Уникальные исследования наших специалистов по использованию наночастиц кремнезема и диоксида церия (CeO2 NP) заинтересовали престижное медицинское издание Biomedicine & Pharmacotherapy. Работа получила название «Противовоспалительное и антиоксидантное действие наночастиц диоксида церия, иммобилизованных на поверхности наночастиц кремнезема в экспериментальной пневмонии крысы».

Исследование вызвало интерес, поскольку биологические свойства наночастиц данных минералов привлекают внимание в различных областях биомедицины. В общем объеме публикаций, посвященных CeO2 NP, можно проследить две тенденции:

1. Токсичность CeO2, которая содержится в метаболических побочных продуктах и других загрязнителях, которые могут накапливаться в тканях, особенно в легких и печени, вызывая повреждение.
2. Потенциальное применение CeO2 NP в сельском хозяйстве и медицине. В частности, в противораковом лечении: исследования показали, что CeO2 NP токсичен для раковых клеток, проявляя минимальную токсичность для нормальных тканей. Цитопротекторная эффективность CeO2 NP также обнаружена в клетках сердца, моноцитах и клетках поджелудочной железы.

Наше исследование будет интересно как специалистам, так и производителям агропромышленного сектора. Публикацию можно прочитать на страницах Biomedicine & Pharmacotherapy, а также на нашем сайте (на английском языке).

 

Anti-inflammatory and antioxidant effect of cerium dioxide nanoparticles immobilized on the surface of silica nanoparticles in rat experimental pneumonia.

To be published in: Biomedicine & Pharmacotherapy

 

1. Serebrovska¹,R.J. Swanson², V. Portnichenko¹, A. Shysh¹,S. Pavlovich¹, L. Tumanovska¹, A.Dorovskych³, V. Lysenko⁴, V. Tertykh⁵, Y. Bolbukh⁵,V.Dosenko¹
2. Bogomoletz Institute of Physiology, National Academy of Sciences, 4 Bogomoletz St. Kyiv01024, Ukraine
3. LibertyUniversityCollege of Osteopathic Medicine in Lynchburg, 306 Liberty View Lane Lynchburg, VA24502, USA
4. Integrative Medicine Clinic «SmartMed» 16 Luteranska St. Kyiv01024, Ukraine
5. Lashkariov Institute of Semiconductor Physics, National Academy of Sciences, 41 Nauki Ave. 03028, Kyiv, Ukraine
6. Chuiko Institute of Surface Chemistry, National Academy of Sciences, 17 Generala Naumova St., 03164, Kyiv, Ukraine

 

Abstract

A massage with the potent counter-inflammatory material, cerium dioxide nanoparticles, is promising and the antioxidant properties of CeO₂ are considered the main, if not the only, mechanism of this action. Nevertheless, the elimination of ceria nano-particles from the organism is very slow and there is a strong concern for toxic effect of ceria due to its accumulation. To overcome this problem, we engineered a combined material in which cerium nanoparticles were immobilized on the surface of silica nanoparticles (CeO₂ NP), which is shown to be easily removed from an organism and could be used as carriers for nano-ceria. In our study particle size was 220±5 nm, Zeta-potential -4.5 mV (in water), surface charge density -17.22 mC/cm² (at pH 7).

Thirty-six male Wistar rats, 5 months old and 250-290 g were divided into four groups: 1) control; 2) CeO₂ NP treatment; 3) experimental pneumonia (i/p LPS injection, 1mg/kg); and 4) experimental pneumonia treated with CeO₂ NP (4 times during the study in dosage of 0.6 mg/kg with an orogastric catheter). Gas exchange and pulmonary ventilation were measured four times: 0, 1, 3 and 24 h after LPS injection in both untreated and CeO₂ NP-treated animals.  The mRNA of TNF-α, Il-6, and CxCL2 were determined by RT-PCR.  ROS-generation in blood plasma and lung tissue homogenates were measured by means of lucigenin- and luminol-enhanced chemiluminescence.

