Synthesis and Characterization of a Water-Soluble Nanomaterial via Deep Nitration of Light Fullerene C₆₀

Abstract

A direct non-catalytic synthesis of a new water-soluble polynitro-hydroxylated fullerene derivative, C₆₀(NO₂)₁₈(OH)₂, was carried out using a mixture of concentrated nitric and sulfuric acids. The resulting poly-nitro adduct was comprehensively characterized by elemental C-H-N analysis, energy-dispersive X-ray spectroscopy, infrared (IR) and electron spectroscopy, nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), and thermogravimetric analysis (TGA). A detailed investigation of the physicochemical properties of aqueous solutions of C₆₀(NO₂)₁₈(OH)₂ demonstrated that the synthesized compound is a previously undescribed mixed polynitro-hydroxyl adduct of light fullerene C₆₀, featuring a high degree of nitration (18 nitro groups per fullerene core). The composition and structure of the adduct were confirmed by spectroscopic and refractometric analyses. In terms of redox behavior, the compound exhibits significant reducing and antioxidant properties. These physicochemical characteristics suggest the potential of C₆₀(NO₂)₁₈(OH)₂ for further development as a biocompatible nanomaterial suitable for medical applications.

1. Introduction

Fullerenes have been intensively studied for almost four decades, and interest in these carbon nanostructures continues to grow. Owing to their high chemical reactivity and unique cage geometry, carbon-based nanoparticles display outstanding tribological, mechanical, and electrical properties across a wide range of morphologies and sizes [1,2,3,4]. Among them, the low-density fullerene C₆₀ is especially promising for biomedical applications [5,6,7].

Recent work shows that incorporating C₆₀ into nanocomposite coatings markedly improves anticorrosion performance and mechanical integrity relative to the corresponding neat polymers [1]. A comprehensive review of fullerene-based nanocomposites highlights their broad biomedical potential, including corrosion-resistant polymers and antimicrobial materials [8,9]. In addition, C₆₀ exhibits neuroprotective activity by scavenging superoxide and hydroxyl radicals [10] and possesses pronounced antioxidant properties [11,12]. These effects are observed not only for pristine C₆₀ but also for low-molecular-weight derivatives such as fullerenols and for polymeric conjugates [13,14].

The possibility of exploiting fullerene coatings in joint endoprostheses is therefore attracting considerable attention [15]. A key challenge in implantology, however, is peri-implant infection, which may necessitate revision surgery and threaten patient survival [16]. Fullerene-modified biopolymers offer a promising avenue for antimicrobial surfaces and medical devices [16]. Moreover, C₆₀ coatings have been shown to enhance cell adhesion and viability on complex dental implants, indicating anti-inflammatory potential and improved biocompatibility [17].

Although pristine C₆₀ is essentially water-insoluble, its solubility can be tailored by chemical functionalization with hydrophilic groups [6,18,19]. Water-soluble, nitrogen-containing derivatives are of special interest for nanomedical diagnostics and therapy [6]. As early as 1996, the high reactivity of C₆₀ toward nitrogen-dioxide radicals was reported, leading to polyhydroxylated fullerenes (fullerenols) [20].

Selective nitration of C₆₀ remains a challenging topic; relatively few studies address it, and the available data are not always consistent [21,22,23]. Reported nitration methods employ reagents such as concentrated HNO₃/NaNO₂, N₂O₄, fuming HNO₃, or aqueous NaNO₂/FeSO₄/H₂SO₄ under air [22]. Polynitrofullerenes subsequently undergo slow hydrolysis in water, yielding mixed poly(hydroxynitro) derivatives.

A recent survey discusses the major classes of multifunctionalized fullerenes, emphasizing structure–property relationships [24]. In general, organic chemistry, nitration of hydrocarbons yields two principal product families: nitroadducts R(NO₂)ₙ and nitro-esters R–O–NO₂. Classic nitrating agents include HNO₃ itself, its protonated form NO₂⁺, various nitrogen oxides (NO₂•, NO•, N₂O₅, N₂O₃), and mixed systems containing additional oxidants or Lewis-acid catalysts such as AlCl₃, ZnCl₂, or BF₃ [24,25]. These nitro compounds are indispensable intermediates in chemical manufacturing, pharmacology, and materials science (e.g., nitrobenzene for aniline synthesis).

Fullerenes (C₆₀, C₇₀, etc.) are formally polyolefins, and their nitration parallels that of unsaturated hydrocarbons [25]. When C₆₀ is treated with nitric acid, nitrates, or NO₂•, followed by alkaline hydrolysis, polyhydroxylated fullerenes C₆₀(OH)ₙ (n ≈ 16–20) are obtained [20,26]. Consequently, nitration pathways that would normally generate poly-nitroadducts often converge to fullerenols after base treatment.

Nitric acid is the most accessible nitrating reagent; NO₂⁺ generated in situ acts as the electrophile, while other active species arise as secondary products. For unsaturated systems, six mechanistic routes are possible:

• Electrophilic H–NO₂ substitution (infeasible for hydrogen-free C₆₀)
• H-substitution via N₂O₅ (also impossible for C₆₀)
• Addition of HNO₃ across C═C to give nitro-alcohols
• Addition of pure HNO₃ to yield nitro-ethers
• Radical addition of NO₂•, N₂O₃, or N₂O₅ affording mono- or di-nitroadducts, nitro-alcohols, nitro-esters, or their mixtures [18]
• Multi-step sequences combining these elementary reactions

The present work aims to synthesize a water-soluble polynitro derivative of C₆₀ and to elucidate its structure and physicochemical properties by a suite of analytical techniques. The resulting material is intended for selective etching and surface modification of titanium medical implants, with the goals of enhancing hardness, tribological performance, antioxidant capacity, electrical conductivity, and corrosion resistance. Because the grafted adduct displays limited yet significant solubility in physiological fluids, beneficial modifications of the surrounding biological milieu (blood, lymph, cerebrospinal fluid, etc.) are anticipated. We therefore hypothesize that the C₆₀ polynitro-adduct obtained herein is a promising biocompatible nanomaterial for medical applications [27].

Selective nitration of fullerenes and their derivatives by novel synthetic approaches and reagents is an important yet challenging topic. The number of papers devoted to the study of nitration of the lightest fullerene, C₆₀, is relatively small [11,28,29,30], and the data presented by the authors do not seem to be fully consistent.

