Nanopolymer GluLa-DPG-PEG600-F Can Penetrate into Cells and Deposite in Rats Body

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Nanopolymer GluLa-DPG-PEG600-F Can Penetrate into Cells and Deposite in Rats Body

Bogdan Chekh, Maria Ferens, Natalia Susol, Sergiy Varvarenko, Dmytro Ostapiv, Vasyl Vlizlo

Abstract

Via fluorescence microscopy we found that nanopolymer GluLa-DPG-PEG600-F can penetrate bovine sperm cells via binding membranes and deposit in the cite of injection in muscle tissues by binding proteins.

Nanopolymer influences on sperm cells metabolic activity decreases oxygen intake and redox activity. We also determined sperm cell survival time, that was decreased of high doses of nanopolymer added to sperm.

Key words: sperm cells, laboratory rats, nanopolymer, pseudo-polyamino acids, metabolic processes.

Анотація

Досліджено нанополімер на основі псевдополіамінокислот GluLa-DPG-PEG600-F. За допомогою люмінесцентної мікроскопії встановлено здатність GluLa-DPG-PEG600-F проникати в клітини (спермії бугаїв), а також депонуватись у тканинах організму щурів за внутрішньом'язового введення. За різних доз полімеру (0,01, 0,05 та 0,1 мл/мл розрідженої сперми) визначено активність окисно-відновних процесів у сперміях і їх виживання за додавання GluLa-DPG- PEG600-F. Нанополімер GluLa-DPG-PEG600-F спроможний зв'язуватися з мембранами та проникати в структурні компоненти клітин. При цьому GluLa-DPG-PEG600-F депонується в м'язах дослідних щурів у місці введення за допомогою зв'язування з білками. Вплив нанополімеру GluLa-DPG-PEG600-F на метаболізм клітин (сперміїв бугаїв) за доз 0,01, 0,05 й 0,1 мл/мл розрідженої сперми характеризується зменшеним споживанням кисню на 26; 40 та 56 % та відновної здатності дихального ланцюга мітохондрій на 73; 80 і 87 % відповідно.

Ключові слова: спермії бугаїв, лабораторні щурі, нанополімер, псевдополі-амінокислоти, метаболічні процеси.

nanopolymer sperm cell metabolic

В работе исследуется нанополимер на основе псевдополиаминокислот GluLa-DPG-PEG600-F. С помощью люминесцентной микроскопии установлена способность GluLa-DPG-PEG600-F проникать в клетки (сперматозоиды быков), а также депонироваться в тканях организма крыс. При введения различных доз полимера (0,01, 0,05 и 0,1 мл/мл разреженной спермы) установлена активность окислительно-восстановительных процессов в спермиях и их выживания. Нанополимер GluLa-DPG-PEG600-F обладает способностью связываться с мембранами клеток и проникать в их структурные компоненты. При этом GluLa-DPG-PEG600-F депонируется в мышцах крыс в месте введения путем связывания с белками. Влияние нанополимера GluLa-DPG-PEG600-F на метаболизм клеток (сперматозоидов быков) при дозах 0,01, 0,05 и 0,1 мл/мл разреженной спермы характеризуется уменьшенным потреблением кислорода на 26 %; 40 % и 56 % и восстановительной способности дыхательной цепи митохондрий на 73 %, 80 % и 87 %, соответственно.

Ключевые слова: сперматозоиды быков, лабораторные крысы, нанополимер, псевдополиаминокислоты, метаболические процессы.

Introduction. Today pharmaceutical industry has several main tasks for developing, in particular with the creation and use novel nanopolymeric systems for drug delivery [1; 2]. Main interest for scientists - are creating novel nanopolymer systems based on pseudo-polyamino acids and its using as adjuvants for vaccines development. The main task of our research work is to create and test novel nanopolymer as drug delivery system especially for adjuvant selection in vaccines development.

