CC BY
11
STUDY OF THE IMMUNOMAGNETOLIPOSOMES' CYTOTOXICITY TO LEUCOCYTES AND ERYTHROCYTES OF HUMAN BLOOD
I.A. Koltakov, E.V. Shilova*, V.G. Artyukhov
Voronezh State University, 1 Universitetskaya square, Voronezh, 394018, Russia * Corresponding author: zinkovae@list.ru
Abstract. In this study, we investigated the toxic properties of synthesized immunomagnetoliposomes of the following composition: phosphatidylcholine - cholesterol - disteroylphosphoethanolamine-polyethylene glycol (2000) - magnetite - antibodies to histone H3. Their dimensional characteristics were studied by the dynamic light scattering method. It was revealed that liposomal nanoparticles have dimensions of 176.4 ± 12.9 nm. Data on the toxicity of the synthesized nanoparticles in relation to human blood cells: liposomes in cells to liposomes ratio from 1/1 to 1/1000 have no toxic effect on lymphocytes; lactate dehydrogenase (LDH) release from human blood erythrocytes has been observed in the case of cells/liposomes ratio 1/100-1/1000 after the incubation with liposomes containing sodium azide (0.03%).
Keywords: toxicity, nanoparticles, magnetoliposomes, erythrocytes, lymphocytes.
List of Abbreviations
CTAB - cetyltrimethylammonium bromide
DSPE-PEG - disteroylphosphoethanola-mine-polyethylene glycol
LDH - lactate dehydrogenase
NETs - neutrophil extracellular traps
Introduction
Targeted drug delivery is a rapidly developing area of medicine now. Researchers are actively looking for the new ways of target delivery and improvement of present analytical methods. Liposomes take a special place among such agents due to the fact that they can be used to neutralize the toxic effect of drugs on healthy tissues (Baryshnikov., 2012; Krasnopol'skij et al., 2012; Nakvasina & Artyuhov, 2015). Currently, research devoted to the creation and possible practical application of liposomes is being actively carried out, including development of magnetically sensitive nanoparticles. For example, liposomes withy incorporated iron oxide nanoparticles are effective in the treatment of iron deficiency anemia in Wistar rats (Fathy et al., 2019). The use of magnetically controlled liposomes for targeted drug delivery seems promising, also iron oxide may act as an MRI contrast agent.
One of the ways of anticancer drugs delivery may be their transfer to the tumor through neu-
trophil extracellular traps (NETs). A study by Cools-Lartigue et al. (Cools-Lartigue et al., 2014) showed that after the stimulation with phorbolmeristacetate neutrophils form reticular DNA around A549 lung cancer cells. It is also known that tumor cells interact with NET structures, being adsorbed on them through the pi-integrin molecule (Najmeh et al., 2017). All these data allow us to consider NETs as possible targets for anticancer drugs delivery. The site of interaction between liposomes and NETs can be histone H3 molecules, which are an integral part of extracellular networks, but do not circulate in free form in the blood stream (Korotina & Gener-alov, 2012). In this regard, immunoliposomes coated with antibodies to histone H3 as a vector have been developed.
In this work we investigated the toxic properties of the immunomagnetoliposomes synthesized by us. Magnetite nanoparticles coated with cetyltrimethylammonium bromide (Shilova et al., 2019) are used as a magnetically sensitive substance, since antibodies to histone H3 were attached as vector.
The purpose of these studies was to study the toxicological properties of immunomagnetoliposomes synthesized in our laboratory earlier in relation to human blood cells.
Materials and Methods
Ethical statement
The study was carried out in accordance with the standards of Good Clinical Practice and the principles of the Helsinki Declaration of 1975 and its revised version from 2013. Prior to inclusion in the study, written informed consent was obtained from all participants, and the study itself was approved by the Local Ethics Committee.
Obtaining immunomagnetoliposomes
Liposomes were synthesized by the hydration / rehydration method (Shilova et al., 2018). A solution of phosphatidylcholine (0.5%), cholesterol (0.5%) and DSPE-PEG (0.1%) in ethyl alcohol was evaporated in a Control RV10 rotary evaporator at a water bath at 60 °C. 0.1 M sodium phosphate buffer with magnetite nano-particles (1 mg / ml) were added to the obtained lipid film and stirred for 1 minute. Antibodies to histone H3 were incubated with Trout's reagent for 1 hour. Thiolated antibodies were added to the synthesized liposomes and incubated for 12 hours at 4 °C. The next step was the dispersion of the obtained liposomes by exposing the solutions to ultrasound with an ultrasonic disintegrator Qsonica Sonicators for 15 minutes (20 kHz, pulse 10 seconds with a break of 3 seconds).
Investigation of the sizes of synthesized na-noparticles by dynamic light scattering
The size of the obtained liposomes was measured on a dynamic light scattering spectrometer Zetasizer Nano ZSP (Malvern, UK). Measurements were performed in a cuvette with an optical path length of 1 cm at 37 °C.
