Intracellular delivery of antioxidant CeO2 nanoparticles via

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Intracellular delivery of antioxidant CeO2 nanoparticles via polyelectrolyte microcapsules Anton Popov, Nelli Popova, Nadezda V. Tarakina, Olga S. Ivanova, Artem M. Ermakov, Vladimir K. Ivanov, and Gleb B. Sukhorukov ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00489 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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ACS Biomaterials Science & Engineering

Intracellular delivery of antioxidant CeO2 nanoparticles via polyelectrolyte microcapsules Anton L. Popov,a Nelli R. Popova,a Nadezda V. Tarakina,b Olga S. Ivanova,c Artem M. Ermakov,a Vladimir K. Ivanov,c,d* Gleb B. Sukhorukova,b a

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Moscow

region, Pushchino, Russia b

Queen Mary University of London, School of Engineering and Materials Science, London, UK

c

Kurnakov Institute of General and Inorganic Chemistry, Moscow, Russia

d

National Research Tomsk State University, Tomsk, Russia

* [email protected]

Keywords: cerium oxide nanoparticles, polyelectrolyte microcapsules, cell culture, antioxidant activity, oxidative stress.

Abstract Cerium oxide nanoparticles (nanoceria) are regarded as one of the most promising inorganic antioxidants for biomedical applications. Considering nanoceria as a potential therapeutic agent, we aimed to develop a robust system of its intracellular delivery using LbL polyelectrolyte microcapsules. We have shown that citrate-stabilized cerium oxide nanoparticles can be effectively incorporated into the structure of polyelectrolyte microcapsules made from biodegradable and non-biodegradable polymers. The structure and morphology of synthesized microcapsules were investigated and analyzed using CLSM, SEM, TEM, EDX and UV/VISspectroscopy. Results of experiments in vitro on B-50 neuroblastoma cells confirmed nanoceria delivery into the cell, while maintaining their antioxidant properties. The results presented confirm polyelectrolyte microcapsules to be an efficient intracellular delivery system for therapeutic nanoparticles.

1 Introduction Nanocrystalline cerium oxide (CeO2) nanoparticles are widely used in various industrial applications, as catalysts, UV filters, oxygen sensors and fuel additives.1-5 More recently, nanoceria have been regarded as one of the most promising materials for biomedical applications.6,7 Nanoceria exhibit antioxidant, radioprotective, anti-inflammatory and other therapeutic properties.8-11 Therapeutic activity of nanoceria is related to its unusual physical and chemical properties.12 First of all, the high level of oxygen non-stoichiometry provides its 1

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remarkable redox activity in inactivating a wide spectrum of free radicals and reactive oxygen species.13 Additionally, СеО2 nanoparticles are able to regenerate their redox activity and act as biocatalysts for an unlimited number of times.14 Furthermore, the toxicity of nanoceria is rather low; they have a high level of biocompatibility. These properties make cerium oxide a very promising material for safety usage in biomedical applications. The cellular uptake of nanoceria depends on their physicochemical characteristics, (i.e. shape, size, charge), which determine the specific type of endocytosis.15 Their ultra-small size and high reactivity of surface limit their effective targeted delivery. Therefore, the development of an effective nanoceria delivery system is an important problem. One of the most common delivery systems of different substances into cells is polyelectrolyte microcapsules able to encapsulate biological macromolecules, proteins, DNA, RNA, pharmaceuticals, nanoparticles and others.16–22 Several types of nanoparticles have been successfully introduced into LbL polyelectrolyte microcapsules (TiO2,16 SiO2,21 Fe3O4,23 etc.). Nanoparticles’ integration into capsules is associated with a specific charge of its surface which enables interaction with oppositely charged polyelectrolyte molecules in the microcapsule structure. For example, it is possible to integrate iron nanoparticles into microcapsules, which allows for controlled movement of the microcapsules under the influence of the magnetic field.23 The use of gold nanoparticles in the structure of microcapsules has allowed controlled destruction of the shells of microcapsules through the focused action of a laser beam and the release of the encapsulated protein.24 The possibility of the incapsulation of therapeutic nanoparticles, such as cerium oxide, has not been demonstrated yet. The most important aspect of a nanoparticles delivery system is control of the final concentration (dose) in predetermined organs (or cells), to preserve native biological activity, and the LbL technique provides this. The main advantage of the polyelectrolyte microcapsule as an intracellular delivery system is its undoubted multifunctionality and modularity. The principle of electrostatic-driven assembly enables the integration of various substances into the microcapsule, such as proteins, nanoparticles, etc. Such a modification, as well as the precise adjustment of the microcapsule’s shell thickness, gives a unique opportunity to change both the mechanical and chemical properties of the microcapsule in a targeted manner. Moreover, the encapsulation of biologically active substances in a polyelectrolyte microcapsule can easily be achieved under mild conditions, avoiding the use of organic solvents or mechanical stresses, which are often used in the preparation of traditional drug delivery systems such as liposomes.25 One major disadvantage of the polyelectrolyte microcapsule is its rather tedious and time-consuming synthesis. The further essential problem arising from the relatively large microcapsules’ size (sizes less than 1 µm are

