CC BY
11
УДК 547.8
M. V. Bazunova, E. I. Kulish, R. F. Tukhvatullin, L. A. Sharafutdinova, V. G. Shamratova, G. E. Zaikov
ULTRAFINE SYSTEMS ON THE BASIS OF CHITOSAN AND ITS DERIVATIVES POLYMER-COLLOID COMPLEXES WITH PARTICLES OF SILVER IODIDE SOLS
AND THEIR HEMOCOMPATIBILITY
Keywords: polymer - colloidal complexes, Mtosan, micelles of silver iodid, hemocompatibility, acid resistance of erythrocytes
This article presents the results of the development process for the preparation of micro- and nano-sized polymer-colloid complexes (РСС) on the basis of water-soluble natural polymer chitosan (С1.Z) and the sodium salt of chitosansuccinylamid(SСTZ) with silver halide sols in aqueous media. Results of research of С1.Z, sodium salt ofSСTZ solutions and РСС of CTZ and SCZ with colloidal particles of silver iodide influence on structurally-functional properties of erythrocytes' membranes on model of acidic hemolisis are presented in the article. Their influence on nature oferythrocytes distribution by degree of their stability and on kinetic parameters (the beginning, intensity and completion ofprocess of their destruction) under the influence of the damaging agent (HCl) is shown. The comparative analysis of results convinces that СТ.Z, SСTZ solutions and disperse systems on the basis of РСС of СТ.Z and SСTZ with colloidal particles of the silver iodide are capable to modulate variously matrix properties of erythrocytes of blood.
Ключевые слова: полимер-коллоидные комплексы, хитозан, мицеллы йодида серебра, гемосовместимость, кислотоустойчи-
вость эритроцитов.
В данной статье представлены результаты процесса развития для подготовки микро- и наноразмерных полимер-коллоидных комплексов (РСС) на основе водорастворимых природных полимеров хитозана (ZTZ) и натриевой соли хитозана сукциниламида (SСTZ) с солью галогенидом серебра в водной среде. В статье представлены результаты исследования СTZ, раствора натриевой соли SСTZ и РСС из СTZ и SСTZ с коллоидными частицами йодистого серебра и влияние на структурно-функциональные свойства мембран эритроцитов на модели кислотного гемолиза. Показаны их влияние на характер распределения эритроцитов по степени устойчивости и на кинетические параметры (начало, интенсивность и завершения процесса их разрушения) под влиянием повреждающего агента (HCl). Сравнительный анализ результатов убеждает, что СТ.Z,, раствор SСTZ и дисперсных систем на основе РСС из СTZ и SСTZ с коллоидными частицами йодистого серебра способны модулировать по-разному матричные свойства эритроцитов крови.
Introduction
The problem of creating of stable disperse systems based on lyophobic sols stabilized by non-covalent interaction of colloidal particles with macromolecules of natural and synthetic polymers istopical in the following task:
- synthesisof metal nanoparticles in polymer-protectorssolutions;
- creating of metal-polymer nanocompositeswith unique catalytic, electrical, optical and magnetic properties [1];
- obtaining of nano- and micro-sized polymeric containers for targeted delivery of slightly soluble drugs [2, 3].
Using of ultrafine systemsis especially effective while introducing new ways to target delivery of drugs to the affected area of the body and new methods of prolonging the therapeutic effect of drugs. As a means of target delivery of drug with prolonged action nanosized systems based on natural and synthetic biodegradable biocompatible polymers, including chitosan (CTZ) and its derivatives are often used.
The positive charge of CTZ macromolecules promotes its penetration through cell membranes and dense layers of the epithelium, provides good adhesion to mucosa and determines its bacteriostatic properties.
Spontaneously formed aggregates of macromoleculesof CTZ and its derivatives are arousing particular interest during the creating of drug carriers based on these polysaccharides. It's possible to obtain polymeric carriers with desired sizedirectly knowing the laws of aggregates of CTZ and its derivatives formation,
and the factors that determine their size.The theory describing the behavior of polyelectrolytes being associated in dilute aqueous solutions predicts that the stabilization of intermolecular aggregates is determined by the competition between attraction and repulsion (caused by the charged groups on the polymer chain) of groups being associated and the osmotic pressure of counterions which prevents the aggregation. The aggregates sizes depend on the content of associating and charged groups. However,
theavailableexperimentaldataarecontradictory. Also, there is evidence of instability of associated systems based on CTZ and its derivatives[4].
The size of polymeric nanoparticles for targeted drug delivery isequally important.It is known that polymeric carriers with sizes of 100 - 200 nm may provide directional transportation ofantituberculosis drugs directly into macrophages, as macrophages are capable of absorbing foreign objects exactlywith such sizes [5]. Particles of smaller size (30-40 nm to 200 nm) can accumulate passively in antitumor foci by a mechanism known as increased permeability and retention. This happens because of the increased blood supply and lower lymphatic drainage in tumor [6].
