RO134540A2 - Antimicrobial systems containing silver nanoparticles and antibiotic for biomedical applications: preparation and characterization - Google Patents

Abstract

The invention relates to a process for preparing a colloidal system based on Ag nanoparticles functionalized with an antibiotic, with enhanced antispetic/antimicrobial activity. According to the invention, the process consists in preparing a mixture of trisodic citrate and tannic acid, which is heated to a temperature of 100°C, a AgNO3 solution is admixed while vigorously stirring, is maintained for 15 min, after which the Ag nanoparticle suspension is cooled to the ambient temperature, it is subjected to purification, resulting in silver nanoparticles which are dispersed in deionized water, forming a colloidal solution of Ag concentration of 0.25 mM which is mixed, in an amount of 2...2.8 ml, with 0.2...0.5 ml of vancomycin hydrochloride solution, in a concentration of 50 μg/ml, resulting in an antimicrobial colloidal system with synergistic effect on the antibiotic, so that it becomes active at lower concentrations, of 2 μg/ml, in pharmaceutical preparations.

Description

STATE OFFICE FOR INVENTIONS AND TRADEMARKS Patent application

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Antimicrobial systems containing silver nanoparticles and antibiotic for biomedical applications: preparation and characterization

The invention relates to antimicrobial systems for external use based on silver nanoparticles, NpAg, functionalized with vancomycin with significant antibacterial activity against Gram-positive bacteria of the genera Staphylococcus (S. aureus, S. epidermidis, S. schleiferi), Streptococcus (S. bovis S. viridans), Enterococcus (E. faecalis and E. faecium), Micrococcus (M. luteus), including a certain Di activity against Gram-negative bacteria, genus Escherichia (E. coli), compared to pure VCM.

The antiseptic activity of silver has been known since antiquity. The importance of silver nitrate in the medical field, in the treatment of infections and certain diseases such as constipation or heartburn has been highlighted in various studies [1]. Recent research in the field [2-4] has demonstrated the antibacterial, antiviral and antifungal activity of silver, which is the basis for the development of several types of antiseptic materials, different types of silver dressings, as well as creams, sprays or lotions. , used to treat wounds from burns or those caused by chronic ulcers [5-9]. The use of silver as an agent with antiseptic activity is fully justified on the one hand by the broad antimicrobial spectrum which includes mainly Gram-positive and less Gram-negative species [10 - 15], as well as by its low toxicity to large organisms [5, 6] .

The biological mechanism of action of silver is determined by its ability to associate or adhere to the cell wall of microorganisms, respectively the bacterial membrane, or the cytoplasm, where it interacts with proteins that enter the structure of the bacterium that thus distorts them. The changes suffered by the proteins in the structure of bacteria are practically made by the interaction of silver ions with the thiol group (-SH) in the structure of enzymes inhibiting their development, the thiol groups becoming (-SAg) [6]. Moreover, silver ions can catalyze the oxidation reactions of the thiol group (-SH) with the formation of disulfide bonds (-SS-) between two amino acid molecules, thus causing the structural modification of proteins and enzymes involved in the process of cellular respiration. , thus causing the death of the bacterium [16]. Through the reactions that lead to the appearance of disulfide groups (-S-S-) and (-SAg) is affected both the ATP and the DNA of bacteria.

From the above it results that the antibacterial, antiviral and antifungal activity of silver can be achieved practically only through Ag ⁺ ions, the metallic silver having no antiseptic activity. In this context, silver nitrate seems to be a very effective antiseptic agent due to its ability to release, in a short time, a large number of Ag ⁺ ions that interact rapidly with the cell walls of fungi and bacteria ( Staphylococcus and Streptococcus), but at the same time they can and even interact with the cell walls of healthy cells, which makes the action of silver nitrate short-lived. in this way silver can also become toxic to large organisms. Hence the need for silver to be administered in a form that releases Ag ⁺ ions over a longer period of time and in a concentration that on the one hand destroys bacteria (staphylococci and streptococci), but which not to reach the dose toxic to large organisms. This goal is achieved by using Ag ° of the smallest possible size, ie in the form of silver nanoparticles, NpAg. Numerous methods have been proposed to obtain silver nanoparticles by reducing Ag ⁺ with different reducing agents [2, 3, 17, 18], \ * ^r ' ¹ of 2019 00279

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Thus, commercial products such as Argentum colloidal were made, which come in the form of a suspension with a content of 70-75% Ag, used mainly for diseases such as: rhinitis, conjunctivitis, blepharitis, urethritis, vaginitis. Another commercial silver-based product Targesin is a colloidal complex with an organic part, consisting of diacetyltin, silver albuminate, with a content of 4-7% colloidal silver, indicated for use in gastritis, duodenitis, gastric ulcer and duodenal , ophthalmology and various infections.