(A)

Let us first focus on the article [28], which most fully corresponds to the thematics of our article, i.e., the direct nitration of fullerene C₆₀. Nitration of fullerene molecules has been carried out under different conditions and nitration reagents, including a mixture of conc. HNO₃ and sodium nitrite, dinitrogen tetroxide, fuming nitric acid, and a mixture of aqueous sodium nitrite, FeSO₄, and H₂SO₄ in the presence of air. Hexaanilino [60]fullerenes (${\mathrm{C}}_{60}({\mathrm{N}\mathrm{O}}_2{)}_6$), as the predominant nitration product, have been described [28]. In an aqueous alkaline solution, rapid and complete hydrolysis of poly-nitrofullerenes was observed to produce fullerenol molecules containing at least 16 hydroxy- groups per C₆₀ core [28]. The latter, in our opinion, indirectly indicates a deeper course of fullerene nitration up to 16 nitro groups, at least for a part of the mixture of products obtained in this work. The mixed composition of poly-nitroadducts during nitration is also indirectly confirmed by the results of HPLC studies, where the yield of a mixture of products from the column is characterized by a mixture of 4–7 chromatographic peaks [28] with a mixture output time of several minutes. At the same time, with an increase in the synthesis time from 24 h to 144 h of HPLC, the picture is significantly simplified. From the latter, it can be assumed that either longer synthesis times, more intensive mixing, or a higher temperature could have been used to complete the nitration process.

(B)

Article [11] is also directly devoted to the direct fullerene nitration. The nitration of fullerene molecules has been carried out under different conditions and nitration reagents (very close to [28]), including a mixture of conc. HNO₃ and sodium nitrite (7), dinitrogen tetroxide, fuming nitric acid, and a mixture of aqueous sodium nitrite, FeSO₄, and H₂SO₄ in the presence of air. However, polynitrofullerenes react slowly with H₂O to yield partially hydroxylated products of poly(hydroxynitro)fullerenes. In the presence of an aqueous alkaline solution, rapid and complete hydrolysis of poly-nitrofullerenes was observed to produce fullerenol molecules containing at least 16 hydroxy groups per C₆₀ core. The result of nitration in this work is also very close to the article [28], the formation of two main poly-nitroadducts, tetra- and hexa-derivatives, was found. However, the authors of the review [11] come to more cautious conclusions, noting that in some studies reported, C₆₀ was nitrated by the multiple addition of NO₂ and the product isomerized partly to the nitrito form with subsequent hydrolysis by atmospheric moisture to yield nitrofullerols consisting of 6–8 nitro and 7–12 hydroxy groups per C₆₀.

(C)

Authors [28] summarize the recent progress of functionalized fullerene materials (i.e., fullerene derivatives) that have been applied in PSCs, focusing on chemical functionalization strategies. Despite the completeness and undoubted value of the presented review, the last one is generalized and non-specific and has no direct relation to the topic of our article related to the direct nitration of fullerene C₆₀. The goal of the authors [28] was to propose an outlook on the future development of fullerene derivatives in realizing high-performance PSC devices.

(D)

Article [30] is devoted to the consideration of the major types of multi-functionalized fullerenes through selected examples with a link to the structural assignments. At the same time, the structural features of fullerene derivatives are the main subject of this review. At the same time, this aspect of the review is not the main and essential determinant in our work.

When comparing our results with the above works, we can say that as a result of the synthesis, the authors were able to obtain and isolate a poly-nitroadduct of fullerene C₆₀ with a very high degree of addition of nitro groups (18 instead of a maximum of 6 [11,28,29,30]). The authors attribute this fact to the simultaneous use of strong mineral acid as a catalyst, the duration of synthesis at elevated temperatures, and, most importantly, the prevention of hydrolysis by maintaining low pH values of the medium.

2. Results

2.1. Synthesis of the Polynitro-Hydroxylated Adduct C₆₀(NO₂)₁₈(OH)₂

A direct, one-pot nitration–hydroxylation of pristine fullerene C₆₀ was performed in mixed fuming nitric and sulfuric acids (20:1 v/v; ρ ≈ 1.50 g cm⁻³) under vigorous stirring at 50 °C for 7 days. Trace SO₃ in the medium acted as a Brønsted super-acid catalyst, generating NO₂⁺ electrophile in situ. The overall stoichiometry can be summarized as:

$${\mathrm{C}}_{60}+16{\mathrm{N}\mathrm{O}}_2·+2\left[\mathrm{O}\mathrm{H}{\mathrm{N}\mathrm{O}}_2\right](\mathrm{c}\mathrm{a}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{y}\mathrm{s}\mathrm{t}=\mathrm{O}\mathrm{H}\mathrm{S}{\mathrm{O}}_3\mathrm{H})\to {\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2$$

(1)

After reaction completion, the dark green solution was repeatedly diluted with ice water, neutralized to pH ≈ 6, and dialyzed (MW 1000) to remove acid residues. Rotary evaporation afforded a brown microcrystalline solid in 62% isolated yield.

The control of the reaction was indeed carried out by the authors using standard methods: electron spectroscopy, thin-layer chromatography, and turbodimetry (the latter for tracking the formation of associates in solution). The analysis was performed once a day. Experience has shown that there were no qualitative changes in the composition of the solution, and the quantitative reaction was completed after about 4–5 days. We did not observe the destruction of the fullerene core, including according to the results of mass spectrometric analysis, which in this case would show molecular weights less than M/z < 720 (even if the nitro groups were destroyed under experimental conditions). Nevertheless, the authors undoubtedly recognize the possibility and high probability of the formation of isomeric and closely related forms during synthesis.

2.2. Structural Characterization

To streamline the presentation, all analytical data are grouped into a single subsection; Figure 1 gives a schematic overview of the workflow.

Elemental and Spectroscopic Evidence

The results of elemental analysis of poly-nitroadduct ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2$ are summarised in

Table 1. Elemental analysis of synthesized poly-nitroadduct ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2$.

Element	Theoretical Amount Ctheor, %, wt	Experimental Amount— Cexp, %, wt
C	45.51	45.51 ± 0.1
H	0.13	0.13 ± 0.1
N	16.0	16.0 ± 0.1
O	38.43	38.43 ± 0.1

.

Based on the results of the elemental analysis, it was found that the composition in atomic ratio (per 1 fullerene core ${\mathrm{C}}_{60})$: C = 60; N= $18\pm 2$; H = 2 $\pm 1;$ O = 38 $\pm 4$. Formula: ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2.$

The obtained data show the correspondence between calculated and experimental data. Infrared spectrum poly-nitroadduct ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2$ shown in Figure 2.

The infrared (IR) spectrum displays characteristic features: a prominent absorption band corresponding to the O–H stretching vibration is observed at 3449 cm⁻¹. Fullerenols synthesized by various methods exhibit similar IR patterns, typically showing strong hydroxyl group absorption near 3400 cm⁻¹ [21].

The identification of key absorption bands in the IR spectrum is as follows (see Figure 2; ṽ in cm⁻¹):

• ν(O–H): 3449
• ν(NO₂): 1647, 1348, 1560
• δ(NO₂) (“scissoring”): 733, 638, 617
• ν(C–O): 1790, 1647, 1152, 1063
• ν(C–N): 1113 (strong), 833
• ν(C–O–N): 2982
• Fullerene core vibrations: 997, 733, 484, 457 [9]

Notably, the typical cage-related vibrational bands of pristine C₆₀ at 528, 578, and 1183 cm⁻¹ are absent, which indicates significant functionalization of the fullerene surface.

