In vitro studies of titanium dioxide nanoparticles modi�ed with glutathione as a potential drug delivery system

Nowadays, the development of medicine is inextricably linked with the use of new products, often with unique features. Nanotechnology deals with the creation of materials with speci�c properties in order to meet the requirements. This paper addresses issues related to the use of nanoparticles as drug delivery carriers, with a particular focus on titanium dioxide nanoparticles. To reduce the release of harmful titanium ions from the carrier surface, the materials were modi�ed. In the �rst stage of research work, TiO 2 nanoparticles were prepared by sol-gel method. Their surface was coated with an organic substance, i.e. glutathione. The properties of the obtained materials were then investigated, including particle size, speci�c surface area and microscopic morphology. In the next stage of the study, the amount of titanium ions released from the modi�ed carriers was determined. For this purpose, leaching of nanoparticle samples in deionized water environment was performed. Another stage included the assessment of an active substance releasing froth the prepared materials loaded with tadala�l. In vitro studies were also performed. Cytotoxic and mutagenic properties of the obtained materials in relation to CHO cells were investigated. The results obtained con�rmed a decrease in the amount of titanium ions released in comparison to the reference material in which no modi�er was used. In addition, the obtained materials show satisfactory purity and surface morphology allowing the formation of carrier-drug interfaces. The results of in vitro studies let us to claim that the prepared modi�ed titanium dioxide nanoparticles have a great potential for being applied as drug carrier.


