RU2842966C1 - Method of producing biocompatible and biodegradable hydrogels by step-by-step solvent replacement - Google Patents

Abstract

FIELD: medical science.

SUBSTANCE: invention refers to regenerative medicine. Disclosed is a method of producing biocompatible and biodegradable hydrogels by step-by-step solvent replacement, characterized by that it includes: stage of preparation of 15-20 wt.% of a solution or solutions of amphiphilic block copolymers of polylactides or polylactones with polyethylene glycol in an organic solvent, followed by introduction of the resulting solution into a dialysis bag with a pore size of 3.5 to 5.0 kDa and diameter of 6-10 mm, stage of dialysis of the obtained block copolymer solution in 80 wt.% solution of an organic solvent with distilled water with specific electrical conductivity of not more than 10^-4 S/cm in a glass with volume of 0.5-1 l for 5-7 days with constant stirring with step-by-step reduction of concentration of organic solvent by 5-10 wt.% per hour until complete replacement with distilled water and completion of self-organisation process with formation of physical hydrogel network and removal of residual organic solvent, wherein the solvent must meet 2 conditions: dissolving both blocks well in the copolymer, i.e. be a non-selective solvent, and miscible with water in any ratios.

EFFECT: obtaining biocompatible and biodegradable hydrogel materials based on amphiphilic ternary block copolymers through step-by-step replacement of the organic solvent with a wide range of modulus of elasticity from 7 kPa to 0.5 MPa.

2 cl, 1 dwg, 5 ex

Description

Field of technology

The invention relates to the field of high-molecular chemistry and regenerative medicine, and is aimed at developing biocompatible and biodegradable polymer hydrogel materials that can be used to create tissue-engineered structures, drug carriers, as well as wound dressings and other medical products.

State of the art

Currently, the development of regenerative medicine requires an increasing number of biocompatible materials for the restoration of human tissues and organs. The main idea is to create a matrix (framework) that is compatible with the biological environment of the body and is similar in its physical and mechanical properties to native tissues. The inclusion of cells and growth factors in such a matrix, which stimulate their proliferation and differentiation, will contribute to the regeneration of native tissue. Spongy, fibrous or hydrogel materials can be used for such purposes.

Hydrogels are colloidal systems consisting of more than 80-90% water, the dispersed phase of which is a three-dimensional framework of a high-molecular compound of natural or synthetic origin. Such materials potentially have excellent biocompatibility due to their high water content, as well as soft and elastic structure. Various high-molecular compounds can be used as a polymer base for hydrogels, such as polyacrylamide, polyvinyl alcohol, cellulose, collagen, chitosan, block copolymers of ethylene glycol with hydrophobic polymers, alginic acid, organosilicon polymers, etc.

Hydrogels can be physically or chemically cross-linked, both options have their advantages and disadvantages. In chemically cross-linked hydrogels, the nodes of the network are linked by covalent bonds, which makes the materials stronger, but this type of cross-linking involves the use of cross-linking agents, catalysts, or UV exposure. All this makes the production method quite complex and also raises the question of the biocompatibility of the materials, since cross-linking agents and catalysts are often highly toxic. In the case of physically cross-linked hydrogels, the forces holding the three-dimensional network are not covalent in nature, but, as a rule, adhesion occurs due to hydrophobic interactions, hydrogen bonds, electrostatic attraction, etc. All this suggests simpler production methods and the absence of potentially toxic cross-linkers, but a significant disadvantage of such systems is their low physical and mechanical properties. Often, such materials are not able to maintain shape and have fairly low elastic moduli, not comparable with those of native body tissues. In this regard, the development of new approaches to obtaining hydrogels and the search for new materials for creating biosafe, durable systems for potential use in biomedicine are relevant.

An analysis of Russian and foreign patents showed that at this stage of development of the field, globally, it is possible to identify 3 main methods for obtaining hydrogel materials:

1) Chemical cross-linking (about 29% of the patents studied).

2) Mixing the components to form a hydrogel (52%).

3) Freeze-thaw cycles to obtain cryogels (8%).