It should be emphasized that the IR spectrum of C₆₀(NO₂)₁₈(OH)₂ is highly informative and can be reliably used for compound identification.

The electronic absorption spectrum of C₆₀(NO₂)₁₈(OH)₂ in aqueous solution was recorded in the UV–Vis–NIR range to investigate the optical properties of the synthesized compound. The spectra at various concentrations are presented in Figure 3. A distinct absorption maximum is observed at λₘₐₓ = 297–300 nm, which becomes more intense with increasing concentration. This peak is characteristic of π–π* electronic transitions associated with the conjugated fullerene structure modified by nitro and hydroxyl groups.

To verify the applicability of the Bouguer–Lambert–Beer law, the optical density at 300 nm was plotted against the concentration of the compound. As shown in Figure 4, a clear linear relationship is observed within the concentration range of 0.06 to 0.48 g/dm³, confirming the validity of the law in this system. The slope of the linear fit corresponds to an empirical coefficient of 0.178 g/dm³ per unit of D₃₀₀, allowing for reliable spectrophotometric quantification of C₆₀(NO₂)₁₈(OH)₂ in aqueous media.

A single, well-defined absorption peak is observed in the visible and near-UV region at λ = 300 ± 3 nm. The absorbance at this wavelength follows the Bouguer–Lambert–Beer law, allowing for precise quantitative analysis. The relationship between concentration and absorbance at 300 nm is described by the following empirical equation:

$${\mathrm{C}}_{{\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2}\left({\displaystyle \frac{\mathrm{g}}{{\mathrm{d}\mathrm{m}}^3}}\right)=\left[0.178\pm 0.003\right]{\mathrm{D}}_{300}\left(\mathrm{l}=1\mathrm{c}\mathrm{m}\right)$$

(2)

This linear relationship holds in the concentration range of 0.06 to 0.48 g/dm³. At shorter wavelengths, an increase in absorbance is observed, indicative of higher energy π–π* transitions. The observed spectral behavior confirms the utility of UV–Vis spectrophotometry for determining the concentration of C₆₀(NO₂)₁₈(OH)₂ in aqueous media with high sensitivity.

To further evaluate the chemical purity of the synthesized compound, high-performance liquid chromatography (HPLC) was performed. The analysis was carried out under the following conditions:

• Column—Agilent Zorbax SB-C18;
• Eluent—CH₃CN/H₂O (1:20, v/v);
• Detection—spectrophotometric monitoring at λ = 330 nm.

The resulting chromatogram is presented in Figure 5.

As shown in Figure 5, the HPLC chromatogram of the synthesized C₆₀(NO₂)₁₈(OH)₂ reveals a single sharp peak at t = 3.6 min, corresponding to the main product. A minor impurity peak, constituting approximately 3% of the total signal area, is detected at t = 2.7 min. The half-width of the primary peak (d₁/₂ ≈ 0.45 min) and its symmetrical shape indicate high chromatographic purity. The authors realize that the chromatographic purity of the poly-nitroadduct, estimated at 97–98 wt%, is rather arbitrary. It is clear that the peak of impurity yield is very diffuse and wide. It probably corresponds to a mixture of closely related and partially isomeric products, which are also probably lower in molecular weight than poly-nitroadduct. We did not isolate this impurity individually. Nevertheless, we believe that the quantities are small enough not to significantly affect the physicochemical properties of the target product.

To further confirm the chemical structure, nuclear magnetic resonance (NMR) spectroscopy was performed. The ¹³C and ¹H NMR spectra of the product, recorded at a frequency of 100.4 MHz and a temperature of 25 °C, are presented in Figure 6. These spectra confirm the presence of the expected functional groups: nitro (–NO₂), hydroxyl (–OH), and sp² carbon–carbon bonds characteristic of the fullerene core.

In the ¹³C NMR spectrum, distinct signals are observed at δ = 142–143 ppm, corresponding to the unmodified cage carbon atoms (C=C); at δ ≈ 76 ppm, attributed to hydroxylated carbon atoms (C–OH); and at δ ≈ 53 ppm, assigned to carbon atoms bonded to nitro groups (C–NO₂). The ¹H NMR spectrum shows a weak signal at δ ≈ 5 ppm, with a very faint broad peak at δ ≈ 8 ppm, likely arising from exchangeable hydroxyl protons.

The thermal stability of the polynitro adduct was assessed using thermogravimetric and differential thermal analysis under atmospheric conditions. The results are presented in Figure 7.

The thermal behavior of C₆₀(NO₂)₁₈(OH)₂ was investigated using complex thermal analysis, including thermogravimetric (TG), derivative thermogravimetric (DTG), and differential thermal analysis (DTA), as shown in Figure 7. The TG/DTG curves reveal a multistage decomposition profile under an air atmosphere from 0 to 900 °C.

According to the data, the compound undergoes:

• 60–100 °C: decomposition of crystallohydrates due to external dehydration;
• 170–250 °C: breakdown of hydroxyl groups, corresponding to internal dehydration;
• 400–650 °C: gradual thermal degradation of relatively stable nitro groups;
• 680–740 °C: active decomposition of residual nitro substituents;
• 800 °C and above: explosive destruction of remaining nitro groups and oxidative combustion of the fullerene core.

These results confirm the moderate thermal stability and reactive nature of the polynitro adduct, with a clear transition from dehydration to progressive denitration and eventual carbon skeleton oxidation.

To assess the solid-state structural features of the synthesized material, X-ray diffraction analysis was performed. The diffraction pattern obtained under standard conditions (Co Kα radiation, 30 kV, 10 mA, 0.2 s exposure, 2θ range 10–80°) is presented in Figure 8.

As illustrated in Figure 8, the X-ray diffractogram of C₆₀(NO₂)₁₈(OH)₂ demonstrates a unique fingerprint distinctly different from those of pure C₆₀, fullerenols (C₆₀(OH)_n, where n = 8–40), fullerene oxides (C₆₀O_n), and other known derivatives in existing diffraction databases. The difference in peak positions and intensities confirms that the synthesized compound represents a structurally unique form. Although no direct reference patterns for polynitro-hydroxylated fullerenes have been reported in the literature to date, the observed diffractogram provides strong indirect evidence supporting the structural individuality of the new adduct.

Here and everywhere after, authors insert all the numerical data on the properties of the poly-nitroadduct and its solutions into Supplement (1) in the form of 13 tables.

Building on the established molecular structure and thermal properties, the next stage of the study focused on the physicochemical behavior of C₆₀(NO₂)₁₈(OH)₂ in aqueous solution, which is of central importance for its prospective biomedical applications. Specifically, information regarding solubility, colloidal state, aggregation, and volumetric properties is crucial, as these parameters significantly affect biological activity, delivery, and interaction with physiological media.

