WO2024099063A1

WO2024099063A1 - Articular cavity injection gel and method for preparing same

Info

Publication number: WO2024099063A1
Application number: PCT/CN2023/126089
Authority: WO
Inventors: 李晓盟; 翟晖; 江艳; 牛睿; 代金明
Original assignee: 北京大清生物技术股份有限公司
Priority date: 2022-11-08
Filing date: 2023-10-24
Publication date: 2024-05-16
Also published as: CN115671405A; CN115671405B

Abstract

The present invention provides an articular cavity injection gel and a method for preparing same, which relate to the field of bio-tribology. Specifically, polyethylene glycol active ester and carboxymethyl chitosan for crosslinking are mixed and subjected to a crosslinking reaction to obtain a gel, then the gel is comminuted and mixed with a non-crosslinked carboxymethyl chitosan solution, and the mixture is sterilized and cooled to obtain the articular cavity injection gel, wherein the mass ratio of the polyethylene glycol active ester to the carboxymethyl chitosan for crosslinking is 1:20 to 20:1. The articular cavity injection gel prepared by the method features better flexibility, absence of granular sensation, small friction coefficient, better lubricating performance, good thermal stability, and higher safety performance; moreover, using the polyethylene glycol active ester as a crosslinking agent enables fast reaction speed and short reaction time.

Description

A kind of joint cavity injection gel and preparation method thereof

Technical Field

The invention belongs to the field of biotribology, and in particular relates to a joint cavity injection gel and a preparation method thereof.

Background technique

Osteoarthritis is a non-inflammatory degenerative joint disease, often manifested as joint pain and stiffness, especially after long-term activities. The disease is more common in people over 50 years old and can start at the age of 20, but most are asymptomatic and generally difficult to detect. The prevalence of osteoarthritis increases with age and is more common in women than in men. The main pathology of osteoarthritis is cartilage degeneration and disappearance, as well as reactive hyperplasia of the ligament attachments at the joint margins and subchondral bone to form osteophytes, which cause joint pain, stiffness, deformity and dysfunction.

Joints include cartilage, synovium and joint capsule. The basic components of joint cartilage are chondrocytes and extracellular matrix, of which chondrocytes account for only 1% and extracellular matrix accounts for 99%. About 3% of the extracellular matrix is glycosaminoglycan. Synovial fluid is secreted by the synovial membrane of the joint bursa and tendon sheath. It contains a transparent viscous lubricant similar to mucin. It has a lubricating effect and is a secretion of human organs and tissues. It plays a role in lubricating, moisturizing organs and expelling toxins. If the synovial fluid decreases or becomes viscous, metabolic products will remain in the body, causing various diseases: for example, the synovial fluid decreases with age, the joints lack lubricants, and the joints will develop degenerative arthritis, bone spurs, osteoporosis, etc. due to wear and tear. Long-term lack of synovial fluid in the joint cartilage will also cause osteoarthritis.

At present, degenerative arthritis is mostly treated by injecting viscoelastic supplements in the early stages of its occurrence. Commonly used viscoelastic supplements include hyaluronic acid and carboxymethyl chitosan (also known as carboxymethyl glucosaminoglycan, chitosan). As a traditional viscoelastic supplement, hyaluronic acid has been used for many years with good results, but carboxymethyl chitosan, as a new type of viscoelastic supplement, has a better therapeutic effect than hyaluronic acid as a viscoelastic supplement. Patent application CN105709274A discloses a hyaluronic acid-like joint capsule thermosensitive gel for artificial joints and a preparation method thereof. A hyaluronic acid-like joint capsule thermosensitive gel for artificial joints can be obtained. The gel can be a sol, i.e., a liquid state, at a temperature below body temperature. After being injected into the human body, it is evenly distributed around the artificial joint to gel, forming a septum similar to the joint capsule. The porous network after gelation forms a sustained-release environment for transporting artificial synovial fluid to the friction surface of the artificial joint. Chinese patent CN107349476B discloses a bionic Synovial fluid and its preparation method, specifically, carboxymethyl chitosan is used as the main raw material, and carboxymethyl chitosan with different cross-linking degrees and non-cross-linked carboxymethyl chitosan are respectively used to mix by physical method to prepare bionic synovial fluid, which can form bionic synovial fluid with viscoelasticity and fluidity index reaching normal synovial fluid, and can be used as a viscoelastic supplement when synovial fluid is missing or viscoelasticity decreases due to degenerative or traumatic arthritis, effectively slowing down the progression of degenerative or traumatic arthritis and relieving pain. However, its friction coefficient is large, the gel granularity is heavy, and the lubrication performance is poor.