The most frequently used high-molecular compounds for obtaining hydrogels, according to the results of the analysis of the known level of technology, are: chitosan (about 25% of the studied patents), amphiphilic block copolymers of ethylene glycol and caprolactone and/or lactide and/or glycolide (22%), collagen (13%), polyvinyl alcohol (10%), cellulose (10%), polyacrylamide (7%).

Patent US 11680107 B2 discloses a method for producing thermosensitive hydrogels based on amphiphilic triple and double block copolymers of ethylene glycol and lactide-co-glycolide of various compositions. Mixtures of the said copolymers were dissolved in phosphate-buffered saline (PBS) or tris-buffered saline (TBS) at 4°C (concentrations of 15-25%). The resulting solutions were thermostatted at temperatures from 15 to 45°C to establish the sol-gel transition point. According to the results of rheological tests for all samples with a copolymer concentration of 15%, the sol-gel transition is above 25°C. This method for preparing hydrogels is fairly simple, but is only suitable for copolymers with a low molecular weight. In addition, the results of rheological tests demonstrate very low elastic moduli (less than 100 Pa). As the authors of the patent propose, such material can be used as injectable carriers of medicinal preparations, however, these systems are too fragile for creating tissue-engineered structures.

In the articles [Bhatia SR, Yin X., Hewitt DR O., Quah SP, Zheng B., Mattei GS, Khalifan PG, Grubbs RB Soft Matter. V. 14. P. 7255-7263 (2018)] and [Surita R. Bhatia, Gregory N. Tew. Degrad. Polymers and Mater. V. 1114 (18). P. 313-324 (2012)], hydrogel materials were obtained based on a synthesized series of triple block copolymers P(D,L)LA-PEG-P(D,L)LA (M _w = 16.5-19.6 kDa, PDI = 1.1-1.3) with different ratios of D/L units. The production method involves dispersing the copolymers in water, followed by maintaining the resulting suspensions at an elevated temperature. Changing the D/L ratio results in a nonlinear change in the storage modulus: at ratios of 75/25 and 85/15, the hydrogels are more rigid and have a higher modulus than at 50/50 and 100/0. The authors explain this rheological behavior by the competition between the strength due to the formation of PLLA crystallites and the decrease in the proportion of PEG bridging chains. In the case of ratios of 75/25 and 85/15, the nodes in the physical network of the hydrogel are semicrystalline, i.e. both amorphous domains and small or weakly ordered crystallites are present. Such a structure provides sufficient mobility to the through chains of the hydrophilic block to form bridging structures. Depending on the composition and concentration, the storage modulus values were in the range of 1-10 kPa.

Patent US 11884765 B2 discloses a method for producing chemically cross-linked hydrogels based on amphiphilic double and triple block copolymers of ethylene glycol and caprolactone modified with acryloyl chloride. Water-soluble lithium phenyl (2,4,6-trimethylbenzoyl)phosphinate (LAP) was used as an initiator. The modified block copolymers were dissolved in distilled water and mixed with an aqueous solution of LAP, the resulting mixture was placed in an appropriate mold and the process was initiated by exposure to visible radiation (395-405 nm). As a result, transparent and elastic hydrogels were obtained. This cross-linking method has a number of advantages. LAP is a biosafe cytocompatible water-soluble photoinitiator, in addition, this initiator starts polymerization under the influence of visible radiation, which is safer than UV. However, in general, this method of production is quite complex and expensive, since it includes the process of modification of the end groups of copolymers with acryloyl chloride, as well as the synthesis of LAP (as indicated by the authors of the patent). Although LAP is a commercially available initiator, its use will greatly increase the cost of such hydrogels. Also, despite chemical crosslinking, the elastic moduli of these systems still remain low: up to 32 kPa according to the results of compression tests.

