Ceria nanoparticles-decorated microcapsules as a smart drug delivery

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Biological and Medical Applications of Materials and Interfaces

Ceria nanoparticles-decorated microcapsules as a smart drug delivery/ protective system: Protection of encapsulated P. pyralis luciferase Anton Popov, Nelli Popova, David James Gould, Alexander B. Shcherbakov, Gleb B. Sukhorukov, and Vladimir K. Ivanov ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19658 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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ACS Applied Materials & Interfaces

Ceria nanoparticles-decorated microcapsules as a smart drug delivery/protective system: Protection of encapsulated P. pyralis luciferase

Anton L. Popov1, Nelli Popova1, David J. Gould2, Alexander B. Shcherbakov3, Gleb B. Sukhorukov1,4, Vladimir K. Ivanov5,6* 1

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

Pushchino, Moscow region, 142290, Russia 2

Queen Mary University of London, William Harvey Research Institute, London, EC1M 6BQ,

U.K. 3

Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine,

Kyiv D0368, Ukraine 4

Queen Mary University of London, School of Engineering & Materials Science, London, E1

4NS, U.K. 5

Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences,

Moscow, 119991, Russia 6

National Research Tomsk State University, Tomsk, 634050, Russia

e-mail: [email protected] Key words: layer-by-layer assembly, polyelectrolyte microcapsules, cerium oxide nanoparticles, luciferase, antioxidant activity

ABSTRACT The design of novel, effective drug delivery systems is one of the most promising ways to improve the treatment of socially important diseases. This paper reports on an innovative approach to the production of composite microcontainers (microcapsules) bearing advanced protective functions. Cerium oxide (CeO2) nanoparticles were incorporated into layer-by-layer (LbL) polyelectrolyte microcapsules as a protective shell for an encapsulated enzyme (luciferase of Photinus pyralis), preventing its oxidation by hydrogen peroxide – the most abundant type of reactive oxygen species (ROS). The protective effect depends on CeO2 loading in the shell: at a low concentration, CeO2 nanoparticles only scavenge ROS, while a higher content leads to a decrease in access for both ROS and the substrate to the enzyme in the core. By varying the

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nanoparticle concentration in the microcapsule, it is possible to control the level of core shielding – from ROS filtering, to complete blocking. A comprehensive analysis of microcapsules by transmission electron microscopy (TEM), scanning electron microscopy (SEM), atomic force microscopy (AFM), confocal laser scanning microscopy (CLSM) and energy-dispersive X-ray spectroscopy (EDX) techniques was carried out. Composite microcapsules decorated with CeO2 nanoparticles and encapsulated luciferase were shown to be easily taken up by rat B50 neuronal cells; they are non-toxic and are able to protect cells from the oxidative stress induced by hydrogen peroxide. The approach demonstrated that the active protection of microencapsulated substances by CeO2 nanoparticles can be used in the development of new drug delivery and diagnostic systems.

INTRODUCTION Many therapeutic agents are subjected to a degradation in aggressive surrounding media, which reduces the efficiency of medical treatment. Sensitive organic molecules can be damaged by different physical and chemical factors, (heating, irradiation, oxidation), affecting their pristine activity. Thus, the development of artificial microenvironments providing protective properties during drug delivery to the target zone, through the aggressive media, and providing the possibility of controlled release of the protected contents, is of great interest in terms of designing new drug delivery systems. The polyelectrolyte microcapsule is one of the most promising means of controlled drug delivery.1-8 The possibility of encapsulation has been demonstrated already, for both macromolecules9 and small molecules,10 proteins,11 DNA,12 different pharmaceuticals,13-14 cells15 and other substances.16 The LbL technique allows the production of multifunctional microcapsules for biomedical application, ensuring the safety of the encapsulated substance and its controlled release.17-18 For example, it has been shown previously that hollow aliphatic poly(urethane-amine)/sodium poly(styrene sulfonate) multilayer microcapsules with smart nanomembranes possessed pH- and temperature-dependent drug release properties.19 Hybrid hollow hydroxyapatite microparticles with a chitosan/hyaluronic acid multilayer shell have been demonstrated to be capable of controllable drug release.20 Similarly, silica composite microcapsules assembled using biodegradable polymers (poly-L-arginine hydrochloride/dextran sulfate sodium salt) can be loaded with small molecules (Rhodamine B) providing their efficient intracellular delivery and release.21 However, the polyelectrolyte shell of microcapsules provides only «passive» protection of the encapsulated substances, being unable to resist aggressive environmental conditions. ACS Paragon Plus Environment