The use of silver for medical purposes requires that it be in the form of very small particles (on the order of nanometers) which ensures a very large surface area of contact with the plasma liquid and thus the conditions for an adequate reaction rate by which it is released. an amount of Ag ⁺ ions that provide the antiseptic effect, but which is not toxic to large organisms.

In order to reduce the negative effects and to attenuate its toxicity, a series of “organo-silver” compounds have been developed, such as: silver sulfodiazine (DERMAZIN), silver zinc allantoanate (known as AZAC), silver proteinate, dextron nitrate. silver, characterized by a high content of Ag ° silver used mainly in the manufacture of sterile dressings, disinfectant soaps, urinary catheters. In the veterinary field we mention the product Agipiu Vet Spray, used as a disinfectant, for external use, for any skin lesion.

The shortcomings of commercial silver-based products stem from the fact that Ag ⁺ ions form strong bonds at the cell surface that can cause a toxic effect by inhibiting the respiratory chain and healthy cells. Another disadvantage of existing silver-based products is that they can cause burns and necrosis, especially in the case of high concentration of Ag and repeated application over long periods of time. Such applications can cause the phenomenon of cell intoxication [19]. On the other hand, various antibiotics are available to fight infections [20], but due to their misuse some microorganisms have developed resistance to antibiotics or multiple drugs (multiple drug resistance: MDR). This situation has become difficult for the medical field, limiting the choice of antibiotics. In general, any use of antibiotics can cause an increase in resistance to some bacteria and this allows resistant bacteria to survive and only bacteria that are likely to die.

Vancomycin hydrochloride (VCM) is indicated for the treatment of infections [21-23] with methicillin-resistant staphylococcal strains (MRSA), in patients allergic to penicillin, for patients who do not respond to other medicines and for other infections caused by sensitive microorganisms. to vancomycin, but resistant to other antimicrobial drugs. Vancomycin is also effective in treating bone infections, infections of the skin and its structures [20-22],

The effects of using vancomycin as a pharmacological product with antibacterial activity may be altered and influenced over time by various factors including the production of bacteriocins [23]. Prolonged use of vancomycin at the indicated doses may lead to the development of staphylococcal strains that become over time resistant to this antibiotic. On the other hand, prolonged use of vancomycin can lead to superinfections with microorganisms with much increased antibiotic resistance [24],

The antimicrobial system containing silver nanoparticles (NpAg) and vancomycin according to the invention, eliminates the disadvantages of existing products in the field in that it is composed of NpAg with dimensions of 5- ^ 40 nm preferably 1025 nm, obtained by reducing Ag ⁺ (concentration 0.001 - 0.005% gr (0.1 - 0.5 mM), preferably 002-0.003% gr (0.2 - 0.3

and 2019 00279

13/05/2019 mM) at Ag ° with a reducing agent consisting of a solution containing trisodium citrate, CTS (within 0.1 - 0.6% gr. (4 - 23 mM), preferably 0.1- 0.2% by weight (4-8 mM) and tannic acid AT (within 0.003 - 0.1% by weight) (0.02 - 0.6 mM), preferably 0.004-0.01% by weight. [0.025 - 0.06 mM) wherein the mass ratio Ag: trisodium citrate: tannic acid, Ag: CTS: AT = 1: (10-60) :( 1-20), functionalized with vancomycin in the mass ratio NpAg: VCM = 1: (0.1 + 1.5), preferably 1: (0.13-0.45), the concentration of the VCM @ NpAg-CTS-AT suspension varying within the limits: for Ag 10 - 25 pg / ml preferably 15 -20 pg / ml respectively for VCM 2 + 20 pg / ml, preferably 3 + 10 pg / mL

The resulting system, described in the example of the invention, was characterized from a physico-chemical point of view by UV-VIS spectroscopy (Fig. 1), scanning electron microscopy, SEM and X-ray dispersion spectroscopy, EDX (Fig. 2) and atomic force microscopy, AFM (Fig. 3). They all highlight the presence of silver nanoparticles and give an image of their size distribution.

