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Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

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Open Access Full Text Article   Research Article

Humic nanoparticles as a tool for eliminating the toxicity of zinc L-valinate.

Vladimir N. Tumasov ¹*, Alla V. Marukhlenko ² and Anton V. Syroeshkin ²

¹ Department of Pharmaceutical Chemistry and Organization of Pharmaceutical Business, Faculty of Medicine, Lomonosov Moscow State University, GSP-1, Leninskie Gory, Moscow, 119991, Russian Federation

² Department of pharmaceutical and toxicological chemistry, Medical Institute, RUDN University, 6 Miklukho-Maklaya St, Moscow, 117198, Russian Federation

 

 

INTRODUCTION

Modification of the dispersion properties of a dosage form has long been the key to changing the pharmacokinetic properties of drugs ^1-3. The inclusion of an active pharmaceutical ingredient (API) in dosage forms of nanoparticles, nanomicelles, microemulsions, and liposomes enables improving the pharmacokinetic parameters (absorption, bioavailability, distribution, targeted delivery) of the drug ^4,5. 

For example, the inclusion of surface-active lipids in the composition of the drug allowed obtaining liposomal dosage forms that are distinguished by their high therapeutic efficacy rates achieved through stabilization, increased permeability through natural barriers and targeted delivery of the API ⁶. An important consequence of targeted delivery of the API in this case is a decrease in the systemic toxicity of the drug. These characteristics of liposomal drugs, together with biodegradability and reduced toxicity of the liposomes themselves, determine the interest in them. 

Modification of the physicochemical properties of nanoparticles, such as size, shape, structure, zeta potential and surface functionality, is achieved by obtaining a drug with targeted delivery of the API ⁷. Stabilization of dispersed phase particles by regulating the zeta potential allows for an increase in the drug shelf life, as well as an increase in its bioavailability, as has been shown in the example of self-nanoemulsifying drug delivery systems ⁸.

Natural polyelectrolytes are of interest in the development of new drug delivery systems as a replacement for synthetic carriers, which are more expensive and less environmentally friendly ⁹. An example of such natural polyelectrolytes is humic acids (HAs) and fulvic acids (FAs), which have great potential for medical use ^10,11. Humic substances (HS) are heterogeneous, polydisperse mixtures consisting of polymer structures that are formed in soils, bottom sediments, and natural waters during the destruction of microbial and plant residues ¹². HS are characterized by a large number of polar groups in their structures, such as carboxyl, hydroxyl, and phenolic ones; thereby humic substances are anionic polyelectrolytes. This allows humic substances to chelate metals, forming structures with high bioavailability ¹³. As some studies show ¹⁴, humic substances are also able to increase the bioavailability of organic molecules. The main components of humic substances are fulvic acids, which are soluble in the entire pH range, while humic acids precipitate at pH <2 ¹⁵. In recent years, various data have appeared on the pharmacological activity of humic and fulvic acids in relation to HIV-1 ¹⁶, influenza virus ¹⁷, herpes simplex virus-1 ¹⁸, tick-borne encephalitis virus ¹⁹, Enterococcus faecalis, and Klebsiella pneumoniae ²⁰. The specific virucidal activity of the HAs and FAs complex against SARS-CoV-2 was also established ²¹. Thus, humic and fulvic acids can be used as drug delivery systems and also have their own pharmacological effect: antiviral or antibacterial. 

Zinc micronutrient deficiency in the population is a public health problem in a wide range of countries ²². Zinc deficiency is associated with severe diseases of various etiologies ^23,24. Chelated zinc compounds with amino acids are noteworthy owing to the high bioavailability of the metal and the absence of an irritant effect on the esophagus compared to inorganic salts ^25,26. Therefore, the development of effective, safe, and low-cost approaches to obtain zinc delivery systems is an important task in the fight against zinc deficiency conditions. This study investigated the possibility of using a humic-fulvic acid complex to reduce the toxicity of zinc valinate through the specific interaction with HAs and FAs nanoparticles that changes their dispersion.

MATERIALS AND METHODS

Chemicals

HAs and FAs

We used a liquid concentrated complex of humic and fulvic acids isolated from lowland peat according to the patented technology of the company VimaVita (LLC Sys-tem-BioTechnologies, Moscow, Russia). Concentrated humic complex (HC) containing purified water and active components HAs, hymatomelanic acids, FAs and structural analogs of humic substances, was obtained by oxidative-hydrolytic degradation of lignin-containing raw materials followed by high-intensity acoustic cleaning. The test solution was a concentrated dark brown viscous liquid with pH = 7.98 ± 0.1 and dry matter content 7.34×10⁻² g/mL [21]. To study the properties of the humic complex as a delivery system for antiviral drugs the original preparation was not used, but its aqueous dilutions in the ratio from 1:400 to 1:800 by volume. For dilution, highly purified water was used, obtained using the Milli-Q® purification system (Merck, Darmstadt, Germany). All investigated solutions were stored at room temperature for no longer than 24 h.