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barely achievable) and high degree of aggregation is their limited use for drug delivery through the bloodstream. Meanwhile, other drug delivery systems, for example polymer-based nanocapsules,26 could be advantageous over micronized microcapsules, due to their small size and low degree of aggregation in biological liquid media. Another current synthetic delivery system is the cationic lipid nanocapsule, which has been considered as a novel and versatile family of carriers.27 Intracellular antibody delivery has also been achieved by the design of ‘smart’, self-organizing protein structures. Lim et al.28 synthesized an all-protein, self-assembled nanocarrier, by combining an α-helical peptide that self-assembled into a hexameric coiled-coil bundle and an Fc-binding Protein A fragment, which were capable of delivering functional antibodies to the cytosol. The self-assembly of peptides and proteins is also considered to be a good technique for the fabrication of an effective delivery system of phototherapeutic nanomaterials for antitumor photodynamic and photothermal therapy.29 Today, there are several different approaches to creating functional polymer matrices, scaffolds or delivery systems based on the LbL technique. In particular, Drachuk et al.30 have shown the possibility of using silk fibroin for encapsulating S. cerevisiae yeast cells without compromising their viability. The use of silk fibroin allowed for the formation of a highly porous shell on the cell’s surface, unlike polyelectrolyte-based materials. Such a porous shell provided almost 100% cell survival. Deng et al.31 encapsulated bacteria in chitosan/alginate biomicrocapsules for pyrene biodegradation under harsh environmental conditions. The results of the biodegradation experiments revealed that the 95% pyrene could be removed by the bacteria encapsulated in chitosan/alginate bio-microcapsules in 3 days, which was much more effective than for free bacteria (59%). Ochs et al.32 prepared capsules whose degradation rate depended on the intracellular concentration of glutathione. Microcapsules were synthesized by LbL deposition of the thiol-functionalized poly(methacrylic acid) and poly(vinylpyrrolidone) on silica particles, followed by cross-linking of thiol-functionalized poly(methacrylic acid) layers, followed by removing poly(vinylpyrrolidone) and the silica template. The disulfide cross-links provided a redox-active trigger for microcapsules’ degradation initiated by intracellular concentration of glutathione. The route of nanoparticles’ administration in vivo is a determinant of their pharmacological kinetics and distribution in organs and tissues.33 The ultra-small size of nanoparticles allows them to overcome the blood-brain barrier;34 due to their high specific surface area, nanoparticles absorb different proteins,35 peptides36 and ions,37 changing their circulation time in the bloodstream and their final localization. In turn, the interaction of nanoparticles with proteins and peptides determines the bioactivity of nanoparticles. This 3



interaction leads to the formation of a “protein corona” on their surface and can affect intracellular uptake processes, namely rates of accumulation, degradation and excretion.38 Protein adsorption on nanoparticle surfaces can cause conformational changes in peptide molecules and also affect the biological activity of nanoparticles. For example, as previously shown, the interaction of human carbonic anhydrase (one of the most stable proteins) with silica nanoparticles of various size and shape has resulted in significant conformational changes in the secondary structure of the protein.39 Also, the biological activity of nanoparticles depends on the ionic composition of the microenvironment. It has been shown that the catalytic activity of nanoceria is strongly dependent on the composition of the culture medium or the buffers.40 The use of a phosphate buffer for dispersing nanoceria leads to a significant reduction in SOD mimetic activity and an increase in catalase activity in a dose-dependent manner. On the contrary, the use of sulfate and carbonate solutions did not affect the enzyme-like activity of nanoceria. The presence of phosphate in the microenvironment of nanoceria alters its surface chemistry and catalytic activity.41 Therefore, the design and development of new, effective delivery systems for nanoceria is required, allowing it to preserve its unique bioactivity. The aim of the current work was to develop a targeted, controlled delivery system of antioxidant ceria nanoparticles in order to retain their native biological activity. In this work, we propose a method of encapsulating citrate-stabilized cerium oxide nanoparticles into polyelectrolyte microcapsules made from biodegradable or non-biodegradable polymers. The properties of CeO2 composite microcapsules were investigated and analyzed using CLSM, SEM, TEM, EDX and UV/VIS-spectroscopy. In addition, the biocompatibility of the CeO2 composite microcapsules was confirmed by different cytological assays in vitro, and their antioxidant activity was examined in a model of hydrogen peroxide induced oxidative stress.

