TRITC-BSA Atomic force microscopy – AFM Scanning electron microscopy – SEM Transmission electron microscopy – TEM Laser scanning confocal microscopy – CLSM Quantitative real-time polymerase chain reaction – qPCR No template controls – NTC TATA-box binding protein – TBP Abelson murine leukemia viral oncogene homolog – ABL Immunocytochemistry – ICC Tris-Buffered saline – TBS Regions of interest – ROI Stromal cell-derived factor 1 – SDF-1 Messenger ribonucleic acid – mRNA Dextran conjugated with Alexa Fluor 647 dye – AF647 Phalloidin conjugated with Alexa Fluor 488 dye – AF488 Poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) silica coated capsules with average size of 0.65 ± 0.21 μm – Caps_0.6 Poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) silica coated capsules with average size of 2.12 ± 0.45 μm – Caps_2 Poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) silica coated capsules with average size of 5.45 ± 1.03 μm – Caps_5 Poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) silica coated capsules with average size of 0.65 ± 0.21 μm loaded with vincristine – VCR@Caps_0.6 ACS Paragon Plus Environment


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Human mesenchymal stem cells associated with poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) silica coated capsules with average size of 0.65 ± 0.21 μm loaded with vincristine – VCR@Caps_0.6/hMSCs

1. Introduction During the past decade, cell-based therapies have received an increased attention as a promising tool for diseases that are hard to treat with conventional methods.1 Among the accepted cell platforms, mesenchymal stem cells (MSCs) are employed in more than 700 registered clinical trials. As an example, MSCs can be used for treatment of graft-versus-host-diseases,2 cancers,3,4 cardiac damage,5–7 muscular dystrophy.8 MSCs have a number of unique functional properties: these cells are adherent, they are able to differentiate into osteoblasts, adipocytes, and chondrocytes. Moreover, MSCs possess well-characterized immunophenotype: positive for CD73, CD90, CD105 while negative for CD45, CD34, CD14/CD11b, CD19/CD20/CD79α, and HLA‐DR.9,10 Beside of this, one of the key features of this cell population is that MSCs can act as universal cell donor, because of their immunocompatibility, what makes them useful for allogeneic transplantation.11 Another important property of MSCs is their intrinsic ability for migration to the sites of inflammation and tumor environment.12 In the recent studies, MSCs showed ability to migrate specifically to the tumor regions including gliomas3 and breast,13 colon,14 ovarian,15 and lung carcinomas, as well as many other primary and metastatic tumors, including melanomas.16–18 In these in vitro and in vivo models, MSCs have successfully homed to the tumor areas from a large variety of administration routes including the tail vein, carotid artery, femur, tibia, and trachea. MSC tumor migration is motivated by many factors including tumor cell-surface specific receptors and soluble tumor derived factors. Only a few of these factors have been studied, for example, stromal cell-derived factor-1 (SDF-1), tumor necrosis factor (TNF)-α, and interleukins (IL).15 MSCs demonstrate a range of tumor inhibition properties in sarcomas19 and leukemias20, and can suppress breast cancer cell growth and lung metastasis in vivo.21 This cell population can also inhibit primary tumor growth22 and malignant colony formation.23 However, several studies


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reported that the number of factors, such as secretion of chemokines (CXCL1, CCL5 and CXCL12), release of angiogenic and growth factors (angiopoietins, EGF, galectin‐1, KGF, VEGF, IGF‐1), and local immunosuppressive effect of MSCs may influence on the potential ability of this cell type to potentiate tumor growth.24,25 Despite these concerns, there are only a few reports regarding the induction of tumor growth by MSCs in clinics.26 In fact, the clinical trials related to the MSCs infusions have proved this treatment to be generally safe with good toxicity profile.24,25 To date, the main drawback of MSCs implementation in clinical practice is associated with their low treatment efficiency. Moreover, the migration properties of MSCs during cultivation period can be changed. In order to increase the efficiency of MSC-based antitumor therapy, MSCs should be combined with antitumor agents. There are two general approaches for inhibition of tumor growth using MSCs. The first one is transfection of MSCs with transgenes defining the expression of proteins with antitumor activity. In the previous works, following proteins with antitumor activity were expressed in MSCs: IL2, IL12,27 IL18,28 IFNβ,29 TRAIL.30 All these studies demonstrated the massive migration of MSCs into the tumor regions with subsequent regression of tumor. Nevertheless, the transfection approach has certain limitations, since only proteins can be used as antitumor agents. Moreover, non-viral cell transfection is usually poor and in case of viral gene delivery vehicles can induce malignant transformation of injected cells.31 Another approach to use MSCs in cell-based antitumor therapy is introduction particles previously loaded with drugs into MSCs. Since MSCs possess mechanisms of active micro- and nanoparticle internalization,32–34 such cell-delivery vehicles can effectively transfer antitumor drugs into tumor regions. It is required that particles with encapsulated highly toxic drugs should not significantly influence cell functionality, such as viability and migration abilities. There are some works describing the modification of MSCs of different nanoparticles based on liposomes,35 silica,36 iron oxide37 but there are some limitations in employment of these carriers as drug-containers associated with low loading capacity, which is necessary to create high local concentrations of antitumor drugs in the tumor areas.