Determination of the nanoparticles' toxicity to human erythrocytes
The suspension of erythrocytes obtained from the blood of donors has been used as the research object at the day of the blood draw. Erythrocytes have been washed 3 times with centrifugation in physiological solution at 1500 rpm during 10 minutes. The obtained suspension of erythrocytes was adjusted to the optical density (D412) 0.8 and then used in experi-
ments. The erythrocytes suspensions were preliminary incubated for 1 hour with empty liposomes and liposomes containing 0.03% sodium azide in erythrocytes/liposomes ratios 1/1, 1/10, 1/100, 1/1000. Then erythrocytes were centri-fuged at 3000 rpm using the MPV-340 centrifuge. The destruction of erythrocytes was estimated based on the lactate dehydrogenase release by determining its activity in supernatant (Artyukhov & Nakvasina, 2000).
2.8 ml of supernatant and 0.1 ml of NADH were added into a quartz cuvette for spectro-photometer to obtain the final concentration 5.4 x 10-5 M. The optical density D1 was measures at 340 nm with the Shimadzu UV-2401 spectrophotometer. Then 0.1 ml of sodium pyruvate has been added and its final concentration in the reaction mixture was 1.5 x x 10-3 M, the optical density D2 has been measured 30 seconds later. The catalytic activity of lactate dehydrogenase was estimated using the formula:
A = (D1 - D2) x Vf / (s x l x t),
s - molar extinction coefficient of NADH, that equals to 6,22x103 mole-1 cm-1, l - optical path length, t - incubation time (30 sec), Vf - final volume of the reaction mixture (3 ml).
Determination of the nanoparticles' toxicity to human blood lymphocytes
Human blood lymphocytes were obtained with Ficoll-verografin density gradient centrif-ugation (p = 1.077 g/cm3). Nanoparticles were added to the lymphocytes in the same ratios as in the experiment with erythrocytes.
Viability determination of the obtained cell fraction has been performed with the flow cyto-photometer Guava easyCyte 8 HT (MerkMilli-pore Group, USA) according to GUAVA VIA COUNT kit protocol. The cell samples with the viability not less than 98% have been used in this research.
Statistical data processing
Statistical processing of the results was carried out using the software packages Microsoft Excel 2010. The significance of differences between the control and experi-
mental values was established using the Student's t-test. In the procedures of statistical analysis, the achieved level of significance (p) was calculated, while the critical level of significance in this study was taken equal to 0.05. The obtained data was presented as the arithmetic mean and its standard deviation (M ± Sd).
Results
The obtained liposomal nanoparticles (Fig. 1) had the following structure.
The liposome size was controlled by dynamic light scattering. The study of the size of the obtained liposomes showed that the liposo-mal nanoparticles are 176.4 ± 12.9 nm in size (Fig. 2). This size allows us to consider the obtained liposomes as possible agents for targeted drug delivery.
For the estimation of the toxicity of the resulting liposomes, it is necessary to control their concentration. We calculated the concentration of liposomal nanoparticles as follows.
The number of molecules per liposome was calculated based on the formula proposed by Huang and Mason (1978).
where: Ntot is the total number of molecules, d is the liposome diameter, h is the bilayer thickness (about 5 nm), a is the lipid head area of 0.71 nm2.
The number of liposomes in the solution was calculated based on the molar mass of lipids included in the liposome and their concentration by the formula:
where Nlipo is the number of liposomes; ni, m, m - amount of lipids (mol); Ntot is the number of molecules in one liposome; Na - Avogadro's number - 6.02 x 1023.
The toxic effect of liposomes was evaluated by adding the synthesized nanoparticles in ratios of chlorella cells / liposomes from 1/1 to 1/000. The effect of «empty» liposomes and liposomes containing sodium azide in their composition at a concentration of 0.03% was also investigated.
The toxic effect of immunomagnetolipo-somes on erythrocytes was estimated based the lactate dehydrogenase release from the cells. During the research in was observed that there is no increase of lactate dehydrogenase concentration in supernatant compared with control in case of incubation of the cells with empty liposomes at cells/liposomes ratio 1/1 -1/1000 (Fig. 3).
In case of the incubation of erythrocytes with liposomes containing sodium azide the increase of lactate dehydrogenase release from cells has been observed at ratio 1/100-1/1000.
Lymphocytes viability was determined with flow cytofluorimetry. It was shown that liposomes have no toxic effect on human blood lymphocytes (Fig. 4).
Fig. 1. The membrane's schematic structure of synthesized magnetoimmunoliposomes
Fig. 2. The size distribution of soy lecithin liposomes with incorporated magnetite nanoparticles
1/1000 cell/lipo some ratio Fig. 3. Release of LDH from human erythrocytes during their incubation with liposomes
Fig. 4. Viability of human blood lymphocytes upon addition of nanoparticles
Discussion
Data on the toxicity of the synthesized nanoparticles in relation to human blood cells: liposomes in cells to liposomes have no toxic effect on lymphocytes; LDH's release from human blood erythrocytes has been observed in case of cells/liposomes ratio 1/100-1/1000 after the incubation with liposomes containing sodium az-ide (0.03%). Sodium azide is a toxic chemical that accumulates in the mitochondria and causes the disjunction of oxidative phosphorylation. We used this substance as a model system for the estimation of liposomes stability and the yield of a drug in them. It is known that inhibition of catalase activity by sodium azide, including the action of iron chloride, the rate increases erythrocyte hemolysis. Hemolysis of erythrocytes under the influence of hydrogen peroxide can be the result of not only damage to the lipid component of membranes. Sulfhy-dryl group's oxidation of membrane proteins can also lead to lysis of red blood cells (McMillan et al, 2005).