Patent US 10011689 B2 discloses a method for producing chemically cross-linked hydrogels based on amphiphilic triple block copolymers of ethylene glycol and lactide-co-glycolide modified with acrylate end groups. A fairly toxic and explosive compound, azobisisobutyronitrile (AIBN), is used as a cross-linking initiator, and the radical polymerization process itself takes quite a long time, after which long-term purification from residual monomer, initiator, etc. is required. Also, the obtained hydrogels have relatively low elastic moduli of no more than 5 kPa.

Similar approaches to obtaining chemically cross-linked systems are disclosed in patents US 2004077797 A1, CN 101845120 A.

In the article [Li S., Molina I., Martinez MB, Vert MJ Mater. Science: Mater. Medicine. V. 13. P. 81-86 (2002)] a method for obtaining hydrogel materials through an organic phase based on synthesized block copolymers P(D,L)LA-PEG-P(D,L)LA (M _n =45.8 kDa and M _n =78.9 kDa) with sufficiently long hydrophilic and hydrophobic blocks was disclosed. For this purpose, the copolymers were dissolved in tetraethylene glycol upon heating, after which they were cooled to room temperature and washed from the initial solvent in distilled water. The hydrogels obtained in this way are capable of maintaining a disc-shaped shape, which indicates sufficiently high values of the elastic modulus for such systems. However, the physicomechanical characteristics of the obtained hydrogels were not studied in this work.

The closest to the present invention is patent US 6,350,812 B1, which discloses a method for producing physically cross-linked hydrogels based on triple block copolymers of lactide and ethylene glycol by selecting the composition of the high-molecular compound. The method includes dissolving the block copolymers in a minimum amount of acetone (0.2 g of sample in 0.4 or 0.8 ml of solvent), adding distilled water (4 ml) and cleaning from residual organic solvent by replacing the water, by dialysis or by evaporating the acetone. As a result, a soft hydrogel was obtained. This method is similar in meaning to the claimed patent, since it similarly assumes dissolving the block copolymer in an organic solvent and gradually replacing it with distilled water by gradually reducing the concentration of the solvent. Disadvantages of the prototype: 1) the volume of the block copolymer solution partially limited by the vessel walls can lead to the formation of a rather loose and/or incapable of maintaining the shape of the hydrogel material; 2) a high concentration gradient, since the contact with water will be predominant, on the one hand, as a result of which the hydrogel network will not be formed in the entire volume at once. When replacing the solvent in the dialysis bag, firstly, limiting the volume of the organic phase allows obtaining materials of a certain shape, which they are able to maintain for a long time. Secondly, a stepwise decrease in the concentration of the organic solvent in the external solution allows the polymer chains to remain mobile for a long time, which contributes to their greater ordering and a more thermodynamically equilibrium flow of the process of hydrogel network formation. Thirdly, the diffusion of the solvent through the pores of the dialysis bag occurs more uniformly throughout the volume.

Disclosure of invention

The technical task of the invention is to develop biocompatible and biodegradable hydrogels by the method of step-by-step replacement of an organic solvent based on amphiphilic triple block copolymers with a wide range of physical and mechanical characteristics for potential applications in biomedicine.

The technical result is the production of biocompatible and biodegradable hydrogel materials based on amphiphilic triple block copolymers by the method of step-by-step replacement of an organic solvent with a wide range of elastic moduli from 7 kPa to 0.5 MPa.

In order to achieve the technical result, a method for producing biocompatible and biodegradable hydrogels by a step-by-step solvent replacement method is proposed, characterized in that it includes: a stage of preparing a 15-20 wt. % solution or solutions of amphiphilic triple block copolymers of polylactides or polylactones with polyethyleneglycol in an organic solvent, followed by introducing the resulting solution into a dialysis bag with a pore size of 3.5 to 5.0 kDa and a diameter of 6-10 mm, a stage of dialysis of the resulting block copolymer solution in an 80 wt. % solution of an organic solvent with distilled water with a specific electrical conductivity of no more than 10 ^-4 S/cm in a 0.5-1 l beaker for 5-7 days with constant stirring with a step-by-step decrease in the concentration of the organic solvent by 5-10 wt. % per hour until complete replacement with distilled water and completion of the self-organization process with the formation of a physical hydrogel network and removal of residual organic solvent, while the solvent must meet 2 conditions: dissolve both blocks in the copolymer well, i.e. be a non-selective solvent, and mix well with water in any proportions.