2 Experimental section 2.1. Materials Poly(sodium 4-styrenesulfonate) (PSS, MW ≈ 70 kDa, #243051), poly(allylamine hydrochloride) (PAH, MW ≈ 56 kDa, #283223), calcium chloride (CaCl2, #223506), sodium carbonate (Na2CO3, #S7795), ethylenediaminetetraacetic acid disodium salt dihydrate (Na2EDTA 2H2O, #E5134), dextran sulfate sodium salt (DS, MW≈10 kDa, #D4911), poly-Larginine hydrochloride (PArg, MW≈70 kDa, #P3892) and hydrogen peroxide solution (30 wt.% H2O2 #216763) were purchased from Sigma-Aldrich. All chemicals were used as received. Ultrapure water with a resistance greater than 18.2 MΩ cm−1 was used for all experiments.

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2.2. Preparation of CeO2 nanoparticles Aqueous colloid solutions of citrate-stabilized cerium oxide nanoparticles were synthesized using a previously reported technique.42 According to transmission electron microscopy data, the sol obtained consisted of slightly aggregated CeO2 particles sized 2.0–2.5 nm, having a nearly spherical shape (Fig. S1 a). Zeta potential was found to be negative (–57 mV) (Fig. S1 b). The average CeO2 hydrodynamic radius was found to be 5.3 nm (Fig. S1 c).

2.3. Composite microcapsule preparation Calcium carbonate templates were fabricated as described previously.43 The process was initiated by rapid mixing of equal volumes of 0.33 M aqueous solutions of CaCl2 and Na2CO3 at room temperature. After intense agitation with a magnetic stirrer for 30 seconds, the precipitate was separated by sedimentation and rinsed with water three times. The sedimentation of CaCO3 microparticles was performed by centrifugation at 1000 rpm for 1 minute. This procedure formed an aqueous suspension containing spherical CaCO3 microparticles with an average diameter ranging from 2 to 3 µm. Calcium carbonate microparticles were used as a template for fabrication of the nanocomposite shells. The first polyelectrolyte layer was made by adsorption of the positively charged PArg from 1 mg mL_1 solution in 0.15 M NaCl (15 minutes of incubation and shaking) on CaCO3 microparticles dispersed in this solution. The second layer was prepared by absorption of the negatively charged DS from 1 mg ml-1 solution in 0.15 M NaCl (15 minutes of incubation and shaking). The core / polyelectrolyte particles were washed three times with deionized water after each adsorption step. A colloidal solution of CeO2 nanoparticles (10–5 M) was used to construct the 4th layer in the microcapsule. The calcium carbonate cores were dissolved in 0.2 M ethylenediamine tetraacetic acid (EDTA⋅2H2O) for 30 minutes; next, the microcapsules were centrifuged and rinsed three times with EDTA, and then three times using water. The synthesis procedure of non-biodegradable microcapsules was absolutely the same. Two types of the microcapsule shells were manufactured. The first one was composed of biodegradable polyelectrolytes (PArg and DS) with CeO2 nanoparticles in the middle layer (PArg/DS)(PArg/CeO2)(PArg/DS); the second type was composed of nonbiodegradable polyelectrolytes (PAH and PSS) with CeO2 nanoparticles in the middle layer (PAH/PSS)(PAH/CeO2)(PAH/PSS).

2.4. Zeta-potential measurements The surface potentials of CeO2-containing microcapsules were measured from aqueous suspensions on a Zetasizer Nano-ZS analyzer (Malvern). Each value of the zeta-potential was

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obtained under ambient conditions by averaging three independent measurements of 100 subruns each.

2.5. Scanning electron microscopy (SEM) studies The morphology of the microcapsules was studied by SEM (FEI Inspect-F), using an accelerating voltage of 20 kV. The diluted microcapsule suspension was dropped on a glass slide, air dried and coated with gold before observation. The capsule size was calculated and determined using an Image-Pro Plus Version 6.0 analysis software (Media Cybernetics Inc.). The microcapsule diameter and distribution were expressed as mean ± SD by randomly averaging the diameters of at least 35 capsules per sample from the SEM data.

2.6. Energy dispersive X-ray spectroscopy (EDX) Elemental analysis was performed by means of an EDX Oxford Inca X-act detector attached to the SEM, operating at an accelerating voltage of 20 kV.

2.7. Transmission electron microscopy (TEM) Further, the morphology and structure of microcapsules, as well as the distribution of CeO2 particles and shell thickness, were studied using a JEOL 2010 transmission electron microscope with a LaB6 filament, operated at 200 kV. For TEM experiments, the diluted microcapsule suspension was dropped on a copper grid with a holey carbon film and left to dry for 5 minutes.