The available data must be taken into account when assessing the possible toxic effect of liposomes on human blood cells.
Conclusion
During the carried research it the effect of synthesized immunomagnetoliposomes on human blood erythrocytes and lymphocytes has been studied. It has been established that empty liposomes have no toxic effect on the cells at studied concentrations (cell to liposomes ratios from 1/1 to 1/1000); in case of liposomes with 0.03% sodium azide the destruction of erythrocytes is observed at cells/liposomes ratio 1/100-1/1000.
Acknowledgements
This work was supported by the Ministry of Science and Higher Education of the Russian Federation within the framework of the state assignment to universities in the field of scientific activity for 2020-2022, project № FZGU-2020-0044.
References
ARTYUKHOV V.G., KOLTAKOV I.A. & SHILOVA E.V. (2017): Method for obtaining liposomes. Pat. 2621145, Russia.
ARTYUKHOV V.G. & NAKVASINA M.A. (2000). Biological membranes: structural organization, functions, modification by physical and chemical agents. Voronezh. Gos. Univ., 230-273.
BALOYAN B.M., KOLMAKOV A G., ALYMOV M.I. & KROTOV A.M. (2007): Nanomaterials. Classification, features of properties, application and production technology [in Russian]. Moscow: Mezhdu-narodnyj universitet prirody, obshchestva i cheloveka "Dubna", Filial "Ugresha", 125 pp.
BARYSHNIKOV A.Y. (2012): Nanostructured liposomal systems as transport agent for anticancer grugs. Vestnik RAMN 3, 23-31.
COOLS-LARTIGUE J., SPICER J., NAJMEH S. & FERRI L. (2014): Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. Cell Mol Life Sci. 71(21), 4179-4194.
FATHY M M., FAHMY H.M., SAAD O A. & ELSHEMEY W.M. (2019): Silica-coated iron oxide nanoparticles as a novel nano-radiosensitizer for electron therapy. Life Sci. 234, 116756.
HUANG C.H. & MASON J.T. (1978): Geometric packing constraints in egg phosphatidylcholine vesicles. Proceedings of the National Academy of Sciences 75(1), 308-310.
IVANISHCHEV V.V. (2019): Role of iron in plant biochemistry. Izvestiya Tul'skogo gosudarstvennogo universiteta, Estestvennye nauki 3, 149-159.
KOROTINA O.L. & GENERALOV I.I. (2012): Neutrophil extracellular traps: mechanism of formation, function. Immunopatologiya, allergologiya, infektologiya 4, 23-32.
KRASNOPOL'SKIJ Y.M., BALABAN'YAN V.Y., SHOBOLOV D.L. & SHVETS V.I. (2012): Prospective clinical applications of nanosized drugs. Rossijskij Himicheskij zhurnal (Zhurnal Rossijskogo himich-eskogo obshchestva im. D.I. Mendeleeva) LVI(3-4), 11-32.
MCMILLAN DC., POWELL C.L., BOWMAN Z.S., MORROW J.D. & JOLLOW D.J. (2005): Lipids versus proteins as major targets of pro-oxidant direct-acting hemolytic agents. Toxicological sciences 88(1), 274-283.
NAJMEH S., COOLS-LARTIGUE J., RAYES R.F., GOWING S., VOURTZOUMIS P., BOURDEAU F., GIANNIAS B., BERUBE J., ROUSSEAU S., FERRI LE. & SPICER J.D. (2017): Neutrophil extracellular traps sequester circulating tumor cells via ß1-integrin mediated interactions. Int. J. Cancer 140, 2321-2330.
NAKVASINA M.A. & ARTYUHOV V.G. (2015): Bionanotechnology: achievements, problems, development prospects [in Russian]. Voronezh: Voronezh. gos. un-t., Izdatel'skij dom VGU, 151 pp.
SHILOVA E.V., KOLTAKOV I.A. & ARTYUKHOV V.G. (2019): Ways of inclusion of magnetite nanoparticles in the composition by lipos on the basis of soya lecithin. In: Collection of Scientific Papers of the VI Congress of Biophysicists of Russia: Sciproceedings Vol. 2., p. 273. Krasnodar: Publishing Printing Association «Plekhanovets».
SHILOVA E.V., ARTYUKHOV V.G., SKORBACH ED., TOLOLINA M.V., BLIZNETSOVA G.N. & KOLTAKOV I.A. (2018): On the creation of magnetic-controlled lipos for address drug delivery. Nan-otekhnologii: razrabotka, primenenie: XXI vek 4, 9-14.