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In this paper, we propose the use of cerium oxide nanoparticles (CeO2) as an «active» component of the protective polyelectrolyte shell. Previously, it has been shown that CeO2 nanoparticles can act as an effective antioxidant,22 capable of scavenging a wide range of reactive oxygen and nitrogen species,23-27 protecting cells from UV- and X-ray irradiation.28-29 Nanocrystalline ceria can be judged as a promising constituent of smart hybrid materials. For example, Zholobak et al. designed a novel type of panthenol-stabilized cerium dioxide nanoparticle which protected cells efficiently from reactive oxygen species (ROS) and UVirradiation.30 Ceria/calcein complex was successfully used for direct ROS-monitoring in living cells.31 This complex penetrates easily into a living cell and is decomposed readily by endogenous or exogenous ROS, releasing brightly fluorescent calcein, which can be observed using conventional fluorescent microscopy. This complex is less cytotoxic than individual cerium oxide nanoparticles, and is capable of providing an efficient cell protection from oxidative stress. Another example of cerium-based smart hybrid materials is a delivery system for

human

carbonic

anhydrase

(hCAII)

inhibitors.32

This

hybrid

system

contains

carboxybenzenesulfonamide, an inhibitor of the hCAII enzyme, which is linked to cerium oxide nanoparticles by epichlorohydrin. Along with the inhibitor, a carboxyfluorescein fluorophore was attached to the nanoparticles to enable their tracking. It has been shown that such a hybrid system can efficiently deliver an inhibitor to the affected area in vivo, with real-time fluorescence-imaging capabilities. Additionally, CeO2 nanoparticles are non-toxic for normal mammalian cells33-34 and have great therapeutic potential,35-39 which offers opportunities for their safe usage in multifunctional drug delivery systems. In our study, the bioluminescent enzyme luciferase from fireflies (Photinus pyralis) was selected as a model of encapsulated protein. This enzyme has previously been introduced into polyelectrolyte capsules and its activity has been monitored in vitro.40 The aim of this study was to examine the effect of CeO2 nanoparticles placed in the shell of microcapsules as an «active» protector of encapsulated protein (luciferase) oxidation by the most common type of ROS, hydrogen peroxide. As a result, we designed a new type of hybrid intracellular delivery system which is capable of being taken up by various types of cell, to effectively protect the encapsulated enzyme from external destructive factors. This result opens up new opportunities for the controllable intracellular delivery of biologically active substances.



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EXPERIMENTAL SECTION Materials and reagents Calcium

chloride

(CaCl2,

#223506),

sodium

carbonate

(Na2CO3,

#S7795),

ethylenediaminetetraacetic acid disodium salt dehydrate (Na2EDTA, #E5134), citric acid (HOC(COOH)(CH2COOH)2, #C0759), cerium (III) chloride heptahydrate (CeCl3⋅7H2O, #22300), dextran sulfate sodium salt (DS, MW ≈ 10 kDa, #D4911), poly-L-arginine hydrochloride (PArg, MW≈70 kDa, #P3892), hydrogen peroxide solution (30 wt.%, #216763), rhodamine B isocyanate (RBITC) labelled PArg, phalloidin fluorescein isothiocyanate labelled (#5282) and bisBenzimide H 33342 trihydrochloride (#14533) were purchased from SigmaAldrich. All chemicals were used as received. Ultrapure water with a resistance greater than 18.2 MΩcm−1 was used for all experiments. Recombinant American firefly luciferase (Roche Diagnostics GmbH, Mannheim, Germany), luciferase assay system, D-luciferin K+ salt from Promega Corp (Madison, WI, USA) and Dulbecco’s Modified Eagles Media (DMEM), Fetal Bovine Serum (FBS), penicillin, streptomycin and trypsin, were purchased from Lonza.