Therefore, there is currently a lack of an injectable gel for joint cavity with a small friction coefficient, better thermal stability, strong safety and small swelling degree.

Summary of the invention

In view of the problems existing in the prior art, the present invention provides a joint cavity injection gel and a preparation method thereof. The joint cavity injection gel obtained by the preparation method has a small friction coefficient, better lubrication performance, stronger safety performance and thermal stability. The viscoelasticity and fluidity indicators of the formed gel meet the standards of normal joint cavity synovial fluid, effectively increase bone growth factor and have the effects of relieving pain and inflammation.

To achieve the above purpose, the technical solution adopted by the present invention is as follows:

The invention provides a method for preparing a gel for joint cavity injection, wherein the active ester of polyethylene glycol is mixed with cross-linking carboxymethyl chitosan to produce a cross-linking reaction to obtain a gel, the gel is crushed and then mixed with a non-cross-linked carboxymethyl chitosan solution, sterilized, and cooled to obtain the gel for joint cavity injection;

Preferably, the mass ratio of the polyethylene glycol active ester to the cross-linking carboxymethyl chitosan is 1:20-20:1.

Preferably, the molecular weight of polyethylene glycol in the polyethylene glycol active ester is 2000Da-10000Da, 2-8 arms, and its chemical structure is as follows:

Using polyethylene glycol active ester (PEG-NHS) as a cross-linking agent has a fast reaction speed and a short reaction time, which can reduce the growth of bacteria and endotoxins in the preparation process. Compared with small molecule epoxy and aldehyde cross-linking agents, it has lower toxicity and a long and controllable molecular chain. If the molecular weight is too small, the molecular chain is not long enough; if the molecular weight is too large, the molar ratio of the active group used for cross-linking is too low.

Further preferably, the molecular weight of polyethylene glycol in the polyethylene glycol active ester is 2000Da-5000Da, 2-4 arms;

Most preferably, the molecular weight of the polyethylene glycol in the polyethylene glycol active ester is 3500 Da, with 4 arms.

Preferably, the carboxymethyl chitosan used for cross-linking has a deacetylation degree of 30-80%, a molecular weight of 500-1500 kDa, a substitution degree of 80%-200%, and a viscosity of 300-1000 mPa.s (at a mass fraction of 1%).

Further preferably, the carboxymethyl chitosan used for cross-linking has a deacetylation degree of 40-60%, a molecular weight of 600-800 kDa, a substitution degree of 100%-180%, and a viscosity of 300-500 mPa.s (at a mass fraction of 1%).

Most preferably, the carboxymethyl chitosan used for cross-linking has a deacetylation degree of 50%, a molecular weight of 700 KDa, a substitution degree of 120%, and a viscosity of 360 mPa.s (at a mass fraction of 1%).

Preferably, the non-cross-linked carboxymethyl chitosan solution has a deacetylation degree of less than 30%, a molecular weight of 500-1500 kDa, a substitution degree of 80%-200%, and a viscosity of 300-1000 mPa.s (at a mass fraction of 1%).

Further preferably, the non-cross-linked carboxymethyl chitosan solution has a deacetylation degree of 10-28%, a molecular weight of 600-800 kDa, a substitution degree of 100%-180%, and a viscosity of 300-500 mPa.s (at a mass fraction of 1%).

Most preferably, the non-cross-linked carboxymethyl chitosan solution has a degree of deacetylation of 25%, a molecular weight of 700 kDa, a degree of substitution of 120%, and a viscosity of 360 mPa.s (at a mass fraction of 1%).

