Lidija Matija

To overcome the negative effects of the biochemical application of nano-substances in medicine (toxicity problem), using the example of fullerene C₆₀’s first derivative (fullerenol, FD-C₆₀), we show that their biophysical effect is possible through non-covalent hydrogen bonds when around FD-C₆₀ water layers are formed. SD-C₆₀ (Zeta potential is −43.29 mV) is much more stable than fullerol (Zeta potential is −25.85 mV), so agglomeration/fragmentation of the fullerol structure, due to instability, can cause toxic effects. When fullerol in solution was exposed to an oscillatory magnetic field with Re (real) part [250/−92 mT, H(ωt) = Acos(ωt)], water layers around FD-C₆₀ (fullerenol) are formed according to the Penrose process of 3D tiling formation, and the second derivative, SD-C₆₀ (or 3HFWC), is self-organized. However, when Im (imaginary) part [250/−92 mT, H(ωt) = Bisin (ωt)] of the external magnetic field is applied in addition to SD-C₆₀, ordered water chains and bubbling of water (“micelle”) are formed as a third derivative (TD-C₆₀). Fullerol (FD-C₆₀) interacts with biological structures biochemically, while the second (SD-C₆₀) and third (TD-C₆₀) derivatives act biophysically via non-covalent hydrogen bond oscillation. SD-C₆₀ and TD-C₆₀ significantly increased water solubility and reduced toxicity. The paper explains the synthesis of SD-C60 and TD-C60 from FD-C60 (fullerol) as a precursor by the influence of an oscillatory magnetic field (“Yin-Yang” principle) on hydrogen bonds in order to create water layers around fullerol. Examples of biomedical applications (cancer and Alzheimer’s) of this synergetic complex are given. This study shows that the “Yin-Yang” machinery, based on the nanophysics of C₆₀ molecules and non-covalent hydrogen bonds, is possible. The first attempt has been composed to synthesize nanomaterial for biophysical vibrational nanomedicine.

Objectives: Today's pharmacy and its application in medicine, including the current nanotechnological approach, is based on the biochemical laws of covalent bonds. Due to their changed chemical potential, most nanosubstances, compared to classical ones, as well as tissue penetration, have distinct toxic properties. To overcome the negative effects of the biochemical application of nano-substances in medicine, using the example of fullerene C60 and its derivatives, we show that their biophysical effect is possible through non-covalent hydrogen bonds; Methods: In the first step, we used the molecule C60, which is very poorly soluble in water and toxic under certain conditions, and then we added C60 with 36 OH groups (first derivative, FD-C60 or fullerol) with increased water solubility and reduced toxicity. To reduce higher toxicity and increase solubility and effect, fullerol in solution was exposed to an oscillatory electromagnetic field with Re (real) and Im (imaginary) parts. Three to nine stable water layers around the fullerol are formed when Re part of the electromagnetic field is used (second derivative, SD-C60 or 3HFWC). However, in addition to SD-C60, ordered water chains and bubbling of water in the solution (third derivative, TD-C60) are formed when both Re and Im part of the external electromagnetic field are applied. Results: A comparative structural and physicochemical characterization of fullerene C60 and its derivatives was performed. Fullerene C60 and fullerol (FD-C60) interact with biological structures biochemically, while the second (SD-C60) and third (TD-C60) derivatives act biophysically via hydrogen bond oscillation. The paper explains the mechanism of action of SD-C60 and TD-C60 via hydrogen bonds and gives examples of biomedical applications citing our published experimental results. Conclusions: This study shows that the Yin-Yang machinery, based on the nanophysics of non-covalent hydrogen bonds, is possible and that the first attempt has been made to establish nanomedicine based on non-covalent hydrogen bonds of water, DNA and proteins.

In order to maximally reduce the toxicity of fullerenol (the first derivative of C₆₀, FD-C₆₀), and increase its biomedical efficiency, the second derivative SD-C₆₀ (3HFWC, Hyper-Harmonized Hydroxylated Fullerene Water Complex) was created. Several different methods were applied in the comparative characterization of FD-C₆₀ and SD-C₆₀ with the same OH groups in their core. FD-C₆₀ as an individual structure was about 1.3 nm in size, while SD-C₆₀ as an individual structure was 10–30 nm in size. Based on ten physicochemical methods and techniques, FD-C₆₀ and SD-C₆₀ were found to be two different substances in terms of size, structure, and physicochemical properties; FD-C₆₀, at 100 °C, had endothermic characteristics, while SD-C₆₀, at 133 °C, had exothermic characteristics; FD-C₆₀ did not have water layers, while SD-C₆₀ had water layers; the zeta potential of FD-C₆₀ was −25.85 mV, while it was −43.29 mV for SD-C₆₀. SD-C₆₀ is a promising substance for use in cosmetics and pharmaceuticals.