Pseudo-polyamino acids (PPA) - class of polymeric compounds based on aminoacids, which structure doesn't include peptide bonds. Class of nanopolymers described in this article consist of pseudo-polyamino acids which are derived from natural a-dicarboxylic acids and polyols that has advantages of the polymer that are known by the brand name «Pluronic». The resulting nanopolymer has the ability to self-stable aqueous dispersions like «Pluronic» molecules [7]. Nanopolymer consist of polyols of polyoxyethylene and polyoxypropylene with much lower molecular weight that «Pluronic». This nanopolymer refers to pseudopolyamino acids, it means that its components include natural, inherent human body amino acids which doesn't contain peptide bounds. New class of amphiphilic pseudo-polyamino acids of polyester type based on natural dicarboxylic acids are untoxic and don't cause side effects for laboratory rats. Additionally, scientists can bind fluorescein molecules that are biologically neutral to nanopolymers that allow to visualize their penetration and deposit in cells, tissues and organs of laboratory animals [3]. The main purpose of our research work is to test nanopolymer GluLa-DPG-PEG600-F for its future use as drug delivery system. There were several tasks of our research, first - to find out the ability of nanopolymer GluLa-DPG-PEG600- F to penetrate mammalian cells tissues and after explore its effect on sperm cells redox activity and sperm cell survival time.

Methods. For our investigation we created nanopolymer GluLa-DPG-PEG600-F, that consist of glutamic and lauryl acids (GluLa), dipropylene glycol (DPF), polyethylene glycol (PEG600) and fluorescein (F) - which covalently attached to mail macromolecule chain (Fig. 1).

Fig. 1. Structure of Nanopolymer System GluLa-DPG-PEG600-F

For studying the ability of nanopolymer GluLa-DPG-PEG600-F to penetrate inside sperm cells, it were collected bovine sperm ejaculates in volumes of 3-5 ml and concentration of 0,71 - 1,12*10⁹ cells/ml with 65-85 % of living sperm cells.

To study the ability of nanopolymer to spread in organs and tissues - we were collected mature rats Rattus norvegicus var. Alba, line Wistar, weighing 250-300 g.

To study the ability of nanopolymer GluLa-DPG-PEG600-F to penetrate inside mammalian cells, sperm ejaculates were mixed with 2 % water dispersion in ratio of 10:1 and applied to a glass slide.

To find out the localization of GluLa-DPG-PEG600-F in rats tissues, experimental groups of rats were intramuscularly injected by 2 % water dispersion of nanopolymer, rats from control group were intramuscularly injected by saline.

On 1-st, 4-th and 18-th hours of experiment, experimental and control groups of rats were decapitated in softly chloroform condition. After we made histological analysis of muscles tissues in the site of injections, other muscles tissues, spleen, liver, brain and kidneys tissues using cryostat.

The ability of GluLa-DPG-PEG600-F to penetrate cells and bind tissues structure components were investigated by luminescent microscopy. We studied the influence of nanopolymer on metabolic activity of sperm cells, samples were divided on: control group of sperm cells - dilutioned in ratio of 1:4 with nutrient medium «Optydyl» and experimental groups with additional of 2 % water dispersion of GluLa-DPG- PEG600-F in doses of 0,01, 0,05 and 0,1 ml/ml sperm cells ejaculates. It were researched: ex tempore - respiratory activity (using polarograph; ng 0/0,1 ml sperm x min) in thermostatted tube (t = 38,5°C), tube volume = 1,0 ml with automatic registration of process, redox activity (using polarograph; mV/0,1ml sperm x min) with open microelectrodes system and activity of succinate-coenzyme Q reductase SQR (E/0,1 ml sperm x hour) [4; 5]; also we studied sperm cell survival time (hours) to the termination of rectilinear forward movement of sperm cells preserved at sperm in temperature of 2-4 ° C.

Statistical analysis of the results were conducted by statistical methods to determine the coefficient of reliability.

Results and Discussion. We found that nanopolymer GluLa-DPG-PEG600-F can penetrate cells via binding membranes and get into nucleus, acrosome, mid and tail of sperm (Fig. 2).

Fig. 2. Bovine Sperm Cell Under the Influence of GluLa-DPG-PEG600-F; 1 - Acrosome; 2 - Nucleus; 3 - Mid, 4 - Axoneme, 5 - Tail; (Total Magnification - 900x)

Intensity of the glow indicates on the interaction between nanopolymer and components of sperm: high level of penetration into membranes, nucleus, mid and tales and lower level of nanopolymer penetration into acrosome and axoneme in comparison with other components of sperm cell (Fig. 2).

We studied GluLa-DPG-PEG600-F localization in muscle tissues on 1-st and 4-th hours after injection of nanopolymer (Fig. 3).