Generally speaking, the larger the molecular weight of the carboxymethyl chitosan, the greater the viscosity, the closer it is to normal synovial fluid, the smaller the friction, and the longer the degradation time.

Preferably, the pH of the cross-linking reaction is 7-8.5.

Preferably, the preparation method specifically comprises the following steps:

S1, dissolving carboxymethyl chitosan for cross-linking in a phosphate buffer solution to obtain a mixed solution 1;

S2, adding active ester of polyethylene glycol to the mixed solution 1 to carry out a cross-linking reaction, and allowing to stand to obtain a gel;

S3, crushing the gel and adding non-cross-linked carboxymethyl chitosan solution to obtain mixed solution 2, sterilizing and cooling to obtain intra-articular injection gel.

Preferably, the concentration of the phosphate buffer solution in step S1 is 0.2 mol/L and the pH is 7.2.

Preferably, the mass fraction of the carboxymethyl chitosan used for cross-linking in step S1 is 1%-20%.

Preferably, the conditions for the cross-linking reaction in step S2 are: temperature of 20-25° C., stirring speed of 60-90 rpm, and reaction time of 10-30 min.

Further preferably, the conditions for the cross-linking reaction in step S2 are: temperature of 25° C., stirring speed of 80 rpm, and reaction time of 15 min.

Preferably, the standing time in step S2 is 2-4 hours.

Further preferably, the standing time in step S2 is 2 hours.

Preferably, the polyethylene glycol active ester described in step S2 needs to be dissolved in PBS first.

More preferably, the concentration of PBS is 0.2 mol/L and the pH is 7.2.

Preferably, the gel in step S3 needs to be diluted by adding PBS first.

Preferably, the mixed solution 2 described in step S3 also includes bone growth factor (SGF), anti-inflammatory and analgesic drugs such as lidocaine, moxifloxacin hydrochloride, vitamins, and polypeptide nutrients.

Preferably, the mass ratio of the gel in step S3 to the non-cross-linked carboxymethyl chitosan solution is 1:1-9:1.

Further preferably, the mass ratio of the gel in step S3 to the non-cross-linked carboxymethyl chitosan solution is 4:1.

Preferably, the concentration of the non-cross-linked carboxymethyl chitosan in the mixed solution 2 in step S3 is 10-50 mg/ml.

Preferably, the sterilization conditions in step S3 are: temperature 115°C-121°C, time 12-45 min; the cooling temperature is 18-25°C.

Further preferably, the sterilization conditions in step S3 are: temperature of 121° C., time of 15 min; and the cooling temperature of 25° C.

The present invention also provides a gel for joint cavity injection, which is prepared by the above-mentioned preparation method.

The high cross-linked gel of the invention has better flexibility and no granularity. The increase of carbon chains leads to increased hydrophobicity, so that the swelling degree of the gel is lower, and the tissue compression caused by the increase of swelling volume is reduced. The polyethylene glycol in the cross-linking agent forms reversible hydrogen bonds between carboxymethyl chitosan and polyol molecules, thereby enhancing the stability of the molecular structure of carboxymethyl chitin, reducing the damage of carboxymethyl chitin molecular chains by high temperature and high pressure, improving the sterilization stability of carboxymethyl chitin, and extending the degradation time.

The deacetylation degree of the non-cross-linked carboxymethyl chitosan solution is required to be less than 30 to avoid the immune response that may be caused by its amino group, which in turn affects the safety of the organism, and also to avoid the Maillard reaction with the carboxyl group on the molecular chain to cause self-cross-linking, destroy the product stability, and make the product yellow. The deacetylation degree of the cross-linked carboxymethyl chitosan is required to be 30%-80%, because its amino group needs to undergo a cross-linking reaction with the active ester in the cross-linking agent to form a stable amide bond, and the amino group cannot be too much after cross-linking. The carboxymethyl substitution degree of all carboxymethyl chitosans should be optimally 80%-200%, and have better water solubility within this substitution range.