One of the approaches for establishing stable nanostructured systems with adjustable sizes may be using of the ability ofmacromolecules to self-assembly by intermolecular association vianon-covalent bonds-on the example of a CTZ and its derivatives polymer-colloid complexes (PCCs), such as sodium salt of chitosan
succinylamide(SCTZ) with inorganiccolloidal particles of lyophobic sols, e.g.,sol of silver iodide.Dispersed system on the basis of sols in the presence of the polymer solution is essentially organic-inorganic nanocomposite in which the polysaccharide macromolecules form shields around inorganic nanoparticles.
The proposed approach is interesting because of:
- the resulting complexes may retain aggregate stability for a long time, that allows to predict the possibility of practical application of composites;
- inorganic colloidal particles of lyophobic sols can act as nucleus for encapsulation of slightly soluble drugs by using nano- and micro-sized containers [7].
An additional advantage of developing methods of targeted delivery of drugs based on the PCC ofCTZ and SCTZ with colloidal particles of silver iodide is antiseptic properties of Agl.
Colloidal systems of drugs' delivery are usually designed both for oral and intravenous administration of drugs [8].Therefore, the study of the biocompatibility of initial polysaccharides CTZ and SCTZsolutions and aqueous dispersions of PCCsof CTZ with blood cells gains special importance.Blood as one of the body fluids serves as a kind of informative biomarker for organism resistance to the action of various factors, allowing to establish the critical points of transition from a pronounced toxic to the stimulating effect.Erythrocytes, like other blood cells respond to changes in the external and internal environment, but due to the morphological simplicity their pathological reactions are slightly informative. Due to this, the action of certain chemicals in subtoxicdoses, usually does not cause microscopically observed changes.However, one of the manifestations of their influence may be changes in cell resistance to the effects of hemolytic agents. Dysfunction of biological membranes under the influence of various exogenous substances is in many cases not only the result, but the cause of pathological changes in the cell and the organism as a whole [9].
Purpose of the work
In connection with the foregoing, it is appropriate to obtain the PCC on the basis of water-soluble natural CTZ and SCTZ polymers with silver halidesols in aqueous media and to study the influence of the sample of CTZ, SCTZsolutions and the resulting dispersion PCC on the structural and functional properties of eryth-rocyte membranes.Taking into account that the main reaction of erythrocytes in contact with a foreign surface is lysis [10], as a model for evaluating the effects of these drugs on cells acid hemolysis selected.
Experimental part
We used CTZ samples withdeacetylation degree of 82% and Mw = 80000 a.m.u. and SCTZ with Mw = 207000a.m.u.with the substitution degree of 75% (produced by "Bioprogress"Russia, Shchelkovo).
The crossover concentration of CTZ and SCTZ determined from the dependence of logarithm ofpolysaccharides solutions dynamic viscosity on theconcentration logarithm was 0.4 wt% and 0.7% wt%. respectively.
AgI sols prepared by standard methods from 7 ml of 0.01 N silver nitrate solution and 10 ml of 0.01 N potassium iodide.
TheinitialAgI micelle particles' size, defined by two independent methods - turbidimetry at a wavelength of 440 and 490nm byFEC-56 instrument, and by particle size analyzer «Shimadzu Salid - 7101» - was between 95 and 120 nm.
Disperse systems on the basis of SCTZPCC with colloidal particles of silver iodide are obtained by mixing of an aqueous solution of SCTZ with 0.4% concentration with freshly prepared AgI sol in volumetric ratio of 1: 1 and 2: 1 (hereinafter - 1: 1SCTZ:AgI sol and 2: 1SCTZ:AgIsol).
Disperse systems on the basis of CTZPCC with colloidal particles of silver iodide are obtained by mixing of CTZ dissolved in 1% acetic acid with concentration of 1 wt.%with freshly prepared AgIsol in volumetric ratio of 1: 1 (hereinafter - 1: 1CTZ:AgI sol).
The1: 1SCTZ:AgI sol, 2: 1 SCTZ:AgI sol and1: 1CTZ:AgI sol dispersions particles' size defined by the particle size analyzer «ShimadzuSalid - 7101»is between 100 and 375 nm.
Quantitative studies of CTZ and SCTZ complexes with AgI micelles are carried out by turbidimetric titration ofAgIsols by polymers' solutions so that the [Ag +]: unit of the polymer ratio is from 0.175: 1 to 2: 1.
Rheological measurements of CTZ, SCTZ solutions and PCC dispersionconducted on a modular dynamic rheometerHaake Mars III at 25°C. Flow curves andviscosity curves were obtained in a constant shear stress mode at a shear rate of 0.1 to 100 s-1.