The combination of the above essential features leads to the fact that the hydrogels obtained by these methods have a predominantly cylindrical shape with clear boundaries, which confirms the possibility of obtaining materials of any shape and size depending on the potential application. By varying the degree of polymerization of the hydrophilic and hydrophobic blocks of the copolymer, its degree of crystallinity, it is possible to obtain materials with a wide range of elasticity moduli from 7 kPa to 0.5 MPa. The structure of the hydrogels is stable under normal conditions.

The step-by-step replacement of the solvent promotes long-term preservation of the mobility of polymer chains and their greater ordering, bringing the conditions of the process of formation of the hydrogel network closer to more equilibrium.

Brief description of the drawings

Fig. 1 - Scheme for obtaining hydrogel materials.

Implementation and example of implementation of the invention

The method for obtaining biocompatible and biodegradable hydrogels by the method of step-by-step solvent replacement consists of the following stages:

1) Preparation of solutions of amphiphilic triple block copolymers in an organic solvent.

2) Dialysis in a mixed solvent.

3) Extraction of hydrogels.

4) Control of material characteristics.

Amphiphilic triple block copolymers include biocompatible hydrophilic and hydrophobic blocks. Amphiphilic triple block copolymers with amorphous hydrophobic blocks, semicrystalline hydrophobic blocks or semicrystalline hydrophobic blocks with opposite configurations are used as amphiphilic triple block copolymers of polylactides or polylactones with polyethyleneglycol.

A polymer from the following group can be selected as a hydrophobic block: polyglycolide (PGA), polylactide (PLA), polycaprolactone (PCL) and their random copolymers poly(lactide-co-glycolide) (PLGA), poly(lactide-co-caprolactone) (PCLA). Polyethyleneglycol (PEG) can be selected as a hydrophilic block. In a preferred embodiment, triple block copolymers of lactide and ethylene glycol (PLA-PEG-PLA) are used. P(L or D or D,L)LA-b-PEG-b-P(L or D or D,L)LA with different degrees of polymerization of the hydrophilic and hydrophobic blocks are used as amphiphilic triple block copolymers.

Organic solvents that can be used to prepare solutions of block copolymers must meet two conditions: dissolve both blocks in the copolymer well, i.e. be non-selective solvents, and mix well with water in any proportions. Possible solvents: 1,4-dioxane, DMSO, acetone, THF. In the preferred embodiment, 1,4-dioxane is used.

Preparation of solutions of amphiphilic triple block copolymers in an organic solvent

To prepare a solution of the amphiphilic triple block copolymer in an organic solvent, take an Eppendorf tube or a vial with a lid, in which the amphiphilic triple block copolymer is dissolved in an organic solvent to obtain a solution with a block copolymer concentration of 15-20 wt.%. The resulting, as a rule, viscous solution is placed in a dialysis bag with a pore size of 3.5 to 5.0 kDa and a diameter of 6-10 mm.

To prepare a solution of a mixture of amphiphilic triple block copolymers with opposite configurations of hydrophobic blocks (L and D) in an organic solvent, two different Eppendorf tubes or vials with a lid are used, in which the amphiphilic triple block copolymers are dissolved in an organic solvent to obtain 15-20 wt. % solutions, which are then mixed to obtain stereocomplex structures. The resulting, as a rule, viscous solution is placed in a dialysis bag with a pore size of 3.5 to 5.0 kDa and a diameter of 6-10 mm.

Mixed solvent dialysis

A dialysis bag with a solution of amphiphilic triple block copolymer(s) is placed in a 0.5-1 l beaker with 80 wt. % solution of organic solvent with distilled water with a specific electrical conductivity of no more than 10 ^-4 S/cm. Dialysis is carried out for 5-7 days with constant stirring with a gradual decrease in the concentration of organic solvent by 5-10 wt. % per hour until complete replacement with distilled water and completion of the self-organization process with the formation of a physical hydrogel network and removal of residual organic solvent. The step-by-step replacement of the solvent contributes to the long-term preservation of the mobility of polymer chains and their greater ordering, bringing the conditions of the process of formation of the hydrogel network closer to more equilibrium.

