RU2699045C1 - Method of modifying surface of biodegradable polymer materials - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to a method of modifying the surface of biodegradable polymers used in the field of chemistry of polymers and medicine. Method includes treatment of polymer surface with solution of aliphatic diamine in 60 % aqueous solution of isopropyl alcohol at temperature of 24–30 °C for 30–60 minutes, then with 10 mM solution of hydroxyl-succinimide ester of linoleic (9,12-unoxadienoic) acid in isopropyl alcohol containing 0.1% triethylamine at temperature of 37–40 °C for 60 minutes, then polymer surface is treated with aqueous solution of human serum albumin (HSA) conjugate with corresponding maleimide derivatives of biologically active compound for 15–30 minutes. Method comprises preliminary modification of HSA with required maleimide derivatives of biologically active compound.

EFFECT: method increases efficiency of albumin sorption and increases stability of modified surfaces of polymer materials.

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Description

The invention relates to the field of polymer chemistry and medicine, and in particular to a method for modifying the surface of biodegradable polymer materials to give them the desired properties (hemocompatibility, thromboresistance, accelerated endothelization, antibacterial or antiviral properties, etc.) for use in the medical industry to create, for example, prosthetic blood vessels, vascular patches for arterial reconstruction or the creation of dosage forms of drugs with a prolonged duration.

In modern medicine, various biodegradable polymeric materials are widely used. In particular, biodegradable polymers are used to reconstruct blood vessels. Nanosized particles from biodegradable materials are widely used in the development of targeted drug delivery systems. The use of polymers in the circulatory system puts forward a number of requirements for them, the main of which is hemocompatibility. At the same time, other requirements may be imposed on polymers depending on the particular tasks being solved.

Currently, to give the polymeric materials in contact with blood the desired properties, various approaches are used. So, for example, to give the polymer of which the vascular prosthesis is made antibacterial, the product is immersed in a saturated alcohol-soluble antibiotic solution for 24-72 hours, before use, it is removed and placed in a 1% solution of sulfacrylate glue (patent RU 2141280 C1 , published on November 20, 1999).

A similar approach to the introduction of biologically active substances into a vascular prosthesis made by the electrospinning method is proposed in patent application US 2014/0309726 A1, publ. 10/16/2014. The presented vascular graft contains an inner layer made of a biodegradable polyester compound - polyglycerolsebacate, and an outer membrane made of polycaprolactone and / or polymers or copolymers of glycoic and lactic acid. To reduce the thrombogenic properties of the implant and increase its biocompatibility, the inner surface is treated with a heparin solution, and the outer shell is impregnated with any biologically active components that promote tissue regeneration (for example, stem cell growth factor (SCF), vascular endothelial growth factor (VEGF), etc.) .

The disadvantage of this method is the uncontrolled release of adsorbed components. In addition, direct adsorption of heparin on polymers does not provide sufficient thromboresistance, since, on the one hand, the interaction “hydrophobic polymer surface - hydrophilic heparin molecule” is weak, and, on the other hand, heparin has a large affinity for a number of blood components, which leads to its rapid removal from the polymer surface.

To slow down the release of the drug from the composition of the polymer material, a method for producing a microfiber vascular prosthesis by the electrospinning method is proposed, where the drug in a solution of dimethyl sulfoxide (paclitaxel, sirolimus or diclofenac) is directly introduced into the initial polymer solution (patent RU 2669344 C1, publ. 10.10.2018).

However, the use of dimethyl sulfoxide can lead to a change in the physical characteristics of the polymer, which can be critical when creating vascular prostheses or other products for arterial reconstruction.

A number of studies have proposed a method for covalently attaching cell adhesion factors — peptides containing an RGD fragment to biodegradable surfaces of tissue-engineered vascular grafts based on polycaprolactone (Gabriel M, et. Al. Biomater. Direct grafting of RGD-motif-containing peptide on the surface of polycaprolactone films. // Journal of Biomaterials Science. Polymer Edition, 2006, Vol. 17, No. 5, pp. 567-577). The method includes a two-stage surface modification, where in the first stage the polymer is treated with a 40% aqueous solution of ethylene diamine, and in the second stage, the surface is modified with a peptide containing an RGD fragment (Gly-Arg-Gly-Asp-Ser), using glutaric as a cross-linking agent aldehyde.