Reagents

L-valine (98.0%, Sigma-Aldrich Co., Massachusetts, United States), zinc sulfate monohydrate (99.0%, Acros Organics, Barcelona, Spain) and all other chemicals used (potassium hydroxide) were of analytical grade.

 

Preparation of solutions

Dilutions of humic and fulvic acids were obtained by dissolving the HAs and FAs concentrate (dry matter content 7.34×10⁻² g/mL [21]) in MQ water (v/v) with constant stirring on a magnetic stirrer.

Zinc valinate solutions were obtained by dissolving L-valine and zinc sulfate (in a molar ratio of 2:1) in the obtained dilutions of HAs and FAs, with constant stirring on a magnetic stirrer. After dissolving the powders of the substances, the pH of the solution was adjusted to a value of 6.00 by adding 0.01 M potassium hydroxide aliquots with constant stirring.

Methods

Dynamic Light Scattering (DLS)

A Zetasizer Nano ZSP (Malvern Panalytical, Worcestershire, UK) based on dynamic light scattering was used to measure the size of nanoparticles in the aqueous solutions of zinc chelate complexes with valine. Disposable polystyrene cuvettes, filled with 1 mL of sample, were used. For each size determination, three replicate measurements were performed, and the average size value was calculated. Each measurement consisted of 12 runs. The refractive index value was 1.3400. To measure the zeta potential, laser Doppler microelectrophoresis was used, based on determining the velocity of movement of nanoparticles using electrophoresis. Each sample was measured 5 times.

Cellular Biosensor Spirostomum ambiguum for Testing the Biological Activity

Spirostomum ambiguum is the protozoan ciliate that is widely used as a test culture for toxicological and pharmacological studies of individual and combined biological activity of medicines ²⁷. 

The advantage of this model is due to the fact that the sensitivity of the eukaryotic cell makes it possible to interpret the obtained toxicity results in relation to multicellular organisms and humans. Under favorable conditions in a low-mineralized environment, cells do not die for a period exceeding their cell cycle (about 20 h). When it is introduced into an environment with chemical compounds, it dies within a time interval that is a function of concentration and temperature. ²⁸

The experiment was carried out in a temperature range of 22 – 32 °С (in increments of 2 °С). The experimental installation included a thermostatically controlled 5-hole plate (Lauda Alpha A6 termostat, Göttingen, Germany) and an MBS-10 binocular. Low-power fluorescent daylight lamps (10 W) were used for additional lighting.

One test infusoria S. ambiguum and 250 µl of the test solution and were introduced into each of the plate holes. Five measurements were carried out at each test temperature for each solution of the test sample. The cell lifetime was calculated as the interval from the moment of introducing to the solution to cell death. The cell death was determined by immobilization with no contractile reaction to mechanical irritation or by the rupture of the membrane with the release of the contents of the protoplasm outwards.

Molecular modeling and data processing

Calculations, statistical processing and visualization of measurements were performed using the OriginPro 2021 software (OriginLab, USA).

RESULTS AND DISCUSSION

Dispersion characteristics of colloids of humic dilutions containing dissolved substances.

As was shown earlier ²⁹, the humic and fulvic acids complex forms a polydisperse system in water with a tendency to fragment the particles of the dispersed phase upon dilution. Thus, large particles disintegrate upon dilution, releasing smaller particles. We managed to reproduce these results – the results obtained are presented in Figure 1. 

 

 

 

Figure 1: Size (a) and volume (b) distributions of particles in nanodispersion of HAs and FAs dilutions (v/v): 1:400 (18.4*10^-3 g/mL) – black, 1:800 (9.18*10^-3 g/mL) – red (n=5).

 

The obtained results characterize the 1:400 and 1:800 HAs and FAs dilutions (obtained from the HS concentrate with a dry residue of 7.34×10⁻² g/mL) as monodisperse systems with a submicron particle size²¹. The pH of the solvent (HAs and FAs) is 6.00 according to the conditions for the formation of zinc chelates determined in one of the previous studies ⁴⁹. The change in the size spectra of HAs and FAs with the formation of zinc chelates in solution is shown in Figure 2.

 

 

Figure 2: Size (a) and volume (b) distributions of particles of a 20mM zinc valinate solution in HAs and FAs dilution (1:800) (n=5). 

 

As shown in Fig. 2, when zinc valinate is formed in a 1:800 HAs and FAs dilution, the solution has a bimodal distribution of particles: 20 and 300 nm. It is noteworthy that the most abundant particles in the solution, in terms of volume, are formed in the nanoscale range. The dispersion characteristics of the 1:400 dilution were also studied by the DLS method and are presented in Figure 3.

 

Figure 3: Size (a) and volume (b) distributions of particles of a 20mM zinc valinate solution in a HAs and FAs dilution (1:400) (n=5).