Extraction of hydrogels

After completion of dialysis, the resulting hydrogels are removed from the dialysis bags for further studies. Control of material characteristics.

Control of material characteristics is carried out in accordance with technical requirements for manufactured products.

The examples below illustrate variants of the claimed invention, but do not limit it.

Example 1

In a plastic vial, 0.8 g of the amphiphilic triple block copolymer P(D,L)LA ₁₀₁ -PEG ₁₃₆ -P(D,L)LA ₁₀₁ , where the subscript indicates the degree of polymerization of the block, with a molecular weight of 20.5 kDa and amorphous hydrophobic blocks is dissolved in 3.2 g of 1,4-dioxane (Komponent-reaktiv). The resulting 20 wt. % solution is placed in a dialysis bag with a diameter of 6 mm and a pore size of 3.5 kDa (Orange Scientific), which is immersed in a 1 L beaker with a mixed solvent of 80 wt. % 1,4-dioxane in distilled water with a specific conductivity of 10 ^-4 S/cm. Dialysis is carried out for 5 days with constant stirring with a step-by-step decrease in the concentration of the organic solvent by 10 wt. % per hour until complete replacement with distilled water and completion of the self-organization process with the formation of a physical hydrogel network and removal of residual organic solvent. After completion of the dialysis process, the resulting hydrogel is removed from the dialysis bag. The elastic modulus of the resulting hydrogel material was 16 kPa.

Example 2

In a plastic vial, 0.8 g of the amphiphilic triple block copolymer P(L)LA ₆₆ -PEG ₁₃₆ -P(L)LA ₆₆ , where the subscript indicates the degree of block polymerization, with a molecular weight of 15.5 kDa and semicrystalline hydrophobic blocks is dissolved in 3.2 g of 1,4-dioxane (Komponent-reaktiv). The resulting 20 wt. % solution is placed in a dialysis bag with a diameter of 10 mm and a pore size of 3.5 kDa (Orange Scientific))), which is immersed in a 0.5 L beaker with a mixed solvent of 80 wt. % 1,4-dioxane in distilled water with a specific conductivity of 10 ^-4 S/cm. Dialysis is carried out for 7 days with constant stirring with a step-by-step decrease in the concentration of the organic solvent by 5 wt. % per hour until complete replacement with distilled water and completion of the self-organization process with the formation of a physical hydrogel network and removal of residual organic solvent. After completion of the dialysis process, the resulting hydrogel is removed from the dialysis bag. The elastic modulus of the resulting hydrogel material was 140 kPa.

Example 3

In a plastic vial, 0.8 g of the amphiphilic triple block copolymer P(L)LA ₂₆ -PEG ₁₀₄ -P(L)LA ₂₆ , where the subscript indicates the degree of block polymerization, with a molecular weight of 9.0 kDa and semicrystalline hydrophobic blocks is dissolved in 3.2 g of 1,4-dioxane (Komponent-reaktiv). The resulting 20 wt. % solution is placed in a dialysis bag with a diameter of 10 mm and a pore size of 3.5 kDa (Orange Scientific))), which is immersed in a 1 L beaker with a mixed solvent of 80 wt. % 1,4-dioxane in distilled water with a specific conductivity of 10 ^-4 S/cm. Dialysis is carried out for 6 days with constant stirring with a step-by-step decrease in the concentration of the organic solvent by 7 wt. % per hour until complete replacement with distilled water (10 ^-4 S/cm) and completion of the self-organization process with the formation of a physical hydrogel network and removal of residual organic solvent. After completion of the dialysis process, the resulting hydrogel is removed from the dialysis bag. The elastic modulus of the resulting hydrogel material was 187 kPa.