Compared with the prior art, the present invention has the following beneficial effects:

1. Use polyethylene glycol active ester as cross-linking agent, which has fast reaction speed, short reaction time and reduces pollution;

2. The prepared high-crosslinked intra-articular injection gel has better flexibility and no granularity. The increase in carbon chains leads to an increase in hydrophobicity, which makes the gel swelling lower and reduces tissue compression caused by the increase in swelling volume;

3. The intra-articular injection gel of the present invention has a small friction coefficient and better lubrication performance;

4. Lysozyme is used to degrade the intra-articular injection gel of the present invention, and the degradation time is long;

5. The intra-articular injection gel prepared by the present invention has good thermal stability and is safer.

Detailed ways

The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

Example 1

Weigh 1g of polyethylene glycol active ester PEG-NHS (PEH molecular weight 3500, 4 arms) and dissolve it in 10ml of 0.2mol/L pH7.2 3g of carboxymethyl chitosan (deacetylation degree 50%, substitution degree 1.2, molecular weight 700kDa, viscosity 360mpa.s) was weighed and dissolved in 50ml PBS and fully dissolved. The above two solutions were quickly stirred and mixed evenly, sealed and placed in a 25°C environment for reaction for 2h, and after the reaction, the gel was cut into pieces and added to PBS to dilute to 30mg/ml. A carboxymethyl chitosan (deacetylation degree 25%, substitution degree 1.2, molecular weight 700kDa, viscosity 360mpa.s) solution was prepared with a concentration of 20mg/ml and a pH of 7.2. The gel and solution were crushed and dispersed by compressed air through a 70-mesh sieve according to a mass ratio of 4:1, mixed 15 times, and the mixed gel was filled into a pre-filled syringe, sterilized with high-temperature steam at 121°C for 15min, and the sample was obtained after cooling.

Example 2

Weigh 1.5g of polyethylene glycol active ester PEG-NHS (PEH molecular weight 5000, 4 arms) and dissolve it in 10ml of 0.2mol/L pH7.2 PBS, weigh 3g of carboxymethyl chitosan (deacetylation degree 45%, substitution degree 1, molecular weight 500kDa, viscosity 300mpa.s) and dissolve it in 50ml PBS to fully dissolve. The above two solutions were quickly stirred and mixed evenly, sealed and placed at 25℃ for reaction for 2h, and after the reaction, the gel was cut into pieces and added to PBS to dilute to 30mg/ml. Prepare carboxymethyl chitosan (deacetylation degree 30%, substitution degree 0.85, molecular weight 500kDa, viscosity 300mpa.s) solution with a concentration of 20mg/ml and pH 7.2. According to the ratio of 3:1, the gel and solution were crushed and dispersed by compressed air through a 70-mesh screen, mixed 15 times, and the mixed gel was filled into a pre-filled syringe, sterilized with high-temperature steam at 121℃ for 15min, and the sample was obtained after cooling.

Example 3

Weigh 3g of polyethylene glycol active ester PEG-NHS (PEH molecular weight 2000, 2 arms) and dissolve it in 10ml of 0.2mol/L pH7.2 PBS. Weigh 3g of carboxymethyl chitosan (deacetylation degree 55%, substitution degree 1, molecular weight 600kDa, viscosity 330mpa.s) and dissolve it in 50ml PBS to fully dissolve. Quickly stir and mix the above two solutions evenly, seal and place them at 25℃ for reaction for 2h. After the reaction, cut the gel into pieces and add PBS to dilute to 30mg/ml. Prepare carboxymethyl chitosan (deacetylation degree 25%, substitution degree 0.85, molecular weight 600kDa, viscosity 330mpa.s) solution with a concentration of 20mg/ml and pH7.2. According to the ratio of 2:1, the gel and solution are crushed and dispersed by compressed air through a 70-mesh screen, mixed 15 times, and the mixed gel is filled into a pre-filled syringe, sterilized with high-temperature steam at 121℃ for 15min, and the sample is obtained after cooling.

