RU2780084C1 - Injectable composite material for the regeneration of inflammatory diseases of bone tissue - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to an injectable composite material for the regeneration of inflammatory diseases of bone tissue in the form of a hydrogel. This material contains high molecular weight polyvinylpyrrolidone, vegetable protein - legumin or zein or animal protein - ovalbumin, nanocrystalline calcium hydroxyapatite, vancomycin and distilled water.

EFFECT: invention makes it possible to obtain a biodegradable and biocompatible injectable composite material for the regeneration of inflammatory diseases of the bone tissue, for example, for the treatment of osteomyelitis, containing an antibiotic and having a slow solidification, while being non-toxic.

1 cl, 2 tbl, 6 ex

Description

The invention relates to medicine, in particular to a pharmaceutical composition for the delivery of a pharmaceutical agent to the focus of a disease, which is an injectable composite material containing an antibiotic. The invention can be used for the regeneration of inflammatory diseases of bone tissue, for example, for the treatment of osteomyelitis, in the form of a hydrogel injected into the affected areas of human bone material.

The injectable hydrogel composite material can be injected into diseased areas of human bone material. Thus, delivery of the required amount of antibiotic without loss during the entire treatment process can be ensured, which will avoid repeated surgical intervention, reduce body toxicity from antibiotic therapy, and can also be a measure for the prevention of antibiotic resistance in patients with osteomyelitis, due to which the claimed biodegradable composite can find widespread use compared to known toxic drugs used in modern therapy.

The technical problem of the claimed invention is the creation of a non-toxic biodegradable and biocompatible injectable composite material containing an antibiotic and providing the possibility of local non-surgical treatment of sites of inflammatory diseases of the bone tissue.

EFFECT: biodegradable and biocompatible injectable composite material for regeneration of inflammatory diseases of bone tissue, containing an antibiotic and having a slow solidification, while being non-toxic.

The use of the inventive biodegradable and biocompatible injectable composite material will lead to an improvement in the non-surgical method of treating osteomyelitis, which in turn will increase the efficiency and reduce the cost of treatment due to the possibility of targeted drug delivery to the lesions. Delivery of the drug to the affected area occurs due to injections. The inventive composition of the composite hardens more slowly compared to the known ones, making it possible to introduce it with a syringe.

The effectiveness of the treatment of osteomyelitis is increased by reducing the toxic effect on the body through the use of high molecular weight polyvinylpyrrolidone, the possibility of prolonged and point action of the drug for a long time.

State of the art:

There are various compositions of composites for drug delivery in the treatment of diseases and defects of bone tissue.

Patent RU 2710252 uses the antibiotic gentamicin, which is highly active against aerobic gram-negative bacteria: Escherichia coli. Its effectiveness against gram-positive bacteria is quite low. The proposed injectable composite material as a composite is focused on the treatment of osteomyelitis, which is caused by gram-positive bacteria Staphylococcus aureus, therefore, vancomycin is used as part of the composite.

Also, gentamicin is used in patent RU 2723588, which is included in a too complicated process for obtaining a film with its content. The production of films not only requires special equipment, but the method has a lower reproducibility of the characteristics and properties of products in comparison with other methods for creating composites. The proposed development uses the traditional process of obtaining a composite in the form of hydrogels - it requires less material costs, and also has a higher reproducibility.

The hydrogel also has advantages over the cement form. For example, CN 104511051 describes a treatment composite in the form of a cement, although this form has better mechanical characteristics, but less ability to fill variously shaped defects and a lower drug recovery rate than a hydrogel.

In the patent RU 2522977, the composition contains albumin, but in the claimed invention, proteins of plant origin are mainly used, since they are much more accessible, they are also low molecular weight and high bioactivity is observed with them.

The technical result is achieved due to the claimed composition of a biodegradable and biocompatible injectable composite material for the regeneration of inflammatory diseases of bone tissue, containing high molecular weight polyvinylpyrrolidone, vegetable or animal protein, nanocrystalline calcium hydroxyapatite, vancomycin, distilled water in the following ratio of components per 100 g of the composition:

high molecular weight polyvinylpyrrolidone 0.304 g ± 0.010 g

vegetable or animal protein 0.608 g ± 0.050 g

nanocrystalline calcium hydroxyapatite 1.388 g ± 0.100 g

vancomycin 0.500 g ± 0.500 g

distilled water 97.2 ml.

As a vegetable protein, legumin or zein can be used.

As an animal protein, ovalbumin can be used.