Endotoxemia in the acute phase was associated with: (1) pathological changes in lung morphology; (2) increase of ROS generation; (3) enhanced expression of CxCL2; and (4) a gradual decrease of V˙o₂ and V˙_E. CeO2 NP treatment of intact animals did not make any changes in all studied parameters except for a significant augmentation of V˙o₂and V˙_E. CeO₂NP treatment of rats with pneumonia created positive changes in diminishing lung tissue injury, decreasing ROS generation in blood and lung tissue and decreasing pro-inflammatory cytokine expression (TNF-α, Il-6 and CxCL2). Oxygen consumption in this group was increased compared to the LPS pneumonia group.

In our study we have shown anti-inflammatory and antioxidant effects of CeO₂ NP. In addition, this paper is the first to report that CeO₂ NP stimulates oxygen consumption in both healthy rats, and rats with pneumonia. We propose the key in understanding the mechanisms behind the phenomena lies in the property of CeO₂ NP to scavenge ROS and the influence of this potent antioxidant on mitochondrial function. The study of biodistribution and elimination of СеО₂NP is the purpose of our ongoing study.

 

Keywords

Anti-inflammatory; antioxidant; cerium dioxide nanoparticles; metabolism; silica nanoparticles; TNF-α;Il-6;CINC-2; oxygen consumption

 

Introduction

Biological properties of cerium dioxide nanoparticles (CeO₂ NP) have recently gained attention in different fields of bio-medical research. Biological activity of CeO₂ NP depends on particle shape, size, lattice features, and technology of production [1 – 3].

Two trends can be traced in the total volume of publications dedicated to CeO₂ NP: (1) the toxicity of CeO₂, which is contained in the metabolic byproducts, and other contaminates that can accumulate in tissues, especially in the lungs and liver, causing damage [4, 5, 6]; and (2) the potential application of CeO₂ NP in agriculture and medicine. One possible application is anti-cancer treatment since data have shown CeO₂ NP to be toxic to cancer cells while displaying minimal toxicity to normal tissues [7 – 9]. Several in vitro studies support the cytoprotective efficacies of CeO₂ NP in cardiac cells [10], monocytes [11], and pancreatic cells [12].

Publications of potent counter-inflammatory action of CeO₂ NP are promising as shown in vivo in peritonitis [13, 14], sepsis [15], age-related macular degeneration [16], light damaged retina [3], and others. Antioxidant properties of CeO₂ are indicated as the main, if not the only mechanism for this effect [17 – 19]. The antioxidant potential of CeO₂ NP is high and comparable to the powerful agent superoxide dismutase [20]. As a regular antioxidant, CeO₂ NP diminishes oxidative disruption of membranes and scavenging superoxide decreases attraction of macrophages and neutrophils in inflamed tissue [21].

Recently published data show that due to its antioxidant properties, CeO₂ NPs are able to provide restoration of cell energy supplies when there is an increase of an external reactive oxygen species (ROS) load [22]. Mitochondrial membrane potential loss, calcium flux, decrease of the NADH/NAD ratio and ATP concentration caused by H₂O₂ treatment of primary cortical cells were restored by introduction of CeO₂ NP [23]. We assume that CeO₂NP can improve energy production also in other physiological and pathological conditions.

Potential clinical application of nano-ceria is promising, nevertheless, the elimination of ceria nano-particles from the organism is very slow [24 ] and there is a strong concern for toxic effect of ceria due to it accumulation[25]. Some authors consider the ability of CeO₂ to be accumulated in tissues as a promising property since once introduced, CeO₂ can remain in damaged cells for a long time, continuing its cytoprotective work [1]. Nevertheless, accumulation of CeO₂ in tissues in uncontrolled concentration is dangerous since higher doses of CeO₂ NP become toxic. To overcome this problem, we engineered a combined material in which cerium nanoparticles were immobilized on the surface of silica nanoparticles . Because of their low toxicity and easy elimination from organisms, silica nanoparticles are widely used as nano-carriers for toxic drugs [26, 27]. Here we describe a pilot study of new engineered particles, namely CeO₂ NP immobilized on commercially available spherical-shaped silica dioxide nanoparticles. In this combination nano-silica were used as potential safe CeO₂ nano-carrier.

We have made a model of lipopolysaccharides (LPS)-induced pulmonary inflammation in rats to study the effects of CeO₂ NP treatment evaluating lung morphology, ROS -production, expression of pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleykin-6 (IL6) , chemokine (C-X-C motif) ligand 2(CxCL2)), oxygen uptake and other characteristics of lung ventilation.