The concentration-dependent behavior of C₆₀(NO₂)₁₈(OH)₂ in aqueous solution was studied by analyzing the size distribution of molecular associates, zeta (ζ) potentials, and electrokinetic mobility at 25 °C. These characteristics were evaluated across a concentration range of 0.001–10 g/dm³, with the results summarized in Tables S1–S3 (Supplementary Materials) and graphically presented in Figure 9.

The analysis of the obtained data allows us to draw the following conclusions:

• Monomeric molecules of C₆₀(NO₂)₁₈(OH)₂, with an estimated hydrodynamic diameter of δ ≈ 2 nm, are dominant at concentrations C ≤ 0.00195 g/dm³.
• In the range of 0.00195–0.50 g/dm³, first-order associates form, with sizes around δ ≈ 50 ± 10 nm.
• Between 0.0624 and 2.0 g/dm³, second-order associates (nanocolloidal particles) are observed, with sizes approximately δ ≈ 170 ± 50 nm.
• At concentrations C ≥ 1.0 g/dm³, third-order associates (microcolloids) with sizes up to δ ≈ 5 ± 0.6 µm coexist with second-order aggregates.

In the concentration range C ≥ 0.125 g/dm³, the zeta potential (ζ) values range from –42 to –73 mV, indicating strong electrostatic stabilization and high colloidal stability of the aqueous system.

The volumetric behavior of aqueous solutions of C₆₀(NO₂)₁₈(OH)₂ was also investigated. The data, obtained at 25 °C in the concentration range 1.95 × 10⁻³ to 16.0 g/dm³, are presented in Table S4 (Supplementary Materials) and visualized in Figure 9.

As shown in Figure 10, the density of the solution increases with concentration, reaching a maximum at C ≈ 2.0 g/dm³. The average molar volume exhibits a shallow minimum in the range C = 0.0624–1.0 g/dm³, while the partial molar volume of the polynitro adduct passes through a broad maximum, and the partial molar volume of water shows a corresponding minimum near C ≈ 4.0 g/dm³.

At low concentrations (C ≤ 0.0156 g/dm³), the partial molar volume of the polynitro adduct is significantly negative, indicating a pronounced structuring or compaction of the solvent matrix around the solute. As concentration increases, the partial molar volume becomes positive and rises steadily. This behavior clearly reflects a shift from monomeric and small associative states to the formation of larger aggregates (associates of order II and III), with corresponding changes in the solution’s physicochemical environment.

The activity and activity coefficients of the polynitro adduct C₆₀(NO₂)₁₈(OH)₂ in aqueous solution were determined using cryometric analysis in the near-freezing temperature range T = 272–273 K. This method is based on the depression of the freezing point of water upon the addition of solutes, which reflects non-ideal solution behavior and allows estimation of thermodynamic parameters.

The measurements were conducted in the binary system C₆₀(NO₂)₁₈(OH)₂–H₂O, with the mole fraction of the polynitro adduct varying from 0 to 1.82 × 10⁻⁴. The onset of ice crystallization was recorded to determine the freezing point depression ΔT (K), and the corresponding values of water activity (ln aₜ) and activity coefficients for both water and the solute were calculated using classical cryoscopic equations.

The results of the cryometric study are presented in

Table 2. Cryometric analysis of aqueous solutions of C₆₀(NO₂)₁₈(OH)₂ in the temperature range T = 272–273 K.

$\mathbf{Molar}\mathbf{Fraction}\mathbf{of}{\mathbf{C}}_{60}\left({\mathbf{N}\mathbf{O}}_2{)}_{18}\right({\mathbf{O}\mathbf{H})}_2$ In Water Solution ${\mathbf{X}}_{{\mathbf{C}}_{60}{(\mathbf{N}\mathbf{O}}_2{)}_{18}(\mathbf{O}\mathbf{H}{)}_2}$ (a.u.)	Liquidus Temperature Decrease ∆T (K) (a.u.)	lnaW ln (Water Activity) (a.u.)	lnγW ln (Water Activity Coefficient) (a.u.)	${\mathbf{l}\mathbf{n}\mathbf{\gamma }}_{{\mathbf{C}}_{60}{(\mathbf{N}\mathbf{O}}_2{)}_{18}(\mathbf{O}\mathbf{H}{)}_2}$ ln (poly-Nitro-Adduct Activity Coefficient) (a.u.)
0.000	0.000	0.00000	0.00000	0.000
2.84 × 10−6	0.065	−0.00063	−0.00063	24.8
5.69 × 10−6	0.11	−0.00106	−0.00106	48.1
1.13 × 10−5	0.19	−0.00184	−0.00183	90.9
2.27 × 10−5	0.27	−0.00262	−0.00259	161
4.55 × 10−5	0.41	−0.00398	−0.00393	248
9.10 × 10−5	0.64	−0.00622	−0.00613	278
1.82 × 10−4	0.91	−0.00886	−0.00868	281

.

[−∆H^f_W∆T − ∆C_P∆T²]/[R (T^f₀ − ∆T)T^f₀] = lna_W

(3)

where ∆H^f_W = 5990 J/mole, ∆C_P = −38.893 J/mole∙K, and T^f₀ = 273.15 K are the heat of fusion, isobar heat capacity change at crystallization (in the temperature range T = 272 ÷ 273 K, the value is practically constant), and H₂O fusion temperature, correspondingly. High positive deviations from the ideality are observed. The results show strong positive deviations from ideality, indicating the presence of significant solute–solvent interactions and non-linear behavior typical of structured or aggregated systems.

To model the non-ideality more accurately, the Virial Decomposition Asymmetric Model (VD-AS) was applied. This model accounts for higher-order interactions in asymmetric binary systems and allows quantification of non-ideality parameters Λ₂, Λ₃, and Λ₄. These values, as well as the concentration range of diffusional instability (corresponding to spinodal decomposition boundaries), are presented in

Table 3. VD-AS model parameters (Λ₂, Λ₃, Λ₄) and concentration range of stability region (Xdiff-instab) in the binary system: ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2-$H₂O (in temperature range $\mathrm{T}=272÷273\mathrm{K}$).

VD-AS Parameters				Diffusional Instability Concentration Region
Λ2 (a.u.)	Λ3 (a.u.)	Λ4 (a.u.)	${\mathrm{R}}^2$(a.u.)	Xdiff-instab-start (a.u.)	Xdiff-instab-finish (a.u.)
4.48 × 106	−2.96 × 1010	6.60 × 1013	0.93	7.9 × 10−5	1.4 × 10−4

.

Using the Virial Decomposition Asymmetric Model (VD-AS), the non-ideality parameters were determined (Table 3):

lnγ₁^ass ≈ 2Λ₂ X₁ + 3Λ₃ X₁² + 4Λ₄ X₁³

(4)

lnγ₂^ass ≈ −Λ₂ X₁² − 2Λ₃ X₁³ − 3Λ₄ X₁⁴

(5)

where 1 is the poly-nitroadduct number and 2 represents the solvent number (w).