Comparative Example 1

Weigh 1g of polyethylene glycol active ester PEG-NHS (PEG molecular weight 1500, 4 arms) and dissolve it in 10ml of 0.2mol/L pH7.2 PBS. Weigh 3g of carboxymethyl chitosan (deacetylation degree 50%, substitution degree 1.2, molecular weight 700kDa, viscosity 360mpa.s) and dissolve it in 50ml of PBS to fully dissolve. Quickly stir and mix the above two solutions evenly, seal and place them at 25℃ for 2h to react. After the reaction, cut the gel into pieces and add PBS to dilute to 30mg/ml. Prepare carboxymethyl chitosan (deacetylation degree 50%, substitution degree 1.2, molecular weight 700kDa, viscosity 360mpa.s). 25%, degree of substitution 1.2, molecular weight 700kDa, viscosity 360mPa.s) solution, concentration 20mg/ml, pH 7.2. Gel and solution were crushed and dispersed by compressed air through a 70-mesh sieve according to a mass ratio of 4:1, mixed 15 times, and the mixed gel was filled into a pre-filled syringe, sterilized with high-temperature steam at 121°C for 15 minutes, and the sample was obtained after cooling.

Comparative Example 2

Weigh 1g of polyethylene glycol active ester PEG-NHS (PEG molecular weight 15000, 4 arms) and dissolve it in 10ml of 0.2mol/L pH7.2 PBS, weigh 3g of carboxymethyl chitosan (deacetylation degree 50%, substitution degree 1.2, molecular weight 700kDa, viscosity 360mpa.s) and dissolve it in 50ml PBS. Stir the two solutions quickly and mix them evenly, seal them and place them at 25℃ for reaction for 2h, cut the gel into pieces after the reaction and add PBS to dilute it to 30mg/ml. Prepare carboxymethyl chitosan (deacetylation degree 25%, substitution degree 1.2, molecular weight 700kDa, viscosity 360mpa.s) solution with a concentration of 20mg/ml and pH 7.2. According to the mass ratio of 4:1, the gel and solution are crushed and dispersed by compressed air through a 70-mesh screen, mixed 15 times, and the mixed gel is filled into a pre-filled syringe, sterilized with high-temperature steam at 121℃ for 15min, and the sample is obtained after cooling.

Comparative Example 3

Weigh 0.1g of polyethylene glycol active ester PEG-NHS (PEG molecular weight 3500, 4 arms) and dissolve it in 10ml of 0.2mol/L pH7.2 PBS, weigh 3g of carboxymethyl chitosan (deacetylation degree 50%, substitution degree 1.2, molecular weight 700kDa, viscosity 360mpa.s) and dissolve it in 50ml PBS to fully dissolve. The above two solutions were quickly stirred and mixed evenly, sealed and placed at 25℃ for reaction for 2h, and after the reaction, the gel was cut into pieces and added to PBS to dilute to 30mg/ml. Prepare carboxymethyl chitosan (deacetylation degree 25%, substitution degree 1.2, molecular weight 700kDa, viscosity 360mpa.s) solution with a concentration of 20mg/ml and pH 7.2. According to the mass ratio of 4:1, the gel and solution were crushed and dispersed by compressed air through a 70-mesh screen, mixed 15 times, and the mixed gel was filled into a pre-filled syringe, sterilized with high-temperature steam at 121℃ for 15min, and the sample was obtained after cooling.

Test Case

Viscoelasticity testing

The rheological data of the gel injected into the joint cavity were tested by referring to the method in paragraphs 0031-0032 of patent application CN107349476A. The results showed that the elastic modulus and viscous modulus of each sample were close to those of normal synovial fluid, as shown in Table 1.

Table 1 Viscoelastic modulus data

Lubrication capacity test

The friction coefficient was measured according to the method in paragraph 0331 of patent CN114634585A to detect the lubricating ability of the gel. The results showed that the friction coefficient of the intra-articular injection gel of the present invention was lower and the lubricating performance was better, and it had a lower friction coefficient than the commercially available product Syvnisc. The specific results are shown in the following table.