The disadvantage of this method is the use of glutaraldehyde leading to the formation of cytotoxic products (Hass, V. et. Al. Collagen cross-linkers on dentin bonding: Stability of the adhesive interfaces, degree of conversion of the adhesive, cytotoxicity and in situ MMP inhibition. / / Dent Mater 32, 732, 2016), as well as facilitating calcification (Fahrenholtz, MM et.al., Development of a heart valve model surface for optimization of surface modifications. Acta Biomater, 2015, V. 26, P. 64) and inflammatory processes (Delgado, LM et. al., To cross-link or not to cross-link? Cross-linking associated foreign body response of collagen-based devices. // Tissue Eng Part In Rev, 2015, V. 21, P. 298).

It is known that the key moment affecting the process of blood coagulation in vascular prostheses and patches is the adsorption of proteins on the surface of polymers of proteins (Smurova E.V., Dobrova NB. Development of polymer materials with thromboresistant properties, Chemistry and Technology of High Molecular Compounds, Moscow, VINITI , 1976, v. 10, pp. 30-60), which is favorable and unfavorable. So, adsorption of human serum albumin (HSA) somewhat inhibits the further stages of blood coagulation on the polymer surface, and fibrinogen adsorption, on the contrary, accelerates the blood coagulation process.

Closest to the claimed method, the prototype, is a method for producing thromboresistant polymer materials in contact with blood by sequentially treating them with a surfactant, then heparin, then glutaric albumin, with a 1-2% solution of human albumin being used as a surfactant or a 0.5% fibrinogen solution, and they are treated at 30-40 ° C for 1.5-2.5 hours, the material is washed with water, followed by sorption of heparin on the albuminized surface and “crosslinking” heparin-albumin by treatment with glutaraldehyde. Treatment with heparin is carried out from an aqueous solution with a concentration of 40-5000 units / ml at 30-40 ° C for 1-1.5 hours, and glutaraldehyde at 50-60 ° C for 15-30 minutes (SU 1097336 A1, publ. 15.06.1984). This method allows you to avoid sorption on the surface of the polymers of unwanted proteins that accelerate blood coagulation.

The disadvantage of this method is the use of a toxic reagent (glutaraldehyde) and the use of high temperature (50-60 ° C). In addition, the adsorption of albumin largely depends on the type of polymer used, and in the case of the use of biodegradable polymers based on polyhydroxybutyrate / valerate (PHBV) and polycaprolactone (PCL), the sorption efficiency under physiological conditions may not be sufficient for the modified layer to exist for a long time.

The objective of the invention is to develop a method that provides the ability to obtain stable under physiological conditions albuminized surface with the desired biological properties.

Effect: increase the efficiency of sorption of albumin and increase the stability of the modified surfaces of polymeric materials.

The task is achieved by sequentially treating the surface of biodegradable polymers first with aliphatic amino groups, then with an activated polyunsaturated fatty acid ester, then with albumin, with a maleimide derivative, attached to the SH cystine 34 HSA group, necessary to give the polymer the desired properties.

The proposed method is as follows. At the first stage, the polymer surface is treated with a solution of aliphatic diamine in a 60% aqueous solution of isopropyl alcohol at a temperature of 24 ° C to 30 ° C for 30-60 minutes. After that, the surface is thoroughly washed successively with a 50% aqueous solution of isopropyl alcohol, then with deionized water, a 0.3% solution of non-ionic detergent Tween-20 in deionized water. Hexamethylenediamine, 4,7,10-trioxa-1,13-tridecandiamine and the like can be used as an aliphatic diamine.