Concentration region of solution diffusional instability was calculated for the system ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2-{\mathrm{H}}_2\mathrm{O}$ (the region of instability are determined by inequality):

12Λ₄ X₁³ + 6Λ₃ X₁² + 2Λ₂ X₁ +1 > 0

(6)

The model parameters allowed us to calculate from water activity, activity, and activity coefficients of polynitro addcuts, as well as the diffusion stability regions of liquid solutions (i.e., the boundaries of delamination regions—the so-called spinodals). The latter coincide with the concentration boundaries of the existence of III-order associates with linear dimensions δ several μm.

The dependence of refractive indices of aqueous solutions of poly-nitroadduct in ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2$ (n_D²⁰) was determined by refractometry under the following conditions: $\mathrm{T}\approx 21\pm 2°\mathrm{C};$atmospheric pressure 748 mm. Hg; relative humidity of air—81%).

The experimental values of the refractive indices of aqueous solutions, the dependence of the refractive index on concentration, and the specific and molar refractive indices of the poly-nitro-adduct solution ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2-$ H₂O are given in Table S5 (in Supplement Materials) and Figure 11.

As shown in Figure 11, the refractive index of aqueous solutions of C₆₀(NO₂)₁₈(OH)₂ exhibits a distinct maximum at a concentration of approximately C ≈ 2.0 g/dm³. Both the specific refraction and molar refraction of the solution demonstrate significant changes at and above this concentration, indicating nonlinear behavior related to structural transformations in the system.

The molar refraction of the polynitro adduct itself aligns well with the theoretical value calculated by the additivity rule:

$${\mathrm{R}}_{\mathrm{a}\mathrm{d}\mathrm{d}}\approx 419{\mathrm{c}\mathrm{m}}^3/\mathrm{m}\mathrm{o}\mathrm{l}\mathrm{e}$$

(7)

This calculation assumes that the total molar refraction of a compound equals the sum of the atomic and bond contributions within the molecule. The experimentally determined molar refraction matches the average values reported in Table S5 (Supplementary Materials), thereby reinforcing the validity of the proposed molecular structure and elemental composition.

In addition, the specific refraction data provide a useful tool for determining the concentration of binary aqueous solutions. According to the additivity rule for specific refraction, the specific refraction of a solution is equal to the weighted sum of the specific refractions of its components, each multiplied by its corresponding mass fraction. This relationship enhances the utility of refractometry as a rapid and non-invasive method for concentration monitoring in fullerene-based nanomaterials.

The acid–base properties of the synthesized compound were further investigated at 25 °C, and the results are summarized in Figure 12. These findings offer additional insights into the dissociation behavior and potential buffering capacity of the polynitro adduct in aqueous environments.

As shown in Figure 12, an evident monotonic acidification of the solution is observed with increasing concentration of C₆₀(NO₂)₁₈(OH)₂, with pH values decreasing from approximately 5.1 to 4.3. This trend indicates progressive dissociation of acidic functional groups in the polynitro adduct.

Extrapolation of the dissociation constant to infinite dilution yields a thermodynamic dissociation constant of:

$${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2\leftrightarrow {\mathrm{C}}_{60}({\mathrm{N}\mathrm{O}}_2{)}_{18}\left(\mathrm{O}\mathrm{H}\right){\mathrm{O}}^{+}+{\mathrm{H}}^{+}$$

(8)

This value confirms that the compound behaves as a weak monoprotic acid in aqueous media. The corresponding dissociation equilibrium can be described by the following reaction:

This partial release of protons supports the presence of ionizable hydroxyl groups in the structure, consistent with prior NMR and IR spectral analyses.

To complement the acid–base study, the electrical conductivity properties of C₆₀(NO₂)₁₈(OH)₂ solutions were evaluated at 25 °C using a quartz conductivity cell equipped with platinum electrodes. Sodium chloride in water (NaCl–H₂O) was used as the reference system. The resulting conductivity data, including specific and equivalent conductivities as well as their extrapolated values at infinite dilution, are presented in Table S6 (Supplementary Materials) and graphically summarized in Figure 13.

As the concentration of C₆₀(NO₂)₁₈(OH)₂ increases, a nearly linear increase in specific electrical conductivity is observed. Simultaneously, a sharp decline in equivalent conductivity occurs, reflecting the typical behavior of weak electrolytes. Extrapolation of the concentration dissociation constant to infinite dilution yields a thermodynamic dissociation constant:

$$\mathrm{p}{\mathrm{K}}_{\mathrm{D}}^{\mathrm{t}\mathrm{h}\mathrm{e}\mathrm{r}\mathrm{m}}=\mathrm{l}\mathrm{i}{\mathrm{m}}_{\mathrm{M}\to 0}{\mathrm{p}\mathrm{K}}_{\mathrm{D}}\approx 5.1$$

(9)

This value indicates that C₆₀(NO₂)₁₈(OH)₂ behaves as a weak acid, comparable in strength to acetic acid (CH₃COOH), and supports the interpretation of partial dissociation in aqueous solution.

The antioxidant capacity of the polynitro adduct was evaluated by potentiometric redox titration, using hydrogen peroxide (H₂O₂) as the oxidizing agent. The experiments were conducted using a platinum working electrode in H₂O₂/NaOH medium, with a reference calomel electrode (Hg₂Cl₂/Cl⁻, saturated KCl, E⁰ = +0.28 V), and the standard potential of the H₂O₂/2OH⁻ system set at E⁰ = +0.40 V. Solutions of 0.01 mol/dm³ C₆₀(NO₂)₁₈(OH)₂ and 0.01 mol/dm³ H₂O₂ were used for mutual titration (see Figure 14).

This redox transformation (Equations (8)–(10)) occurs in quasi-equilibrium mode and reflects the ability of the polynitro adduct to accept electrons and reduce oxidizing agents. These findings confirm that the synthesized nanomaterial possesses notable antioxidant properties, which may be of practical significance in biomedical applications where oxidative stress is a concern.

The redox reaction proceeds through the following electrochemical equilibria:

• At the cathode:

H₂O₂ → H₂O + ½O₂ + 2e⁻ → 2OH⁻

(10)

• At the anode:

C₆₀(NO₂)₁₈(OH)₂ + 2OH⁻ − 2e⁻ → C₆₀(NO₂)₁₈(OH)₄

(11)

• Overall cell reaction:

C₆₀(NO₂)₁₈(OH)₂ + H₂O₂ → C₆₀(NO₂)₁₈(OH)₄

(12)

The potentiometric titration curve exhibits a characteristic sigmoidal (S-shaped) profile, indicating a single-step redox transition. The titration was performed in an acetate buffer (CH₃COOH/CH₃COONa, 1:1) at pH ≈ 4.8. The observed jump in the electromotive force of the galvanic cell was relatively small, |ΔE_G.C.| ≈ 0.091 V, which is indicative of a quasi-equilibrium redox process. The standard Gibbs free energy change for the reaction was estimated to be ΔG⁰ ≈ –17 kJ/mol, confirming the mild antioxidant potential of the polynitro compound through a reversible electron transfer pathway.