Table 2 Lubrication capacity data

In vitro degradation ability test

According to the method for lysozyme degradation of six-fold cross-linked carboxymethyl chitosan gel described in Example 4 of patent CN107179381A, lysozyme was used to degrade the mixed gel, and the degradation product was determined by the third method of potassium ferrocyanide reducing sugar determination method in GB/T5009.7-2016 "Determination of reducing sugar in food in national food safety standard". The specific results are shown in the following table. The articular injection gel of the present invention has a longer complete degradation time and a strong in vitro degradation ability.

Table 3 In vitro degradation time data

Stability testing

The mixed gel was filled into a prefilled syringe and sterilized with high-temperature steam at 121°C for 15 minutes. The appearance of the sterilized sample and the sample before sterilization were compared and the elastic modulus was tested. It was found that the intra-articular injection gel of the present invention had better stability. The specific results are shown in the following table.

Table 4 Stability data

Safety Testing

The samples were tested according to the in vitro cytotoxicity test method of GB/T16886.5-2017. The results showed that the cell survival rate of the intra-articular injection gel of the present invention was higher and safer, and the specific data are shown in the following table.

Table 5 Safety data

Swelling degree test

The swelling degree of the gel was detected according to the method in paragraphs 0154-0155 of CN110643057A, and the swelling rate of the gel was calculated according to the following formula: Swelling rate = (mass of the sample after swelling - sampling amount) × 100% / sampling amount. The results show that the swelling degree of the intra-articular injection gel of the present invention is lower, and the specific results are shown in the following table.

Table 6 Swelling degree data

Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

A method for preparing a gel for intra-articular injection, characterized in that: a polyethylene glycol active ester is mixed with cross-linked carboxymethyl chitosan to produce a cross-linking reaction to obtain a gel, the gel is crushed and mixed with a non-cross-linked carboxymethyl chitosan solution, sterilized, and cooled to obtain a gel for intra-articular injection; wherein the mass ratio of the polyethylene glycol active ester to the cross-linked carboxymethyl chitosan is 1:20-20:1.
The preparation method according to claim 1 is characterized in that the polyethylene glycol active ester comprises polyethylene glycol, and the molecular weight of the polyethylene glycol is 2000Da-10000Da and 2-8 arms.
The preparation method according to claim 1 is characterized in that the carboxymethyl chitosan used for cross-linking has a deacetylation degree of 30%-80%, a molecular weight of 500-1500 kDa, a substitution degree of 80%-200%, and a viscosity of 300-1000 mPa.s.
The preparation method according to claim 1 is characterized in that the non-cross-linked carboxymethyl chitosan solution has a deacetylation degree of less than 30%, a molecular weight of 500-1500 kDa, a substitution degree of 80%-200%, and a viscosity of 300-1000 mPa.s.
The preparation method according to claim 1 is characterized in that it specifically comprises the following steps:

S1, dissolving carboxymethyl chitosan for cross-linking in a phosphate buffer solution to obtain a mixed solution 1;

S2, adding polyethylene glycol active ester to the mixed solution 1 to carry out a cross-linking reaction, and letting it stand to obtain a gel;

S3, crushing the gel and adding non-cross-linked carboxymethyl chitosan solution to obtain mixed solution 2, sterilizing and cooling to obtain intra-articular injection gel.
The preparation method according to claim 5, characterized in that the mass fraction of carboxymethyl chitosan for cross-linking in the mixed solution 1 described in step S1 is 1-20%.
The preparation method according to claim 5 is characterized in that the conditions of the cross-linking reaction in step S2 are: temperature of 20-25°C, pH of 7-8.5, stirring speed of 60-90rpm, and reaction time of 10-30min.
The preparation method according to claim 5 is characterized in that the mass ratio of the gel in step S3 to the non-cross-linked carboxymethyl chitosan solution is 1:1-9:1.
The preparation method according to claim 5, characterized in that the concentration of the non-cross-linked carboxymethyl chitosan in the mixed solution 2 described in step S3 is 10-50 mg/ml.
A joint cavity injection gel, characterized in that it is prepared by the preparation method described in any one of claims 1 to 9, and the joint cavity injection gel also includes growth factors, analgesics and nutrients.