Biofunctional nanomaterials based on ultrafine silica , protein and aminocarbohydrates

Aim. Investigation of adsorptive interactions of N-acetyl-D-glucosamine (GlcNAc) and N-acetylneuraminic acid (NANA) with ultra fine silica (UFS) modified by protein bovine serum albumin (BSÀ). Methods. Adsorption, IR-, Í NMRand laser Doppler spectroscopy. Results. Conditions for obtaining bionanocomposites (BNC) UFS/GlcNAc, UFS/BSÀ/GlcNAc and UFS/BSÀ/NÀNA are developed. Conclusions. Impellent ability of deconservated bovine gametes decreased in the presence of BNC after adsorptive fastening of protein on UFS surface. The H NMR spectroscopy data proved that interaction of GlcNAc with protein was accompanied by essential dehydratation of protein molecules.

Introduction.Ultrafine silica (UFS) is a promising carrier for immobilization of many synthetic and natural compounds which allows creating biologically active nanocomposites (BNC) for biotechnologies on its basis [1,2].Large specific surface area of UFS (S sp = 300 m 2 /g) is conditioned by a small size (4-40 nm) of its primary particles.It determines high adsorptive capability of UFS while binding many substances, including biomolecules.Silane groups (ºSi-OH) are adsorption centres on the surface of UFS.
Hydroxylic surface determines the considerable hydrophilic properties of UFS surface and capability of sorbing polar molecules.
UFS is widely used as an additional substance in the production of many medicinal preparations, as its biological safety has been well established [3].The studies of the Chuiko Institute of Surface Chemistry, NAS of Ukraine, have extended the ideas concerning the properties of this silica.It was proven that the addition of UFS in rather low concentrations to the suspension of cells (yeast, microorganisms, gametes, erythrocytes) stimulates their viability [2,4].Immobilization of some biomolecules (proteins, carbohydrates, vitamins, etc.) on UFS surface allowed creating BNC, increasing this effect [4].
These results served as a basis for the elaboration of BNC, which allow optimizing cryomedia in the technology of long-term storing a gene pool of some agricultural animals.Different carbohydrates (mono-, oligo-and aminosugars) are used in such BNC.Their introduction into the medium with bovine gametes at the stage of deconservation results in considerable prolongatio of the cells survival [5].In these studies the method of non-covalent immobilization was used for sacñharose, lactose, raffinose as well as for aminosugars N-acetyl-D-glucosamine (GlcNAc) and galactosamine.However, this method appeared inefficient for fastening monosugars and N-acetylneuraminic acid (NANA) on the UFS surface [6].Nevertheless, pretreatment of UFS surface with bovine serum albumin (BSA) allowed fastening these carbohydrates on UFS surface and obtaining BNC on their basis [7].
GlcNAc, tied up on the UFS sur face, may be come a prom is ing mod i fier in cre at ing BNC with high level of af fin ity to oligosaccharide struc tures of cell sur face recep tors.For this pur pose the pa ram e ters of GlcNAc ad sorp tion should be stud ied as well as a pos si bil ity to im prove it through the pre lim i nary sur face mod i fi cation with pro tein.There fore, the aim of this work was to com pare an abil ity of GlcNAc and NANA to ad sorb on the sur face of com pos ite ad sor bent UFS/BSA, to study some phys i cal and chem i cal prop er ties of BNC ob tained, and to de ter mine their bioactivity in cryomedium in relation to bovine gametes.