 

Methods

The study protocol was approved by the Animal Care Ethics Committee of Bogomoletz Institute of Physiology, Kyiv, Ukraine. Male Wistar rats were maintainedin a 12 h light:dark cycle, with a temperature of 22±3°C. Food and water were available ad libitum.

Experimental pneumonia.

We have chosen pneumonia induced by LPS specific to the cell membrane of E. coli as the model for our study. The presence of these structures in the tissues is recognized by the immune system as bacterial invasion which initiates an inflammation cascade. In our experiment LPS was administered via intraperitoneal (i.p.) injection. Pneumonia formed in this way is accompanied by sepsis [28, 29, 30, 31, 32].

Rats were injected i.p. with 1 mg/kg of LPS (Escherichia coli 055:B5; Sigma, St. Louis, MO, USA) dissolved in physiological saline, to induce endotoxaemia and lung inflammation.Controls were injected with sterile saline alone.

Study design.

Thirty-six Wistar, 5 month old male rats, weighing 250-290 g were divided into four groups of 9 rats each:  (1) control, (2) CeO₂ NP treatment, (3) experimental pneumonia, (4) experimental pneumonia treated with CeO₂ NP. Animals of all groups were sacrificed 24 h after pneumonia induction (LPS injection) and tissue samples were collected.  The parameters of pulmonary ventilation and gas exchange were measured four times using a respiratory mask in conscious animals as follows: first before LPS injection (time zero), and then 1, 3 and 24 h after LPS injection. In control and healthy CeO₂ NP treated groups, pulmonary ventilation and gas exchange were both measured at these times with no additional treatment. 2,5% water solution of CeO₂ NP was administered 4 times during the study in dosage of 0.6 mg/kg with an orogastric catheter: first at time zero (for groups having pneumonia it was immediately prior to injection of LPS), and then at 1, 3 and 24 hours after time zero.

Synthesis and characterization of particles.

Synthesis of silica nanoparticles with immobilized cerium dioxide (CeO₂/SiO₂) was carried out as described in [33]. We used a deposition precipitation method via thermal treatment of deposited cerium hydroxide obtained by interaction of the dilute solutions of ammonium cerium(IV) nitrate (NH₄)₂Ce(NO₃)₆ (Sigma-Aldrich) with ammonium hydroxide in the presence fumed silica nanoparticles (specific surface area of 300 m²/g, Kalush Research Experimental Plant, Ukraine) all in solution. During thermal treatment at 623K for 2 h, CeO₂ nanoparticles were formed and immobilized on a surface layer of highy-dispersed silica.The presence of CeO₂ nanoparticles was confirmed by X-ray diffraction analysis (diffractometer DRON-4.7, Russia, CuK_a–irradiation) (Fig 1).

 

Fig. 1. X-ray diffraction pattern of cerium dioxide nanoparticles immobilized on silica particles. The XRD diffraction pattern indicated that the synthesized material contains particles of cerium dioxide (JCPDS card #34-0394). The crystallite’s size was determined by the method for polycrystalline solids using the Scherrer equation. Per the data obtained, the average size of the nanoparticles of cerium dioxide deposited on the surface layer of our silica was 6 — 8 nm.

 

Some surface characteristics (isoelectric point, surface charge density, Zeta potential) and average size of particles of the initial carrier (SiO₂) and particles with immobilized cerium dioxide (CeO₂/SiO₂) were estimated by a Zetasizer NanoZS^TM(Malvern Instruments, UK). These data are represented in Table 1.Surface charge densities of nanoparticles (s) were measured in 0.01 M KCl solution at the dispersion concentration (C)of 3 mg/ml.

 

Table1. Surface characteristics of particles of the initial carrier (SiO₂) and particles with immobilized cerium dioxide (CeO₂/SiO₂)

Sample	C, mg/ml	IEP, pH	Surfacecharge density mC/cm2(at pH 7)	Zeta-potential, mV (in water)	Particles size, nm (average)
SiO2	3	2.40	-0,02	-7.6	56±2
CeO2/SiO2	3	3.68	-17.22	-4.5	220±5

 

SiO₂ and CeO₂/SiO₂ particles sizes were estimated by a ZetasizerNanoZS^TM(Malvern Instruments, UK).