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Multifunctional hollow micro- and submicropartiles, so-called capsules, made with Layer-byLayer (LbL) technique can be considered as one of the most promising carriers, which possess necessary requirements for their introducing into clinically relevant cells.38 These capsules demonstrate non-toxicity and biocompatibility (up to 10 added capsules per cell),39 as well as high loading capacity.40 Capsules wall can be functionalized with different nanostructures,41 e.g. silica, what can decrease permeability of capsules,42 so that there will be almost no leakage of drugs before their administration and, thus, no significant influence on cell functions might be observed. Capsules cavity can be loaded with sufficient amount of antitumor drugs; thereby the gradual degradation of silica-functionalized capsules in an intracellular environment will lead to the prolonged release of these drugs. In the recent works, it has been shown that even micrometric sized capsules can be internalized by human MSCs (hMSCs).34 However, despite of the key importance of migratory activity and homing of hMSCs for their therapeutic implementation, to date, there is a lack of information regarding the influence of capsules on hMSCs migration. The aim of this work is to explore the influence of different amounts and types of capsules on internalization, viability, spontaneous, directed migration of hMSCs, and on the ability of these cells associated with capsules to migrate within the intercellular environment of artificial tumor spheroid formed from the primary tumor cells of patients. These data may be crucial for the further development of hMSC-based drug delivery systems in combined cell therapy.

2. Experimental section 2.1. Synthesis of capsules with different sizes Silica (SiO2) coated capsules were synthesized as previously reported.40,43–45 The poly(sodium 4styrenesulfonate (PSS)/poly(allylamine hydrochloride (PAH)) capsules with 3 polyelectrolytes bilayers at three different sizes: 0.65 ± 0.21 µm; 2.12 ± 0.45 µm; 5.45 ± 1.03 µm were obtained. Different bioactive compounds were loaded into the capsules cavity according to the previously published protocols.46 The (i) BSA conjugated with tetramethylrhodamine (TRITC-BSA), (ii)


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Dextran conjugated with Alexa Fluor 647 (AF647) were used for labelling of capsules. Details and the full data set are given in the Supporting Information. 2.2. Structural characterization of capsules The capsules morphology, structure, size distribution and stiffness were performed with scanning electron microscopy (SEM), Quanta 200, FEI, Netherlands, at 10 kV acceleration voltage; JEOL JEM-2100F transmission electron microscope; Carl Zeiss confocal laser scanning microscope (LSM 710); AtoFM Ntegra Aura (NT-MDT, Russia). Sample preparation procedure is presented in the Supporting Information. 2.3. Calculation of encapsulated and released VCR amount Vincristine (VCR) loading as a model commercially available antitumor drug was performed. Calibration curve of VCR was obtained using UV-Vis spectrometer (SmartSpec Plus, BioRad) (Figure S8). Release of cargo to hybrid capsules loaded with VRC was investigated. Amount of released cargo from capsules was also measured with UV-Vis spectrometer (SmartSpec Plus, BioRad). The absorbance of supernatant was checked during 105 hours. Detailed description of encapsulated amount VCR and release data is given in the Supporting Information. 2.4.

Cell culture

2.4.1 Human mesenchymal stem cells (hMSCs) Human mesenchymal stem cells (hMSCs) were derived from the bone marrow of healthy donors who gave their informed consent. Cells were isolated using a direct plating procedure. For this, 1 mL of heparinized bone marrow was resuspended in an alpha-MEM medium (Lonza, Switzerland) supplemented with 100 IU/mL penicillin, 0.1 mg/mL streptomycin (Biolot Russia), 10% of vol. fetal bovine serum (FBS, HyClone, USA), and 2 mM Ultraglutamin I (Lonza, Switzerland). The hMSCs were cultured under standard conditions (37°C, 5% of CO2, humidified sterile environment) to >85% confluency. Subsequently, hMSCs were detached with trypsin-EDTA solution (Sigma-Aldrich, UK) and passaged up to the second passage for further experiments.



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Confirmation of hMSC cell type was performed immunophenotypically using flow cytometry (FACS Aria, BD, USA) with a multicolor antibody panel at accordance consensus criteria of the International Society of Cellular Therapy.47 The analysis shows that cell population expressed low levels ( 95%) of typical hMSC markers, i.e., CD105, CD90, CD73 (Figure S5). 2.4.2 Primary tumor cells Biopsy samples from solid tumor patients (n=11) were collected and further used to develop cell culture in vitro as previously described.48 Cells were cultured in medium containing DMEM/F12 (80%) (Lonza, Switzerland), fetal bovine serum (20%) (FBS, HyClone, USA), insulin (5 μg/ml), transferrin (5 μg/ml), selenium (5 ng/ml) (Sigma-Aldrich, UK), 100 IU/mL penicillin, 0.1 mg/mL streptomycin (Biolot Russia); 37 ° C, 5% CO2, 100% humidity. After reaching 80% confluent monolayer cells were reseeded at vials of 25 cm2 in 5 ml of medium seeded 1x106 cells using 0.25% trypsin-EDTA solution (Sigma-Aldrich, UK). Three types of tumor cell cultures were chosen for further experiments according to their CXCL12 chemokine receptor gene expression levels. Immunohistochemical characterization of cells (differentiating antigens EMA, BerEP4, RCC, Vimentin, CD44, and cytokeratine markers) was carried out and presented in Supporting Information. 2.5. Capsules-cell association/internalization studies Two approaches were used to evaluate the capsule association and internalization efficacy. The pretreated hMSCs with capsules (cell-to-capsule ratios=1:1, 1:3, 1:5, 1:10) were seeded into flasks and incubated overnight. Capsules association with cells was evaluated using flow cytometry (FACSAriaIII, BDBioscience). The internalization efficiency of capsules into hMSCs was performed using CLSM Z-stack option. Details and the full data set are given in the Supporting Information. 2.6. Toxicity studies (LIVE/DEAD assay)