Ad sorp tion of GlcNAc and NANA on UFS/BSA from the wa ter phase was stud ied as de scribed in [8].Ini tial UFS was an ad sor bent for GlcNAc.The ad sorbate:ad sor bent ra tio was 1:10; ini tial con cen tra tions: BSA -1-14 mg/ml; GlcNAc -0.2-1.6 mg/ml, NANA -16-60 µg/ml.The pro tein was ad sorbed at pH 4.8.Ad sorp tion time for BSA and GlcNAc was 2 h, and for NANA -1 h.The pre cip i tate was iso lated by centrifugation for 10 min at 4000 rpm, dried (t = 37°C) and me chan i cally grained for fur ther in ves ti ga tion.The sub stance con cen tra tion in supernatant was mea sured by the fol low ing meth ods: for pro tein - [9]; for GlcNAc - [10], for NANA - [11] with sub se quent calcu la tion of ad sorp tion value ac cord ing to the for mula [2]: A = (C ini tial -C equil ) V/m, where C ini tial and C equil -initial and equi lib rium con cen tra tions in the so lu tion, respec tively, mg/ml; V -so lu tion vol ume, ml; m -ad sorbent mass, g.The mea sure ments were per formed on Lambda-35 spectrophotometer (Perkin-Elmer, USA) and photoelectrocolorimeter KFK-2.The ef fi ciency of ad sorp tive in ter ac tions of biomolecules and ad sor bent was es ti mated by ad sorp tion iso ther mal curves.Cal cula tions of ul ti mate ad sorp tion A ¥ values were conducted as described in [8].
BNC were studied by IR-spectroscopy in the wavelength range of 4000-400 cm -1 on Thermo Nicolet Nexus FTIR spectrophotometer using the attachment for diffused reflection SMART Collector.Their samples were mixed with preliminary dried KBr (Riedel-de Haen, France, AR) in the ratios 1:19 for protein and 1:4 for carbohydrate.The omnic software was used for the processing of spectra.
An efficiency of protein interaction with UFS surface was estimated by intensity of band adsorption at the wavelength of 3750 cm -1 , distinctive for silane groups, prior to and after BSA adsorption [12].
The mech a nism of pro tein in ter ac tion with GlcNAc was stud ied us ing 1 H NMR spec tros copy on the sam ples of hu man se rum al bu min (HSA) (Kyiv Blood Trans fusion Cen tre). 1 H NMR spec tra were mea sured with the high-res o lu tion NMR-spec trom e ter Varian Mer cury 400, the work ing fre quency of 400 MHz and at 90°C-prob ing im pulse for 2 µs.To pre vent the over cooling of bound wa ter, 1 H NMR spec tra were re corded at heat ing the sam ples, pre lim i nary cooled to 200 K.
The temperature of samples was regulated using thermal attachment Bruker VT-1000 with ±1 K precision, integral intensities of signals were determined with ±10% precision.The characteristics of bound water were determined according to [12][13][14].The calculations by cryoporometry method were performed by the procedure, described in [12,14], using Gibbs-Thomson ratio for decrease of water freezing temperature (DT) in cylindrical pores of radius R (DT = k/R).
La ser-dopp ler spec tros copy with Spectrolas Instrumentas Model LDS MQE de vice (Ukraine) was used for de ter mi na tion of BNC bi o log i cal ac tiv ity accord ing to the pa ram e ters of move ment of re pro duc tive cells af ter deconservation of gran ules of bo vine sperm, cryopreserved in the lac tose-glyc erol-yolk (LGY) medium [15].BNC sus pended in 2.9% so lu tion of so dium ci trate was added to deconservated sperm.The range of stud ied con cen tra tions was 0.002-0.6%.The ra tio of vol umes of sperm and BNC sus pen sions was 1:3.The mea sure ments were con ducted af ter sperm in cu ba tion with BNC at t = 37°C for 1 h.The cells were il lu minated by la ser He-Ne at 632.8 nm in 1-mm cuvettes for 3 min.The ac tion of BNC on cells was es ti mated by the num ber of mov ing cells (%), fre quency of their ro ta tion (Hz), speed (µm/s) and motional en ergy (con ven tional units).The pa ram e ter "motional en ergy" (N) is en ergy con sump tion for move ment of cells in vis cous me dium, cal cu lated by the fol low ing for mula: N = g • V 2 , where V -speed of cell move ment; g -co ef fi cient, re lated to the form and sizes of sperm cells, and the me dium features [15].The re sults were ob tained with the con sid eration of de crease in motorial ac tiv ity of cells in time com pared to the con trol sam ple with out BNS taken as 100%.Bioactivity was es ti mated by the ratio of cumulative indices in the sample to the control for the whole period of measurements.