Influence of solutions CTZ, SCTZ and disperse systems based on CTZ and SCTZ PCCs with colloidal particles of silver iodide on the structural and functional properties of erythrocyte membranes was estimated by recording the kinetics of acid hemolysis [2]. In the invitro experiment, used blood in an amount of 0.5 ml obtained from the tail vein of Wistar (n = 20) rats weighing 200-250g in compliance with the European Convention requirements for the Protection of Vertebrate Animals used for experimental and other scientific purposes (Strasbourg, 1986) and the Russian Federal Law "On Protection of Animals on Cruelty" from 01.01.1997. Heparin is used as anticoagulant. Study of the influence degree of ultrafine systems on the basis of chitosan and its derivatives on the physicochemical properties of the lipid-protein complexes of the plasma membranes was performed on the model system CTZ sample-erythrocytes-HCldue to erythrocyte resistance change because of influence of hydrochloric acid (0.08 ml) in iso-osmotic solution ofNaCl (0.85%).Hemolysis was carried out in quartz cuvettes with exterior dimensions of 20*40*10 mm and a volumetric displacementof 5 ml. InitialAgIsol,aqueous solution of SCTZ withconcentration of 0.2 wt.%, CTZ dissolved in 1% acetic acid with concentration of 0.5 wt.%, aqueous dispersion of 1: 1SCTZ:AgI sol and 2: 1 SCTZ: AgIsol, 1: 1 CTZ: AgI solare used asactiveagents. Hemolysis intensity in time recorded photometrically using photoelectrocolorimeterKFK - 3.Determination of extinction was performed every 15 seconds until no further changes. Reducing of extinction - result of the
gradual destruction of red blood cells, and those cells are first broken, which have weaker resistance to hydrochloric acid.According to the obtained values acidic erythrograms depicting the distribution of erythrocytes by resistance were built: on the horizontal axis - hemolysis time, which is a measure of the resistance, delayed, the vertical axis - the percentage of erythrocytes. In the control, the differential distribution curve of erythrocytes by resistance takes the form of unimodal curve with steeply descending branch.Analysis of the kinetics of acidicerythrograms performed using the following parameters: start time, end time and the peak of hemolysis, the width of the interval of the dominant group of erythro-cytes in the population.The proportion of cells with different resistance in the general population of erythro-cytes was calculated.
Mathematical and statistical analysis of the results was carried out in the application package STATISTICA v. 7.0 («StatSoftInc», USA).Comparison of acidicerythrograms was performed using the nonpar-ametric Mann-Whitney test, the null hypothesis of no difference was rejected at p <0.05.
Discussion of the results
The particle sizes of initial AgI micelles, defined according to the turbidity data and measurement using a particle size analyzer «Shimadzu Salid- 7101» are, on average, 95-120 nm. Consequently, one can expect the formation ofpolycomplexeson the basis of AgI sols with water-soluble polymers having a particle size close to the nanometer range.
To find the critical molar ratio of [Ag +]: polysaccharideunit, leading to the formation of an insoluble complex, theturbidimetric method was used at polymer concentrations below the crossover point to avoid the mutual influence of macromolecular coils.The results are shown in Figure 1.
■ ■ • - CTZ-KOH-Agl sol
Fig. 1 - The dependence of the optical density of mixtures of CTZsolutions with AgIsol, of CTZ solutions in the presence of 0.01 mol / lKOH with AgIsol, of SCTZ solutions with AgI sol on composition of the mixture Z = [Ag +] / [polymer unit] at t = 20° C, l = 490 nm
It has been established that for PCC on the basis of SCTZ and Agi sol range of molar ratios wherein aggregate stability of the particles observed is larger than for systems based on CTZ-AgIsol and even particularly neutralized CTZ- AgIsol.This can probably be attributed to the higher density of the CTZ charge distribution on the polysaccharide macromolecules and high ionic strength of solution.
In any case, it was found that the addition of a weak polyelectrolyte increases sedimentation stability of AgI micelles.
The particle sizes of obtained PCC on the basis of SCTZ solution mixtures with AgIsol, ranging from 100 to 375 nm.
Significant increase in the dynamic viscosity SCTZ:AgI sol system at middle concentrations that are lower in comparison with SCTZ's (Fig. 2) indicates the formation of the PCC and the structuring effect of AgI particles inSCTZ: AgI systems.