(1) Background: Since fullerene C60 is insoluble in water and is toxic, it is not applicable in biomedicine and cosmetics. For this reason, the first derivative FD-C60 (fullerenol) was synthesized, which solved the problem of solubility in water and partially reduced toxicity. In order to maximally reduce toxicity and increase biomedical efficiency, the second derivative SD-C60 (3HFWC- Hyper-Harmonized-Hydroxylated Fullerene Water Complex), was created. (2) Methods: In order to obtain the most reliable data, several different methods are applicable in the characterization of FD-C60 and SD-C60 with water layers (NIR, FTIR, 13C-NMR, 1H-NMR, TGA/DTA, XRD, AFM/MFM etc.). (3) Results: FD-C60 as an individual structure is about 1.3 nm in size, while SD-C60 as an individual structure is 1030 nm in size. The aforementioned methods showed that SD-C60 contains water layers, which explains the difference between those two structures in the physicochemical properties, and their effects on biomolecules. (4) Conclusions: Based on the above methods and techniques, FD-C60 and SD-C60 are two different substances in terms of size, structure, and physicochemical properties; FD-C60 at 100C has endothermic characteristics and SD-C60 at 133C has exothermic characteristics, FD-C60 possesses water molecules only from humidity, while SD-C60 has water molecules from both humidity and extra water molecules in layers, the zeta potential of FD-C60 is -25.85 mV, while it is -43.29 mV for SD-C60.

The human body contains 60–70% water, depending on age. As a body fluid, it is not only a medium in which physical and chemical processes take place, but it is also one of the active mediators. Water is the richest substance with non-covalent hydrogen bonds. Water molecules, by themselves (in vacuum), are diamagnetic but when organized into clusters, they become diamagnetic or paramagnetic. Also, biomolecules (DNA, collagen, clathrin, and other proteins) have non-covalent hydrogen bonds in their structure. The interaction, as well as signal transmission, between water and biomolecules is achieved through the vibrations of covalent and non-covalent hydrogen bonds, which determine the state and dynamics of conformational changes in biomolecules. Disruptive conformational changes in biomolecules, cells, and tissues lead to their dysfunctionality, so they are a frequent cause of many disorders and diseases. For example, the rearrangement of hydrogen bonding due to mitochondrial disease mutation in cytochrome bc1 disturbs heme bH redox potential and spin state. In order to prevent and repair the dysfunctional conformational changes, a liquid substance was developed based on the second derivative of the C₆₀ molecule (SD-C₆₀), which has classical and quantum properties. The characterization of SD-C₆₀ by UV-VIS-NIR, FTIR, TEM, and AFM/MFM was performed and it is shown that SD-C₆₀ water layers generate vibrations with near-zero phase dispersion which are transmitted through Fibonacci’s water chains to biomolecules. In comparison with previously published SD-C₆₀ derivate (3HFWC, size until 10 nm, and 1–5 water layers), the improved formulation (3HFWC-W, size 10–25 nm, and 6–9 water layers) showed multiplied cytotoxic activity against melanoma cell lines of different aggressiveness. Apart from this, the mode of action was preserved and based on an induction of senescence rather than cell death. Importantly, high selectivity towards malignant phenotypes was detected. Observed effects can be ascribed to a machinery of hydrogen bonds, which are generated in SD-C₆₀ and transmitted through water to biomolecules. This approach may open a new field in science and healthcare—a “water-based nanomedicine”.