To further evaluate the redox behavior, a second potentiometric titration was conducted using iodine (I₂/I⁻) as the redox couple. The system employed a platinum working electrode (Pt) with a standard potential of E⁰ = +0.54 V and a saturated calomel reference electrode (Hg₂Cl₂/Cl⁻ in 1 mol/dm³ KCl, E⁰ = +0.28 V). A 0.01 mol/dm³ aqueous solution of C₆₀(NO₂)₁₈(OH)₂ was titrated with 0.01 mol/dm³ iodine solution in potassium iodide (I₂/KI), and vice versa (see Figure 15).

This redox transformation (Equations (11)–(13)) reflects a one-step electron transfer involving iodine as the oxidant, further supporting the antioxidant character of the synthesized polynitro fullerenol derivative. The shape and amplitude of the titration curve correspond to a low electrochemical driving force, with the reaction proceeding under near-equilibrium conditions.

The redox equilibria can be described as follows:

• Cathodic half-reaction:

I₂ + 2e⁻ → 2I⁻

(13)

• Anodic half-reaction:

C₆₀(NO₂)₁₈(OH)₂ + 2I⁻ + 2H₂O − 2e⁻ → C₆₀(NO₂)₁₈(OH)₄ + 2HI⁻

(14)

• Overall cell reaction:

C₆₀(NO₂)₁₈(OH)₂ + I₂ + 2H₂O → C₆₀(NO₂)₁₈(OH)₄ + 2HI

(15)

The potentiometric titration curve exhibits a characteristic sigmoidal (S-shaped) form. The titration was conducted in an acetate buffer solution with a 1:1 molar ratio of CH₃COOH to CH₃COONa at a pH of approximately 4.8. The redox reaction proceeded in a single stage and resulted in a relatively small jump in the electromotive force of the galvanic cell, ΔE_G.C. ≈ 0.115 V. This behavior is indicative of a quasi-equilibrium process corresponding to the electrochemical reaction (7), with an associated standard Gibbs free energy change of approximately ΔG⁰ ≈ –22 kJ/mol.

The solubility behavior of the poly-nitrofullerene C₆₀(NO₂)₁₈(OH)₂ was examined in both binary (water) and ternary (C₆₀(NO₂)₁₈(OH)₂–NaCl–H₂O) systems. Experimental data are presented in Tables S7 and S8 (see Supplementary Materials) and illustrated in Figure 16. Saturation was performed isothermally over 8 h in a shaker-thermostat operating at ~2 Hz, with temperature control precision of ±0.2 K at 0 °C, ±0.05 K at 25 °C, and ±0.5 K at 100 °C. Concentrations of the polynitro adduct in solution were determined spectrophotometrically using UV-visible absorption at λ = 300 nm.

To model the solubility behavior, an extended four-parameter version of the Sechenov equation (SEM-4 model) was employed. This model is particularly suited for describing the solubility of weak or non-electrolytes in solutions of strong electrolytes within polar solvents such as water, acetonitrile (CH₃CN), methanol (CH₃OH), and trifluoroacetic acid (CF₃COOH), among others. The SEM-4 model allows for accurate fitting of experimental data and accounts for complex association effects and ionic strength contributions within multicomponent aqueous systems.

The solubility of C₆₀(NO₂)₁₈(OH)₂ was initially modeled using the classical mono-parameter Sechenov model (SM), which is expressed as:

$$\ln ({\mathrm{m}}^0/\mathrm{m})={\mathrm{A}}_{\mathrm{s}}{\mathrm{m}}_{\mathrm{NaCl}}$$

(16)

where m⁰ and m represent the concentrations of C₆₀(NO₂)₁₈(OH)₂ in saturated binary (water only) and ternary (NaCl–water) systems, respectively. However, this model proved to be inadequate for accurately describing the solubility behavior of systems with complex, hierarchical, and multistage association mechanisms, such as those observed in fullerene derivatives [23,25].

To address this limitation, an extended four-parameter version of the Sechenov model (SEM-4) was employed:

$$\ln ({\mathrm{m}}^0/\mathrm{m}){\mathrm{A}}_{\mathrm{s}}{\mathrm{m}}_{\mathrm{NaCl}}+{\mathrm{B}}_{\mathrm{s}}{\mathrm{m}}_{\mathrm{NaCl}}^2+{\mathrm{D}}_{\mathrm{s}}{\mathrm{m}}_{\mathrm{NaCl}}^3+{\mathrm{E}}_{\mathrm{s}}{\mathrm{m}}_{\mathrm{NaCl}}^4$$

(17)

This expanded model significantly improved the fit to the experimental data and provided a more reliable representation of solubility in the ternary system C₆₀(NO₂)₁₈(OH)₂–NaCl–H₂O, accounting for the complex molecular associations and salt effects present in the solution.

3. Discussion

The synthesized compound, C₆₀(NO₂)₁₈(OH)₂, represents a novel polynitro-hydroxyl adduct of light fullerene C₆₀, characterized by an exceptionally high degree of nitration—18 nitro groups per fullerene core. To the best of our knowledge, such a structure has not been previously reported. Its molecular composition and unique structure were thoroughly confirmed using a combination of analytical techniques, including elemental analysis, UV-visible and infrared spectroscopy, NMR spectroscopy, and refractometric analysis.

The adduct exhibits significant thermal instability, which limits the application of mass spectrometry, even under gentle ionization conditions such as soft sublimation. Moreover, the compound shows pronounced chemical lability in alkaline media: irreversible alkaline hydrolysis occurs with the transformation of nitro groups into hydroxyls, forming fullerenol derivatives. It follows from the results of complex thermal analysis (pp. 9–10) that in the natural temperature range for humans, the poly-nitroadduct is quite stable both in the solid phase and in aqueous solutions. Under conditions of normal values of the hydrogen index of physiological fluids (close to neutral, as well as pronounced acidic media)—blood, cerebrospinal fluid, lymph, and gastric juice—the polynitro-adduct is quite resistant to hydrolysis. In alkaline physiological fluids, such as in the oral cavity or, for example, intestinal juice, with increased secretion, more or less irreversible hydrolysis may occur with the substitution of nitro groups for hydroxyl groups.