Particles were dispersed in water (1 mg/ml) by ultrasound. Non-Invasive Back Scatter technology (NIBS) is incorporated to give the highest sensitivity simultaneously with the highest size and concentration range. Particles size was calculated as an average value from three scans which were performed without disturbing the initial water dispersion of the sample. The pH value was in the range of 5.2-6.0 (Fig. 2).

a

b

Fig. 2. Particles size distribution for SiO₂ (a) and CeO₂/SiO₂ (b) samples as a result of three successive measurements (curves 1I, 2I, 3I and 1, 2, 3, respectively) carried out for 10 min.

Significant increase in diameter of particles of CeO₂/SiO₂ samples presumably can occur due to essential changes of s (Fig. 3) and Zeta-potential value (Table 1). Such characteristics can significantly reduce colloidal stability of water dispersions and thus the determined sizes correspond to conglomerates, not to single particles.

 

Fig. 3. Surface charge density of CeO₂/SiO₂particles at different pHvalues.

Histological analysis.

All tissues were collected as 2-4 mm blocks, fixed in 10% neutral phosphate-buffered formalin for 24-48 hours, dehydrated, cleared and embedded in paraffin. Four to five um sections were stained with hematoxylin and eosin (H&E) and evaluated with light microscopy (Scope: Nikon, Eclipse E-200, camera Nikon, ds-FI1) at 200, 400 and 1,000x for morphological characteristics.

 ROS generation.

ROS generation in blood plasma and a lung tissue homogenate was measured by means of lucigenin- and luminol-enhanced chemiluminescence (CL). After decapitation, blood was gathered in a heparinized tube and immediately CL-assayed. Lung tissue homogenized on ice in a Potter-Elvehjem tissue grinder with 5 vol (w/v) of Hanks’ balanced salt solution (100 mg/mL) without phenol red (HBSS).  Samples for CL measurement contained the following ingredients in a total volume of 1 mL: 0.9 ml whole blood or lung tissue homogenate; 0.1 mL of 50 mM lucigenin or 0.1 mL of 20 mM luminol as the final concentration. After 3-5 min of spontaneous chemiluminescence, 0.1 mL of HBSS with 0.1 μg of opsonized zymosan (30 min incubation with rat serum at 37°C) was added to enhance chemiluminescence. CL was then monitored for 15 min (Luminometer ЕА-1, Ukraine) at 37°C and continuous mixing. The sum of light signals at a sampling frequency of 0.25 Hz was calculated and expressed as relative light units.

RNA isolation, reverse transcription, and real-time polymerase-chain reaction.

Total RNA was isolated from lungs using the Trizol RNA-prep kit (Isogen, RussianFederation) according to the manufacturer’s protocol. RNA concentration was determined using a NanoDrop spectrophotometerND1000 (NanoDrop Technologies Inc, USA).

Reverse transcription was performed using a RevertAid^TMH Minus First Strand cDNA Synthesis Kit (Fermentas, Germany), using 1.2–1.5 µl of total RNA and random hexamerprimer. Received single-stranded DNA was used for real-time polymerase chain reaction (PCR).

Real-time PCR for mRNA expression of genes TNFa,  IL6, CxCL2.

We performed amplification in 10 µL of SYBR Green PCR Master Mix (Thermoscientific, USA)containing 20 pM of each primer. For amplifications of TNF-α, IL-6, CxCL2(corresponding toMIP2-alpha), and ACTB (corresponding to b-Actin, housekeeping gene) gene fragments, we used the following primers:

TNF-α Up: 5`-CCTCAGCCTCTTCTCATTCCT-3`

TNF-α Dw: 5`-GGGAACTTCTCCTCCTTGTTG-3`

IL6 Up: 5`-CAAGAGACTTCCAGCCAGTTG-3`IL6 Dw: 5`-TGGGTGGTATCCTCTGTGAAG-3` CxCL2 Up: 5`-AGGCTAACTGACCTGGAAAGG-3`CxCL2 Dw: 5`-ATCAGGTACGATCCAGGCTTC-3`