Preparation and characterization of CeO2 nanoparticles Aqueous colloid solutions of citrate-stabilized cerium oxide nanoparticles were prepared using a previously reported technique.41 Briefly, to obtain a colloid solution of cerium oxide nanoparticles, 0.24 g of citric acid was dissolved in 25 ml of 0.05 M aqueous solution of cerium chloride (III), and rapidly poured into 100 ml of 3M ammonia solution with stirring, then boiled for 2 hours and purified by precipitation-redispersion. The size and shape of CeO2 nanoparticles were determined by transmission electron microscopy (TEM), on a Leo912 AB Omega electron microscope (accelerating voltage 100 kV). UV-absorption spectra were registered using a Cary 100 UV-visible spectrophotometer, in the wavelength range from 200 to 800 nm. The hydrodynamic size and zeta-potential of CeO2 nanoparticles were measured using a Malvern Zetasizer Nano ZS Analyser. Before measurement, the CeO2 sol was diluted in distilled water. The antioxidant activity of CeO2 was investigated by the polarographic method, using a Clark electrode. Preparation of microcapsules Microcapsules were prepared using the LbL technique, with alternate deposition of oppositely charged polyelectrolytes on calcium carbonate particles.42 CaCO3 particles were synthesized by reacting CaCl2 and Na2CO3 in aqueous solution. Luciferase was incorporated into the cores by co-precipitation. To synthesize luciferase-loaded CaCO3 templates, 0.250 mL of 1 ACS Paragon Plus Environment

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g⋅L-1 luciferase solution in phosphate buffered saline was mixed with 2.5 mL of CaCl2, then Na2CO3 (2.5 mL) was added to the mixture. The luciferase loading efficiency was estimated according to previously reported techniques43–44. According to our data, the loading efficiency was 95±2%. Polyelectrolyte microcapsules were prepared immediately after CaCO3 particles synthesis according to previously reported procedure45. First, positively charged PArg was adsorbed on CaCO3 surface, further microparticles were covered with negatively charged DS. Citratestabilized cerium oxide colloidal particles with an average size of 3–4 nm were used as the fourth layer. CaCO3 cores were then etched by ethylenediaminetetraacetic acid, then the resulting polyelectrolyte microcapsules were centrifuged and washed. Biodegradable polyelectrolyte microcapsules were prepared from PArg and DS with cerium oxide nanoparticles in the middle layer (PArg/DS)(PArg/CeO2)(PArg/DS). The preparation of RBITС-labelled microcapsules was carried out in the same way; RBITС-labelled PArg was incorporated as the fifth layer of the microcapsules.

Characterization of microcapsules TEM images were taken using a JEOL-JEM 2010 transmission electron microscope. The specimens were analyzed at an acceleration voltage of 200 kV and magnifications of up to 300000x. Scanning electron microscopy (SEM) and energy dispersive X-ray (EDX) analysis were carried out using a FEI Inspect F microscope. Laser scanning confocal microscopy (LSCM) images were taken using a Leica TS laser scanning confocal microscope, with a 63x f/1.4 oilimmersion lens. Atomic force microscopy (AFM) was carried out using a Smart SPMTM-1000 atomic force microscope (AIST-NT, Russia). Tapping mode with resonance frequency of 115– 170 kHz was used in all experiments. Scanning was performed under normal conditions, at humidity below 60%. The sample was applied to a glass surface. The stability of hybrid microcapsules was studied in buffer solutions with pH 5.0, 7.4 and 9.2, using a Tecan Infinite 200 PRO luminometer. Luminescence intensity was recorded every 10 seconds for 60 min. Luciferase activity assay In

CeO2-containing

microcapsules,

the

luciferase

activity

was

monitored

luminometrically by a standard procedure43 using luciferase assay reagent (Promega Corp, USA) and MLX Microtiter Plate Luminometer (Dynex Technologies, USA).