Results and Discussion.According to [2] adsorption of BSA on UFS is maximal at its isoelectric point -at pH 4.8.The isothermal curve looks like Langmuir's curve (Fig. 1, a).According to Giles classification of isothermal curves [16], this curve belongs to type L2.Maximal adsorption (A) is 410 mg/g which testifies to strong interaction of BSA and UFS.Maximal desorption of BSA from the surface of UFS does not exceed 11.3%.
Preliminary immobilization of BSA on the surface of UFS promotes adsorption of NANA, the isothermal curve of which is presented in Fig. 1, b. Desorption of NANA from the surface of composite BNC/BSA does not exceed 0.015%.
A comparison of isothermal curves of adsorption, presented in Fig. 1, c, demonstrates that GlcNAc is sorbed on UFS/BSA several times worse than on UFS.Relatively high adsorption of GlcNAc on UFS may be related to the formation of salt-like adducts with a transferred proton.
De crease or com plete ab sence of ab sorp tion band of free SiOH-groups (n = 3750 cm -1 , Fig. 2) is ob served in IR-spec tra of car bo hy drates, in ter act ing with UFS or UFS/BSA com pos ite, which proves their bind ing to the func tional groups of ad sorbed mol e cules [2].The shifts in ab sorp tion bands of Amid I-va lence vi bra tions C=O and C-N (from 1650 to 1657 cm -1 ) and Amid II-valence C-N and deformational vi bra tions NH (from 1550 to 1657 cm -1 ), ev i dent in IR-spec tros copy of proteins, were ob served in BNC of UFS/BSA [17].It tes tifies to the for ma tion of H-bond be tween NH-groups of BSA molecule and hydroxyls of UFS [18].
A con sid er able de crease in the in ten sity of ab sorption band at 3750 cm -1 in case of the for ma tion of UFS/GlcNAc (Fig. 2, a) is con di tioned by the par tic i pation of SiOH-groups in hy dro gen bonds of GlcNAc with UFS.At the same time there is a wide ab sorp tion band in the range of 2700-3600 cm -1 , caused by si multa neous ap pear ance of NH bonds in aminogroup (3350 cm -1 ) and va lence vi bra tions of CH-(2960, 3020, 3300 cm -1 ) and OH-groups (3550 cm -1 ) [18].
In IR-spectra of UFS/BSA/GlcNAc composite (Fig. 2, b, spectrum 3) there are an evident shoulder at 1630 cm -1 , corresponding to physically adsorbed water, and an absorption band in the range of 1730 cm -1 , which belongs to vibrations C=O-group of aminosugar (1850-1550 cm -1 ) [19].IR-spectra also show peaks at 1545 and 1650 cm -1 , corresponding to deformational vibrations of NH 2 -group, and an absorption band at 1520 cm -1 of deformational vibrations of NH-group.
In general, IR-spectrum of UFS/BSA/NANA composite is similar to that of GlcNAc.It was observed the vanishing or decrease of absorption bands in the range of 3500-3100 cm -1 , specific for NH-bond in aminogroup of carbohydrates, a band of 1730 cm -1 , as well as a number of bands in the range of 1241-1015 cm -1 , corresponding to frequencies of deformational vibrations of groups CH 3 , CH 2 , and CH [20].Therefore, immobilization of GlcNAc and NANA on the surface of UFS/BSA composite occurs with the participation of hydroxylic, carbonyl and aminogroups of both protein and the carbohydrates studied.
The binding of GlcNAc to albumin globules in solutions was studied in order to reveal the reasons of weak desorption of GlcNAc from the surface of UFS/BSA composite.Fig. 3 demonstrates   water signal, comparing with the intensity of water signal prior to freezing (which is 19 g/g for 5%-solution of HSA); dependences of changes in free Gibbs energy on the concentration of non-freezing water; temperature dependences of changes in concentration of non-freezing water, conditioned by the presence of GlcNAc; distribution of non-freezing water along the radii of nanodrops of strongly associated water; and changes in DC uw depending on the value of DG.The values D 1 were calculated determining the difference in C uw at fixed temperature of measurements.The radii of nanodrops of non-freezing water were calculated by Gibbs-Thomson equation (R = DT/k, where k = 50 degrees ´nm [12]) for the portions of water, frozen at changing temperature in the range from T 1 to T 2 .