Introduction
Non-speci c distribution and uncontrolled release of active substances in conventional drug delivery systems have led to the development of nanocarrier-based drug delivery systems.They can deliver the drug to the target sites with a lower dosing frequency and in a spatially controlled manner [1].Delivery of more drug to the target site by nanoparticles should reduce the required doses of medicinal substances, which in turn will result in a reduction of toxic side effects [2].There are several elements that need to be considered to ensure clinical potential for newly designed nanoparticle-based carriers.On the one hand, the design of such nanomaterials should take into account the following key issues such as: su cient biocompatibility and biodegradability, good stability under physiological conditions, and high loading capacity and low toxicity.On the other hand, in addition to the primary requirement of safety and therapeutic e cacy, industrial scale-up is also a prerequisite for this type of nanomaterial in clinical applications [3].Titanium (IV) oxide nanoparticles, being chemically stable, environmentally friendly and non-cytotoxic, are considered as intelligent drug delivery systems to pathogenic sites.Their small, nanometric size makes them suitable for targeted therapy, e.g.cancer.In such systems, active substances deposited on carriers based on nanomaterials reach sick cells, bypassing healthy cells and tissues.This solution increases the effectiveness of therapy and reduces the possibility of negative side effects.The analysis of TiO2 nanoparticles for use in drug delivery systems is based on the observation of the loading e ciency or the effect of pH on the release of the active substance from the carrier surface.Additionally, uptake by neoplastic cells and cytotoxicity in target cells are assessed [4].The titanium (IV) oxide nanoparticles can deliver anti-cancer drugs such as paclitaxel, doxorubicin or temozolomide on their surface.Moreover, the carrier itself, thanks to its properties, increases their anti-cancer effect [3].
The use of nanometric TiO2 in biosystems may have a number of di culties due to their poor dispersibility and stability in water and biological uids.In order to improve their colloidal properties, it is necessary to study their surface properties and stability.Coating nanoparticles of titanium (IV) oxide with polymeric materials to eliminate aggregation and sedimentation is becoming popular.This treatment also reduces toxicity and increases biocompatibility.Polyethylene glycol (PEG) is successfully used as a modi er.It has a hydrophilic character.This enables the modi cation of the nanoparticle surface in order to eliminate the aforementioned agglomeration and to make the carrier resistant to protein adsorption [4].
Polyethylene glycol forms a speci c, polymeric, thin layer on the surface of the nanoparticle.The in vivo studies carried out so far show that the TiO2-PEG nanoparticle conglomerate can be successfully used as one of the cancer treatment tools.The next step to con rm its effectiveness is the analysis of the effects of nanoparticle therapy as drug carriers along with other treatment methods, such as photodynamic therapy [39].One of the ways to increase the selectivity of TiO2 nanoparticles is to combine them with folic acid, which allows to achieve high selectivity for certain types of cancer.Like antibodies, folic acid increases the a nity of molecules to pathological tissues, increasing their accumulation at the target site [5].
The toxic effects associated with titanium (IV) oxide nanoparticles in humans are mainly long-term effects resulting from chronic exposure via various routes (respiratory system, digestive system, dermal route).Human exposure to TiO2 via various consumer products in Western countries has been estimated at 5 mg per person per day [6].These values may increase by about 10 to 100 times for some risk groups who are exposed to inhalation of large amounts of these particles in the workplace (bleaching of paper, production of paints, etc.), or who consume large amounts of products coated with these particles.After penetration into tissues, TiO2 nanoparticles are not eliminated and accumulate over time, which can lead to very high doses after decades of exposure.It is very di cult to reproduce such chronic exposures e.g. in rodent models with a short lifetime of no more than two years.Therefore, most animal toxicity studies of these nanoparticles use different doses administered at once or for a relatively limited time [7].Studies have been carried out in which mice are administered titanium (IV) oxide nanoparticles intragastrically for 90 days.As a result, macrophage in ltration was formed, resulting in spleen apoptosis.In addition, the entire genome was analyzed.Exposure in the form of nanoparticles caused signi cant changes in the expression of over 1000 genes involved in immune responses, oxidative stress, metabolic processes, ion transport and others [8].The titanium (IV) oxide polymorph in uences the toxicity of the preparation in which it was applied.Anatase with a particle size below 100 nm is more toxic than rutile due to its catalytic properties and high reactivity.Therefore, the use of this ingredient, e.g. in preparations, is more and more cautious for sun protection [9].As a result of chronic inhalation exposure to titanium (IV) oxide nanoparticles, in ammation may occur, which causes brotic and proliferative changes [10].Drug carriers based on titanium (IV) oxide nanoparticles introduced into the patient's body and, therefore, in contact with body uids, decompose.During this process, titanium ions are released.They can react with the molecules of the living tissues of the body.If a patient is treated with a titanium implant, its degradation and the release of titanium ions can directly lead to the diagnosis of metallosis.It is a side effect of the implant insertion and the local effects of decay products on the body's tissues.Clinical studies have shown that TiO2 nanoparticles together with the released ions from the implants accumulate in the periimplant tissues [11].Long-term exposure to titanium ions is one of the causes of the allergic response.Allergic reactions and cytotoxic effects of titanium ions released from TiO2 nanoparticles are directly correlated with their size.The mentioned effects may become visible even after a period of 12 months from the administration of the preparation containing titanium and generate in ammatory reactions.Titanium ions most often accumulate in the liver, lungs and spleen.Increased amounts of titanium have also been observed in the brain in patients who have been exposed to titanium.Studies were carried out on pregnant mice administered titanium (IV) oxide nanoparticles.Titanium compounds have been detected in the placenta, liver and fetal brain.Moreover, mice administered with the nanoparticles had smaller uterus and smaller fetuses than rodents in the control group.When released into the body, titanium may exist as free ions or, due to its unstable nature, will be bound with proteins [12].The speci c properties of titanium (IV) oxide nanoparticles, their use in pharmacy and medicine, and sensitivity to environmental factors cause their degradation and decomposition with the release of titanium ions.This makes it necessary to control this phenomenon already at the stage of obtaining and producing the desired material.
Thus the aim of this study was to obtain titanium dioxide nanoparticles modi ed with glutathione which would inhibit releasing of titanium ions.This approach would reduce the toxic effect of titanium dioxide applied as drug carrier.

Materials
In the processes of formation modi ed titanium dioxide nanoparticles the following compounds were used: titanium(IV) isopropoxide (TIPO) (97.0%), sodium hydroxide (≥ 98.-%) and L-glutathione reduced (GSH) (≥ 98.0%).Tadala l was used as Pharmaceutical Secondary Standard.All compounds were obtained from Sigma Aldrich.All aqueous solutions were prepared using deionized water (Polwater, 0.18 µS).Culture media (F-12K Medium), CHO cell line and supplements (FBS, antibiotics) were also obtained from Sigma-Aldrich.BrdU cell proliferation kit was obtained from Roche and LDH cytotoxicity assay Kit was provided by Thermo Fisher Scienti c. Ketamine, xylazine, buprenorphine, heparin and saline were obtained purchased in Sigma Aldrich.

