pH of Water Intercalated into Graphite Oxide as Determined by EPR Spectroscopy

Two pH-sensitive spin probes 4-(methylamino)-2-ethyl-5,5-dimethyl-4-pyridine-2-yl-2,5-dihydro-1H-imidazol-1-oxyl (DPI, also known as MEP) and 2,2,3,5,5-pentamethyl-2,5-dihydro-1H-imidazol-1-oxyl (MTI) were used to measure pH of water intercalated in Brodie graphite oxide. The pH value was found to be 2.25 ± 0.05 immediately after adding water to graphite oxide and decreased to 1.75 ± 0.05 during ca. 30 h.


Introduction
Graphite oxide (GO) is a layered material in which the graphene-like planes contain oxygen functionalities. At present, GO draws researchers' attention as one of the promising materials for drug delivery systems and other biomedical applications [1][2][3][4]. GO has certain advantages comparing to other drug carrying nanomaterials. First, it has enormous surface area (up to 2600 m 2 /g [3]), providing high drug loading values. For example, doxorubicin loading value as much as 235 wt% was achieved in [5]; it is few times higher comparing to the loading value for nanotubes [6,7]. Second, GO has both hydrophilic and hydrophobic properties, since its planes contain oxidized and deoxidized areas. As result, there are multiple possibilities for interaction between the nanocarrier surface and other components of the drug delivery system. Typical GO-based drug delivery system consists of GO carrier, drug compound molecules, some enhancing additives (grafted polymeric molecules [8][9][10][11], nanoparticles [4]), and water as a liquid phase for dispersion.

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Functionalization of GO with polymers is aimed to improve biocompatibility and stability of the drug delivery system.
One of the most important characteristics of GO-based drug delivery system is pH value of aqueous medium both outside GO flakes and inside the inter-plane space of GO. Acidity of outer water can be used for drug release control [1,5,8]. Acidity of inner (intercalated) water can have a significant effect on properties of the drug compound sorbed on the inner GO surface. Many drug compounds are pHlabile. Drug stability issues are thoroughly developed in works [12][13][14][15][16]. For example, the authors of [15] studied the stability of anticancer agent daunorubicin and its derivatives in a wide range of pH. Some sort of V-shaped dependencies has been revealed. It was found that daunorubicin demonstrates the greatest stability within pH range of 4-6.
At present, it is well known that GO suspensions have acidic properties. Hartono et al. [17] prepared the suspension (0.01 g of GO in 10 ml of water) and found pH of filtrate 4.3. Szabó and coauthors [18] found that pH of the suspension with the same GO concentration was in the range from 3.1 to 3.7 depending on ionic strength created in the solution. The nature of acidity of GO suspensions is still an open issue. Initially, it was supposed that the cause of acidification is elimination of protons by carboxyl and hydroxyl groups of GO [19]. Later Dimiev et al. proposed the "dynamic structural model" which consists in chemical interaction of water molecules with hydroxyl groups of GO with the formation of ketones and enols and continuous acidifications of the solution. The scheme of the processes is given in [20,21].
It should be noted that the local acidity of water in the nanoscale inter-plane space does not automatically follow pH of the surrounding bulk aqueous medium. Indeed, the positive-charged protons can be located mainly near the negatively charged GO layers. In a view of GO-based drug delivery systems, the knowledge of the local pH value of water confined between GO layers seems to be important. To date no such kind of research has been reported.
In early 80s of twentieth century, pH influence on electron paramagnetic resonance (EPR) spectra of imidazoline and imidazolidine stable nitroxide radicals was first described [22][23][24]. Such nitroxides can be easily protonated at the nitrogen atom of heterocycle in the close vicinity to the paramagnetic center. Protonation causes a change in the density distribution of the unpaired electron, that result in a change of the radical spin-Hamiltonian parameters. Consequently, EPR spectra of the imidazoline and imidazolidine nitroxides are sensitive to pH value of the surroundings. pH-sensitive spin probe method is a powerful tool for pH-measurements in systems which cannot be analyzed using macroscopic pH measuring equipment [25][26][27][28]. This technique is extensively used for study of polymers [29,30] biological membranes and proteins [31,32], in vivo objects [33,34], nanomaterials [35,36], etc. In works [26,33,34,37], pH-sensitive nitroxides were used for 3D-mapping of biological tissues and tumors.
Recently, it was shown by Chumakova and coauthors [38][39][40], that nitroxide radicals can be introduced into the inter-plane GO space with various polar liquids. However, pH-sensitive spin probes have never been used for investigation of the swelled GO. The main objective of the present work is to show the possibility of pH of Water Intercalated into Graphite Oxide as Determined… pH measuring of water intercalated into GO using X-band EPR spectroscopy. Two spin probes which are sensitive to acidity of solution in different ranges of pH values were used.