Cell culture B-50 rat neuronal cells were obtained from the European Collection of Animal Cell Cultures (ECACC, Porton Down, UK). These cells represented an ethyl nitrosourea-induced tumor cell line with neuronal morphology. B-50 cells were grown in DMEM supplemented with penicillin (100 U ml−1), streptomycin (100 µg ml−1), glutamine (2 mM) and 10% FBS, in a humidified incubator containing 10 % CO2, at 37 °C. Additionally, cellular uptake of CeO2containing RBITC-labelled microcapsules was analyzed on human breast cancer cells (MCF-7 cell line from the Russian Cell Culture Collection, Institute of Cytology of the Russian Academy of Sciences, St. Petersburg), human cervix epitheloid carcinoma (HeLa cell line from the Russian Cell Culture Collection, Institute of Cytology of the Russian Academy of Sciences, St. Petersburg) and primary mouse embryonic fibroblasts obtained from embryos of SHK white mice on day 13 of pregnancy, according to the protocol.46 MTT assay The viability of B-50 cells incubated with microcapsules was assessed by MTT assay, based on the reduction of colourless tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide), as described previously.47 Six hours after cell seeding, the medium was replaced with a medium containing 1, 10 or 100 microcapsules per сell, and the viability of the cells was assessed after 24 h. 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 microcapsules in the amount of 1, 10 or 100 microcapsules per cell. Cells without the addition of microcapsules were used as a positive control. Triton X-100 (10 µL) was used as a negative control. The level of lactate dehydrogenase (LDH) was determined in the culture medium 24 hours after the addition of microcapsules, according to the manufacturer's protocol (Thermo Scientific™ Pierce™ LDH Cytotoxicity Assay Kit). Oxidative stress induced by hydrogen peroxide Cells were incubated with different concentrations of microcapsules (1, 10 or 100 microcapsules per cell) for 24 hours, with subsequent induction of oxidative stress by the addition of exogenous hydrogen peroxide (1.5 mM) for a period of 30 min. After incubation, cells were washed three times with PBS, and the medium was replaced with a fresh portion. After 24 hours, cell viability was assessed using the standard MTT assay. ACS Paragon Plus Environment

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Statistical data analysis Statistical analysis was performed using variation statistics, with GraphPad Prism 5.0 and Microsoft Excel 2007. The mean values and the standard deviation of the mean were determined. The significance of differences was assessed using the U Mann-Whitney criterion.

RESULTS AND DISCUSSION TEM images (Figure 1a) of citrate-stabilized CeO2 nanoparticles confirmed their ultrasmall dimensions (2-3 nm). The particles were sufficiently monodisperse and well crystallized, and had an isotropic shape. In the UV-absorption spectra, the absorption peak of CeO2 nanoparticles was at ~290 nm (Figure 1b). The mean hydrodynamic diameter of CeO2 nanoparticles diluted in water (MQ) was about 4-7 nm (Figure 1c). The zeta-potential of CeO2 nanoparticles was negative, at about –40 mV (Figure 1d). The antioxidant activity of СeO2 nanoparticles was assessed using a polarography technique, using a Clark electrode (Figure 1e). The addition of СeO2 nanoparticles (1.4 µM) into the system did not lead to an increase in the oxygen concentration, thus confirming the absence of СeO2 nanoparticles’ pro-oxidant activity. The subsequent application of hydrogen peroxide (1.4 mM) resulted in a sharp increase in the oxygen concentration from 270 µM to 520 µM for 50 seconds, demonstrating the active process of hydrogen peroxide decomposition and confirming the enormous antioxidant activity of nanoceria. The addition of a reducing agent (1 mM ascorbate) to the system did not lead to a decrease in the concentration of oxygen, which, additionally, confirmed the absence of pro-oxidant activity in citrate-stabilized СeO2 nanoparticles, even in the presence of a free electron donor. A schematic representation of citrate-stabilized CeO2 nanoparticles and their catalase-like activity is given in Figure 1f.



Figure 1. Cerium oxide nanoparticles: TEM and HRTEM images (a), UV-vis spectra (b), particle size distribution (c), zeta-potential (d), decomposition of hydrogen peroxide by CeO2 nanoparticles (e), schematic representation of catalase-like activity of citrate-stabilized CeO2 nanoparticles (f).