Nanocrystalline calcium hydroxyapatite as part of a biodegradable composite is preliminarily obtained using the coprecipitation method, which consists in obtaining a suspension of calcium hydroxide by dissolving calcium oxide in distilled water cooled to 4°C, followed by the addition of orthophosphoric acid and 0.5±0.1 ml of concentrated ammonia solution to achieve a pH of 8-9 to the resulting suspension of calcium hydroxide, stirring with a magnetic stirrer for three days at a temperature of 70°C, then filtering the resulting calcium hydroxyapatite and drying at a temperature of 120-150°C. The particles of nanocrystalline calcium hydroxyapatite should be of a certain size (no more than 200 nm) and have a lamellar or needle-shaped microcrystals.

The essence of the invention is illustrated by figure 1, as well as other illustrations:

Fig. 1 X-ray pattern of synthesized hydroxyapatite;

Fig. 2 Reaction of proteins with ninhydrin;

Fig. 3 Calibration graph of the dependence of optical density on the amount of albumin in relative units;

Fig. 4 Change in the concentration of calcium cations in SBF (human plasma) composites with zein/legumin + PVP and ovalbumin + PVP during the month;

Fig. 5 Mung bean phytotoxicity;

Fig. 6 Mung bean growth chart;

Synthesis of hydroxyapatite (HAP)

Nanocrystalline calcium hydroxyapatite is obtained using the codeposition method:

Prepare a suspension of calcium hydroxide by dissolving calcium oxide in distilled water, cooled to 4°C. Then orthophosphoric acid and 0.5±0.1 ml of concentrated ammonia solution are added to reach pH 8-9 to the resulting suspension of calcium hydroxide. The resulting solution is stirred with a magnetic stirrer for three days at a temperature of 70°C, then the resulting calcium hydroxyapatite precipitated is filtered off. The filtered precipitate of calcium hydroxyapatite is dried at a temperature of 120-150°C in an oven. The composition of the resulting HAP and its purity are estimated according to X-ray phase analysis. X-ray phase analysis is carried out using an ARLX`TRA diffractometer in a copper Kα cure. Scanning speed 5°/min with phase analysis up to 0.5°/min. For qualitative analysis, a PDF2 X-ray file is used. The figure 1 shows the X-ray pattern of the synthesized hydroxyapatite.

Isolation and purification of proteins occurs according to known methods.

Isolation of proteins

Isolation of ovalbumin

The egg white is separated from the yolk. The volume of protein is determined with a measuring cylinder. For every 10 ml of sample, 3.6 g of NaCl is added. Mix gently until the ovoglobulin precipitates. For filtration, gauze is first used to avoid mixing with the precipitate of undissolved NaCl. The filtrate is stored for further actions. The precipitate is transferred to pre-weighed filter paper and dried in air. Add 2-3 drops of 0.2 M acetic acid to the filtrate and heat to boiling until the ovalbumin precipitates. The precipitate is filtered through gauze and placed on a weighed filter, dried in air. The sample is weighed and the yield is calculated [1].

Isolation of Zein

Zein is prepared by periodic extraction with 70% ethanol from dried, crushed corn at 60°C for 2 hours [2].

Isolation of legumin

A 50 g portion of pea flour was quantitatively transferred into a volumetric flask. Then 50 ml of distilled water and 1M NaOH solution were added to bring the pH to 9.5, stirred for 1 hour on a magnetic stirrer. At the end of the time, the solution was filtered and 1 M HCl was added to the filtrate until a precipitate formed, and the procedure was repeated again with filtration. The precipitate that settled on the filter was carefully collected, poured into molds, and placed in a freezer [3, 4].

Preparation of biodegradable (biocompatible) injectable composite material

1. Reagents used: Polyvinylpyrrolidone (chemically pure); vegetable proteins: zein and legumin; animal protein: ovalbumin, Vancomycin (Kraspharma PAO, Russia), distilled water.

2. Add 20 ml of distilled water to a 100 ml beaker.

3. 0.28125 g of HAP is added and placed on a magnetic stirrer at room temperature for 5 minutes until the HAP is completely dissolved in water.

4. 0.125 g of protein (zein\ovalbumin\legumin) is added and left on a magnetic stirrer for 10 minutes until the solution is completely homogenized.

5. Add 0.0625 g of PVP and place on a magnetic stirrer for 30 minutes at room temperature.

6. 0.1 g of Vancomycin is added and this homogeneous mixture is stirred again for 15 minutes, increasing the number of revolutions on the stirrers to maximum.