In this paper, a relatively new hyper convolution spectra for characterization liquid, viscoelastic and solid samples is presented. Special attention is paid to the biophysical characterization of epithelial tissues and early detection of cancer of the skin, cervix, colon, and oral cavity. The method is based on the use of two types of white light, diffuse and polarized, from the same LED light sources. Based on the subtraction of the intensity values of these two lights and using the hyper convolution algorithm, the ratio of paired and unpaired electrons of tissue is obtained. This ratio correlates well with the state of the tissue: normal, irritating, precancerous, and cancerous. First, a proof-of-concept studies were performed, and later clinical studies on samples from one thousand two hundred fifteen people were performed. It showed that hyper Opto-Magnetic Imaging Spectroscopy (OMIS) can detect epithelial tissue cancers with an accuracy of 85-96%. Bearing in mind that the OMIS method also acquires the biophysical characteristics of the deeper skin layers (epidermis, basement membrane, dermis) by analysing convolution spectra of RGB channels of both diffuse and polarized white light, it is possible to apply this method in cosmetics (skin hydration, collagen, and elastin states, etc.). During OMIS applications, we noticed places where improvements could be made to increase method’s accuracy using advanced machine learning methodologies.

The human body contains 60-70% of water, depending on age. As a body fluid, it is not only a medium in which physical and chemical processes take place, but it is also one of the active mediators. Water is the richest substance with non-covalent hydrogen bonds. Water molecules, by themselves (in vacuum), are diamagnetic, but when organized into clusters, become diamagnetic or paramagnetic. Also, biomolecules (DNA, collagen, clathrin, and other proteins) have non-covalent hydrogen bonds in their structure. The interaction, as well as signal transmission, between water and biomolecules is achieved through the vibrations of covalent and non-covalent hydrogen bonds, which determine the state and dynamics of conformational changes of biomolecules. Disruptive conformational changes of biomolecules, cells, and tissues lead to their dysfunctionality, so they are a frequent cause of many disorders and diseases. For example, the rearrangement of hydrogen bonding due to mitochondrial disease mutation in cytochrome bc1 disturbs heme bH redox potential and spin state. In order to prevent and repair the dysfunctional conformational changes, a liquid substance was developed based on the second derivative of the C60 molecule (SD-C60), which has classical and quantum properties. Characterization of SD-C60 by UV-VIS-NIR, FTIR, TEM, and AFM/MFM was done and it is shown that SD-C60 water layers generate vibrations with near-zero phase dispersion which are transmitted through the Fibonacci's water chains to biomolecules. In comparison to previously published SD-C60 derivate (3HFWC, size until 10 nm, and 1-5 water layers), improved formulation (3HFWC-W, size 10-25 nm, and 6-9 water layers) showed multiplied cytotoxic activity against melanoma cell lines of different aggressiveness. Apart from this, the mode of action was preserved and based on induction of senescence rather than cell death. Importantly, high selectivity toward malignant phenotype was detected. Observed effects can be ascribed to a machinery of hydrogen bonds, which are generated in SD-C60 and transmitted through water to biomolecules. This approach may open a new field in science and healthcare - a “water-based nanomedicine".

Multiphoton microscopy (MPM) is a cutting-edge nonlinear microscopy technique known for its exceptional capabilities in imaging dense tissues, making it an ideal tool for exploring human kidney tissue. This study delves into the application of MPM in evaluating renal cell tumors and characterizing tumor capsules. MPM encompasses two core methods: two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG). It leverages femtosecond lasers to provide deep tissue imaging while minimizing phototoxicity, making it well-suited for live tissue analysis. The study analyzed 100 formalin-fixed paraffin-embedded (FFPE) kidney tissue samples, ensuring compliance with ethical standards. Using a Ti:Sapphire laser for femtosecond pulses and a variety of optical components and filters, the researchers captured TPEF and SHG signals, facilitating image analysis. Normal kidney tissue exhibited distinctive structures like glomeruli and tubules under MPM. Renal cell carcinoma (ccRCC) displayed unique TPEF patterns with eccentrically located nuclei and indistinct cytoplasmic membranes. Papillary RCC (pRCC) exhibited a papillary pattern with dark nuclei, while chromophobe RCC (chRCC) showcased distinct features like thick cell membranes, strong TPEF signal in nuclei, and granular cytoplasm. Renal oncocytoma (RO) presented strong TPEF signals in cuboidal cells with microcyst-like arrangements. Overall, MPM proved invaluable in assessing human kidney tissue, offering distinct TPEF patterns for different renal cell tumor types. This non-invasive technique has the potential to enhance pathological examinations and advance our understanding of complex biological processes, ultimately contributing to improved diagnostics and treatment strategies for renal diseases.