Obtaining of modi ed titanium dioxide loaded with tadala l
Process for titanium oxide obtaining had three stages and involved basic hydrolysis, polycondensation and dehydration.In the rst step hydrolysis of titanium isopropoxide occurred.In the presence of sodium hydroxide the formation of titanium hydroxide took place.In the second step the polycondensation of titanium hydroxide occurred.It led to releasing of water.The last stage involved the dehydration of condensation product to titanium(IV) oxide and water.The amounts of all reagents were so calculated as the nal mass of TiO 2 was equal to 2.6978 g (this mass results from the TIPO volume which was taken into the processes (10 ml)).Also, in some cases the additional amount of base was used so the fold of NaOH vs. stoichiometric amount required was equal to 1, 2 or 3.
Figure 1 presents the schematic diagram of the process for obtaining titanium dioxide modi ed with glutathione loaded with tadala l.A series of 9 products was obtained.Brie y, titanium isopropoxide was added dropwise into the Te on vessel in which aqueous solution of sodium hydroxide had been introduced already.The obtained mixture was then homogenized with using an ultrasound homogenizer, Hielscher UP400St, Germany (40 W).After 2 minutes of homogenization an aqueous solution of glutathione was added dropwise to the mixture and the whole was homogenized for further two minutes.The concentration of glutathione solution was so calculated as the molar ratio of GSH to TiO 2 differed and it was equal to 0.02, 0.11 or 0.20 (provided in Table 1).As a result suspension 1 was obtained.In the next step the active substance was added.For this purpose the proper amount of tadala l in a solid form was introduced into the suspension 1 and the whole was stirred for 5 minutes (C-MAG HS 7, IKA).The mass of tadala l was so calculated as the mass ratio of tadala l to the whole TiO2-Tad complex was equal to 1.0:3.5.Next, the Te on vessel was placed in the microwave reactor, Magnum v2, Ertec, Poland.The process temperature differed and it was equal to 120, 150 or 180°C.After reaching the required temperature, the sample stayed in it always for 5 min.The obtained mixture was ltered (0.45 µm).The solid phase was washed with deionised water and the liquid phase was discarded.The solid product was dried in a laboratory drier at 80˚C for 24 h.Pure titanium oxide nanoparticles loaded with tadala l were the reference material.The speci c values of input parameters are provided in Table 1.The physicochemical properties of the obtained products have been analysed.XRD technique (X'Pert PW 1752/00, Philips) has been used in order to assess the crystallographic structure of prepared titanium dioxide nanoparticles.Based on the Scherrer equation the crystalline size was calculated.In order to con rm the organic matter coating the surface of titanium dioxide nanoparticles the ATR-FTIR analysis has been performed (Nicolet 380 spectrophotometer, Thermo Fisher).This technique served to con rm the presence of glutathione built in the structure of titanium dioxide particles.The analysis of nanoparticles size was conducted with using DLS technique (Zetasizer Nano ZS, Malvern Instruments Lt).Also, this method served to assess the stability of the aqueous suspensions by providing the elecrokinetic potential, ζ.The concentration of the analysed suspension was equal to 10 mg/L.The suspensions were homogenized for 1 minute prior to analysis (Hielscher UP400St, Germany, 40 W).The speci c surface area, pore volume and size were assessed using the low-temperature nitrogen sorption (ASAP2010 apparatus from Macromeritics USA).The samples were desorbed at 200°C before measurement in helium ow for 6 hours and then under vacuum to a nal pressure of 0.001 torr.The size and shape of the prepared materials were analysed based on TEM-EDS microscopy (Tecnai TEM G2 F20X-Twin 200 kV, FEI).