Materials
The spin probes 4-(methylamino)-2-ethyl-5,5-dimethyl-4-pyridine-2-yl-2,5-dihydro-1H-imidazol-1-oxyl (DPI), and 2,2,3,5,5-pentamethyl-2,5-dihydro-1H-imidazol-1-oxyl (MTI) were provided to us by Prof. I. A. Grigoriev and Dr. I. A. Kirilyuk (Novosibirsk Institute of Organic Chemistry SB RAS), and the procedures for the synthesis of DPI and MTI nitroxides were described elsewhere [41,42]. GO prepared according to the Brodie synthetic procedure [43] was provided to us by Dr. A. T. Rebrikova (Lomonosov Moscow State University). The GO powder was dried to a constant mass in desiccator with P 2 O 5 for 3-5 days [44]. The C/O ratio after drying as measured by X-ray Photoelectron Spectroscopy was 2.7 ± 0.2. The interplane distance measured by X-ray diffraction was 6.7 ± 0.2 Å. The GO powder was washed repeatedly with acetonitrile to remove residual oxidants. The procedure of washing is described in detail in [38]. HPLC grade acetonitrile was used for washing of the material and for introducing of the nitroxide radicals into the inter-plane space of GO.

Samples' Preparation
The solutions of the nitroxide spin probes in acetonitrile were added to 10-15 mg of GO powder, then acetonitrile was evaporated by airflow accompanying with intensive mixing of the suspension. After evaporation of the solvent, the samples were loaded into glass tubes with inner diameter of 1 mm, and the traces of acetonitrile were eliminated from the samples using evacuation at 10 -4 -10 -5 mm Hg for 3-6 h. All samples contained approximately 2·10 15 radicals per 1 mg of GO.
The equilibrium sorption value for the Brodie GO at 298 K was measured using isopiestic method [44] and was found to be 0.33 ± 0.03 g of water per 1 g of GO.
Deionized water in amount of approximately 90% of the equilibrium sorption value was added into the tubes containing the GO powder with radicals. The sample tubes were sealed and centrifuged for 10-15 min at 3000 rpm to provide complete impregnation of GO powder with water.

EPR Measurements
EPR spectra were recorded at 295 K using X-band (9.5 GHz) Bruker EMX Plus spectrometer, equipped with a high-sensitive resonator ER4119HS. Microwave power was 1-2 mW. The magnetic field modulation frequency was 100 kHz, and the modulation amplitude was 0.2-1.0 G.

Calibration of the Spin Probes
The spin probes were dissolved in buffer solutions with pH 4.01; the concentrations of the nitroxides were 0.1-0.2 mM. pH of the solution was stepwise changed by adding of KOH or HCl solutions. The pH was monitored using pH-meter "Expert-pH" equipped with a glass electrode. The measurements accuracy was 0.05 pH units. At each step of pH, the solution sample was taken for EPR measurement. EPR spectra of solutions were recorded in glass tubes of 1 mm inner diameter. The 14 N hyperfine splitting constant a N was determined as the distance between low-field (m I = + 1) and central (m I = 0) lines of the spectra with accuracy 0.02 G. For the purposes of this work, we also measured distances between low-field (m I = + 1) and high-field (m I = − 1) components of the spectra, referred to as 2a N .

pH-Sensitivity of the Nitroxides
Protonation schemes of the pH-sensitive spin probes are shown in Fig. 1. MTI can be protonated in one position and is characterized with one value of pK. As for DPI, two consecutive steps of protonation take place [41].
pH-dependencies of a N for MTI and DPI are presented in Fig. 2. The EPR spectra recorded at different pH values are shown in Fig. 1SI. The calibration curves were fitted with the following titration equations [41], for MTI and DPI, respectively: Values of pK determined for DPI were pK 1 = 5.31 ± 0.03, pK 2 = 3.31 ± 0.05 (5.08 and 2.86 in [41]), and for MTI, pK was found to be 1.10 ± 0.02 (1.16 in [45]).