2.8. UV-Visible spectroscopy A UV-visible spectrophotometer (LAMBDA950, Perkin-Elmer) was employed to investigate the UV absorption of the microcapsule suspension. Measurements were made using quartz cuvettes (Sigma, S10C).

2.9. Confocal laser scanning microscopy (CLSM) CLSM images were captured with a Leica TS confocal scanning system (Leica, Germany) equipped with a 63×/1.4 oil immersion objective.

2.10. Cell culture B50 rat neuronal cells were obtained from the European Collection of Animal Cell Cultures (ECACC, Porton Down, UK). These cells were an ethyl nitrosourea induced tumor cell line that had a neuronal morphology. 6


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2.11. MTT assay Determination of the activity of mitochondrial and cytoplasmic dehydrogenases of living cells was performed by an MTT test based on the reduction of colourless tetrazolium salt (3-[4,5dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide). Cells were seeded in 96-well plates and cultured in an atmosphere containing 5% CO2 at 37°C. Six hours after cell seeding, the medium was replaced with the medium containing the microcapsules in an amount of 1, 10 or 100 microcapsules per cell. Within 24, 48 and 72 hours of the addition of the microcapsules, viability was determined.

2.12. LDH assay Cells were seeded in 96-well plates and cultured in an atmosphere containing 5% CO2 at 37°C. Six hours after cell seeding, the medium was replaced with the medium containing the microcapsules in an amount of 1, 10 or 100 microcapsules per cell. As a positive control, cells were used without the addition of the microcapsules. Triton X-100 (10 µL) was used as a positive control. Within 24, 48 and 72 hours of the addition of the microcapsules, the level of lactate dehydrogenase in the culture medium was determined according to the manufacturer's protocol (The Thermo Scientific™ Pierce™ LDH Cytotoxicity Assay Kit). Absorbance of the solution was measured at the wavelengths λ = 490 nm and λ = 640 nm, using Microplate Reader Thermo Multiskan Ascent 96 & 384 (Thermo Fisher Scientific, UK). We determined LDH activity according to the protocol described elsewhere44 by subtracting the 680 nm (background) value from the 490 nm (maximum formazan absorbance) value.

2.13. Determination of reactive oxygen species (ROS) Determination of the level of intracellular reactive oxygen species was performed using 2',7'-dichloroflurescein diacetate (DCF-DA) and dihydroethidium (DHE). Cells were preincubated with microcapsules, at various concentrations, for 24 hours, in a black, 96-well plate. After incubation, the culture medium was replaced with Hank's Balanced Salt Solution (HBSS) containing 2,7-DCFH-DA (60 µM) and dihydroethidium (30 µM). The detection was performed using a Fluostar OPTIMA fluorescence plate reader (BMG LABTECH, Germany).

2.14. Oxidative stress induced by hydrogen peroxide Oxidative stress was induced by hydrogen peroxide, as follows. First, microcapsules were added to the B-50 cells in an amount of 1, 10 and 100 microcapsules per cell. 24 hours later, the 7



cells were washed three times with PBS to remove non-uptaken microcapsules. Next, hydrogen peroxide (1.5 mM) was added to the cells and they were incubated for 30 minutes with H2O2. After that the cells were washed three times with PBS to remove hydrogen peroxide, and the medium was replaced with a fresh one. After 24 hours’ incubating, cell viability was assessed using a standard MTT test.

2.15. Statistical data analysis Statistical analysis was performed using the methods of variation statistics. We determined mean values and the standard deviation of the mean. The significance of differences was determined by the Student’s t-test. The significance of differences in cell assays was assessed using the Mann-Whitney U-criterion.

3. Results and discussion 3.1. Composite capsule formation and characterization The formation of microcapsules’ shells is based on the electrostatic interaction between oppositely charged functional groups of polyelectrolytes, which can be conditionally divided into “strong” and “weak”. The conformation and charge of “weak” polyelectrolytes’ functional groups depends on the acidity of the medium (pH). Conversely, charged groups of “strong” polyelectrolytes remain unchanged upon pH variation. Here, we decided to use “strong” polyelectrolytes to ensure structural stability of composite microcapsules. We chose both nonbiodegradable (polystyrene sulfonate / polyallylamine hydrochloride) and biodegradable (dextran sulfate/poly-L-arginine) polyelectrolytes, as they are actively used in the design of intracellular delivery systems.45 To prepare biodegradable microcapsules, we used poly-Larginine and dextran sulfate polyelectrolytes, due to their excellent properties, including good biodegradability and dispersibility.46–48 PArg-containing polyelectrolyte microcapsules possess excellent dispersibility and demonstrate low aggregation, which is inherent to, for example, polylysine-based microcapsules.49 Such advantageous properties facilitate the synthesis of microcapsules and should favour their further in vitro and in vivo studies. The number of polyelectrolyte layers forming microcapsules was chosen to ensure their structural stability, including shape and size stability. For the polyelectrolytes used in our work, the thickness of the layers was 5–6 nm per layer.50 Varying the number of layers, we can change the chemical and mechanical properties of microcapsules: for example, by increasing the thickness of biodegradable microcapsules’ shell, we can significantly increase the time of their intracellular biodegradation.51 We decided to place CeO2 nanoparticles in the third layer of the microcapsules’ shell to prevent their detachment during the synthesis stage and to prevent the 8