6.1 Check the pH of the carrier system every 5 minutes (pH should be between 4.5 and 8.0).

7. After mixing, the homogeneous solution of the composite is carefully transferred into silicone molds and placed in a freezer at a temperature of -18°C for 2 days until completely solidified.

The study of the duration of hardening and the microstructure of composites of the composition "calcium phosphate-polymer - protein", depending on the ratio of these components

Composite composition: hydroxyapatite - 60%, polyvinylpyrrolidone (PVP) - 20%, vancomycin - 15%. For complete solidification, the obtained composite samples were placed in a refrigerator for several hours:

high molecular weight polyvinylpyrrolidone 0.304±0.010 g vegetable or animal protein 0.608±0.050 g nanocrystalline calcium hydroxyapatite 1.388±0.100 g vancomycin 0.500±0.500 g distilled water 97.2 ml

For 100 g of composite.

Table 1. Composition of composite samples Component content,
in g per 100 g of composition sample 1 Sample 2 Sample 3 High molecular weight polyvinylpyrrolidone 0.304±0.010 g 0.304±0.010 g 0.304±0.010 g Zein 0.608±0.050 g - - Legumin - 0.608±0.050 g - Ovalbumin - - 0.608±0.050 g nanocrystalline calcium hydroxyapatite 1.388±0.100 g 1.388±0.100 g 1.388±0.100 g vancomycin 0.500±0.500 g 0.500±0.500 g 0.500±0.500 g distilled water 97.2 97.2 97.2 Total 100 100 100

Table 2. Physical properties of the resulting composite samples Sample Sample height, mm bundle Other Poliden 1) 6.51
2) Fell apart when removed -
- Dipped, a funnel formed inside PVP 200
1ml 1) 3, 66
2) 4.20 -
- Crashed when frozen
Do not stick together PVP 200
2 ml 1) 3.10
2) 4.0 +
+ Do not stick together PVP 500
1ml 1) 4.27
2) 2.70 +
+ Didn't stick much PVP 500
2ml 1) 3.95
2) 3.76 +
+ Stick to walls and to each other PVP 1000
1ml 1) 2.64
2) 2.97 +
+ Strong delamination, do not stick together PVP 1000
2ml 1) 3.38
2) 3.10 +
+ Strongly adhere to each other and walls

Investigation of the activity of composites and their resistance to destruction in model media simulating blood plasma

To study bioactivity in the in vitro laboratory, a technique based on the study of degradation / resorption in model fluids was used: SBF (blood plasma), HBSS (Hanks' saline solution), a solution that mimics the composition of human saliva, synovial fluid. Methods for preparing model liquids are described below.

Method for preparing SBF Model Fluid (Blood Plasma)

To prepare the SBF solution, the following components are taken:

NaCl - 5.26 g; KCl - 0.373 g; CaCl ₂ *2H ₂ O - 0.368 g; MgCl ₂ *6H ₂ O - 0.305 g; Na ₂ SO ₄ -0.071 g; NaHCO ₃ - 2.268 g; Na ₂ HPO ₄ * 2H ₂ O - 0.178 g; Lactic acid - 2.41 ml; Sodium lactate - 3.1 ml.

The above components are sequentially added to a 1-liter volumetric flask using a glass spoon, and lactic acid and sodium lactate are added dropwise to the latter to avoid the precipitation of doubly charged metal lactates, after which the solution is brought to the mark with distilled water.

HBSS Model Fluid Preparation Method (Hanks Salt Solution)

To prepare the HBSS solution, the following components are taken:

NaCl - 8 g; KCl - 0.4 g; CaCl ₂ - 0.14 g; MgSO ₄ * 7H ₂ O - 0.2 g; NaHCO ₃ - 0.342 g; KH ₂ PO ₄ - 0.06 g; Na ₂ HPO ₄ * 2H ₂ O - 0.06 g; glucose - 10 g.

The above components are sequentially added to a 1 liter volumetric flask using a glass spoon and brought to the mark with distilled water.

Method for preparing a solution that mimics the composition of human saliva

To prepare the solution, you will need the following components:

KCl - 0.72 g; CaCl ₂ * 2H ₂ O - 0.220 g; NaCl - 0.6 g; K ₂ HPO ₄ - 0.680 g; NaH ₂ PO ₄ *10H ₂ O - 0.866 g; Na ₂ CO ₃ - 1.5 g; KSCN - 0.060 g; citric acid - 0.03 g.