Example 4

In two plastic vials, 0.4 g of amphiphilic triple block copolymers P(L)LA ₃₈ -PEG ₁₃₆ -P(L)LA ₃₈ and P(D)LA ₃₈ -PEG ₁₃₆ -P(D)LA ₃₈ , where the subscript indicates the degree of block polymerization, with a molecular weight of 11.5 kDa and semicrystalline hydrophobic blocks of the opposite configuration are separately dissolved in 1.6 g of 1,4-dioxane (Component-reagent). The resulting 20 wt. % solutions are mixed in a weight ratio of 1:1, placed in a dialysis bag with a diameter of 10 mm and a pore size of 5 kDa (Orange Scientific), which is immersed in a 1 L beaker with a mixed solvent of 80 wt. % 1,4-dioxane in distilled water with a specific conductivity of 10 ^-4 S/cm. Dialysis is carried out for 6 days with constant stirring with a gradual decrease in the concentration of the organic solvent by 7 mass % per hour until complete replacement with distilled water and completion of the self-organization process with the formation of a physical network of the hydrogel and removal of the residual organic solvent. After completion of the dialysis process, the resulting hydrogel is removed from the dialysis bag. The elastic modulus of the resulting hydrogel material was 73 kPa.

Example 5

In a plastic vial, 0.8 g of the amphiphilic triple block copolymer P(L)LA ₂₁₀ -PEG ₂₇₂ -P(L)LA ₂₁₀ , where the subscript indicates the degree of block polymerization, with a molecular weight of 42.2 kDa and semicrystalline hydrophobic blocks is dissolved in 3.2 g of 1,4-dioxane (Komponent-reaktiv). The resulting 20 wt. % solution is placed in a dialysis bag with a diameter of 6 mm and a pore size of 5 kDa (Orange Scientific), which is immersed in a 1 L beaker with a mixed solvent of 80 wt. % 1,4-dioxane in distilled water with a specific conductivity of 10 ^-4 S/cm. Dialysis is carried out for 5 days with constant stirring with a step-by-step decrease in the concentration of the organic solvent by 10 wt. % per hour until complete replacement with distilled water and completion of the self-organization process with the formation of a physical hydrogel network and removal of residual organic solvent. After completion of the dialysis process, the resulting hydrogel is removed from the dialysis bag. The elastic modulus of the resulting hydrogel material was 330 kPa.

Thus, the proposed method for obtaining biocompatible and biodegradable hydrogels allows obtaining hydrogels of predominantly cylindrical shape, with clear boundaries, the structure of which is stable under normal conditions, with a wide range of elastic moduli from 7 kPa to 0.5 MPa, which expands the range of possible applications of such materials in the biomedical field.

Claims (2)

1. A method for producing biocompatible and biodegradable hydrogels by a step-by-step solvent replacement method, characterized in that it includes: a stage of preparing a 15-20 wt. % solution or solutions of amphiphilic block copolymers of polylactides or polylactones with polyethyleneglycol in an organic solvent, followed by introducing the resulting solution into a dialysis bag with a pore size of 3.5 to 5.0 kDa and a diameter of 6-10 mm, a stage of dialysis of the resulting block copolymer solution in an 80 wt. % solution of an organic solvent with distilled water with a specific electrical conductivity of no more than 10 ^-4 S/cm in a 0.5-1 l beaker for 5-7 days with constant stirring with a step-by-step decrease in the concentration of the organic solvent by 5-10 wt. % per hour until complete replacement with distilled water and completion of the self-organization process with the formation of a physical hydrogel network and removal of residual organic solvent, while the solvent must meet 2 conditions: dissolve both blocks in the copolymer well, i.e. be a non-selective solvent, and mix well with water in any proportions. 2. A method for producing biocompatible and biodegradable hydrogels by the step-by-step solvent replacement method according to claim 1, characterized in that amphiphilic triple block copolymers of polylactides or polylactones with polyethyleneglycol are used as amphiphilic triple block copolymers with amorphous hydrophobic blocks, semi-crystalline hydrophobic blocks, or semi-crystalline hydrophobic blocks with opposite configurations.