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adsorption of serum proteins on their surface, while providing their effective intracellular delivery. The Layer-by-Layer (LbL) technique provides a unique ability to control the size of the microcapsules. By varying incubation times, one can change the size of CaCO3 microparticles, thus changing the size of the microcapsules.52 The incorporation procedure of citrate-stabilized СеО2 nanoparticles in the polyelectrolyte microcapsule structure is shown in Scheme 1. Positively charged PAH (or PArg) and negatively charged PSS (or DS) were assembled alternately onto the freshly synthesized CaCO3 cores. After 6 polymer layers were formed, the template was dissolved by 0.2 M EDTA solution, to obtain hollow polyelectrolyte microcapsules. We used the minimum number of layers of polyelectrolytes (six layers), which ensured stable structure and shape of microcapsules after dissolution of the CaCO3 core. A sufficiently high zeta potential of citrate-stabilized ceria nanoparticles (–53 mV) ensured formation of a CeO2 layer in the microcapsule structure. The structure of non-biodegradable microcapsules is as follows: PAH/PSS/PAH/СеО2/PAH/PSS; for biodegradable microcapsules: PArg/DS/PArg/СеО2/PArg /DS. The morphology of the composite microcapsules formed was characterized by SEM, TEM and CLSM. The morphological analysis of microcapsules using confocal laser scanning (Fig. 1 a-f) and scanning electron microscopy (Fig. 2 a, b, d, e), as shown below, identified the typical spherical shape of the microcapsules, with some folds which were formed during the preparation of samples for analysis. The size of the microcapsules slightly varied, with biodegradable microcapsules being smaller (2–3 µm) in comparison with the non-biodegradable ones (3–4 µm). There was a slight aggregation of biodegradable microcapsules. Energy dispersive spectroscopy (EDX) analysis of the composite capsules indicated the presence of large amounts of Ce (Fig. 2 c, f). The amount of ceria, as estimated from the EDX data (Fig. S2), was around 20-30 wt%. No traces of calcium were seen in the spectrum, revealing that CaCO3 was completely removed by EDTA. According to the estimations made using UVvis spectroscopy, one biodegradable microcapsule contained 6 ± 1.3 pg of CeO2 nanoparticles, one non-biodegradable microcapsule contained 8 ± 1.9 pg of CeO2 nanoparticles. In order to analyze the morphology, size and spatial distribution of CeO2 nanoparticles in the shell of the capsules, high resolution transmission electron microscopy (TEM) was used (Fig. 3). TEM analysis showed the presence of СеО2 nanoparticles in the structure of polyelectrolyte microcapsules. More dense areas are typical of the localization of СеО2 nanoparticles. СеО2 nanoparticles’ lattice fringes are clearly visible in the high magnification images, in the form of distinctive structured lines (Fig. 3 c, f). 9



Figure 1. Schematic representation of the CeO2 composite microcapsules formation process by the LbL technique. The fourth layer of polymer was replaced by the sol of citrate-stabilized nanoparticles of CeO2. The zeta potential of the capsules is an indicator of their overall surface charge related to its composition.53 The zeta potential of polyelectrolyte microcapsules made from biodegradable polyelectrolytes (PArg/DS) was -25 mV (Fig. S3 a), while, for non-biodegradable microcapsules (PAH/PSS), it was -40.6 mV (Fig. S3 b). The absorption spectra of biodegradable and synthetic microcapsules are presented in Fig. S4. The absorption peak of microcapsules coincided with the adsorption maximum of CeO2 nanoparticles (304 nm). The control of the dispersibility of a microcapsule is the most important parameter for its potential in vivo uses. The size of a microcapsule upon introduction into a living organism significantly increases due to the formation of a so-called “protein corona” intended to the rapid elimination of microcapsules from the bloodstream by specialized cells (macrophages). This effect is hazardous, due to the possible development of thrombosis. The LbL technique limits the microcapsule’s minimum size to approximately 1 µm, which is the upper boundary for safe intravenous administration. In these circumstances, intravenous administration seems to be not an effective route for LbL microcapsules’ use for drug delivery. Yet, the elaboration of a secure and efficient LbL microcapsule-based delivery system (e.g. for local administration) requires studies of their hemolytic activity. Our preliminary results have shown that CeO2-containing composite microcapsules did not exhibit hemolytic activity, even at high concentrations (Fig. S5). The mechanical properties of a microcapsule play an important role, not only in the cellular uptake mechanism, but also in cellular metabolic activity54,55, thus they require careful adjustment for further biomedical applications. We investigated the effect of the amount of CeO2 nanoparticles incorporated in the microcapsule’s shell on its mechanical properties using an osmotic pressure measurement technique.56 No significant effect of CeO2 nanoparticles on microcapsules’ stiffness was observed.