A little distilled water is added to a 1 liter volumetric flask, then all of the above components are added with a glass spoon and brought to the mark.

Synovial fluid model preparation method

To prepare the solution, the following components were needed:

NaCl - 8 g; KCl - 0.2 g; NaH ₂ PO ₄ - 1.44 g; K ₂ HPO ₄ - 0.24 g; hyaluronic acid - 3 g.

A little distilled water is added to a 1 liter volumetric flask, then all of the above components are added with a glass spoon and brought to the mark.

Study of mechanical and chemical properties of materials: porosity and sorption, assessment of phytotoxicity of composites - PVP-Polydon.

Extraction of the drug is carried out in a buffer solution (Hanks' saline solution). Samples with the drug are placed in test tubes, each of the test tubes contains 50 ml of salt solution. The test tubes are placed in glasses, and all this is placed in an autoclave. Withdrawal of 5 milliliters is carried out according to time: after 20 minutes, 50 minutes, 80 minutes and 120 minutes. Further, 20 milliliters of salt solution was added to each sample.

To study bioactivity in the in vitro laboratory, a technique based on the study of degradation / resorption in model fluids is used: SBF, HBSS, a solution that mimics the composition of human saliva, synovial fluid.

HBSS (Hanks' Salt Solution)

To prepare the HBSS solution, the following components are taken:

NaCl-8 g; KCl - 0.4 g; CaCl ₂ - 0.14 g; MgSO ₄ * 7H ₂ O - 0.2 g; NaHCO ₃ -0.342 g; KH ₂ PO ₄ - 0.06 g; Na ₂ HPO ₄ * 2H ₂ O - 0.06 g; glucose - 10 g

The above components are sequentially added to a 1 liter volumetric flask using a glass spoon and brought to the mark with distilled water. To develop the color, an alcohol solution of ninhydrin is added to the solution and heated. In this case, the reaction illustrated in Fig. 2 proceeds.

The same reaction is given by primary amines, imino acids and ammonia, without the release of CO, the amino acids proline and hydroxyproline also interact with ninhydrin, however, the resulting complex has a yellow color. The violet complex is found at 550 nm and the yellow complex at 440 nm. The ninhydrin reaction is highly sensitive, allowing the detection of micrograms of amino acids in chromatograms, as well as column fractions.

Reagents:

1. 0.5 M acetate buffer, pH 5.5.

2. Ninhydrin reagent: dissolve 2 g of ninhydrin in 30 ml of acetone and add 20 ml of 0.5 M acetate buffer. Prepare before each experience. Store in brown glass bottles.

3. Amino acid stock solution: 1 mg/ml in distilled water.

Order of execution:

1. Pipette 5 ml of amino acid stock solution.

2. Make up with distilled water.

3. Add 1 ml of freshly prepared ninhydrin reagent.

4. Cover the tubes with aluminum foil and place them in a boiling water bath for 10 minutes.

5. Cool to room temperature in a container with cold water and add 1 ml of 50% ethanol to each tube.

6. Leave for 5 minutes and then read the absorbance at 550 nm.

Optical density measurements were carried out on the KFK-2 Photocolorimeter, different wavelengths of 540 nm and 590 nm were used, the data were entered into a table, and on their basis a calibration graph of the dependence of optical density on the amount of albumin in relative units was built (figure 3).

Based on the calibration curve, it is possible to calculate the protein concentration in the solution. The calculation is based on the Bouguer-Lambert-Beer Law. For this, the resulting equations are used:

C _x \u003d ((D-0.1413) / 9.4667) * 2 - equation for a wavelength of 540 nm

C _x \u003d ((D-0.079) / 9.9282) * 2 - equation for a wavelength of 590 nm

D/L + R1 - Zein\Legumin + PVP

O + P - Ovalbumin PVP

In order to evaluate the behavior of the sample in a longer term, a study of 2 samples (with animal and vegetable protein) was carried out for a month in plasma simulating blood (SBF). The general plots presented in FIG. 4 is that after 12 days the maximum crystallization is reached (points 3 and 3'), further resorption is observed (sections 4 and 4'). Up to this point, animal and vegetable protein behave somewhat differently, in the case of animal protein (ovalbumin). Crystallization begins almost immediately, while at point 1` primary resorption occurs, which is then again replaced by crystallization (2`), so area 4` can be called repeated resorption. The vegetable protein initially does not show any activity, or we can talk about a slight resorption up to point 1. At point 1, crystallization begins with slight deviations at point 2, and the actual resorption begins only after point 3.