ACTB Up: 50-TCATCACTATCGGCAATGAGC-30

ACTB Dw: 50-GGCCAGGATAGAGCCACCA-30

Sample volume was brought to 20 µL with deionized water. Amplification was performed on a 7500 Fast Real-Time PCR System(“Thermoscientific”, USA).The amplification program consisted ofinitial AmpliTaq Gold_ DNA polymerase activation step at95 ºC for 10 min over the following 50 cycles: denaturation (95 ºC for 15 s), annealing, and elongation (56 ºC for60 s). For control of specificity, we performed dissociation stage—sequential increase of temperature from 56 to 99 ºC with registration of the drop in the double-stranded DNA-SYBR Green complexes fluorescence strength. We performed calculations using the 7500 Fast System SDS software provided. The cycle threshold is defined as the number of cycles required for the fluorescence signal to exceed the detection threshold. We calculated the expression of the target gene relative to the housekeeping gene as the difference between the threshold values of the two genes.

Respiration.

The arrangement for measuring ventilatory and gas exchange parameters in rats included a one-way valved mask, a pneumotachograph for small laboratory animals with a pressure sensor, MPX5050, and a mass spectrometer type MH6202, Ukraine [34].  Signals from the pressure sensor and the mass spectrometer were processed by an analog-digital converter delivered to a computer and analyzed on the custom-written software. As was previously shown [34], the inertia of the mass spectrometer sensor does not affect measurement accuracy under respiratory rate lower than 150 breaths per min. Special calibration curves were used when the respiratory rate reached higher values.

We measured respiratory frequency (Freq), tidal volume (V_T) and calculated minute ventilation (V˙_E). On the bases of expired O₂ and CO₂ curves we calculated rate of oxygen uptake per minute (V˙o₂).

These data were expressed in mL/min/kg of body weight to BTPS (body temperature and pressure, saturated system) for respiratory volume and to STPD (standard temperature and pressure, dry system) for V˙o₂. To exclude circadian metabolism oscillation influences on our results all measured parameters were compared with respective results from intact animals. Data are represented as % in comparison with control group.

 

Results and Discussion

 

Morphological study.

The introduction of CeO₂ NP to intact animals caused minor changes in lung tissue (Fig. 4, E).

LPS injection alone led to primary injuries in lung tissue: (1) formation of emphysematous and atelectatic fragments; (2) pneumocytic dystrophy and loss; (3) bronchial epithelium desquamation; (4) vessel dilation; (5) damage of vessel walls; (6) perivascular edema; and (7) thrombosis. Thus, we conclude that intraperitoneal LPS injection (1 mg/kg) causes pulmonary inflammation in experimental rats (Fig. 4 A, B).

Micrographs from the LPS-injected animals treated with CeO₂ NP showed fewer pneumocytes, bronchioles and vessel damages (Fig. 4 C). These results support a reduced severity in the inflammatory process.

A) Lung tissue (x100) of rat from LPS group.Emphysematous(*)and atelectatic areas(↑), lymphohistiocytic infiltration (lhi) of interalveolar walls, destructive changes of bronchioles (br) with desquamation of single epithelial cells,vessel (V), alveoli (a)	B) Lung tissue (х200) of rat from LPS group. Blood clots (bc) in small vessel. Perivascular infiltration (pvi) and emphyzematous dilatations of alveoli(*).
C) Lung tissue (x100) of rat from LPS injection treated with CeO2NP group shows diminished exudative and prolipherative changes in parenchyma and destructive changes in bronchioles and vessels. Bronchioles (br), alveoli (a).	D) Normal lung tissue (x100) of rat from control group.Bronchioles (br), alveoli (a), vessels (V).

 

 

E) Section of lung tissue of intact rat treated with CeO2NP Bronchioles (br), alveoli (a), alveolar duct (ad), vessel (V).

 

Fig 4. Morphology of lung tissue of rats from different group.

 

ROS generation

ROS generation in whole blood of rats from the CeO₂ NP group did not differ from the control (Fig. 5).  LPS injection caused dramatic increase of ROS production in blood (by 554% compared to control), while in the LPS + CeO₂ NP group ROS was significantly lower (by 67% compared to the LPS group). Luminol-enhanced chemiluminescence reflects the total pool of ROS (O₂^—, ^•OH, H ₂O₂ and other peroxides) generation, thus, CeO₂ NP treatment powerfully diminished oxidative stress induced by LPS.  Interacting with macrophages, LPS initiates a positive feedback loop, involving cytokine expression, neutrophil and macrophage attraction, NAD(P)-H oxidase activation and ROS production.  CeO₂ NP treatment limits one or more links in this process.