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Cell viability was defined using two methods: AlamarBlue assay and LIVE/DEAD assay. AlamarBlue assay was used to investigate the influence of VCR loaded capsules on hMSCs viability. LIVE/DEAD assay was applied to study tumor cells resistance against VCR and viability of 3D cell culture. Detailed protocols are presented in the Supporting Information. 2.7. Spontaneous migration study To study spontaneous migration of hMSCs two different approaches were employed: (i) lifetime observation of hMSCs with microscopy and (ii) scratch wound assay. Cell-IQ experimental analytical system based on optical microscopy was used to study hMSCs mobility. For this, hMSCs were pretreated with TRITC-labeled capsules at different cell-to-capsule ratios (1:1, 1:3, 1:5, 1:10), further seeded in 48-well plates and incubated overnight. To perform scratch wound assay, hMSCs pretreated with capsules were seeded in the 48-well plates to form cell monolayer. Then, a sterile pipette tip was used to scratch the bottom of each well containing hMSCs. As control, cells without capsules were used. After that, the cells were observed with the Cell-IQ imaging system at 37˚C and 5% CO2. The Cell-IQ analyzer software was used to follow the cells throughout the image series providing graphical results of their trajectories. In addition to single cell trajectories, cell speed and migration rate were also determined. The detailed protocols and obtained data are presented in the Supporting Information. 2.8. Directed migration of hMSCs 2.8.1. Inverted invasion assay (transwell invasion assay) To study invasion of hMSCs towards a gradient of SDF-1, commercially available Transwell permeable inserts (Corning) were used. Capsules labeled with TRITC-BSA were added to the hMSCs at cell-to-capsule ratio of 1:10 in 24-well plate. After that, SDF-1 was placed to the lower part of the membrane. Cells with associated capsules in growth medium were added to the upper membrane part. As controls, (i) SDF-1 was added to lower membrane part together with hMSCs without capsule pretreatment and (ii) SDF-1 was not added to the lower part of the membrane. Cells were then incubated overnight at 37˚C and 5% CO2. After incubation, migrated hMSCs at ACS Paragon Plus Environment


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lower part of the transwell were stained with 0.2 µM of calcein AM. Cells were observed under CLSM (Carl Zeiss LSM 710) and hMSCs were counted manually. The full dataset is presented in the Supporting Information. 2.8.2 2D chemotaxis assay To study directed migration of hMSCs, commercially available μ-Slide Chemotaxis chambers (Ibidi) were used. For this, cells were treated with capsules loaded with TRITC-BSA/VCR at cellto-capsules ratio of 1:10, seeded into 24-well plates and incubated overnight. After washing with PBS, directed migration assay was performed following manufacturer instructions. Detailed data are described in the Supporting Information. 2.9.

Experiments with tumor spheroids

2.9.1. Formation of spheroids Spheroids were formed via the “hanging drop” technique49. Briefly, 15 μL of culture medium containing 2700 tumor cells together with 300 hMSCs were dropped onto the inside cover of a 35 mm petri dish, which was filled with 1 mL PBS to maintain humidity. The cover of the dish was placed upside down onto the petri dish, and incubated at 37 °C and 5% CO2. The cell culture medium inside the drop was changed each 3 day. After 6 days, formed tumor spheroids were gently washed twice with DMEM and PBS for application in further experiments. 2.9.2. Migration and invasion of hMSCs loaded with AF647 into spheroid 3D To study migration and invasion of hMSCs associated with capsules labeled by AF647, hMSCs were seeded into 25 cm2 culture flask and incubated overnight with capsules as described previously. After that, cells were washed 3 times with PBS, stained with PKH26 and detached with trypsin-EDTA solution. After counting with hemocytometer, 1000 hMSCs and 1 tumor spheroid (stained with Calcein AM) were mixed together with 1 mg/mL of collagen solution. After that, collagen-cell mixture was added to the previously coated with thin layer of agarose (1%) 35 mm petri dish and incubated for 2 hours at 37 ˚C, 5% CO2. Migration and invasion of cells was then observed with CLSM at different time points (0, 24, 48 h). The total number of hMSCs ACS Paragon Plus Environment

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associated with tumor spheroids was evaluated using Z-stack option. Detailed data and are described in the Supporting Information. 2.9.3. Toxicity of capsules loaded with VCR on spheroid The efficiency of capsules loaded with VCR delivered by hMSCs was evaluated on 3D tumor spheroids model. For this, hMSCs were seeded into 25 cm2 culture flask and incubated with capsules loaded with VCR for 24 h. After that, cells were washed by PBS and detached with trypsin-EDTA solution. After counting with hemocytometer, different amounts of hMSCs with capsules, corresponding to 1, 4, 8 ng/mL of VCR were added to previously formed spheroids (150250 µm) of each tumor cells (melanoma cells_P1, melanoma cells_P4, carcinoma cells_P11). Cell culture medium from drop containing spheroid was replaced with new cell growth medium containing different amounts of hMSCs. After 24 h, cell viability of tumor cells was evaluated with LIVE/DEAD assay as described before. For this, spheroids were stained with calcein AM (live dye) and propidium iodide (dead dye). Note that hMSCs are resistant against several antitumor drugs,50 therefore, it is assumed that VCR was toxic mostly to tumor cells. After staining, spheroids were immediately observed under CLSM using Z-stack option. Images were then analyzed with FIJI open source image analysis software. Finally, percentage of living cells were plotted versus added amount of VCR. 2.10. Statistics Statistical analyses were performed using Prism5 software (Graph Pad, La Jolla, CA, USA). All values were plotted as averages, including standard deviations of the means. The Student’s t-test and ANOVA were used to determine the significant differences between multiple sets of experimental data. P values of * < 0.05 and ** < 0.001 were considered statistically significant.