Fig. 2 - Increase of the dynamic viscosity of the SCTZ: AgI sol system in comparison with solution SCTZ with the polymer concentration increasing, 200C, AgI particle concentration of 0,004 mol / l
The results of studies on the hemocompatibility (Table 1) showed that the studied solutions of samples ofCTZ, SCTZ and PCC dispersions of CTZ and SCTZ with colloidal particles of silver iodide have ambiguous effect on the functional state of therat blood cell mem-branes.Comparative analysis confirms that the tested samples are capable tochangethe character of the distribution of red blood cells in terms of their stability and kinetic parameters of hemolysis indifferent ways. Particularly 0.2% SCTZ solution had no significant effect on the initial capacity of red blood cells to resist the action of hemolytic agent.At the same time, a sample of the disperse systemof 2: 1 SCTZ: AgI sol discovered the ability to change the functional state of erythrocyte membranes. During all periods of observation destruction intensity of red blood cells by the action of hydrochloric acid and the totallysis duration markedly differed from the original characteristics, increased for resistant erythrocytes. Apparently, this SCTZ composition has a stabilizing effect on the erythrocyte membranes.
Estimating theresistance degree after the incubation of red cells with dispersion of 1: 1HTZ: AgI sol,it should be noted that despite the slight decrease erythrocyte fraction with average stability (41.6%), the number of red blood cells with a high and increased stability in comparison with the control (24 6 ± 2,9% and 21,2 ± 2,9% respectively, p <0,01) significantly increases.More later involvement of red blood cell in the process of hemolysis was found, that could indicate a protective effect of the compound.
Comparison ofstudies results of the effect of ultrafine systems based on chitosan and its derivatives on the integrity of red blood cells demonstrated the ability of some (2: 1SCTZ: AgI sol, 1: 1 CTZ: AgI sol, 1: 1
SCTZ: AgI sol) to provide a stabilizing effect on the membrane of red blood cells and others (0.2% aqueous SCTZ solution) to cause a decrease in resistance of erythrocytes. The observed effects may be determined by the variety of features of their impact on the nature of the bonds between the protein and lipid components of the membrane, the level of activity of cell enzyme systems, the value of the negative charge of the mem-Table 1 - Indicators of acidic resistance of erythrocytes solutions, sodium salt of chitosan succinylamide (SCTZ)
brane surface [11]. Taking into account that the acid resistance of erythrocytes is mainly determined by erythrocyte membranesphospholipid bilayer state, it can be assumed that the observed effects are specified by reorganization of the lipid component of erythrocytes membranesunder the exposure of ultrafine systems on the basis of chitosan and its derivatives.
in the control group and after exposure of chitosan (CTZ)
and polymer-colloid complexes (PCCs) of CTZ and SCTZ
Indicators Control 0,2 % SCTZ solution 0,5 % CTZ solution in 1 %acetic acid The aqueous dispersion of 2: 1 SCTZ: AgI sol The aqueous dispersion of 1: 1 SCTZ: AgI sol The aqueous dispersion of 1: 1 CTZ: AgI sol
Time of the beginning of hemolysis, min 0,5±0,01 1,00±0,1 1,5±0,4 1,00±0,1 1,5±0,2 1,5±0,3
Time of the ending of hemolysis, min 5,5±0,1 6,5±0,2 6,5±0,4 6,5±0,3 6,5±0,2 6,5±0,4
Peak of the acidicerythrogram, min 2,00±0,3 2,00±0,1 2,00±0,1 3,00±0,2* 2,0±0,1 3,00±0,2*
Erythrogramsbasewidth, min 5,00±0,1 5,5±0,18 5,00±0,2 5,5±0,1 5,0±0,2 5,5±0,3
The percentage of highly resistant erythrocytes, % 12,9±1,6 11,5±1,4 13,16±2,1 34,1±4,7* 24,9±3,8* 24,6±2,9*
The percentage of erythrocytes with increased stability,% 4,78±0,31 8,6±1,1* 6.96±0,8 19,1±2,6* 13,00±2,1* 21,2±2,9*
The percentage of erythrocytes with average stability,% 73,19±5,86 63,3±4,7 72,5±5,2 35,8 ±3,7* 52,3±4,6* 41,6±4,1*
The percentage of erythrocytes with low stability,% 9,13±0,87 16,6±1,4* 7,38±0,6 11,0±1,8 11,29±2,3 12,6±2,5
Note: * - statistically significant differences in comparison to the control group (p<0,01).
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Conclusion
Thus, the method of obtaining of stable nano-and micro-sized PCC son the basis of water soluble natural polymers CTZ and SCTZ with sols of silver halide in aqueous media was developed. It was found that CTZ and SCTZ solutions and dispersions of polymer-colloid complexes ofCTZ and SCTZ with colloidal particles of silver iodide have a stabilizing or destabilizing effect on erythrocyte membranes depending on the composition of the sample,that is show their different hemocompatibility and points on the urgency of further search of chitosan its and derivatives complexes with inorganic colloidal particles having potentially protective properties.The results of these studies are promising at the obtaining of nano- and micro-sized polymeric containers for targeted delivery of slightly soluble drugs.
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