Fig. 5. Chemiluminescence of whole blood

Values are means ± SE, ^aр<0.05 compared to control, ^b р<0.05 compared to LPS group

 

ROS production in lung tissue was similar to whole blood values. CeO2 NP treatment itself made some enhancement of ROS production, but these changes were statistically insignificant (FIG. 6). LPS injections caused enhancement of ROS production and the addition of CeO2 NP treatment of rats with pneumonia produced a statistically significant (p<0.05) decrease of ROS.

LPS injections caused enhancement of ROS production and the addition of CeO₂ NP treatment of rats with pneumonia produced a significant (p<0.05) decrease of ROS. In lung tissue homogenate, we measured lucigenin-enhanced chemiluminescence, which primarily reflects O₂^—, a major ROS product of mitochondria and activated neutrophils. LPS induces O₂^— production in lung tissue and CeO₂ NP treatment inhibits this process.

 

Fig. 6. Chemiluminescence of lung tissue homogenate. Values are means ± SE, ^aр<0.05 compared to control, ^b р<0.05 compared to LPS group.

 

Cytokine expression

We did not observe any differences of TNF-α, Il-6 and CxCL2 expression in the CeO₂ NP group in comparison with the control. LPS injection caused enhanced expression of CxCL2 (by 68%) while TNF-α and Il-6 remained unchanged.  In the LPS + CeO₂ NP group, a significant decrease of TNF-α, Il-6 and CxCL2 expression was observed.  The level of expression of all three cytokines was lower than the control but not statistically significant (Fig 7-9).

Fig. 7. Expression of TNF-alpha.  Values are means ± SE, ^ap = 0.05compared to LPS.

 

Fig 8. Expression of IL-6. Values are means ± SE, ^ap < 0.05compared to LPS.

 

Fig 9. Expression of CxCl2.Values are means ± SE, ^aр<0.05 compared to control, ^bp=0.001 compared to LPS group

 

Respiration

Treatment of intact rats with CeO₂ NP (CeO₂ NP group) caused an increase of V˙o₂ at the 2^nd and 3^rd measurement. Values returned to baseline in the 4^th measurement. Development of pneumonia in the LPS group caused gradual decrease of oxygen consumption with minimum values in the 4^thmeasurement, 24 hours after LPS injection (Fig. 10).  In the LPS +CeO₂ NP group the V˙o₂ did not differ from the control group in the 2^ndmeasurement but then significantly increased in the 3^rd measurement, returning to control levels in the 4^th measurement. The superposition of two potent factors: pneumonia and CeO₂ NP treatment can be observed in this group. Nevertheless, CeO₂ NP treatment almost eliminated the effect of the LPS injection.

 

 

Fig. 10. Dynamic of V˙O₂ in different groups. Data are represented as % in comparison with control group, indicated by dashed line. Values are means ± SE, ^aр<0.05 compared to control, ^bp<0.05 compared to LPS group.

 

Changes in lung ventilation were similar to those in oxygen consumption. In the LPS group V_T was reduced in all measurements with minimal values obtained in the final measurement. In the CeO₂ NP group V_T increases with the most pronounced effect in the 2^rd measurement (Fig. 11).

Fig. 11. Dynamic of V_T  in different groups. Data are represented as % in comparison with control group, indicated by dashed line.Values are means ± SE, ^aр<0.05 compared to control, ^bp<0.05 compared to LPS group

 

Changes in V˙_E were largely due to tidal volume changes. In the LPS + CeO₂ NP group, V˙_E in the 2^nd and 3^rd measurements with a decrease to baseline values in the 4^th measurement (Fig. 12).

Fig. 12 Dynamic of V˙_E in different groups. Data are represented as % in comparison with control group, indicated by dashed line.Values are means ± SE, ^aр<0.05 compared to control, ^bp<0.05 compared to LPS group.

 

We can conclude that CeO₂NP increases oxygen consumption and lung ventilation in healthy rats and in animals with LPS pneumonia.