3. Results and discussion We investigated the potential of silica-coated capsules to transfer antitumor drugs with human mesenchymal stem cells (hMSCs) for combined cell therapy by introducing the encapsulated antitumor drug into intracellular compartments. Moreover, the influence of capsules on migration ACS Paragon Plus Environment


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behavior of hMSCs was systematically studied in order to reveal possible side effects of drug carriers on hMSCs functional properties and to optimize the protocols for construction of celldelivery platform based on hMSCs and associated capsules. To achieve this goal, the present study was divided into several steps, including complex study of uptake efficiency of capsules, their influence on spontaneous and directed migration of hMSCs, ability of hMSCs with capsules to penetrate into 3D tumor spheroids derived from solid tumor biopsy samples. The road map of this work can be found in the Figure 1.

Figure 1. Road map of work. A. Human mesenchymal stem cells (hMSCs) derived from bone marrow of patients, B. Synthesis of silica coated capsules of different sizes with further drug loading (Capsules were added to the cell suspension at different cell-to-capsule ratios), C. Influence of capsule sizes on their internalization into hMSCs, D. Influence of capsules on spontaneous and directed migration of hMSCs, E. Migration and invasion of hMSCs associated with capsules into tumor spheroids obtained from biopsy samples of patients. 3.1. Preparation of silica-coated capsules with different sizes and their characterization


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Compared to other conventional dosage forms, such as polymeric nanoparticles, capsules offer much higher drug loading capacity, which is crucial for antitumor therapy. The high drug loading amount is beneficial for increasing the drug concentration in tumor region. The large sized capsules should possess higher loading capacity.51 However, the size of capsules play important role in their internalization with the cells. Therefore, it is of special interest to find a balance between size of the capsules and amount of encapsulated drug. To address this issue, silica-coated capsules with different sizes were synthesized in order to examine the percentage of internalized and associated capsules with cells. The silica coating was applied to reduce capsule permeability allowing to encapsulate low-molecular weight drug and prevent its leakage from capsules before their administration.33 The template core-particles based on calcium carbonate (CaCO3) determine the final size of capsules. Size of CaCO3 particles can be varied by changing molarity and mixing ratio of initial salts solutions (CaCl2 and Na2CO3) used for the co-precipitation reaction.52 CaCO3 coreparticles of different sizes were synthesized using different conditions of co-precipitation reaction (see Supporting Information). After the combination of LbL technique with sol-gel method and subsequent dissolution of CaCO3 core-particles, silica coated composite capsules could be obtained (Figure 2). The SEM micrographs of capsules (Figure 2A1-A3) demonstrate that after sol-gel synthesis, these capsules fully covered by a dense layer of silica, making them more robust and stiff unlike simple polymer capsules.53 Additionally, capsules were characterized with TEM and AFM analysis (see Supporting Information). According to the TEM images, it can be seen that capsules are not transparent additionally proving successful deposition of silica shell as previously reported.40 AFM analysis revealed that smaller capsules are stiffer than bigger ones. TRITC-labeled capsules with different sizes were also imaged with confocal laser scanning microscopy (CLSM). Obtained images demonstrate non-aggregated samples what is important for future cells studies (Figure 2B1-B3). Based on SEM images, capsules with following average



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sizes were detected: 0.65 ± 0.21 m (Caps_0.6), 2.12 ± 0.45 m (Caps_2), 5.45 ± 1.03 m (Caps_5) (Figure 2C1-C3).

Figure 2. Representative (A) SEM and (B) CLSM images of silica-coated capsules with different sizes. (C) Size distribution of capsules. A1, B1, C1 – Caps_0.6 (0.65 ± 0.21 µm); A2, B2, C2 – Caps_2 (2.12 ± 0.45 µm); A3, B3, C3 – Caps_5 (5.45 ± 1.03 m). Scale bars: A1-A3, 2 µm, B1B3, 10 µm (insets 5µm). 3.2 Internalization versus capsule association The effect of capsule size on cell association and internalization was investigated by incubating hMSCs with TRITC-labeled capsules of different sizes at several cell-to-capsule ratios: ACS Paragon Plus Environment

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1:1, 1:3, 1:5, and 1:10. The cellular association was monitored using flow cytometry. For the cell association, the amount of fluorescence from each cells originating from TRITC-labeled capsules was quantified. The cellular association depends on the cell-to-capsule ratio while the size of capsules does not significantly influence on cellular association (Figure S6). The cell-to-capsule ratio of 1:10 mediated the highest association efficiency for all types of capsules, which is in a good agreement with previously published works.40 The average fraction of cells associated with capsules is 20.3 – 22.9 % (cell-to-capsules ratio = 1:1), 43.8 – 49.1 % (cell-to-capsules ratio = 1:3), 65.8 – 69.0 % (cell-to-capsules ratio = 1:5) and 89.6 – 94.2 % (cell-to-capsules ratio 1:10). Internalization of capsules with hMSCs was examined using Z-stacking in CLSM. For this, cytoskeleton of cells was fluorescently stained with phalloidin conjugated AlexaFluor488 (AF488) and cell nuclei with DAPI. The co-localization of TRITC signal originating from labeled capsules with AF488 fluorescence signal from cytoskeleton confirmed the intracellular location of capsules inside hMSCs. According to the Z-stack analysis, the capsule internalization rate from CLSM shows different trend than flow cytometry analysis and reveal that the percentage of internalized capsules is strongly related to their size (Figure 3). The highest internalization rate is observed for capsules with the smallest size diameter (Caps_0.6). At cell-to-capsule ratio of 1:10, the internalization percentage of Caps_0.6 is 53 % whereas for Caps_2 and Caps_5 the internalization rate is lower (28% and 17%). Among physicochemical characteristics of capsules, the main contribution in cell interaction is the size of capsules.52 These observations can be explained by several assumptions. First, a higher amount of capsules with smaller size can adhere to the cell plasma membrane what leads to higher probability of capsules internalization compared to bigger capsules, i.e. small capsules increase the cell membrane area exposed to the cell-capsule contact.34 Second, it can be associated with the multiple internalization pathways that are involved in the capsule uptake. Based on previous data for particle internalization, we assume that phagocytic internalization pathway is more dominant in case of bigger capsules. Micropinocytosis is more specific for primary human epithelial cells,54



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such as hMSCs, and is engaged in internalization process of smaller capsules. As the result, we observe higher internalization rate for smaller capsules. Third, non-targeted capsules are adsorbed to the cell plasma membrane through electrostatic interactions and gravity. Most likely, smaller capsules can interact with hMSCs in suspension for longer periods via electrostatic interactions than bigger capsules, which faster settle down to the dish bottom.