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Figure 2. Laser scanning confocal images of non-biodegradable (a, b, c) and biodegradable (d, e, f) CeO2 composite microcapsules.

Figure 3. SEM images (a, b, d, e) and EDX analysis (c, f) of the non-biodegradable and biodegradable CeO2 composite microcapsules.

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Figure 4. TEM images of the biodegradable (a, b, c) and non-biodegradable (e, f, g) CeO2 composite microcapsules, and the selected area electron diffraction (SAED) pattern of CeO2 composite microcapsules (d, h).

3.2. Intracellular delivery study of CeO2-containing microcapsules It is well known that larger particles (larger than 2 µm) can enter the cell only through classical or receptor mediated endocytosis (e.g. caveolin dependent endocytosis).54 The rate and the efficiency of microcapsules’ uptake depends on both the type of the cells (specialized macrophage or other types of nonphagocyte cells) and the microcapsule’s shape, composition and mechanical properties.57–59 Previously, it has been shown that cells take up polyelectrolyte microcapsules predominantly via phagocytosis.60 Both types of the synthesized CeO2 composite microcapsules are rapidly taken up by the B50 cell line (in about 3-4 hours) and localized in the cytoplasm. Upon uptake, biodegradable microcapsules are degraded by the proteolytic action of endogenous enzymes that is followed by the release of nanoceria, as proved by the confocal microscopy images (Fig. 4). The biodegradable microcapsules which were not taken up by the cells retained their structure (indicated by arrows), while staying outside the cells. The non-biodegradable microcapsules were localized in the cytoplasm and did not undergo degradation for a long period of time (over 48 hours), maintaining their original shape and structure (Fig. 4 a, b). Threedimensional laser scanning confocal analysis was carried out to confirm that microcapsules were not attached to the cell surface, but were really localized directly in the cytoplasm (Fig. S6). Both types of microcapsule successfully delivered nanoceria into the cells, with their different behaviour in the cell, which may be used for different biomedical applications.

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Figure 5. Uptake of non-biodegradable (PAH/PSS/PAH/СеО2/PAH/PSS) and biodegradable

(PArg/DS/PArg/СеО2/PArg /DS) CeO2 composite microcapsules by В-50 cell line, after 24 and 48 hours of incubation.

We estimated the cellular uptake of both biodegradable and non-biodegradable CeO2composite microcapsules (Fig. S7). No significant difference was observed between the uptake of biodegradable and non-biodegradable microcapsules, according to the Mann-Whitney Ucriterion, p ≥ 0.05. The efficiency of microcapsules’ cell uptake depended on the initial concentration of microcapsules. When 10 microcapsules per cell were added, a 40% absorption efficiency was registered.

3.3. Cytotoxicity analysis of CeO2-containing microcapsules in vitro The comprehensive analysis of cytotoxicity showed no acute toxicity of synthesized polyelectrolyte microcapsules, in a wide range of concentrations (1, 10 or 100 capsules per cell). The highest concentrations (100 microcapsules per cell in the cell media) caused a slight decrease in the MTT signal level, which may have been associated with the mechanical action of microcapsules on cellular structures. After 24 hours of incubation, there was no increase in the level of free LDH in the culture medium, which confirmed the integrity of membranes and the absence of apoptosis. A significant (p < 0.05) increase in the MTT signal after 24 and 48 hours of incubation (Fig. 5 a b) was observed with a biodegradable microcapsule concentration of 1 capsule per cell. Apparently, this effect is associated with the ability of nanoceria to stimulate cell proliferation.61 It should be noted that these unique properties of nanoceria can be used in the 13