The LbL synthesis technique makes it possible to wrap up the charged substances between microcapsule layers, including CeO2 nanoparticles, due to the presence of the negative charge formed by citrate ions on their surface (Figure 2). In this study, we encapsulated luciferase in the core of microcapsules and modified their shells using citrate-stabilized CeO2 nanoparticles. Luciferase was used because it is a convenient model protein with which activity can easily be monitored using luminescence measurement techniques.48 To prepare microcapsules, we used poly-L-arginine and dextran sulfate polyelectrolytes, due to their excellent properties, including good biodegradability.49-51 PArg-containing polyelectrolyte microcapsules possess excellent dispersibility and demonstrate low aggregation, which is inherent to e.g. polylysine-based microcapsules.52 Such advantageous properties facilitate the synthesis of microcapsules and their further use for in vitro and in vivo studies. The number of


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polyelectrolyte layers forming microcapsules was chosen to ensure their structural stability, including shape and size stability.53

Figure 2. The scheme of synthesis of biodegradable polyelectrolyte microcapsules modified by citrate-stabilized CeO2 nanoparticles and encapsulated luciferase.

Citrate-stabilized CeO2 nanoparticles exhibit pronounced laser-excited luminescence, which is associated with the presence of electronic defects in crystal structure or oxygen nonstoichiometry, and the emission band is located in the near UV or visible region, depending on the nanoparticles’ size:54 a decrease in size from 50 nm to 2 nm leads to an eightfold increase in the intensity of luminescence and a red shift in the emission band (390 nm → 450 nm), associated with the 5d → 4f transition of the Ce3+ ion.55 This enables labelling of microcapsules without using any special fluorescent dyes. Confocal microscopy showed that CeO2 nanoparticles were localized in the shell of microcapsules, and formed a specific layer (Figure 3a). Such localization of CeO2 nanoparticles provides maximum protection for the encapsulated protein from external aggressive agents (e.g. hydrogen peroxide). TEM analysis showed that CeO2 nanoparticles were uniformly distributed over the surface of polyelectrolyte microcapsules, ACS Paragon Plus Environment


and that there were only a few small areas with a high degree of aggregation (Figure 3b, Supporting Information, Figure S4). SEM and AFM analysis showed a characteristic change in the microcapsule surface morphology, due to the presence of small aggregates of CeO2 nanoparticles (Figure 3c, 3d, Supporting Information, Figure S5, S11). Apparently, the aggregation of CeO2 nanoparticles with the formation of irregularities on the surface of the microcapsules is associated with the process of sample drying. Energy dispersive X-ray microanalysis confirmed the presence of CeO2 nanoparticles in the shell of the microcapsules (Figure 3e). The amount of СeO2 estimated from the TGA data (Figure S6, Supporting Information) is about 20 wt%. We also investigated the stability of polyelectrolyte microcapsules at various pHs (5.0, 7.4 and 9.2) (Supporting Information, Figure S10 a, b). According to our experiments, luciferase encapsulated in unloaded (without CeO2) microcapsules showed high activity, with a maximum activity at 10 mins of incubation (Figure S10a) in neutral media (pH 7.4). Activity of luciferase encapsulated in CeO2-loaded microcapsules in neutral media increased gradually, and the maximum was not observed. At pH 9.2, the difference in luciferase activity in unloaded and CeO2-loaded microcapsules was only marginal. At pH 5.0, the luciferase activity in unloaded microcapsules was negligible. In contrast, CeO2-loaded microcapsules preserved the activity of the encapsulated luciferase for at least 60 min. In summary, the integration of CeO2 nanoparticles into the microcapsules shell prevents the inhibition of the encapsulated enzyme activity in acidic media and preserves its high activity in alkaline and neutral media.