Figure 3. Internalization of capsules of different sizes into hMSCs: A1, B1, C1) Three-dimensional

reconstructions of consecutive focal plane CLSM images of hMSCs incubated for 24 h with capsules of different sizes. Capsules are labeled with TRITC. A2, B2, C2) Average percentage of capsules associated/internalized with hMSCs. A1, A2 – Caps_0.6 (0.65 ± 0.21 µm); B1, B2 – Caps_2 (2.12 ± 0.45 µm); C1, C2 – Caps_5 (5.45 ± 1.03 µm). Scale bars: A1, B1, C1, 20 µm.


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(Data are presented as mean ± standard deviation, * represents p < 0.05 and ** represents p < 0.001). To date, several strategies have been developed for the construction of cell–drug carrier systems, such as the non-covalent and covalent coupling of nano- and micro carriers with cell membrane, the internalization of drug carriers into the cells and anchoring drug carriers on the surface via antibody–antigen interactions. From all available approaches for carrier association with cells, the internalization pathway is seemed to be the simplest way to introduce drug carriers into the cell and can be considered as the most optimal in our case, because this strategy does not require any cell preconditioning or chemical modification of cells, ensuring minimal cell damage and leads to implementation in pre-clinical practice. 3.3. Drug loading capacity, cumulative release and cytotoxicity of capsules on hMSCs Vincristine (VCR) is a widely used antitumor drug in clinics. VCR was loaded into the silica coated capsules of different sizes (Figure S8-S9). Doxorubicin is a common model drug used in research for drug delivery, however, usual dose of doxorubicin ranges from 60-75 mg/m2, while for VCR ranges 1.5-2 mg/m2.55 Therefore, less amount of VCR is needed to be encapsulated for effective tumor treatment compared with doxorubicin, which served as a key point in the choice of this drug. As shown in Figure S9, Caps_5 demonstrated the highest drug loading capacity (~ 173 µg/mL) compared to Caps_0.6 (~ 99 µg/mL) and Caps_2 (~ 145 µg/mL). The VCR showed a pH-sensitive and sustained release from capsules up to 3 days, with more completed release at acidic conditions (Figure S10). The cytotoxicity of capsules containing VCR and influence of VCR release on cell viability were evaluated using the standard AlamarBlue assay. After examining cytotoxicity of capsules containing VCR at different cell-to-capsule ratios, we found negligible adverse effect on hMSCs viability (Figure S11). The results showed that neither VCR nor capsules have relatively mild adverse effect on cell viability. As reported in literature,34 toxic effects of capsules are observed at



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only high cell-to-capsule ratio (> 1:50). Also, according to the works,36,56 hMSCs have drugresistance ability against several cytostatic drugs. The main explanation of their drug-resistance can be associated with ATP-binding cassette (ABC) transporters. Basically, as reported in the literature56 ABC transporters are expressed in normal stem cells to maintain a relatively stable intracellular environment and to keep them in a quiescent state. In addition, these transporters have certain other notable roles in normal physiology in the transport of drugs across the placenta and the intestine. By using the energy from ATP hydrolysis, these transporters actively efflux drugs from cells, serving to protect them from cytotoxic agents and simultaneously may provide the release of drug from cell into surroundings. 3.4. Study of spontaneous migration of hMSCs with capsules Apart from the cytotoxicity of capsules with antitumor drug, migration ability of hMSCs associated with capsules is important for developing combined cell-based therapy. Cell-based therapy propose to exploit site-directed migration properties to transfer therapeutic cargo (in our case, antitumor drug/VCR) to sites of tumor growth.57,58 Prior the directed migration, the influence of capsules on the ability of cells to move needs to be investigated in order to filter out drug carriers, which may possess side effects. In other words, spontaneous migration of hMSCs with associated capsules of different sizes is required to be evaluated. The spontaneous migration of hMSCs with and without capsules was recorded via time-lapse microscopy (Figure 4A, S12-S14). By tracking the hMSC migration paths, the cell speed could be calculated for each cell with capsules and compared to that of control cells without capsules. No statistical difference for all types of capsules at the cell-to-capsule ratio of 1:1 was detected (Figure 4B). However, with the increase of cell-to-capsule ratio, the speed rate of hMSCs decreased for Caps_2 (almost 40% for cell-to-capsules=1:10) and Caps_5 (almost 46% for cellto-capsules=1:10), whereas for the Caps_0.6 only slight reduction of hMSCs speed was observed (9% for cell-to-capsule=1:10). This suggests that capsules of bigger size had more significant impact on the spontaneous migration of hMSCs. ACS Paragon Plus Environment

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Figure 4. Spontaneous migration activity of hMSCs with capsules: A) Time-lapse microscopy images of hMSCs pretreated with TRITC-labeled capsules of different sizes during 24 h of incubation. The trajectory over time of the hMSCs is indicated with the colored lines in the last panel (Track). Scale bars are 100 µm. B) Cell migration speed of hMSCs pretreated with capsules at different amounts shown as a box. (Data are presented as mean ± standard deviation, N ≥ 45



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single cells were analyzed per condition, ns – non-significance, * represents p < 0.05 and ** represents p < 0.001).