design and development of specific substrates for culturing mesenchymal stem cells in vitro, while polyelectrolyte microcapsules may be used as a high precision system of delivery into stem cells residing in the body. The analysis of intracellular ROS level (Fig. 5 c, d) after 24 hours of incubation with microcapsules showed no significant difference in comparison with the control values. These data indicated that polyelectrolyte microcapsules loaded with nanoceria did not cause oxidative stress in the cell. Adding biodegradable microcapsules in concentrations of 1 or 10 capsules per cell significantly (p < 0.05) reduced the DCF level (Fig. 6 d), which correlates with earlier publications showing the ability of СеО2 to inactivate hydrogen peroxide and other types of ROS.62 We also estimated the intracellular ROS level in the presence of LbL microcapsules without CeO2 nanoparticles and found that there was no statistically significant difference with the untreated control (Mann-Whitney U-criterion, p ≥ 0.05). We also analyzed the level of mitochondrial membrane potential (MMP) upon the uptake of microcapsules. The results showed only an insignificant decrease in MMP for both types of microcapsules, even at maximum concentrations (100 microcapsules per cell). Low concentrations of microcapsules (5 microcapsules per cell) did not cause any decrease in the MMP level (Fig. S8).

Figure 6. Cytotoxicity tests and ROS levels. MTT assay of B50 cell culture after 24, 48 and 72 hours of incubation with CeO2-containing microcapsules (a). LDH assay 24 hours after incubation with microcapsules (b). ROS level determination by staining with dihydroethidium (c) and 2,7-DCFH-DA (d). Green columns – biodegradable microcapsules. Blue columns – non14


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biodegradable microcapsules. The ROS level in the presence of the microcapsules without CeO2 nanoparticles was statistically identical to the untreated control (Mann-Whitney U-criterion, p ≥ 0.05). * Significant differences were compared to the control using the Mann-Whitney U-criterion, p < 0.05. Control – without the addition of microcapsules.

The study of the metabolic activity of the most sensitive type of cells, human mesenchymal stem cells (hMSCs), showed that CeO2 composite microcapsules were captured by hMSCs and localized in the cytoplasm (Fig. S9). Viability analysis after CeO2 composite microcapsules uptake showed no toxic effect on hMSCs in the presence of no more than 20 microcapsules per cell. The expression level of the key genes analyzed using the PCR-RT method showed that the incubation of the hMSCs with biodegradable microcapsules (at the concentration of 20 microcapsules per cell) caused no changes in the expression of most of the selected genes (Fig. S10, Table S1). We observed only a slight increase in KAT2A and RB1 (chromatin and chromosome modulators) and AXIN and CDK4 expression. Note that biodegradable microcapsules provided a significant decrease in the mRNA concentration of the necrosis marker genes (FOXI1, JPH3 and RAB25, cancer cell markers CD24 and GATA3), as well as of the SOX2 and LIN28B genes (pluripotency and proliferation marker, respectively). Non-biodegradable microcapsules (at a concentration of 20 microcapsules per cell) also did not affect the expression of most of the selected genes. We observed a slight increase in the expression level of the NOTCH1, NOTCH2, BGLAP, BMP1, IGF2 and VDR (the markers of osteogenic differentiation), CCNA2, AURKB, TP53, LIN28B and MCM2 (the proliferation markers), and POU5F1 and SOX2 (pluripotency markers) genes. A significant activation of FOXA2 (a marker of stem reduction) and NOS2 (an anti-apoptotic marker) genes transcription was noticed. A decrease in transcriptional activity for the single genes of different clusters (PARD6A, BMPR1A, MSX1, SIRT1, CD24, BMP7, TRAF2, TNFRSF1) was also detected. Despite this, we can conclude that the internalization of both types of microcapsules into hMSCs had no significant effect on their metabolic activity, resulting only in a slight activating of a few genes.

3.4. Antioxidant protective study of CeO2-containing microcapsules in vitro Next, we evaluated the protective effect of the microcapsules in order to confirm that the integration process of cerium oxide nanoparticles in a polyelectrolyte structure did not alter their physical and chemical properties. Particular attention was paid to their antioxidant activity. This was done using a model of hydrogen peroxide induced oxidative stress. As shown previously, 15