In the second stage, the polymer surface containing aliphatic amino groups is treated with a 10 mM solution of hydroxysuccinimide ester of linoleic (9,12-unctadiene) acid in absolute isopropyl alcohol containing 0.1% triethylamine at a temperature of 37-40 ° C for 60 minutes. The modified polymer surface is thoroughly washed successively with isopropyl alcohol, a 50% aqueous solution of isopropyl alcohol, a 0.3% solution of Tween-20 in deionized water, deionized water.

Preliminarily, a modification of human serum albumin (HSA) is performed with the required maleimide derivative of a biologically active compound (BAS) in a known manner (Popova TV, et. Al, Bioorg. Med. Chem. Lett. - 2018. - V. 28. - P. 260- 264). As a biologically active compound, an antibiotic, an RGD peptide, heparin, and the like can be taken.

For this, to a 0.001 M solution of HSA in phosphate buffer (PBS) with a pH of 7.4 add a 0.01 M solution of a maleimide derivative of a biologically active substance in water or a 0.03 M solution in dimethyl sulfoxide, the mixture is incubated at 37 ° C for 18 hours, low molecular weight components the reaction mixture was separated using a Centricon concentrator at 9000 g. The resulting HSA conjugate with the corresponding maleimide derivative (HSA conjugate) is used in the next step.

The polymer surface containing the residues of linoleic acid is treated with an aqueous solution of the HSA conjugate for 15-30 minutes, then the modified polymer surface is washed with water, three times with 0.9% sodium chloride solution, then again with water.

The proposed method is simple and lacks toxic reagents, more specifically, there is no surface treatment of the polymer with glutaraldehyde, leading to disruption of the native protein structure and the formation of toxic products. The introduction of a polyunsaturated fatty acid onto the polymer surface significantly increases the efficiency of HSA binding due to the presence of a specific binding center in HSA, and the preliminary modification of HSA with various biologically active compounds allows introducing various required components (one or several) onto the polymer surface using the general procedure.

The determining essential features of the proposed method, in comparison with the prototype, are:

1) The surface of the polymer is sequentially treated first with a 10% solution of diamine (mainly hexamethylenediamine or 4,7,10-trioxa-1,13-tridecandiamine) in a 60% aqueous solution of isopropyl alcohol at 24-30 ° C for 30-60 minutes, and then with a 10 mM solution of N-hydroxysuccinimide ester of linoleic (9,12-unctadiene) acid in isopropyl alcohol, which can significantly increase the strength of the binding of HSA to the polymer surface by introducing polyunsaturated fatty acids onto the surface through the linker group.

2) The surface of the polymer containing linoleic acid residues is treated with an aqueous solution of pre-modified HSA for 15-30 minutes, which allows the use of a wide range of commercially available or easily obtained biologically active compounds (RGD peptides, antibiotics, anticoagulants, dyes, etc. .).

Due to the fact that a search by sources of patent and scientific and technical information did not reveal a similar technical solution, we can conclude that the claimed method of modifying the surface of biodegradable polymeric materials to give them the desired properties meets the criteria of patentability, namely, it has “novelty” and "Inventive step".

The invention is illustrated by the following figures:

Figure 1. The structure of the RGD peptide containing the maleimide fragment and a fluorescent dye.

Figure 2. Kinetics of leaching of the HSA conjugate from a surface modified with linoleic acid residues (row 1) and an unmodified surface (row 2).

The invention is illustrated by the following examples of specific performance.

Example 1

BUT). A polymer plate based on polyhydroxybutyrate / valerate (PHBV) and polycaprolactone (PCL) (1: 1) with an area of 10 cm ^{2 was} treated with a 10% solution of 4,7,10-trioxa-1,13-tridecandiamine in a 60% aqueous solution of isopropyl alcohol at 24 ° C for 60 minutes After that, the surface was thoroughly washed successively from an excess of starting reagents with a 50% aqueous solution of isopropyl alcohol (3 × 50 ml), then deionized water (2 × 50 ml), and a 0.3% solution of Tween-20 in deionized water. Amino groups were quantified using the ninhydrin test. A fragment of 1 cm ^{2 was} cut from the modified surface, placed in 1.5 ml tubes and treated with 1 ml of a 1% solution of ninhydrin in ethanol in the presence of 20 μl of 0.05% ascorbic acid. The reaction was carried out at 80 ° C for 30 minutes, the samples were thoroughly washed with ethanol, dried, and dissolved in 0.5 ml of chloroform. 0.5 ml of isopropanol was added to the resulting solution, and the optical density (L = 1 cm) was measured at a wavelength of 568 nm. The number of amino groups per 1 cm ² was 8.4 ± 0.2 × 10 ^-9 M / cm ² .