Additionally, scratch wound assay was performed to estimate the influence of capsules on the proliferation rate of hMSCs. It should be noted that this method mimics to some extend cell migration in vivo.59 The number of hMSCs associated with capsules migrated into the scratch areas was compared to control group (hMSCs without capsules). The number of cells migrating within monolayer defect was estimated at several time points (6, 12, 24 hours) for different types of capsules in increasing cell-to-capsule ratios (1:1, 1:3, 1:5 and 1:10).


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Figure 5. Scratch wound assay: A) Scratch assay images of hMSCs cultivated with capsules of different sizes at cell-to-capsule ratio of 1:10 during 24 hours (scale bar represents 50 μm). B) Cell migration quantification of pictures was assessed by counting the number of cells in the central gap. Cell migration was represented as number of cells filling the central gap. (Data are presented as mean ± standard deviation, * represents p < 0.05 and ** represents p < 0.001). The scratch wound assay data clearly demonstrate that cell migration activity depends on capsule size and cell-to-capsule ratio, what is in agreement with previous data (spontaneous migration). Significant decrease in wound healing can be seen for Caps_2 and Caps_5 at cell-to-



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capsule ratios of 1:3, 1:5 and 1:10, while Caps_0.6 demonstrated the minimal influence on the speed of hMSCs. With the increase capsules size and cell-to-capsule ratio, hMSCs speed was reduced and the number of cells migrated into the scratch area was lower compared to the control group (Figure 5). Even at cell-to-capsules ratio of 1:10, the Caps_0.6 did not significantly affect hMSCs speed. To summarize all the obtained results on capsules internalization, cytotoxicity and spontaneous migration assay, we can conclude that Caps_0.6 are considered to be the most optimal drug carriers for association with hMSCs compared to bigger capsules due to their low effect on cells activity. Therefore, they were chosen for further experiments. 3.5. Study of directed migration of hMSCs with capsules forward to chemokine gradient (SDF-1) To evaluate the influence of Caps_0.6 with and without antitumor drug (VCR) on the capacity of cell motility and invasiveness toward a chemoattractant gradient, in vitro transwell assay has been applied. The transwell migration assay may be used to analyze the ability of single cells to directionally respond to various chemoattractants whether they are chemokines or growth factors. As a rule, hMSCs are placed in the upper compartment and allowed to migrate through the pores of the membrane into the lower compartment, where chemotactic agent is presented (Figure 6A). CXCL12 (stromal cell-derived factor-1 (SDF-1)), a member of the CXC family of chemokines, is thought to have crucial role in migration of hMSCs at the sites of inflammation or tumor growth.60,61 The CXCR4 receptors, which are expressed on surface of hMSCs (Figure S15), are for CXCL12 (SDF-1), and therefore, CXCL12 (SDF-1)/CXCR4 signaling can be considering as a master regulator for stem cell migration.60 The migration of hMSCs without pretreatment and cells treated with VCR-loaded capsules towards a SDF-1 gradient, including negative control hMSCs, was measured. Figure 6B shows the images of hMSCs that migrate through the membrane pores (8.0 m) towards SDF-1 gradient. The statistical results (Figure 6C) show that hMSCs pretreated with capsules had similar invasiveness through the pores of transwell membrane toward SDF-1


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compared to hMSCs without capsules. At the same time, the VCR loaded into capsules did not influence the invasive ability of hMSCs.

Figure 6. The migration of hMSCs towards stroma-derived factor-1 (SDF-1) was observed using a Transwell assay: A) Illustration of the transwell migration assay. B) CLSM images of hMSCs migrated though the transwell membrane to the lower side. Scale bars correspond 100 µm. C) Quantitative analysis of the migration assay. (Data are presented as mean ± standard deviation, * represents p < 0.05 and ** represents p < 0.001). Since hMSCs in the transwell assay showed a directed migration towards to SDF-1, we investigated a chemotactic effect of capsules with and without VCR on hMSCs in more details using specific -slide chemotaxis chambers. One of the side channels in the chemotaxis chamber was filled with SDF-1 (100 ng/mL), and the other side was filled with blank medium without SDF-1. The middle channel was filled with cell medium containing hMSCs (Figure S16). The cell movement was recorded for 24 hours (Figure 7). Cell tracks were analyzed to determine the percentage of migrating cells and the migration directionality (chemotactic index). As it can be seen from Figure 7B, 68 -74 % of hMSCs with and without capsules have migrated. The results of chemotaxis assay clearly demonstrated that capsules as well as VCR did not reduced cell directionality (the values of chemotactic index are almost the same for hMSCs, Caps_0.6/hMSCs



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and VCR@Caps_0.6/hMSCs), indicating that both gradient sensing and motility functions of cells are not impaired.

Figure 7. The migration of hMSCs towards SDF-1 was observed using a -slide chemotaxis assay: A) Images of 1 h and 24 h from the experiment (Scale bars correspond 100 µm.). B) Migration tracks of hMSCs without capsules (left), hMSCs with capsules (middle), hMSCs with vincristine loaded capsules (right) for 24 h experiment. The tracks were normalized to a common origin (0.0) and plotted to visualize pattern of movement in two dimensions (the chemokine gradient is in the


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y dimension as indicated). Red tracks represent cells migrating toward the gradient and black tracks are for the cells migrating against the gradient. C) Calculated forward migration index of slide chemotaxis assay for hMSCs without capsules (left), hMSCs with capsules (middle), hMSCs with vincristine loaded capsules (right). (N ≥ 42 single cells were analyzed per condition; Data are presented as mean ± standard deviation, * represents p < 0.05 and ** represents p < 0.001).