Fig. 3. Luminescent Microscopy of Muscle Tissue After the Injection of GluLa-DPG-PEG600-F (Total Magnification - 900x): a - Muscle Tissue of Control Group of Animals; b - Muscle Tissue on 1st Hour After Injection of GluLa-DPG-PEG600-F; c - Muscle Tissue on 4th Hour After Injection of GluLa-DPG-PEG600-F; d - Muscle Tissue on 18th Hour After Injection of GluLa-DPG-PEG600-F.

Our investigation show intensive luminescence of nanopolymer only in muscle tissue in sites of injections. In spite of this, there were found no specific luminescence of GluLa-DPG-PEG600-F in spleen, liver, brain and kidneys tissues of experimental rats.

The Analysis of luminescence intensity shows higher in 5 and 4,5 times GluLa-DPG-PEG600-F luminescence in muscle tissue in sites of injections on 1st and 4st hours after the injection of nanopolymer. Localization of GluLa-DPG-PEG600-F in site of injection shows its ability to bind protein in muscle tissue [6]. The Intensity of nanopolymer luminescence on 18th hours after injection was lower in 3,1 and 2,9 times compared to 1st and 4th hours of experiment.

In spite of the decreasing luminescence intensity in cite of injection on 18th hours after injection, it was higher on 53 % compared to luminescence intensity of muscle tissue in control rats. Muscle tissue of the control group of animals had only low autoluminescence.

Low luminescence of GluLa-DPG-PEG600-F on 18th hour of experiment compared to luminescence intensity on 1st and 4th hours of experiment shows gradual degradation of nanopolymer.

In conclusion, nanopolymer GluLa-DPG-PEG600-F is able to bind muscle cell proteins that provide its deposit in the cite of injection in muscles.

We researched possible impact of GluLa-DPG-PEG600-F on the intensity of oxidative processes in sperm cells. We studied respiratory activity of sperm cells and discovered, that sperm cell oxygen intake is 0,8 ng 0/0,1 ml sperm(C) x min and after nanopolymer addition to sperm cells, oxygen intake decreased on 26 % after adding 0,01 ml of GluLa-DPG-PEG600-F, decreased on 40 % after adding 0,05 ml, and on 56 % after adding 0,01 ml of nanopolymer (Fig. 4). The same changes were detected in mitochondrial redox activity which was lower that in control group on 73 % after adding 0,01 ml of GluLa-DPG-PEG600-F, lower on 80 % after adding 0,05 ml and on 87 % after adding 0,1 ml of nanopolymer, redox activity in control group was 0,15 mV/0,1ml sperm x min.

Fig. 4. Respiratory Activity of Sperm Cells and Mitochondrial Redox Activity After Additing of NanopolymerGluLa-DPG-PEG600-F in Doses of 0,01, 0,05 and 0,1 ml/ml Sperm, Nutrient Medium «Optydyl» - Control

Despite of decresed cell respiration and mithochondrial redox activity of sperm cells, after adding 0,01 ml of nanopolymer to 1 ml sperm - SQR activity and time of sperm cell survival were in the range of control group. (Tab. 1).

Table 1 Time of Sperm Cell Survival and SQR Activity Under the Influence of Nanopolymer GluLa-DPG-PEG600-F, n = 6; M ± m

Note. Coefficient of reliability - * р < 0,05.

After adding of GluLa-DPG-PEG600-F in doses of 0,05 and 0,1 ml/ml sperm - SQR activity decreased on 31 % and 56 % and time of sperm cell decreased on 18 % and 71 % compared to control group (p <0,05). Probably, nanopolymer GluLa-DPG-PEG600-F interacts with components of membrane (lipids and proteins) and with organelles (mitochondria) and affects on sperm cells ability to use substrates of mithochondrial ETC.

Conclusions and Further Perspectives. Nanopolimer GluLa-DPG-PEG600-F can penetrate cells via binding membranes and get into nucleus, acrosome, mid and tail of sperm. Nanopolymer GluLa-DPG- PEG600-F is able to bind muscle cell proteins that provide its deposit and gradual degradation in the cite of injection in muscles. Nanopolymer GluLa-DPG-PEG600-F effect on bovine sperm cell metabolism characterized by reduced oxygen consumption on 26 %, 40 % and 56 % and mitochondrial redox activity on 73 %, 80 % and 87 % by proportionally increasing doses (0,01; 0,05 and 0,1 ml/ml sperm). In our further research we will study the impact of nanopolymer GluLa-DPG-PEG 600-F on rats immunity.

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