Table 1 compares the efficacy of prepared colloidal systems against different bacterial strains depending on the NpAg and vancomycin content. The evaluation of the efficacy of the NpAg - VCM colloidal systems was performed according to the agar gel diffusion method and the measurement of the diameter of the inhibition zone (Fig. 4). They were compared with the effect of VCM solutions of the same concentrations, but without NpAg and with the effect of the 30 pg VCM disk.

The analysis of the results in Table 1 and Figure 4, respectively, shows the superiority of VCM @ NpAg-CTS-AT dispersions over vancomycin hydrochloride solutions of the same concentration, both in terms of the area of inhibition (higher for variant a) and the spectrum of activity. , the former being also active for E. faecalis and E. coli for 0.36 pg Ag / dose and in an amount starting from 0.067 pg vancomycin / dose.

Example

The following is an embodiment of the invention.

The microbial strains were seeded on glucose media (nutritious broth and agar), the incubation being done in a thermostat at 37 ° C, in aerobic conditions, for 18-24 hours. To determine the antibacterial effect, we worked according to the model of performing the antibiogram (diffusimetric method in nutrient agar gel), with the necessary adaptations for testing conditioned products in liquid form, making wells with a diameter of 6 mm, in which 20pl of the different variants of the product [2].

NpAg is prepared by reducing Ag ⁺ to Ag ° with a mixture of trisodium citrate - tannic acid at pH 6.8 + 7.1 for which purpose 100 ml of solution was prepared with 0.153 g of trisodium citrate dihydrate, 0.00425 g of tannic acid (1 , 25 ml 0.34% solution) and heated to 100 ° C. After intense stirring, add 0.0027 g of Ag ^{+ I} (2.5 ml of 10 ' ² M solution of AgNO ₃ ). Stirring is continued for 15 minutes after which the NpAg suspension is cooled to room temperature. After cooling, the reaction mass thus prepared is subjected to purification to remove excess reagents and reaction by-products (NaNO ₃ ). Purification is performed by separating NpAg in an ultracentrifuge at 10,000 rpm for 20 min. and washing twice with 100 ml of distilled water, followed by ultracentrifugation. Ultracentrifuged and washed NPAg are redispersed in deionized water, using an ultrasound unit, so as to form a colloidal solution of 0.25 mM Ag concentration. A solution of vancomycin hydrochloride with a concentration of 50 pg / ml (0.0336 mM; 0.005% gr) was prepared separately. Different amounts of colloidal solution of NpAg (2 + 2.8 ml) were taken from the samples thus prepared and mixed with the vancomycin solution (0.2 + 0.5 ml). From the resulting mixtures, 20 μl samples were taken for testing the activity of 2019 00279

13/05/2019 antibacterial preparations (figure 4a, b, for two concentrations of the colloidal solution). It is observed that the areas of inhibition are larger for the colloidal system VCM @ NpAg-CTS-AT compared to solutions containing only vancomycin at the same concentration (figure 4c). These areas of inhibition are comparable to those of the vancomycin 30 pg vancomycin hydrochloride disc (Figure 4d). It is also found that the spectrum of antibacterial activity is wider, the product inducing positive results for Escherichia coli and Enterococus faecalis.

Below we present patents / patents from the US, EP, WO and China that use various NpAg systems with a wide applicability [1-19]. These documents disclose methods of obtaining NpAg that are completely different from the present invention.

The application of the patent results in the following advantages:

The functionalization of NpAg with vancomycin has a synergistic effect on the antibiotic, potentiating its antibacterial activity, so that it becomes active at lower concentrations, 2 pg / ml, compared to those currently used, 8 pg / ml [25,26], in pharmaceutical preparations current.

- The dose of vancomycin in the pharmaceutical preparation containing vancomycin-functionalized silver nanoparticles is substantially reduced by 30-70%.

- The presence of silver in the colloidal system, which contains vancomycin silver nanoparticles, NpAg - VCM, causes irreversible changes in the structure of bacteria and other pathogenic microorganisms, thus eliminating the possibility of their adaptation to vancomycin.