Analysis of titanium elution from the prepared complexes
The analysis of titanium elution from the prepared samples was conducted in aqueous environment.For this purpose, a speci c amount of the tested sample (0.1500 g) was taken to the glass beaker with an analytical accuracy.Next, the speci c amount of deionised water which played the role of the leaching agent was introduced to the powder.The ratio of substance mass to volume of the leaching agent ratio was equal to 0.1 g : 2.0 mL.The prepared suspensions were mixed on a magnetic stirrer with a temperature control.The elution process was led in 37°C.After the speci c time of mixing (0, 1, 3, 5, 10, 20, 40 and 80 minutes) the suspension was ltered through the syringe lters (φ = 0.45 µm).The concentration of the leached titanium was analysed by Atomic Absorption Spectrometry (Perkin Elmer)

Analysis of active substance elution from the prepared complexes
The analysis of tadala l releasing from the prepared samples was conducted in aqueous environment.
For this purpose, a known mass of the tested sample (0.1500 g) was taken to the glass beaker with an analytical accuracy.The appropriate amount of the leaching agent (Ringer's uid or simultaneous body uid (SBF)) was later introduced to this.The mass to volume of the leaching agent ratio was equal to 0.1 g : 2.0 mL.The obtained suspensions were mixed on a magnetic stirrer with a temperature control.The elution process was led in 37°C.After the speci c time of mixing (0.5, 1, 3, 5, 10, 20, 30, 40, 50, 60, 120, and 180 minutes) the suspension was ltered through the syringe lters (φ = 0.45 µm).The concentration of the eluted tadala l in the obtained ltrates was analysed with using spectrophotometer (Rayleigh UV-1800) at λ max against the reference sample (pure Ringer's uid or SBF).

In Vitro Cell viability assay
In this study the in uence of prepared titanium dioxide nanoparticles (both in basic and modi ed form) on cytotoxicity and proliferation of Chinese hamster ovary (CHO) cells was analysed.In the studies the following materials were used: CHO cells (Sigma-Aldrich, catalogue no.85051005), grown according to the producer's instructions, were used, F-12K medium (Sigma-Aldrich, catalogue no.N4888) supplemented with fetal bovine serum (Thermo Fisher Scienti c no. 10270106)) and antibiotics (Sigma-Aldrich, catalogue no.P4333).The investigated cultures were grown at 37°C and 5% CO2, cells were passaged when reaching 80% con uence 2-3 times per week.The lactate dehydrogenase (LDH) test which is a colorimetric method of analyzing the cytotoxic effect of materials on cells was conducted.
Thanks to this test it was possible to measure the amount of colored formazan formed from tertazoline ( measurement of absorbance at 490 nm).This reaction is catalyzed by lactate dehydrogenase, in the presence of NAD+.As a result of disruption of the integrity of the cell membranes, lactate dehydrogenase is released from dead cells into the culture medium.Analysis of cytotoxicity was performed as follows.
CHO cells were plated in 96-well plates at 9×10 3 cells per well in 150 µL of madium.Cultures were stabilized for 24 hours and after that time the medium was replaced with fresh one which contained the tested nanomaterials.The concentration of the nanoparticles in the applied suspensions were: 30, 50, 70 and 80 µg/mL (control sample did not contain any nanoparticles).The cytotoxicity analysis of the prepared materials was performed using the Pierce LDH cytotoxicity kit (Thermo Fisher Scienti c, Cat.No. 88954), according to the protocol provided by the manufacturer with using Multiskan GO microplate reader (Thermo Fisher Scienti c) at two wavelengths − 490 nm (formazan absorbance) and 680 nm (background absorbance).IN order to calculate the cytotoxicity the following equation was used: The BrdU proliferation analysis assay serves as a colorimetric test for measurement of the amount of 5bromo-2'-deoxyuridine (BrdU) incorporated into DNA.Chemically, BrdU is a synthetic analogue of thymidine nucleoside which is incorporated into the DNA of a dividing cell during the S phase of division.
In order to measure the amount of incorporated BrdU, the enzymatic reaction of the enzyme conjugated with the anti-BrdU antibody and the substrate in previously xed cells is carried out.A low signal indicates an inhibition of division, while a high signal indicates a high proliferative activity of cells.
For proliferation analysis, CHO cells were plated in 96-well plates at 9 × 10 3 cells per well in 150 µL of medium.Cultures were stabilized for 24 hours and after that time the medium was replaced with fresh one that contained the tested nanomaterials.Control sample did not contain any nanoparticles.Cells were let to grow for 24, 48 and 72 hours.Assessment of cell proliferation in the presence of nanomaterials was performed using the Cell Proliferation ELISA kit, BrdU (Roche, Cat # 11647229001) according to the protocol provided by the producer.The readings were made at two wavelengths − 450 nm (product absorbance) and 690 nm (background absorbance) (Multiskan GO microplate reader, Thermo Fisher Scienti c).
The comet test enables the detection of DNA damage at the level of a single cell.The analyzed cells are mounted in agarose on a microscope slide.DNA remains after the proteins are digested.The slide is electrophoresed and stained with a uorescent substance.One obtains an image in the form of "comets".
The "head" is where the cell immobilizes before lysis, the "tail" is the damaged DNA fragments.The measure of the level of DNA damage is the length of the tail and the amount of DNA it contains.In the analysis CHO were seeded in 12-well plates cells in the amount of 80,000 cells per well and grown in 1 ml of culture medium for 24 hours.Then the medium was replaced with fresh, containing nanomaterials and cultured for 24 hours.After incubation, cells were harvested and suspended in 1% low melting point agarose (Eurx, Cat # E0303) and poured onto glass slides pre-coated with 1% agarose (Eurx, Cat # E0301).Slides with cells suspended in congealed agarose were placed in lysis buffer (pH 10, 2.5 M NaCl, 100 mM EDTA, 10 mM Trizma base, 200 mM NaOH) for one hour at 4°C.The slides were then placed in an electrophoresis chamber containing buffer (pH > 10, 300 mM NaOH, 1 mM EDTA).The electrophoresis was performed at 18 V (0.5 V/cm) for 1 hour.After the separation was completed, the slides were washed with a distilled water and placed in a solution containing SYBR™ Gold dye (ThermoFisher Scienti c cat. No. S11494).Detection was performed with a ZOE Fluorescent Cell Imager uorescence microscope and genotoxicity assessment was performed with CometScore 2.0 software.