B) The amino group-treated polymer plate was dried, washed with absolute isopropyl alcohol (3 × 20 ml) and placed in a 10 mM solution of N-hydroxysuccinimide ester of linoleic (9,12-unctadiene) acid in absolute isopropyl alcohol containing 0.1% triethylamine. Processing was carried out in a shaker at 40 ° C for 60 minutes. The treated sample was washed successively with isopropyl alcohol (3 × 50 ml), a 50% aqueous solution of isopropyl alcohol, a 0.3% solution of Tween-20 in deionized water (50 ml), deionized water (30 × 50 ml).

The number of unreacted amino groups per 1 cm ² does not exceed 0.4 ± 0.1 × 10 ^-9 M / cm ² .

C) Modification of HSA. To 1 ml of a 0.001 M solution of HSA (66.4 mg) in phosphate buffer (PBS) with a pH of 7.4, 7 mg of the maleimide derivative of the RGD peptide containing the fluorescent dye Cy3 (Fig. 1) in 50 μl of dimethyl sulfoxide (DMSO) was added. The reaction mixture was incubated at 37 ° C for 18 hours. After incubation, the reaction mixture was purified from low molecular weight substances using a Centricon concentrator (Amicon Centriprep YM30, Millipore, Bedford) at 9000 g. For this, the protein solution was concentrated to the minimum possible volume (~ 0.2 ml), PBS buffer (0.4 ml × 4) was added, and then water (0.4 ml × 6), after adding each portion of the buffer or water, the solution was concentrated . The volume of the HSA conjugate solution with the maleimide derivative of the RGD peptide obtained at the final stage was adjusted with water to 20 ml and used in the next stage.

D) The processed sample of the polymer material obtained in stage B was placed in a solution of the conjugate of HSA with the maleimide derivative of the RGD peptide obtained in stage B (albumin concentration of 3.3 g / l). The sample was kept on a shaker at 37 ° C for 15 minutes, the plate was washed with water (2 × 50 ml), 0.9% sodium chloride solution (3 × 50 ml), water (50 ml).

To determine the amount of sorbed protein, a sample of the modified polymer material (1 cm ² ) was dissolved in hexafluoroisopropyl alcohol and the optical density (L = 1 cm) was measured at a wavelength of 550 nm. The amount of sorbed protein was 3.1 ± 0.2 μg / cm ² .

Example 2

BUT). A polymer plate based on polyhydroxybutyrate / valerate (PHBV) and polycaprolactone (PCL) (1: 1) with an area of 10 cm ^{2 was} treated with a 10% solution of hexamethylenediamine in a 60% aqueous solution of isopropyl alcohol at 30 ° C for 30 min. After that, the surface was thoroughly washed successively with a 50% aqueous solution of isopropyl alcohol (3 × 50 ml), then deionized water (2 × 50 ml), 0.3%) with a solution of Tween-20 in deionized water. Quantitative determination of amino groups was carried out using ninhydrin test as described in example 1. The number of amino groups per 1 cm ² was 7.5 ± 0.2 × 10 ^-9 M / cm ² .

B) The amino group-treated polymer plate was treated with a 10 mM solution of linoleic acid N-hydroxysuccinimide ester at a temperature of 37 for 60 minutes, as described in Example 1.

The number of unreacted amino groups per 1 cm ² does not exceed 0.6 ± 0.1 × 10 ^-9 M / cm ²

C) Modification of HSA was carried out as described in example 1.