We noted a decrease of VO₂, V_T and V˙_E in the 4^th measurement in the CeO₂ NP treated groups. The data patterns in the present study are a consequence of measuring the ventilatory and gas exchange parameters before the CeO₂NP introduction, 1 and 3 hours after CeO2 NP introduction and 21 hours after the last introduction of CeO₂NP. The decreased metabolism in the 4^th measurement in the CeO₂NP and LPS + CeO₂NP groups would thus be due to the decrease in CeO₂NP concentration as a result of normal metabolic clearance from the animal’s body. These results conform with the desirable properties for investigating nano particles: we combined cerium dioxide with silica to promote particle elimination and prevent accumulation in the organism. The pharmacodynamics of CeO₂NP s is the focus of our ongoing research. We have changed the experimental design, excluding the possibility of a habituation effect by introducing the experimental substance prior to all measurements.

 Morphological analysis confirmed the development of pneumonia in rats after LPS injection. Observed pulmonary injury was associated with significant increase of ROS generation in blood and lung tissue and enhanced expression of the pro-inflammatory cytokine CxCl2_. The development of pneumonia was also accompanied by a gradual decrease in oxygen consumption and lung ventilation, which is possibly related to lung function lesion, as well as to the inhibition of metabolism due to intoxication [35].

CeO₂NP treatment of intact animals has not created any changes in the lung morphology, in ROS generation or in cytokine expression. However, oxygen consumption and pulmonary ventilation were significantly increased: oxygen consumption was augmented by 70% an hour after the CeO₂NP introduction. More intensive pulmonary ventilation would be expected to satisfy the enhanced demand of gas exchange.

CeO₂NP treatment of rats with pneumonia created positive changes: diminished lung tissue injury, decreased ROS generation in blood and lung tissue and pro-inflammatory cytokine expression of TNF-α, Il-6 andCINC-2. Oxygen consumption in this group was increased compared to LPS group: it did not differ from the control group in the 2^nd measurement and was significantly higher, than in LPS group in the 3^rd measurement.  We assume a superimposition of the metabolic effect of CeO₂NP with depressed oxygen consumption due to acute pneumonia. Thus, CeO₂NP treatment partially eliminated the effect of the LPS injection.

Our data suggest that the counter-inflammatory effect of СеО₂NP is associated with antioxidant properties of this material. Prior research has shown a major regulatory role which ROS plays in the LPS inflammatory response. Introduction of antioxidant N-acetylcysteine suppressedLPS-inducedexpression ofpro-inflammatory cytokines in gingivalfibroblasts [36].

Metabolic effects of СеО₂NP should be associated with the scavenging of ROS produced in mitochondria. As mentioned, СеО₂NPs increases the mitochondrial membrane potential and ATP synthesis under the influence of ROS[23]. We assume that in our research, СеО₂NP affected the negative feedback mechanism in mitochondrial superoxide production.

Literature data suggest that after i/v introduction CeO₂ nanoparticles accumulate mainly in liver and spleen, with less concentration in lung and kidneys [24]. The absorption of CeO₂ nanoparticles from gastro-intestinal tract is very low [37], that is why inhalation or i/v injections are used to obtain the effects of nano-ceria in organism. In our study we used silica nano-particles, which are shown to be well absorbed from intestine [38] as carrier for ceria. The biodistribution of silica nano-particles is similar to ceria [27 ], that is why we assume that our combined particles after absorption from gastrointestinal tract accumulate in different organs, including lungs, to make their biological effects.

Our study has shown that this new combined material produces effects which are similar to properties described for nano-ceria alone. The next step to possible clinical application of СеО₂NP is to prove, if this material conserves the qualities known for silica nano-particles and can be easily eliminated from an organism. The study of biodistribution and elimination of СеО₂NP is the purpose of our ongoing study.

We do not know yet the exact mechanism of obtained effects: presumably it can be the effect of ceria, whereas silica works exclusively as carrier, but we cannot exclude the possible effect of SiO2 itself, or in combination. Future investigations are needed to answer this question.

 

Conclusion

Our study has shown anti-inflammatory and antioxidant effect of CeO₂NP. In addition, we show that introduction of CeO₂NP stimulates oxygen consumption both in healthy rats, and in rats with pneumonia. We propose the key in understanding the mechanisms behind the phenomena lies in the property of CeO₂ NP to scavenge ROS and the influence of this potent antioxidant on mitochondrial function.

The study of biodistribution and elimination of СеО₂NP is the purpose of our ongoing study.

 

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