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Figure 3. LSCM (a), TEМ (b), SEМ (c), AFM (d) and EDX analysis (e) of CeO2 composite polyelectrolyte microcapsules with encapsulated luciferase. Luciferase activity in the microcapsules without CeO2 nanoparticles is shown in Figure 4a. The peak of enzyme activity was observed at 25 minutes, wherein the treatment of the microcapsules by hydrogen peroxide (1 mM) resulted in a total suppression of luciferase activity. Such an effect is apparently due to the oxidation of the shell material (PArg, DS), which leads to the loss of microcapsules’ integrity, controlled permeability and to the destruction of the polyelectrolyte shell at high H2O2 concentrations. The activity of luciferase in the microcapsules containing CeO2 nanoparticles (10-3 M) was somewhat lower than that in the microcapsules without nanoparticles. Apparently, a high concentration of CeO2 nanoparticles in the shell of the microcapsules influences access to the substrate, and this was confirmed by an almost 10-fold decrease in luciferase luminescence intensity, as compared to the control (Figure 4b). However, the decrease in CeO2 nanoparticles concentration by up to 10-5 M resulted in a three-fold increase in luciferase activity (Figure 4c). In this case, the addition of hydrogen peroxide to the reaction mixture did not inactivate ACS Paragon Plus Environment


luciferase, and the enzymatic activity for both types of microcapsules (with either 10-3 M or 10-5 M CeO2 nanoparticles) was easily registered. CeO2 nanoparticles had no inhibitory effect on the luminescence intensity of luciferase, even at a maximum concentration (10-2 M) (Supporting Information, Figure S1). The choice of the concentration range for the oxidizing agent (H2O2) reflected the fact that 1 mM hydrogen peroxide drastically inhibits luciferase activity (Supporting Information, Figure S2, S3). Importantly, 1 mM concentration of hydrogen peroxide is ten times higher than the possible physiological concentration.56 This ensures that the encapsulated substance is adequately protected.

Figure 4. Luciferase activity in polyelectrolyte microcapsules without CeO2 nanoparticles (a), with 10-3 M CeO2 (b) and 10-5 M CeO2 (c). Each point is a mean of triplicate recording, the standard error is given as error bars. Thus, we demonstrated that the concentration of CeO2 nanoparticles in the shell of a microcapsule influences the activity of luciferase. A slight decrease in this activity was observed with a higher concentration of nanoparticles in the shell. The decrease in CeO2 nanoparticles concentration in the shell of microcapsules led to an increase in the activity of luciferase in the core. This observation is consistent with previous studies,57-59 which have demonstrated a correlation between the number of layers of polyelectrolytes and enzyme activity in the core.60 Interestingly, the decrease in CeO2 nanoparticles concentration in the shell of microcapsules did


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not affect the efficiency of hydrogen peroxide inactivation, which, additionally, confirmed their strong antioxidant activity. It was previously shown that catalase can be used as a protective agent for the encapsulated protein.61 Nanocrystalline cerium dioxide can exhibit even higher catalase-like activity than the natural enzyme.62 Thus, even low concentrations of CeO2 nanoparticles in the microcapsules’ shell can provide “active” protection against hydrogen peroxide. The use of relatively high concentrations of CeO2 nanoparticles accounts for the formation of a dense catalytically active layer in the microcapsules’ shell, which provides both “passive” and “active” protection simultaneously. The process of polyelectrolyte microcapsules uptake by different cell types has been well studied.63-64 It has previously been shown that this process may take no longer than 1.5-2 hours.65 Synthetic and biodegradable microcapsules demonstrate different behaviour in the cell cytoplasm after uptake.66-67 Microcapsules made from synthetic polymers do not undergo enzymatic degradation, and are located in the cell cytoplasm for a long time.68 Microcapsules made from biodegradable polymers are gradually destroyed by endogenous enzymes, and the degradation rate depends on the number of layers, polymer composition and deformability.69 In our study, biodegradable microcapsules, which contained CeO2 nanoparticles, were quickly (4-5 hours) taken up by rat neuronal cells, and were localized in the cytoplasm. Following uptake, microcapsules were destroyed and CeO2 nanoparticles were released into the cytoplasm, as confirmed by characteristic fluorescence in the cell cytoplasm (Figure 5e, f). Some of the microcapsules did not penetrate the cell, but attached to the cell surface, retaining their structure and fluorescence. The cytotoxicity analysis of CeO2 composite microcapsules by MTT assay (Figure 6a) 24 h after application showed no acute toxicity of the microcapsules, in a wide range of concentrations (1, 10 or 100 microcapsules per cell). The highest concentration (100 microcapsules per cell) caused a slight decrease in the MTT signal level, which may be associated with the mechanical action of microcapsules on the cellular structures and higher concentration of nanoparticles in the cytoplasm. However, the level of free LDH (Figure 6b) in the culture medium did not exceed the control value, which means that the reduction of the MTT signal was not related to cell membrane damage. Additionally, using an advanced labelling technique (RBITC-labelling), we studied the microcapsules’ cell internalization process. We showed that CeO2-containing, RBITC-labelled microcapsules were actively taken up by both normal (primary mouse embryonic fibroblasts) and cancer cells (MCF-7 and HeLa cell lines). Upon 6 hours of incubation they were localized in the cytoplasm (Supporting Information, Figure S7, S8, S9). It should be noted that cancer cells took up CeO2-containing microcapsules more efficiently than normal cells. ACS Paragon Plus Environment