Modi ed titanium dioxide loaded with tadala l
Based on XRD results (Fig. 2A) one may observe that not all of the prepared products had the crystalline structure.Samples 2, 3, 4, 5, 9 and Base exhibited strong diffraction peaks at 25.1, 37.4, 47.5 and 54.1° of 2θ angle.The amorphic structure had samples 1, 6, 7 and 8. Samples 1, 6 and 8 were obtained in the lowest temperature which suggests that 120°C was not enough to for the crystalline structure of titanium dioxide.In the case of sample 7 which was obtained in 150°C one may observe that a weak diffraction peek around 25° of 2θ angle appears.This suggests, that despite the highest content of glutathione, the crystallization process begins in this temperature.The size of crystallites has been calculated based on Scherrer equation: where d Sch -crystallite size, k constant -depends on the shape of the crystallite size, β -the width at half maximum peak describing the material, λ -the wavelength of CuKa radiation, θ -the Bragg diffraction angle.Figure 2B presents the results of the analysis.All crystallites are below 10 nm which suggests that the speci c surface area is highly expanded.However, one may observe that the size of crystallites in basic sample (not modi ed) is less than in the rest of the materials.That may indicate that the modi er molecules may clog the pores and thus make the surface area not much expanded.
Figure 3 presents the results of ATR-FTIR spectroscopy.The presence of Ti-O is con rmed by peaks around 700 cm − 1 .This is region is characteristic for metal-oxygen bonding.Weak peaks in the region around 3300 cm − 1 may be attributed to the hydroxyl group of adsorbed water.The presence of glutathione is con rmed by peaks at 1385 and 1516 cm − 1 which correspond to the δ N−H and ν C=O stretching bands in NH 3 + and carboxylic groups [13].
Results of size of nanoparticles measurement by DLS technique are presented in Fig. 4. The size of nanoparticles in most prepared samples was between 40 and 50 nm.Only in sample 8 the nanoparticles were smaller and their dimension was 20 nm.Also, the electrokinetic potential of some of them was measured.It ranged between − 40.7 and − 50.8 mV.The absolute values of this parameter is far higher than 20 mV, which con rms that the aqueous suspensions of the obtained products are kinetically stable.
Figure 5 presents the hysteresis loops along with type of pores identi ed based on hysteresis loop type.
Based on hysteresis loops one may assign their types to the shape of pores.Reference sample and A4 material had the same hysteresis loop type which was H2.A1 material had H3 hysteresis loop.That means that reference and A4 materials had neck-like and wide body pores or ink bottle-like pores.Contrary to that, material A1 had groove pores of nonrigid generation formed by aky particles which was con rmed by H3 hysteresis loop type [14,15].Table 2 shows the surface properties by indicating the speci c surface area, pores volume and pores size.The results are in line with above described observations.Material A1 was characterised by the most developed speci c surface area.It also had the largest pores volume and the smallest pores size.The speci c surface area in all materials was above 230 m 2 /g which suggests that the obtained products have a great load capacity. Figure 6 presents the results of TEM analysis.This was performed for A/Base (Fig. 6A), A1 (Fig. 6B) and A4 (Fig. 6C) samples.It may be clearly seen that material A1 is characterised by the smallest particles (around 5 nm) which is in line with the results obtained via surface properties analysis.Both reference product and A4 material have TiO 2 nanoparticles whose size do not exceed 10 nm.A1 material was obtained in the lowest temperature (120°C).And this parameter seems to be essential in the nanoparticles formation process.
Figure 7 shows results of TEM-EDS analysis.It has revealed that pure titanium dioxide nanoparticles which were not modi ed were consisted of titanium and oxygen, only (Fig. 7A).Based on this analysis one may observe that titanium oxide which were modi ed with glutathione (material A4) had in its structure titanium, oxygen and carbon which origins from the organic matter (Fig. 7B).