Such substitution will not lead to the formation of solid-phase precipitates, since with an increase in such substitution, the solubility of the substituted adducts only increases, and for fully substituted fullerenols (C₆₀(OH)₂₀ in our case), it is several tens of g/dm³. Salting of the poly-nitroadduct in aqueous solutions of electrolytes (sodium chloride, first of all) undoubtedly occurs (see, for example, p. 21); however, it is also insignificant if low concentrations of poly-nitroadduct (up to units of g/dm³) are used (the phrase is inserted in p. 22).

In aqueous solutions, the adduct demonstrates a pronounced hierarchical self-association behavior, forming aggregates of increasing complexity—monomeric units, first-order (nanocluster) associates, second-order (nanocolloidal) aggregates, and ultimately third-order (microcolloidal) structures. These higher-order associations correspond to a microheterogeneous phase state. The observed negative zeta potential across all aggregate types imparts relative colloidal stability, especially at low to moderate concentrations. However, at concentrations exceeding several grams per liter, the system progressively loses diffusion and sedimentation stability due to the formation of large third-order associates.

Electrokinetic measurements indicate that the primary charge carriers in solution are hydrated protons and small- to medium-sized associates. This interpretation is further supported by volumetric data, including concentration-dependent average and partial molar volumes, which confirm structural transitions and aggregation behavior in solution.

From an acid-base perspective, C₆₀(NO₂)₁₈(OH)₂ behaves as a weak acid in aqueous media, with a dissociation constant (pK_D ≈ 5.1) comparable to that of acetic acid (CH₃COOH). In terms of redox behavior, the adduct displays notable antioxidant and reducing properties, though slightly weaker than those of ascorbic acid. Importantly, the redox activity is multi-step and reversible, depending sensitively on the pH and redox potential of the surrounding medium.

C₆₀(NO₂)₁₈(OH)₂ demonstrates high water solubility, reaching tens of grams per liter, and this solubility increases markedly with temperature. The compound is also highly compatible with physiological fluids such as saline, blood plasma, lymph, cerebrospinal fluid, and gastric juice, highlighting its potential for biomedical applications. However, the introduction of strong electrolytes (e.g., NaCl, HCl) into solution leads to a pronounced decrease in solubility due to the well-known salting-out effect.

4. Materials and Methods

The synthesis of the mixed polynitro-hydroxylated adduct C₆₀(NO₂)₁₈(OH)₂ (see Figure 1) was performed using a classical nitration procedure with a strongly oxidizing nitrating mixture composed of concentrated nitric acid (HNO₃) and sulfuric acid (H₂SO₄). The acid volumes were as follows: HNO₃—approximately 250 cm³ (65 wt.%, chemically pure, GOST 4461-77, manufacturer: Magna, Magna International, Orora, ON, Canada); H₂SO₄—approximately 50 cm³ (94 wt.%, extra pure, GOST 14262-78, manufacturer: ChemExpress, Saint-Petersburg, Russia).

A quantity of 500 mg of high-purity C₆₀ fullerene (≥99.9 wt.%, ILIP Ltd., St. Petersburg, Russia) was combined with the nitrating mixture. The reaction mixture was stirred magnetically (rotation speed ~2 Hz) for 7 days at a temperature of 80 ± 5 °C using silicone oil as the heat-transfer medium. During the reaction, the initial black-blue opaque suspension gradually turned into a translucent green-brown viscous solution.

The reaction mixture was then filtered through a Schott glass filter to obtain a clear, light-yellow filtrate.

To remove excess nitric acid, concentrated hydrogen peroxide (H₂O₂, 37 wt.%, chemically pure, GOST 177-88, manufacturer: Lenreactive, Saint-Petersburg, Russia) was added dropwise at room temperature. This resulted in the evolution of brown nitrogen dioxide gas according to the reaction: 2 HNO₃ + H₂O₂ → 2 NO₂ + 2 H₂O + O₂↑.

Subsequently, colorless oxygen was slowly released. An additional 10 cm³ of H₂O₂ was introduced to ensure the complete decomposition of residual HNO₃, and the solution was left to stand for 24 h.

The remaining sulfuric acid was neutralized using an aqueous sodium hydroxide solution (30 wt.%, analytical grade, GOST 4328-77, Chem. Express., Saint-Petersburg, Russia) until the pH reached approximately 7 ± 1.

The resulting solution was evaporated using a rotary vacuum evaporator at 80 ± 5 °C. During evaporation, colorless sodium sulfate (Na₂SO₄) crystals precipitated and were removed by filtration. The remaining transparent yellow solution had a final volume of approximately 100 ± 10 cm³.

Residual inorganic salts were removed by dialysis using regenerated cellulose dialysis tubing (MD25, molecular weight cut-off ~1000, 77 mm diameter, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The dialyzed solution was then evaporated to dryness in a vacuum drying oven at 50 ± 5 °C and a residual pressure of 18 ± 3 mm Hg.

This procedure yielded approximately 475 mg of brown, finely dispersed crystalline polynitro adduct. Based on the assumed molecular formula C₆₀(NO₂)₁₈(OH)₂, the reaction yield was estimated at ~44% of the theoretical maximum. Authors tried to obtain reproducible mass spectra, using soft ionization methods in mass spectrometry (which is usually used for nanocluster investigation): MALDI Express, electrospray ionization, and chemical ionization at atmospheric pressure. However, unfortunately, the result was irreproducible; during sublimation-ionization, a large number of uninterpretable fragments were formed. The authors realize that in the absence of reliable mass spectrometric data, the established formula of poly-nitroadduct is not justified, and the established output is relatively conditional.

Grapher software package ORIGIN (Version OriginPro 7.0 with subprogram package Nonlinear curve fit) was used (Electronic Arts, Redwood City, CA, USA).

Main characteristics of used research methods in the present investigation are represented below in

Table 4. Research methods and instruments used in the identification of ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2.$

No	Research Method	Device
1	Elemental analysis	C/H/N analyzer 5E-CHN2200 and electronic microscope VEGA-3 TESCAN (XRF analyzer, Brno, Czech Republic)
2	Infrared spectroscopy	Spectrophotometer Shimadzu FTIR-8400S ($\tilde{\mathsf{\nu }}$ = $400÷4000{\mathrm{c}\mathrm{m}}^{-1},\mathrm{K}\mathrm{B}\mathrm{r}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{t}\mathrm{s})$
3	Electronic spectroscopy	UV-VIS-NIR Shimadzu, Kyoto, Japan; λ = 200–1110 nm; standard—H2O (distilled)
4	High-performance liquid chromatography	HPLC Agilent 1200, San Clara, CA, USA; Column-Agilent Zorbax SB-C18; Eluent: ${\mathrm{C}\mathrm{H}}_3\mathrm{C}\mathrm{N}+-{\mathrm{H}}_2\mathrm{O}({\displaystyle \frac{1}{20}})$; Detection-spectra-photometry at $\mathsf{\lambda }=330,280\mathrm{m}\mathrm{n}$
5	Nuclear magnetic resonance	13C; 1H; AVANCE-II 400WB Bruker, Berlin, Germany; TMC; $\mathsf{\nu }=$100.4 MHz.
6	Complex thermal analysis	Shanghi Jiahang Instruments Co., Ltd., Shanghai, China; Device JHDTA332; Atmosphere—air, pressure—1 atm., temperature range T = 25 ÷ 950o C, rate: 5 °C/min.
7	X-ray diffraction	Powder diffractometer (Bruker, Berlin, Germany). The anode material of the X-ray tube is cobalt (${\mathrm{K}}_{\mathsf{\alpha }},$ λ = 1.78897 Å); the exposure time at the point is 0.2 s; the angular range is 2θ—10–80 °; the accelerating voltage is Uacc. = 30 kV; and the current of the anode circuit is I = 10 mA. Sample preparation: 1. The poly-nitroadduct was placed in an agate mortar and crushed by grinding for 10 s to a medium dispersed state. 2. By adding ethanol, a suspension was obtained. 3. The resulting suspension was applied with a brush to a mono-crystalline Si substrate (standard cuvette from the D2 Phaser kit) 4. The suspension was dried in a cuvette using infrared radiation from a halogen lamp at a temperature $\mathrm{T}\le 40 °\mathrm{C}$.