As seen from the data in Fig. 4, c, dependences D 1 (T) are complex.At T = 270 K D > 0, i.e. the presence of GlcNAc increases the concentration of non-freezing water in the system.The increase in C uw is 50-100 mg/g (up to 15% from C uw max ) at C GlcNAc = 0.05-0.5%.One of the reasons of increase in C uw may   be decrease in freezing temperature of the solution, conditioned by the decrease in the density of saturated vapour (Raul's law).However, taking into consideration the fact that C GlcNAc does not exceed 1 [´]10 -4 mg/l, freezing temperature cannot decrease more than by 0.1 degree, i.e. this is not the main reason.At T < 270 K the value D 1 < 0, with a wide minimum at T = 240 K in the dependences D 1 (T).Maximal decrease in the concentration of non-freezing water was observed for the sample, containing 0.05% GlcNAc.
The regularities obtained may be explained by efficient interaction of GlcNAc and albumin molecules.As protein molecules are highly hydrated, the molecules of organic admixture (GlcNAc in our case) are capable of replacing a part of hydrate water.It is reasonable to expect such decrease in hydration degree that corresponds to the replacement of the volume of hydrate water, equal to the volume of molecules of organic admixture [12].The data in Fig. 4, c show that maximal decrease in C uw volume reaches 160 mg/g which is an order higher than this volume.Therefore, the main reason of the observed peculiarities of behaviour of dependences D 1 (T) should be considered altering of the interaction parameters in microgel structures of protein which may become either stronger (which results in decrease in C uw value) or weaker in accordance to C GlcNAc .The use of Gibbs-Thomson equation allows determining a size distribution of water clusters or cavities (compartments), filled with water in frozen aqueous solutions of albumin, and the influence of organic admixture molecules (GlcNAc) on them (Fig. 4, d).Several compartments, the number of which is determined by the number of points on temperature dependence C uw (T)), with the radii in the range of 0.7-11 nm can be identified.According to the data in Fig. 4, b, water in clusters with the radii of 6 and 11 nm may be related to weakly bound water.The transition area is conditioned by clusters with the radii of 2 and 4 nm; strongly bound water (SBW) -by the clusters with R < 2 nm.
As seen from the data in Fig. 4, d, GlcNAc admixtures have different influence on the amount of water, located in clusters of different size.The volume of SBW, localized in small cavities, decreases while the volume of water in cavities with the radii of 2 and 6 nm increases considerably.Therefore, there are two opposite tendencies in the change in volumes of water structures, which determine a relatively weak dependence of the interphase energy in a wide range of concentrations of GlcNAc admixtures, which distinguishes it from plain sugars (glucose and fructose) [12].While interacting with the fragments of protein molecules, GlcNAc is likely to block the centres of their intermolecular binding.Taking into consideration that GlcNAc molecules contain electron-donating atoms of nitrogen and oxygen, it is possible to assume its interaction with the protein in acid centres mainly.The data in Fig. 4, d demonstrate that at T = 270 k water in cavities with the radii of 6 nm freezes.It is most likely that the cavities of this size are located in spaces between closely located albumin molecules (the size of protein globule does not exceed 7 nm).Therefore, the breaking of protein-protein bonds results in the formation of clusters of this very size in albumin microgel.In BNC of UFS/BSA, the biopolymer molecules cover a considerable area of silica surface.Thus, it is possible to assume that protein fraction on the surface of silica acts in a way, similar to the behaviour of microgel structures in the solution.
The study of parameters of mobility of deconserved bovine sperm gametes in the presence of BNC demonstrates a biological activity of the gametes that becomes evident through the increase in studied indices for experimental samples compared to the control.There is also an evident dependence of this result on the BNC concentration in the medium with cells.In this respect an obvious parameter is motional energy which is proportional to the force of the moving cell (which depends on its friction in viscous medium) and the speed of movement [15].The increase in mobility of this type of cells testifies to the increase in viability [2].