this model can be used to provide an adequate assessment of the therapeutic potential of cerium oxide nanoparticles.63,64 We used higher concentrations of hydrogen peroxide (1.5 mM for 30 minutes), in comparison with previous reports,65,66 to demonstrate high catalase-like activity of encapsulated nanoceria. The rate of survival of the B50 cell line under different concentrations of hydrogen peroxide was evaluated using the MTT assay (Fig. S11). The addition of 1.5 mM of hydrogen peroxide for 30 minutes resulted in 100% cell death after 24 hours; prior administration of microcapsules increased the number of viable cells: up to 50%, in the case of non-biodegradable microcapsules (Fig. 6 a), and up to 70% in the case of biodegradable microcapsules (Fig. 6 b). It should be noted that one capsule per cell was not enough to effectively protect cells from 1.5 mM hydrogen peroxide. The administration of either 10 or 100 microcapsules per cell protected cells equally. It can be supposed that the optimum concentration is from 5 to 10 microcapsules per cell. Administration of biodegradable microcapsules provided greater protection, in comparison with non-biodegradable ones. This result can be explained by the fact that the biodegradable microcapsules were rapidly degraded by the action of cytoplasmic enzymes releasing nanoceria, spread over the cell cytoplasm; conversely, non-biodegradable microcapsules were localized in the cytoplasm in specific places, and were able to inactivate hydrogen peroxide molecules only in their immediate vicinity (Fig. 6 d). Thus, the integration of nanoceria into a polyelectrolyte matrix did not cause the loss of their antioxidant properties, (in particular, their catalase-like activity (Fig. 6 c)), and so this can be considered to be a safe and efficient intracellular delivery system. A layer-by-layer method provides the possibility to encapsulate biologically active materials (proteins, peptides, pharmaceuticals, etc.), while keeping their native properties. Previously, it has been shown that the integration of various types of nanoparticles into microcapsules provides them with a new functionality, so they can be considered to be effective nanoparticle-based theranostic agents.67,68 Considering nanoceria as a future therapeutic substance, it is necessary to design a new target delivery system. Here, we have demonstrated the possibility of effective integration of citrate-stabilized CeO2 nanoparticles into polyelectrolyte capsules made of non-biodegradable or biodegradable polymers, and their intracellular delivery. Biomedical prospects of such delivery systems are rather good. As shown previously, CeO2 nanoparticles can exhibit strong pro-oxidant activity in an acid microenvironment, due to ROS generation.69,70 For example, as reported by Danhier et al.,71 an active tumour growth is accompanied by a significant pH decrease in the intercellular area. It can be assumed that the loading of biodegradable composite CeO2 microcapsules with anti-cancer drugs would provide their simultaneous delivery to the tumour, with subsequent drug 16


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release and ROS generation by cerium oxide. On the other hand, CeO2 nanoparticles may have synergistic action with other widely used clinical drugs.72 For example, Zholobak et al.73 showed that the use of panthenol as a stabilizer of CeO2 nanoparticles supposedly provides a substantial increase in the efficiency of cell protection from UV-irradiation. Polyelectrolyte microcapsules can be used to improve the accuracy of cellular delivery and controlled release of special drugs and CeO2 nanoparticles.

Figure 7. Protective effects of non-biodegradable and biodegradable CeO2-containing microcapsules after addition of 1.5 mM hydrogen peroxide for 30 minutes in a B-50 cell line (a). Viability of the cells was determined using MTT assay after 24 hours. The yellow marked column refers to biodegradable microcapsules without CeO2 nanoparticles. Possible mechanism of CeO2-containing microcapsules’ protective action under H2O2 exposure (b).

4. Conclusions The targeted delivery of drugs or diagnostic agents into the human body remains one of the challenging issues in modern medicine. In this paper, we have demonstrated that CeO2 nanoparticles can be integrated into polyelectrolyte microcapsules, which are among the most promising delivery systems, with a pronounced therapeutic effect. A complex structural and morphology analysis of synthesized polyelectrolyte microcapsules was carried out, which confirmed a high efficacy of CeO2 integration, while preserving their antioxidant properties. The complex morphological and structural analysis of CeO2-containing microcapsules confirmed the efficiency of integration, while preserving the original biological (antioxidant) activity of nanoceria. A cytotoxicity analysis of CeO2-containing microcapsules showed a high degree of biocompatibility, which suggests the prospect of safe usage of such a delivery system. The delivery system of therapeutic substances (including nanoceria) using biodegradable polyelectrolyte microcapsules is suitable for the pharmaceutical industry because it can meet the

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specific requirements of a pharmaceutical preparation, such as the exact time and place of delivery, and concentration of the active substance.

Acknowledgements This research was supported by the Russian Foundation for Basic Research project № 1634-60248. A.L. Popov is grateful for the President of the Russian Federation’s scholarship for study abroad 2014/2015.

Supporting Information TEM data and ζ-potential values for CeO2 nanoparticles; ζ-potential values, UV-vis spectra, EDX data, confocal images for polyelectrolyte microcapsules; survival data of B50-cells exposed to H2O2; hemolytic activity of composite microcapsules; the results of cellular uptake, mitochondrial membrane potential and gene expression measurements for composite microcapsules.

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Intracellular delivery of antioxidant CeO2 nanoparticles via polyelectrolyte microcapsules Anton L. Popov, Nelli R. Popova, Nadezda V. Tarakina, Olga S. Ivanova, Artem M. Ermakov, Vladimir K. Ivanov, Gleb B. Sukhorukov

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Intracellular delivery of antioxidant CeO2 nanoparticles via

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