Colloidal antimicrobial systems containing silver nanoparticles and an antibiotic, preferably vancomycin, or a combination of antibiotics, become active at lower concentrations than those currently used to prevent bacterial growth and treatment of infections, or in dental and orthopedic medical applications.

Claims (15)

Claims I) Colloidal system containing NpAg-antibiotic (NpAg: AN), preferably NpAg vancomycin, with antibacterial, antifungal, antiviral activity and characterized in that it is composed of NpAg with dimensions 5 - 40 nm preferably 10- ^ 25 nm obtained by reducing Ag ⁺ to Ag ° from a solution with a concentration of 0,001 - 0,005% g, with a reducing agent consisting of a solution containing trisodium citrate of a concentration of 0,1 0,6% g and tannic acid, with a concentration within the limits of 0,003 - 0,1% w / w in which the mass ratio of trisodium citrate: tannic acid varies within the limits (1 - 30): 1, functionalized with antibiotic, preferably vancomycin, in the mass ratio NpAg: VCM = 1: (0,1 + - 1.5) the concentration of the colloidal system VCM @ NpAg-CTS-AT varying in the limits of Ag: 10 25 gg / ml, respectively VCM: 2 + 20 gg / ml. Colloidal antimicrobial system according to claim 1, characterized in that the reduction of Ag ⁺ (AgNOj) of concentration preferably 0.002-0.003% to Ag ° is carried out with a reducing agent consisting of a solution containing trisodium citrate (preferably with a concentration of 0, 1-0.2% and tannic acid with the concentration preferably 0.0040.01%, wherein the mass ratio of trisodium citrate: tannic acid = (15 -25): 1, and the mass ratio NpAg: VCM is 0.15 - 0.5; the colloidal system VCM @ NpAg-CTS-AT being preferably Ag: 15-20 pg / ml and VCM 3-10 gg / ml. Colloidal antimicrobial system according to Claims 1 and 2, characterized in that the reduction of Ag ⁺ to Ag ° is carried out at pH 6.5 + 7.2 and the temperature in the range 65 + 100 ° C, preferably 85 + 95 ° C. Colloidal antimicrobial system according to Claims 1, 2 and 3, characterized in that the reduction of Ag ⁺ to Ag ° is achieved by the gradual addition of the Ag ⁺ solution over that of the reducer, under intense stirring. Colloidal antimicrobial system according to Claims 1, 2, 3 and 4, characterized in that the removal of excess reactants and by-products resulting from the production of NpAg was carried out by ultracentrifugation at a speed of 10000 rpm and resumed twice. with distilled water of the Ag precipitate at room temperature. Colloidal antimicrobial system according to claims 1, 2, 3, 4 and 5, characterized in that the functionalization of NpAg with vancomycin was achieved by adding the vancomycin solution with a concentration within the limits of 50 + 500 gg / ml, with stirring at room temperature, over NpAg colloidal solution whose concentrations varied within the limits of 10-25 gg / ml Ag °, preferably 15-20 pg / ml Ag °. 7) The method of preventing bacterial growth or treating infections which includes a colloidal system containing NpAg-antibiotic according to claims 1, 2, 3, 4. 5 and 6, applied as a topical treatment where polyfunctional NpAg-AN complexes, preferably VCM @ NpAg-CTS-AT, antimicrobial agents act. and 2019 00279 13/05/2019 The method of claim 7, wherein the multifunctional nanoparticles are applied topically and the area of application may be a body cavity, skin, nail bed or mouth. The method of claim 8, wherein the method could be used in treating periodontitis. The method of claim 8, wherein the method could be applied to treating burns or forms of diabetes (diabetic foot). II) The method of claim 8 applied to the treatment of vancomycin-resistant pathogens. The method of claim 7 applied to the disinfection or sterilization of various medical utensils or devices. The method of claim 7, comprising both VCM @ NpAg-CTS-AT and other polyfunctional AN @ NpAg-CTS-AT complexes that can act synergistically (together) as antimicrobial agents having a broad spectrum of action against pathogenic microorganisms. The method of claim 13 applied to the disinfection or sterilization of various medical utensils or devices. The method of claim 7, wherein the polyfunctional nanoparticles, NpAg-AN, are applied (adsorbed) topically on the surface of an orthopedic implant or a dental implant. The method of claim 15, wherein the colloidal AgNp system contains an antibiotic, or a combination of antibiotics, selected from the group of vancomycin, gentamicin and tobramycin.