Fig. 5 demonstrates a histogram which allows comparing bioactivity of obtained BNC by the ratio of cumulative energy of gametes to that of the control cells.An increase in the motional energy of gametes compared to the control was observed in the range of studied concentrations (except for C UFS/BSA/NANA = 0.002%).The highest activity is notable for UFS/GlcNAc composite and the lowest -for UFS/BSA/NANA.Optimal concentrations of nanocomposite in cryomedium were as follows: 0.15% for UFS/GlcNAc and UFS/BSA/NANA, 0.6% for UFS/BSA/GlcNAc.
It is noteworthy that optimal concentrations of BNC, obtained by measuring the motional energy of gametes, are also proven by other parameters, namely, speed and frequency of cell rotation.Maximal number of movable cells was registered at relatively low concentrations of BNC: C op = 0.01% for UFS/BSA/NANA and UFS/GlcNAc, 0.15% for UFS/BSA/GlcNAc.This may be explained by different physiological state of the cells in gamete suspension [21], which means that their sensitivity to the same BNC concentration will be different.
The results obtained prove that all created BNC are capable of increasing the viability of gametes.
However, preliminary immobilization of protein on UFS surface leads to some decrease in the mobility of gametes (it may be conditioned by complete screening of silane groups by protein molecules) as well as in too firm binding of GlcNAc and NANA to BSA, fixed on the surface of BSA adsorbent (which is proven by the results of desorption, NMR-and IR-spectroscopy).There are grounds for the assumption that the action of BNC on cells depends considerably on the character of binding of immobilized biomolecules to the surface of adsorbent.Probably, predominant desorption of aminocarbohydrates from the BNC surface close to the receptor system promotes their integration into the cell metabolism.However, other mechanisms are also possible, for instance, the alteration of the cryomedium properties under the influence of BNC.It is also possible that BNC with immobilized carbohydrates may interact selectively with specific sites of the cell surface and therefore influence the activity of enzymes, built into the membrane.
Conclusions.It was shown that modification of the UFS surface with BSA protein allows immobilizing both GlcNAc and NANA on it.Triple BNC, obtained on this basis, have relatively high biological activity.The molecules of immobilized aminocarbohydrates provide the activity of BNC even when the UFS surface is completely screened by the protein molecules.The parameters of adsorption/desorption of studied carbohydrates on UFS and the data of IR-spectroscopy allow the assumption that their immobilization on the surface of UFS/BSA composite occurs due to the formation of hydrogen-bound complexes with the participation of amino-and amid groups of both protein and carbohydrates.It was demonstrated that the interaction of aminosugar and protein was accompanied by considerable dehydratation of protein structures, caused mainly by the decrease in the efficiency of protein-protein bonds.It was also determined that BNC on the basis of UFS, BSA, GlcNAc and NANA in the range of specific concentrations can be applied for optimization of LGY-cryomedium to stimulate the viability of deconservated bovine gametes.
The granules of cryopreserved bovine sperm were kindly provided by the Institute of Animal Breeding and Genetics, UAAS.
The work was supported by the complex programme of fundamental research of the National Academy of Sciences of Ukraine "Nanostructural systems, nanomaterials, nanotechnologies".
1 H NMR spectra of non-freezing water in 5% solutions of albumin -an initial one and solutions, containing GlcNAc additions.The signal is irregular; when resolved into components in the assumption of Gaussian form of absorption lines, it may be presented by two signals with the chemical shifts d H = 5 and 5.7 parts per million (ppm).The intensities of these signals are in 3:1 ratio.The data in Fig.4demonstrate the temperature dependences of non-freezing water concentration, calculated by temperature changes in the intensity of

Fig. 4
Fig.4 Temperature dependences of concentration of non-freezing water (C uw ) (a), dependences of free Gibbs energy (DG) on C uw (b), changes in C uw , caused by the presence of GlcNAc (c), distribution of non-freezing water along the radii of cavities, filled with water, inside a biopolymer matrix (d) and DC uw according to changes in DG (e).