 

 

According to the results of the dispersion analysis, zinc solution in 1:400 HAs and FAs is a virtually monodisperse colloid with a particle size of about 350 nm. The formation of nanoparticles in it is virtually not observed. Considering the HAs and FAs dilutions as an adjuvant for zinc valinate, it is clear that the 1:800 dilution is more preferable in terms of particle size and the resulting potential for increased bioavailability and colloidal stability. 

To analyze the colloidal characteristics and obtained HAs and FAs dilutions, the zeta potential (ζ) of disperse systems was determined. This indicator is the electrokinetic potential arising at the boundary of the sliding layer of a particle. It is used as a measure of colloidal stability of dispersions ^30-32. Since zinc valinate is synthesized in a HAs and FAs dilution, the zeta potential was measured for the aggregate of components of the target solution and for individual substances separately to assess the contribution of each compound to the stability of the entire sample. The results obtained are presented in Table 1.

 

 

Table 1: Zeta potential of disperse systems with variable composition based on HAs and FAs dilution (1:800).

GC,dilution (water)	Val, mM	Zn2+, mM	ζ, mV
1:800	20	0.25	-24±9
	20	0	0
	0	0.25	-23±8
	0	0	-32±10

 

 

Based on the obtained results, the most stable solution is the 1:800 HAs and FAs dilution. When chelated components are added, the value of the zeta potential decreases. However, although the L-valine solution in HAs and FAs has minimal stability, the value of the zeta potential for zinc valinate is at a level that suggests the stability of the colloidal solution.

Toxicological analysis of zinc valinate in various solvents

A widely studied biological object ^33-36, which has proven itself well in the study of acute toxicity, is the cellular biosensor Spirostomum ambiguum ^37-40. The study of the death kinetics of this ciliate formed the basis of the Spirotox method. S. ambiguum is an obligate inhabitant of oligotrophic natural reservoirs and is completely insensitive to humates ²¹, which, in turn, is explained by the origin of humic substances, which are natural components of natural waters. Thus, we were interested in studying the effect of the solvent nature on the acute toxicity of zinc chelate complexes using the Spirotox method. The results obtained are presented in Table 2.

 

 

 

Table 2: Life time of S. ambiguum in zinc valinate solutions based on dilution of humic and fulvic acids or MQ water.

Temperature, oC	Mean lifetime±SD, c (n=5)
	Zn0.25mM+Val20mM(HAs and FAs1:800)	Zn0.25mM+Val20mM(MQ water)
22	>1800	528±51
24	>1800	321±64
26	>1800	205±4
28	>1800	135±21
30	1491±164	99 ±21
32	508±26	-

 

 

The study of the Spirostomum ambiguum death kinetics revealed a significant difference in the lifetime of the biosensor in solutions containing zinc valinate dissolved in water and in a 1:800 HAs and FAs dilution. The ciliates lived for more than 30 minutes in the zinc valinate medium dissolved in a 1:800 HAs and FAs dilution in the temperature range of 22-28 ^oC. Whereas the ciliates lived from 1.5 to 8 minutes in the aqueous solution of zinc valinate in the same temperature range. However, S. ambiguum died in the aqueous solution at 32 ^oC too fast to obtain a result with a relatively low error. The lifetime of the cellular biosensor was recorded in the humate solution of zinc valinate at 30-32 ^oC, but the obtained values were significantly higher in comparison with the control experiment.

CONCLUSIONS

The use of colloids to increase solubility, bioavailability and improve the dispersion characteristics of substances of various natures is widely presented in the literature ^41-43. There are known examples of the use of humic substances as a disperse system that includes various compounds into the particles of the dispersed phase according to the host-guest principle ⁴⁴. Such colloids can be used as a drug delivery system ^45,46. An important circumstance is the need to control the particle size and colloidal stability of the resulting disperse systems, which were controlled in this work. A method for obtaining a disperse system with preset colloidal characteristics was shown.

The analysis of the biological activity of the dispersed solutions obtained by the Spirotox method showed a possible way to reduce the acute toxicity of metal chelates, i.e. the use of a HAs and FAs dilution as a solvent. This approach can be used as a potential way to reduce the toxicity of chelating compounds and therefore requires more attention. There are also known examples of the use of humic and fulvic acids to reduce the toxicity and increase the bioavailability of d-metals ⁴⁷. Usually, the results obtained by the Spirotox method represent the observation activation energy ²⁸, which is a linear function of LD₅₀ for a wide range of orally administered compounds in rats ⁴⁸. This toxicological indicator has proven itself as a way to avoid inhumane methods in assessing acute toxicity. However, this study did not allow obtaining ^obsE_a due to the low toxicity of the studied sample.

Acknowledgments: This paper has been supported by the RUDN University Strategic Academic Leadership Program.

Conflicts of Interest: The authors declare no conflicts of interest.

Ethical approvals: This study does not involve experiments on animals or human subjects.

Data Availability Statement: Data supporting reported results can be found on request by e-mail vntumasov@ya.ru.

Author Contributions: All authors have equal contribution in the preparation of manuscript and compilation.

Source of Support: Nil

Funding: The authors declared that this study has received no financial support.

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