Study of mechanical and chemical properties of materials: porosity and sorption, assessment of phytotoxicity of composites.

Phytotoxicity Methods

Phytotoxicity is the ability of chemicals, organic and inorganic, to inhibit the growth and development of plants [5].

Mung beans were placed in a glass of ordinary water, T=19°C, a day is given for the beans to open and begin to germinate. Opened beans are placed in Petri dishes with pre-prepared substances, namely: a control sample without reagents and composites, hydroxyapatite, PVP-V200, PVP-Polydon, Composite with PVP-V200 and Composite with PVP-Polydon. The water level in the petri dishes was also noted. Every day a photo was taken with each of the samples and the ruler. Then the photos were transferred to a computer and processed according to the instructions in a special ImageJ program. Thus, the growth length of the beans was measured.

Phytotoxicity Study Results

As mentioned earlier, phytotoxicity assessment is a convenient tool for assessing the possibility of using plant cells in the genetic engineering of implants. On the graph in Fig. 4 and in FIG. Figure 5 shows the results of this toxicity assessment for two composites with PVP-V200, PVP-Polydon, which differ greatly in weight. As can be seen from the obtained data, V200 turned out to be more phytotoxic and almost completely suppressed the growth of beans, in contrast to Polydon. Therefore, it is Polydon that can be recommended for use in drug delivery systems with plant cells and genes. The difference in action can be associated both with different solubility of PVP of different masses and with different organization of the polymer, which requires further studies.

According to the experiment, as expected, the best growth was shown by the control sample, which gave growth both to the stem and to the root. Among the studied samples is PVP-Polydon, in which the root has grown significantly. The worst results were for two samples: HAP and PVP-V200. Very small root processes in hydroxyapatite, even one of the roots detached from the sprout. The samples, where Polydon and V200 are included in the composition of the composites, turned out to be generally better than individual polymers, but retained the same trend. The first showed excellent results in root and stem growth, at the level of the control sample, and the composite with PVP-V200, which gave a small root growth.

In FIG. 6 shows the measurement results of all samples. Particular attention should be paid to the composite with Polydon, which completely bypassed the pure sample in terms of both root and stem growth, which tells us about the feasibility of using this PVP for genetic engineering and the transfer of plant cells in the drug delivery system and the inexpediency of using PVP-V200 for this functions. Most of the samples showed a gradual growth at the beginning, but all of them reached a plateau by the end of the experiment, which tells us about the slowdown in growth.

Sources of information used:

[1] Pereira M.M. et al. Single-step purification of ovalbumin from egg white using aqueous biphasic systems // Process Biochem. Elsevier Ltd, 2016. Vol. 51, No. 6. P. 781-791;

[2] Parris N., Dickey L.C. Extraction and solubility characteristics of zein proteins from dry-milled corn // J. Agric. food chem. 2001 Vol. 49, No. 8. P. 3757-3760;

[3] Hoang H.D. EVALUATION OF PEA PROTEIN AND MODIFIED PEA PROTEIN AS EGG REPLACERS, 2012;

[4] Boye J.I. et al. Comparison of the functional properties of pea, chickpea and lentil protein concentrates processed using ultrafiltration and isoelectric precipitation techniques // Food Research International. 2010 Vol. 43, No. 2. P. 537-546;

[5] Ross S.S. Using Mung Beans as a Simple, Informative Means To Evaluate the Phytotoxicity of Engineered Nanomaterials and Introduce the Concept of Nanophytotoxicity to Undergraduate Students / Ross S.S., Owen M.J., Pedersen B.P., Liu G., Miller W.J.W. // Journal of Chemical Education - 2016. - V. 93 - No. 8 - S.1428-1433;

Claims (7)

Injectable composite material for the regeneration of inflammatory diseases of bone tissue in the form of a hydrogel containing high molecular weight polyvinylpyrrolidone, vegetable or animal protein, nanocrystalline calcium hydroxyapatite, vancomycin, distilled water in the following ratio of components per 100 g of the composition: high molecular weight polyvinylpyrrolidone 0.304 ± 0.010 g, vegetable or animal protein 0.608 ± 0.050 g, nanocrystalline calcium hydroxyapatite 1.388 ± 0.100 g, vancomycin 0.500 ± 0.500 g, distilled water 97.2 ml, moreover, legumin or zein is used as vegetable protein, and ovalbumin is used as animal protein.

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