Titanium elution from the prepared complexes
The results for analysis of titanium releasing from the prepared materials are presented in Fig. 8. Reducing the leaching of titanium was one of the main aim of the studies.This was due to the fact that reduced titanium (in both metallic and ionic form) pose a threat for the living organisms.After being incorporated into tissues they may accumulate it their structure and induce formation of tumours.Thus releasing of metals from the drug carrier systems should be eliminated.Black curve represents the titanium releasing pro le form reference sample (not modi ed).As one may observe, four materials released less titanium than reference sample did (A4, A6, A7 and A8).Samples 7 and 8 had minimal elution of titanium which is most desired result.These were obtained when the molar ratio of glutathione to TiO 2 was the highest and was equal to 0.2:1.0.The same ratio had sample 9 and the pro le releasing is similar to the reference one.That means that coating with glutathione inhibits releasing of titanium indeed.

Tadala l elution from the prepared complexes
Figure 9 presents the results of tadala l elution analysis.The calibration curves are presented in Fig. 9A  and B. Figure 9A shows the curve obtained in Ringer's uid and B -in SBF.The wavelength with peak maximum for tadala l is λ = 284 nm.In both cases the determination factors are above 0.999, which con rms good curve t to empirical points.The tadala l solution in Ringer's and SBF's uid shows linearity in the concentration range from 5 to 60 µg/ml.The linear nature of the tadala l solutions was within the 95% con dence interval.Black curve presents the elution pro les from reference material (not modi ed).In both cases they con rm the strongest elution of active substance from the carrier.Titanium dioxide modi ed with glutathione did not release tadala l as fast as in the case of reference samples (in both tested environments).In case of A4 material which was put into Ringer's uid one may observe curve drop after 120 min.This may result of again adsorption of tadala l on the surface of the material.
There are many known mechanisms by which the active substance is released from the transport system [16].The behavior of the released active substance depends on its stability as well as the physicochemical properties of the nanocarrier.Components of the release process are: (1) desorption of the surface-bound drug; (2) diffusion of the drug from the carrier surface; (3) carrier erosion; and (4) a further combination of erosion and diffusion processes [17].The release pro les shown in Fig. 9C and D follow the typical diffusion pro le that is common for nanoparticle based drug carriers [18].The diffusion mechanism is appropriate for systems in which the diffusion of the drug is faster than the degradation of the carrier, which is consistent with the nature of the type of carrier used in the work (metal oxide).An initial rapid release, called "bursting release", and a further "sequential" release were observed in the studies.This pro le is attributed to complexes in which the drug is adsorbed or weakly bound to the carrier surface.It should be noted that when SBF was used as an acceptor medium, the concentration of eluted tadala l was higher than when Ringer's solution was used.This is in line with the theoretical conditions.The rate of drug release may be in uenced by ionic interactions between the carrier and secondary components present in the acceptor medium.The composition of SBF is much more varied than that of Ringer's.In its environment, the interaction of the active substance with the carrier matrix is weakened, as there is a competitive electrostatic interaction between the carrier and the surrounding ions, which explains the increase in drug release in this environment.