Fluorescence

Merged

6h

Phase contrast

24 h

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Figure 5. Cellular uptake of CeO2-containing microcapsules by B-50 cells after 6 (a-c) and 24 h (d-f). Some of the CeO2-containing microcapsules did not penetrate the cell, but attached to the surface.

Figure 6. MTT (a) and LDH (b) assays 24 h after application of CeO2-composite microcapsules. MTT assay data are presented as a percentage of the untreated control; TRITON X100 was used ACS Paragon Plus Environment

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as a positive control.

As we showed earlier (Figure 1e), citrate-stabilized CeO2 nanoparticles possess enormous antioxidant activity, so we decided to test their antioxidant effect on the cell culture. Preliminary treatment of the culture of neuronal cells with hydrogen peroxide (1.5 mM) resulted in a 20% survival rate of the culture after 24 hours, while the addition of CeO2-composite microcapsules improved the survival rate to 60-70% (Figure 7). These data suggest that the integration of CeO2 nanoparticles in the microcapsule does not affect their antioxidant activity, and so these microcapsules can effectively protect cells from the oxidative stress induced by hydrogen peroxide.

Figure 7. Protective effect of CeO2-composite microcapsules upon addition of 1.5 mM hydrogen peroxide for 30 min in a B-50 cell line. Viability of the cells was determined using MTT assay after 24 hours. The proposed structure and composition of the microcapsule enable the provision of «active» protection of the encapsulated substance from an aggressive external environment (Figure 8) that can be used to design new, multifunctional, smart drug delivery systems, for example, for applications in radiation or photodynamic therapy. This is potentially significant for the preservation of protein and enzyme activity, and may be applicable to other enzymes besides luciferase.



Figure 8. Schematic overview of the “active” protection approaches for nanoengineered polyelectrolyte capsules. Left: hydrogen peroxide penetrates the shell and destroys luciferase in the core of the microcapsule. Right: СeO2 nanoparticles prevent H2O2 from entering the microcapsule core, decomposing it on the surface of the microcapsule.

4. CONCLUSIONS In the present work, we developed protective composite polyelectrolyte microcapsules containing antioxidant CeO2 nanoparticles and encapsulated luciferase. It was shown that CeO2 nanoparticles are capable of providing “active” protection for luciferase against hydrogen peroxide, and “passive” shielding against small molecules. A high concentration of CeO2 nanoparticles in the shell of microcapsules is capable of preventing substrate entry to the enzyme in the core, while a decrease in CeO2 nanoparticles concentration allows an increase in the activity of luciferase, while maintaining the efficiency of protection from hydrogen peroxide. The synthesized microcapsules are easily taken up by rat neuronal cells and are not toxic. The proposed structure of polyelectrolyte microcapsules protects the protein in the core from external oxidative factors, thus enabling the prolonged action of the encapsulated substances.

ACKNOWLEDGMENTS This study was funded by Russian Science Foundation project 17-73-10417.

CONFLICT OF INTEREST The authors declare no conflict of interest.


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Ceria nanoparticles-decorated microcapsules as a smart drug delivery

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