According to the literature,62 the free antitumor drugs have influence on migration capacity of stem cells in vitro while in our work we showed that silica coated capsules (Caps_0.6 added at amount 1:10) loaded with reference drug did not significantly affect migration potential of hMSCs. This can be associated with composite shell of capsules that decreases leakage of VCR and, thus, postpone the released of drug before their association with hMSCs, most probably, excluding the influence of drug on the tropism of hMSCs toward tumor cells. In comparison to previous work,36 submicrometric sized capsules (Caps_0.6) possess perspectives for their further introduction to hMSCs. 3.6. hMSCs/capsules migration toward and penetration into 3D tumor spheroids from obtained from solid tumor biopsy samples Tumor spheroids as 3D culture models are powerful therapy test platforms with a great potential to predict clinical efficacies of investigated drug delivery systems with the possibility to estimate migration and invasion processes of hMSCs associated with capsules (Caps_0.6).63 Moreover, 3D cellular spheroids can be considered as an adequate model, exhibiting a complex microenvironment similar to tumorous in vivo and comparable drug responses.64 As previously reported, tumor cells in 3D spheroids are placed in more physiological conditions than in 2D models. Also, cell-cell interactions in 3D spheroids are similar to those in natural microenvironment.65,66



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It is well known that different types of malignant tumors have different biological features due to the set of various genetic and epigenetic aberrations. Intraspecific tumor variations are playing crucial role in tumor behavior (growth rate, invasion, drug and immune resistance). Not only cells from the various histological types of malignancy have different properties but also tumor cells from the same histological type may differ from each other with the cytokine incl. chemokine expression profile. Aforementioned properties of cells are not typical for immortalized tumor cell lines, therefore, their use do not always provide sufficient result for in vitro studies. Since the CXCL12 (SDF-1)/CXCR4 pathway is mainly responsible for homing effect of hMSCs, we analyzed the expression of CXCL12 mRNA with qPCR in primary tumor cell cultures. For this, biopsy samples derived from 11 solid tumor patients with different form of cancer: P1: melanoma, P2: bladder cancer, P3: bladder cancer, P4: melanoma, P5: melanoma, P6: bladder cancer, P7: bladder cancer, P8: breast cancer, P9: colorectal cancer, P10: renal cancer, P11: renal cancer (Figure S18). From the obtained qPCR analysis, three types of primary cancer cells were used to build-up 3D tumor spheroids. P1: melanoma (melanoma cells_P1) and P11: renal cancer (carcinoma cells_P11), since these cells express the highest level of CXCL12 mRNA. As the control cells P4: melanoma (melanoma cells _P4), because they did not show any expression of CXCL12. The characterization of used tumor cells incl. cancer/testis gene analysis, immunocytochemistry is described in Supporting Information (Figure S19, Table S5-S6). The tumor spheroids with the size range from 150 (carcinoma cells_P11) to 500 µm (melanoma cells _P1, melanoma cells _P4) were formed via hanging drop technique. To evaluate the ability of hMSCs to directly penetrate into 3D tumor spheroids, fluorescently stained tumor spheroids (calcein AM) were co-cultured with hMSCs, which were associated with capsules loaded with AF647 in 3D collagen gel. To detect the hMSCs invasion, CLSM analysis was applied. The tumor spheroids were scanned in several Z-planes during several time periods (Figure 8A, S20-21). Obtained results reveal that invasion of hMSCs associated with Caps_0.6 into melanoma cells_P1 and carcinoma cells_P11 spheroids increased over the time (Figure 8B). In contrast, hMSCs did


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not migrate as far toward the melanoma cells_P4 spheroid. It is worth mentioning that directed migration rate of hMSCs is correlated with CXCL12 (SDF-1) expression level in tumor cells. However, the migration rate of hMSCs into melanoma cells_P1 spheroid is higher than into carcinoma cells_P11, even though the level of CXCL12 expression is the same for both tumor cell types. This observation can be associated with the possible different expression level of other chemokines that were not studied in this work. Despite the CXCR4/SDF-1 axis is considered to be the main pathway for directed migration of hMSCs, other mechanisms should be also taken into account.67,68 Thus, before using hMSCs-based therapy, the individual biopsy material of each cancer patient should be analyzed, what is in favor for development of personalized medicine, which focusses on the tailored treatment to the individual patients based on their predicted response.



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Figure 8. Caps_0.6/hMSCs penetration into solid tumor spheroids (carcinoma cells_P11): A) Zstack CLSM images confirming the hMSCs invasion into 3D tumor spheroid for 48 h (Scale bars correspond 50 µm). B) Quantification of caps_0.6/hMSCs penetration into three types of tumor spheroids (melanoma cells_P1; melanoma cells_P4; carcinoma cells_P11). (Data are presented as mean ± standard deviation, * represents p < 0.05 and ** represents p < 0.001, N.A., not available).