D) The processed sample of the polymer material obtained in stage B was placed in a solution of the conjugate of HSA with the maleimide derivative of the RGD peptide obtained in stage B (albumin concentration of 3.3 g / l). The sample was kept on a shaker at 37 ° C for 15 minutes, the plate was washed with water (2 × 50 ml), 0.9% sodium chloride solution (3 × 50 ml), water (50 ml).

To determine the amount of sorbed protein, a sample of the modified material (1 cm ² ) was dissolved in hexafluoroisopropyl alcohol and the optical density (L = 1 cm) was measured at a wavelength of 550 nm. The amount of sorbed protein was 2.9 ± 0.2 μg / cm ² .

Example 3 A comparative analysis of the effectiveness of the binding of HSA conjugate to a modified, according to the claimed method, and not a modified surface.

Polymer plates based on polyhydroxybutyrate / valerate (PHBV) and polycaprolactone (PCL) with an area of 10 cm ² with an unmodified surface and a surface modified with linoleic acid, as described in Example 1 (stages A and B), were treated with HSA conjugate with RGD peptide, containing in the structure of the fluorescent dye Cy3 (stage B, example 1) as described in example 1 (stage D), without washing with a solution of sodium chloride.

Samples of the modified polymer were placed in a 0.9% sodium chloride solution (saline) at 37 ° C and kept in a shaker. After 0.5, 1, 2, 4, 8, 24, 48, and 72 hours, the optical density of the solution was measured at a wavelength of 550 nm. For 100%, the sorbed protein value, determined as described in paragraph D, Example 2, is taken. The result obtained is shown in FIG. 2.

As can be seen from FIG. 2, in contrast to the unmodified surface, the modification of the polymer surface, in accordance with the claimed method, provides strong binding of albumin conjugates to the polymer surface. After the initial washing with physiological saline, the amount of bound albumin decreases by 6% and practically does not change in the future.

Thus, the use of the proposed method allows to obtain modified polymer surfaces with desired biological properties, which are characterized by increased stability under physiological conditions, and also have due to the presence of albumin conjugates with high bio- and hemocompatibility for use in medical practice. The polymer modification procedure is simple, reproducible and adaptable.

Claims (6)

1. A method of modifying the surface of biodegradable polymeric materials, including surface treatment with albumin, characterized in that at the first stage the surface of the polymer is treated with a solution of aliphatic diamine in a 60% aqueous solution of isopropyl alcohol at a temperature of 24 ° C to 30 ° C for 30-60 minutes, in the second stage, the surface of the polymer containing an aliphatic amine is treated with a 10 mM solution of hydroxysuccinimide ester of linoleic (9,12-unctadiene) acid in isopropyl alcohol containing 0.1% triethylamine at a rate the mixture, 37-40 ° C for 60 minutes, then the polymer surface containing the residues of linoleic acid is treated with an aqueous solution of human serum albumin conjugate (HSA) with the corresponding maleimide derivative of the biologically active compound for 15-30 minutes, after each stage modifications, the surface of polymeric materials is washed from an excess of initial reagents, while the HSA is preliminarily modified with the required maleimide derivative of a biologically active compound. 2. The method according to claim 1, characterized in that hexamethylenediamine or 4,7,10-trioxa-1,13-tridecandiamine is used as an aliphatic diamine. 3. The method according to p. 1, characterized in that after the first stage of modification, the polymer surface is washed successively with a 50% aqueous solution of isopropyl alcohol, then with water, then with a 0.3% solution of Tween-20 in water. 4. The method according to p. 1, characterized in that after the second stage of modification, the polymer surface is washed successively with isopropyl alcohol, then with a 50% aqueous solution of isopropyl alcohol, then with a 0.3% solution of Tween-20 in water, then with water. 5. The method according to p. 1, characterized in that at the last stage the modified polymer surface is washed with water, then with a 0.9% sodium chloride solution, then again with water. 6. The method according to claim 1, characterized in that the biologically active compounds are selected from the group: antibiotic, RGD peptide, heparin.

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