In Vitro Cell Viability Assay
Results of in vitro cell viability analysis are presented in Fig. 10. Figure 10A shows the dependence of cytotoxicity on the concentration of the analysed suspensions.All tested materials modi ed with glutathione induced stronger proliferation of CHO cells than in the case of using reference sample (not modi ed, basic TiO 2 ).One may note that titanium dioxide nanoparticles modi ed with glutathione even promot development of CHO cells.Comparing to the reference material, weaker cytotoxic properties exhibited modi ed materials A4 and A7 (Fig. 10B).It was achieved thanks to the presence on their surface glutathione which inhibited releasing of Ti ions which would induce ROS formation and cell apoptosis.
The comet test (CT), also known as single cell gel electrophoresis or microgel electrophoresis, is used in studies such as: genetics, toxicology, biomonitoring, ecogenotoxicity, molecular epidemiology, nutrigenomics, DNA repair system studies, assessment of genotoxicity and mutagenicity of nanomaterials, integrity assessment DNA in mesenchymal stem cells.The comet test detects breaks in the DNA strands, which are visualized by the increased migration of free DNA segments, resulting in comet-like images.DNA breaks, both double-stranded and single-stranded, are associated with chromosomal aberrations and genome instability.Genome instability is directly related to neoplastic processes.The comet test allows the detection of aneugenic and clastogenic substances with high sensitivity in vivo and in vitro in populations exposed to irritants.Its use allowed for the assessment of genotoxic and mutagenic properties of the obtained products.The advantage of the comet test over the micronucleus test is that the micronucleus test only analyzes genetic damage in mitotic cells, while the comet test detects DNA damage in both the interphase and mitotic cells.Single-cell gel electrophoresis is the basic method of analyzing the degree of DNA fragmentation as a result of genotoxic and mutagenic factors.This method identi es single stranded and double-stranded DNA breaks, and any chemical and enzymatic modi cations that can transform into DNA breaks or chromatids.It was found that compared to the reference material (unmodi ed nanoparticles), the tested products induced shorter comets corresponding to cells undergoing apoptosis (Fig. 11).This means that the modi ed titanium dioxide induce DNA damage to a lesser extent and show lower genotoxicity and mutagenicity.Figures 11D, E and F present these observations in numerical terms.For reference material, the values of all parameters tested (tail length, DNA tail, olive moment) are higher than the values of the modi ed titanium dioxide.It is the expected result of the research indicating the limitation of genotoxic and mutagenic properties of the tested materials.