pH Value of Water in the Inter-plane Space of Graphite Oxide
Typical EPR spectrum of the swelled GO containing spin probe is shown in Fig. 3 (black line). The spectrum consists of three signals, namely the signal of the probes sorbed on the inner surface of GO, the narrow triplet signal of the fast-rotating nitroxides located in the intercalated water, and the native signal of GO which is unstructured singlet.
One can see that the central line of the signal of the fast-rotating nitroxides dissolved in intercalated water overlaps with the nitroxides and the native GO signal. For this reason, to determine pH value, we measured the distance between (2) a N (pH) = p 1 + p 2 ⋅ 10 pK 1 −pH + p 3 ⋅ 10 pK 1 +pK 2 −2⋅pH 1 + 10 pK 1 −pH + 10 pK 1 +pK 2 −2⋅pH low-field and high-field components of the narrow triplet, 2a N . It should be noted that in the case of slow exchange between protonated and deprotonated paramagnetic molecules EPR spectrum is a sum of two signals which manifests itself as a splitting of the high-field component into two lines. However, it was shown that for pH-sensitive nitroxides with pK < 3, the fast proton exchange takes place [24]. We observed that in the case of MPI, the high-field component splitting is not observed at any pH value (Fig. 1bSI). As for DPI, the poorly resolved splitting of the high-field component is observed in pH range approximately 3.4-5.8 (Fig. 1aSI) while pH value of water intercalated into GO was found to be significantly below this range. Measuring of 2a N values for pH study was exploited in works [46][47][48]. Figure 4 shows the calibration dependencies of 2a N on the solution pH for DPI and MTI and the results of pH-measurements for water intercalated into GO inter-plane space. The values of 2a N for GO samples under study are shown by blue horizontal lines. The intersection with corresponding calibration curve gives pH of water in the sample. Value of 2a N for DPI is 27.9 G that corresponds to pH below 2.5. Evidently, DPI is insensitive in this region. MTI returns 2a N = 31.25 G that corresponds to pH 2.25. This value lies in the range of high pH-sensitivity of this nitroxide. Therefore, MTI is a suitable probe for measurements of acidity of water intercalated in GO. It should be noted that the pH values obtained are significantly lower than the values reported before for GO suspensions. For example, Hartono [17], Szabó [18] and Dimiev [20] reported pH values for GO suspensions from 3.1 to 4.3. It means that acidity of intercalated water is noticeably higher comparing with acidity of outer water in suspensions.
It should be emphasized that the amount of water introduced into the samples under study was less than the equilibrium sorption value for Brodie GO. It implies that all introduced water was intercalated into the inter-plane space of the material, provided that the equilibrium distribution of water in the sample was attained. In this case, the radicals reflect just acidity of the intercalated water but not acidity of water located in the pores of the material or on the tube wall. To speed up the impregnation of GO powder with water, we centrifuged the samples; impregnation was monitored by EPR spectra (see Fig. 2SI).  Figure 5A shows the changes of MTI EPR spectrum in the centrifuged sample in time. One can see that the spectrum lines of the nitroxide in liquid phase (Liq LF and Liq HF ) decrease compared to the components of the sorbed radicals (Sorb LF and Sorb HF ). Time dependence of Liq LF /Sorb LF ratio is shown in Fig. 5b; it drops sharply during 10 h. The above observations can be interpreted as gradual redistribution of water in the inter-plane space of GO with decrease in the amount of highly mobile water. The possibility of forming a low-mobile faction of water on the inner surface of GO is discussed in the literature [49,50]. It should be noted that the total amount of nitroxides decreases in ca. 30 times during the 30 h because of irreversible oxidation of the nitroxide group. It is well known that graphite oxide always contains some oxidizing impurities. Since the sorption equilibrium of the remaining radicals is established much faster than the death of paramagnetic molecules, a decrease in the number of radicals does not affect the dependences shown in Fig. 5. The same experiment was performed with acetonitrile instead of water, and no such changes in EPR spectrum has been observed, Liq LF /Sorb LF ratio stayed almost constant in time. Acetonitrile is a polar liquid which readily intercalates into GO, but the permeability of GO membranes for water and acetonitrile differs by many orders of magnitude. Our observations reveal the essential difference between the behavior of water and acetonitrile in the inter-plane space of GO.

Time Evolution of Intercalated Water Properties
Time dependence of pH for the sample MTI/GO/H 2 O is shown in Fig. 5c. It is seen that pH value decreased during 30 h from 2.25 ± 0.05 to 1.75 ± 0.05. The observed increase of acidity of intercalated water is in accordance with dynamic structural model of GO, proposed by Dimiev et al. [20,21]. Chemical reactions of water molecules with GO structural fragments yield hydronium ions and result in continuous acidification of the inter-plane water. As far as the origin of acidity of GO is very intricate and still disputable, pH-sensitive spin probe technique opens a possibility for studying chemical processes occurring inside the GO inter-plane space.

Conclusion
Acidity of water intercalated into Brodie GO was measured using pH-sensitive spin probes DPI and MTI. pH value was found to be 2.25 ± 0.05 for freshly prepared sample and decreases to 1.75 ± 0.05 during ca. 30 h. The pH value of intercalated water determined is significantly lower than the values reported before for GO suspensions. The proposed method can be used for characterization of GO, prepared by different ways and intended for application in drug delivery systems.