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3.7. Comparison of therapeutic efficiency of hMSCs associated with VCR loaded capsules versus individual VCR on 3D tumor spheroid model. We further studied the antitumor effect of our hMSCs-based delivery system on 3D tumor spheroids. For this, VCR loaded capsules associated with hMSCs (VCR@Caps_0.6/hMSCs) were incubated with spheroids at several doses resulting in three different final concentrations of VCR in cell medium. In parallel, individual VCR was used as positive control. The viability of tumor cells was measured with LIVE/DEAD assay after 48 h of incubation. Figure 9 shows the influence of VCR@Caps_0.6/hMSCs on viability of tumor cells. The revealed data demonstrate the dose dependent response of spheroids to the developed delivery system. In case of melanoma cells_P1 and carcinoma cells_P11 spheroids with increasing VCR concentration, the cell viability of the spheroids decreases and at the highest concentration of VCR (8 ng/mL) the tumor cell viability is below 10% for carcinoma cells_P11 and melanoma cells_P1. It should be mentioned that VCR delivered with hMSCs showed more pronounced cytotoxic effect of tumor spheroids than free antitumor drug VCR (Figure 9B). Better therapeutic effect of VCR@Caps_0.6/hMSCs might be associated with different penetration efficiency of individual drug and hMSCs. Due to tight cellcell contact in the tumor spheroid, the free antitumor drug is less diffused into spheroid than hMSCs containing VCR, as it has been showed (Figure 8). However, hMSCs-based delivery systems demonstrated negligible toxic effects on melanoma cells_P4. This can be associated with two factors. First, according to the Figure S18 melanoma cells_P4 did not express CXCL12 (SDF1) and, therefore, formed tumor spheroid did not attract hMSCs via CXCL12 (SDF-1)/CXCR4 pathway. Another reason can be related with resistance of this tumor cell type to VCR. Indeed, the cell viability experiments performed with 2D tumor cells culture confirmed the drug resistance of melanoma cells_P4 (Figure S22).



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Figure 9. The comparison of antitumor efficiency of individual drug (VCR) and hMSCs with capsules examined on tumor spheroids (melanoma cells_P1; melanoma cells_P4; carcinoma cells_P11): A) The representative images using live/dead cells staining taken from confocal microscope of individual spheroids up to two days. Spheroids were treated with different concentration of VCR and hMSCs with capsules (VCR@Caps_0.6/hMSCs). Live cells were stained with Calcein AM; Dead cells were stained with Propidium iodine (melanoma cells_P1; melanoma cells_P4 - scale bars: 100 µm; carcinoma cells_P11 – scale bars: 50 µm);


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B) Quantification of the viability of the cells in the spheroids. (Data are presented as mean ± standard deviation, * represents p < 0.05 and ** represents p < 0.005).

In this part we demonstrate the high potential of hMSCs-based delivery system using submicrometric sized capsules with concomitantly effective antitumor drug efficiency. The silica coated capsules provide a unique platform for high in situ loading capacity of antitumor drugs and combine low toxicity with high efficiency, providing high degrees of flexibility. Due to versatility of capsules modification with various nanostructures (e.g. silica, plasmonic nanoparticles, magnetic nanoparticles, graphene oxide nanoparticles) allow for remote controlled activation of encapsulated drugs using different external stimuli. For instance, capsules coated with silica are sensitive to the ultrasound, while gold- or silver-modified capsules are responsive to laser irradiation. For simultaneous drug activation and visualization capsules can be functionalized with magnetic nanoparticles and placed in the constant magnetic field for MRI measurements69 and in the alternating magnetic field for hyperthermia treatment.70

4. Conclusion In this work, the potential of capsules to carry antitumor drugs with hMSCs for combined cell therapy was investigated for the first time from both fundamental point of view and further application in cancer targeting treatment. It has been shown that differently sized capsules affect cellular internalization and hMSCs migration speed. The capsules demonstrate non-toxicity and enhanced association/uptake efficiency at cell-to-capsule ratio of 1:10. It has been revealed that submicrometric capsules show minimal side effects on the functional properties (spontaneous and directed migration) of hMSCs. Moreover, silica-coated capsules are able to carry low molecular weight drugs, what allows prolonged release of antitumor drugs (e.g. vincristine). According to the revealed data, the 0.6 micrometer capsules are more favorable for stem cell therapy compared to 2 and 5 micrometer capsules. The studies on the tumor spheroid model, which mimic the physiological conditions, demonstrated that hMSCs associated with capsules loaded with ACS Paragon Plus Environment


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antitumor drug superior advantages in increased antitumor efficiency compared with free drug. These experiments provide proof-of-concept on the applicability of developed delivery vehicle on such therapeutically relevant systems and will allow further implementation in targeted drug delivery.

Acknowledgments We thank N.N. Petrov National Medical Research Center of Oncology, especially Baldueva I. A., Danilova A.B. and Nehaeva T.L. for providing assess to Cell-IQ imaging system and gifted primary tumor cells derived from the patient biopsy samples.

Supporting information The detailed description of capsule synthesis, characterization, their association/uptake with hMSCs, toxicity studies, primary tumor cells characterization, spontaneous and directed migration of hMSCs with capsules and their invasion into tumor spheroids are available in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

Author contributions A.S.T. initiated and performed this work and manuscript. O.O.P performed capsule synthesis, studied directed migration of hMSCs, spheroid formation and studied hMSCs invasion. M.V.Z. performed capsules synthesis, capsule characterization, transwell assay studies, flow cytometry analysis, manuscript writing. A.R.M. performed TEM imaging, spontaneous migration, scratch wound assay, formation of spheroids, manuscript writing. T.E.K. prepared tumor and stem cells for biological experiments. O.S.E. conducted flow cytometry experiments. A.I.S. performed qPCR experiments. M.V.Z. performed SEM and AFM analysis. Y.V.T. evaluated uptake of capsules with cells using CLSM. K.V.L. provided materials for biological experiments, co-wrote the manuscript. V.S.S. supported with discussion concerning migration of hMSCs. G.B.S. and B.V.A. analyzed the results and supported with ideas. All authors contributed to discussion and reviewed the manuscript.


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Funding This work was supported by the grant of the Russian Foundation for Basic Research, No. 18-01500100 (A.S.T.). The preparation of capsules and their characterization was partly supported by the Russian Science Foundation, No. 17-73-10023 (A.S.T.). The research is funded from Tomsk Polytechnic University Competitiveness Enhancement Program. M.V.Z. thanks the President’s Scholarship SP-1576.2018.4.

Notes The authors declare no competing financial interests. References (1)

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