Discussion
In order to improve the biocompatibility and tolerances of a speci c material, and thus increase the effectiveness of its potential applications, speci c structures are subjected to modi cation of their surface.This is especially important in the case of biomedical and pharmaceutical applications, where materials used, for example, as drug carriers, must meet a number of restrictive requirements.The use of additional compounds such as proteins, sugars or polymers ensures no accumulation or non-toxicity.In addition, it is often desirable to have speci c functional groups on the surface that will effectively bind the active ingredient to the carrier and maintain its therapeutic properties.The types of surface changes that, for example, drug carriers based on nanoparticles are subjected to, can be broadly divided into physicochemical and biochemical.The former cause changes in the chemical composition of the surface layer, while biochemical methods are based on the attachment of organic compounds to the surface [19].
The properties of the (modi ed) material obtained depend primarily on the substrates used.Their degree of conversion and other process parameters are important.The modi cation is most often performed insitu, and then the modifying substance is added at the stage of the synthesis.The modi er can also be added ex-situ, ie after the synthesis is nished, when the nished, pure product is subjected to modi cation [20].Among the many purposes of modifying materials for biomedical applications is to ensure the ability of the material to function properly in conditions of low pH and body uids.It is also necessary to ensure that the recipient's body is not adversely affected.The use of modi ers also affects the biofunctionality.It consists in ful lling speci c functions in vivo for the assumed time or resistance to degradation.So far, no suitable biomaterial that would meet all the requirements set for it has been developed.Disintegration under relatively aggressive conditions faced by nanoparticle-based carriers is inevitable.Another priority in the use of modi ers is counteracting destruction in order to prevent negative effects caused by decay products, such as: toxic, carcinogenic, mutagenic or in ammatory effects [19].
Covalent carriers linked to an active substance are called conjugates.Depending on the conjugated substance, the following systems are distinguished: carrier-drug, carrier-protein, carrier-DNA.Modifying substances attached to the carriers can, for example, change its solubility.Interestingly, they can play the role of the so-called tropic molecules that are responsible for recognizing target tissues.The rst conjugate of this type to enter the clinical trial phase was the combination of doxorubicin (DOX) with N- (2-hydroxypropyl) methacrylamide (HPMA) and galactosamine, which acted as a tropic molecule [21].
The use of modi cations in the case of nanomaterials is also aimed at reducing agglomeration, controlling hydrophobic properties or facilitating self-organization.Organic compounds are most often used for this purpose.Among them, mention may be made of carboxylic acids, silanes, thiols, surfactants and amines.Polymers for unconventional functionalisation (coating the surface of nanoparticles) are also often used, eg low-molecular polyethylene glycol [21].Galactose can be successfully used as a modi er of drug carriers in anti-cancer therapy.Its effectiveness is mainly based on the fact that carrier molecules loaded with saccharides on the surface are more readily captured by neoplastic cells.This is due to the fact that they have a high energy demand, which ultimately increases the effectiveness of targeted therapy [1].Yu Xia et al. [22] used galactose in their research to modify selenium nanoparticles.
The tumor-targeted "delivery system" created in this way was covered with an anti-cancer drug.Doxorubicin was added to the surface of the modi ed nanoparticles to improve its antitumor e cacy in the treatment of hepatocellular carcinoma (HCC).Further studies showed that the thus prepared, functionalised drug delivery system showed effective cellular uptake and penetrated into them through endocytosis.The selenium-galactose-doxorubicin nanoparticle system showed signi cant activity in inducing apoptosis of HCC cells in vitro [22].Targeted drug delivery can be improved by drug loading on nanoparticles to which an appropriate recognition particle can be added for e cient transport to speci c tumor cells.Galactose is a good example of such a molecule because it enhances uptake by pathogenic cells, but other recognition mechanisms such as ligand-receptor or antibody-antigen interaction can also be used.Also in their research, M. Margarida Cardoso et al. designed nanoparticles coupled with galactose.The anti-cancer drug used in this case was also doxorubicin.Modi cation with galactose showed a huge improvement in the therapeutic effectiveness of its action.The synthesized structures allowed the active base to be trapped in a hydrophobic inner core during the nanoparticle formation process.As a result, a carrier with a high drug content was obtained, which effectively recognizes neoplastic cells [23].

6.
A series of titanium dioxide nanoparticles modi ed with glutathione was prepared.Physicochemical properties of the obtained products were analysed.Also, the analysis of releasing of both titanium ions and active substance was performed.The materials were assessed via in-vitro studies in which the     Results of the TEM-EDS analysis: A) A/Base, B) A4 Pro les of titanium releasing from the prepared materials

Figures
Figures

Figure 9 Results
Figure 9

Figure 10 A
Figure 10

Table 2
Results of analysis of surface parameters