.

The structure and composition of the synthesized C₆₀(NO₂)₁₈(OH)₂ were confirmed using elemental analysis, infrared (IR) spectroscopy, electron (UV–Vis) spectroscopy, high-performance liquid chromatography (HPLC), thermogravimetric analysis (TGA), nuclear magnetic resonance (NMR) spectroscopy, and X-ray powder diffraction (XRD).

Methods and devices used in the study of physicochemical properties of poly-nitroadduct ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2$are shown in

Table 5. Methods and devices used in the study of physicochemical properties of poly-nitroadduct ${\mathrm{C}}_{60}\left({\mathrm{N}\mathrm{O}}_2{)}_{18}\right({\mathrm{O}\mathrm{H})}_2.$

No	Research Method	Device
1	Dynamic light scattering—DLS	Malvern Zetasizer Nano ZS90
2	Density	Quartz pycno-meters
3	Refraction-metry	Refraction-meter HRK 9000 A
4	Electric properties	Immitance RLC-meter—HAMEGHM8118
5	Acid-base properties	Seven Compact pH meter S220, $∆\mathrm{p}\mathrm{H}=\pm 0.005\mathrm{a}.\mathrm{u}.$
6	Potentiometric titration	Ion meter-voltmeter 781 pH/Ion Meter.

.

Authors have not conducted detailed preliminary biocompatibility or antimicrobial tests to support these claims. The authors conducted only a few primary tests on protozoa: Biotesting on chlorella vulgaris growth and toxicity of poly-nitroadduct C₆₀ in this case, Paramecium caudatum. Data are represented in the Supplement to the article. The primary study of antioxidant activity is described on pp. 19–21 of the main text. The authors believe that in the standard conditions of existence of living organisms, it can be argued that super-low and low concentrations of poly-nitroaddct are non-toxic or low-toxic. Undoubtedly, a detailed and thorough study of the possible biochemical and medical aspects of the use of poly-nitroadducts will need to be carried out in the future. However, this was not the direct purpose of this article, which is mainly chemical-oriented.

5. Conclusions

A direct, non-catalytic synthesis of a novel water-soluble polynitro-hydroxylated adduct based on light fullerene C₆₀ was successfully carried out. The resulting compound, C₆₀(NO₂)₁₈(OH)₂, represents a previously undescribed molecular structure with a high degree of nitration—18 nitro groups per C₆₀ core.

The identity and composition of the adduct were confirmed using a range of analytical techniques, including elemental (C-H-N) analysis, energy-dispersive X-ray microanalysis, infrared (IR) and electron spectroscopy, nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), and thermogravimetric analysis (TGA). Refractive, volumetric, cryometric, electrochemical, acid–base, and antioxidant properties were also studied in detail.

Cryometric measurements, interpreted via the Virial Decomposition Asymmetric Model (VD-AS), allowed for the calculation of activity coefficients and identification of diffusion instability zones in binary C₆₀(NO₂)₁₈(OH)₂–H₂O solutions.

The physicochemical data collected are mutually consistent and support the proposed molecular formula. The adduct is thermally unstable, which prevents reliable mass spectrometric analysis, even under mild ionization conditions. In alkaline media, the compound undergoes irreversible hydrolysis, leading to the formation of fullerenols.

In aqueous solution, C₆₀(NO₂)₁₈(OH)₂ exhibits hierarchical self-association. Monomeric molecules form primary (I-order), secondary (II-order), and ternary (III-order) associates, with the latter corresponding to a microheterogeneous phase. These associations are accompanied by uniformly negative electrokinetic potentials, which provide colloidal stability at low to moderate concentrations. However, at higher concentrations (>several g/dm³), the solutions lose both diffusion and sedimentation stability. The formation of associates of the I and II orders (with associate sizes in the tens and first hundreds of nanometers and nanocluster concentrations in hundredths and tenths of g/dm³, respectively) should not significantly limit the biomedical use of poly-nitroadducts. Moreover, as the nanocluster solutions are naturally diluted with physiological fluids, the nanoclusters are crushed with a decrease in their order and size. However, the formation of micro-heterogenic associates of the III order of several μm in size (in solutions with a concentration of several g/dm³) can be dangerous from a medical point of view. They can fundamentally block micro-vessels in living organisms, injure them, serve as centers of subsequent crystallization—plaque growth, etc. The effectiveness of such large associates when used may be very low. That is why the authors believe that for further biomedical applications, the upper limit of the concentration of poly-nitroadducts is a conditional concentration of 1 g/dm³. However, in the future, if medical research establishes the fact of concentration and secondary accumulation of nanoclusters in any tissues, our assessment may change.

Electrochemical mobility data suggest that the primary charge carriers in solution are hydrated protons and I- and II-order associates. These findings are reinforced by volumetric studies showing concentration-dependent variations in average and partial molar volumes.

From an acid–base standpoint, the adduct behaves as a weak acid, similar in strength to acetic acid (CH₃COOH). In redox behavior, the compound exhibits significant antioxidant and reducing activity—though somewhat weaker than ascorbic acid—and can undergo multistep, reversible redox transitions in response to changes in pH or redox potential.

The compound demonstrates high solubility in water (tens of grams per liter), with solubility increasing with temperature. It also dissolves well in physiological fluids, including saline, blood plasma, lymph, cerebrospinal fluid, and gastric juice. However, the introduction of strong electrolytes such as NaCl or HCl causes a marked reduction in solubility due to salting-out effects.

Overall, the comprehensive physicochemical characterization of C₆₀(NO₂)₁₈(OH)₂ suggests strong potential for its application as a medical-grade nanomaterial. These results provide a foundation for further research into its biological compatibility, therapeutic activity, and functional performance in biomedical systems.