Injectable degradable conductive polymer precursor solution and application thereof

Abstract

The invention relates to a precursor solution of an injectable degradable conductive polymer. The precursor solution comprises a pyrrole monomer and / or a pyrrole carboxylic acid monomer, polystyrene sulfonate, hydrogen peroxide and a phosphate buffer salt solution, dissolving a pyrrole monomer and / or a pyrrole carboxylic acid monomer, polystyrene sulfonate and hydrogen peroxide in the phosphate buffer salt solution; the total concentration of the pyrrole monomer and / or the pyrrole carboxylic acid monomer in the precursor solution is 1-10mM, and the pH value of the precursor solution is 7.0-8.0. The precursor solution is prepared by selecting the raw materials with good biocompatibility and solubility, and the degradable conductive polymer with good biocompatibility can be synthesized in situ at a biological tissue interface by using the strongly positive expression of ROS and the reactivity of CAT enzyme (or HRP enzyme) at the damaged part under the action of endogenous polymerization conditions of the damaged tissue, so that the conductive polymer with good biocompatibility can be obtained. Tests show that the conductive polymer is good in conductivity, and the problem that an existing conductive polymer cannot be degraded in vivo is solved.

Description

Technical Field

[0001] The invention belongs to the field of tissue engineering materials, and in particular relates to an injectable degradable conductive polymer precursor solution and application thereof. Background Art

[0002] Conductive polymers (CPs) have been widely used in biomedical fields such as biosensors and tissue engineering, but their non-degradability limits their application scope. Common non-degradable CPs include polyaniline, polypyrrole, and poly(3,4-ethylenedioxythiophene) (PEDOT). In medical devices, such as the integration of sensors or stimulation electrodes, the optimization of mechanical properties and electrode impedance of these materials can improve tissue-electrode interaction, but their non-degradability limits their application in tissue engineering and regenerative medicine. Degradable CPs can be degraded in the short to medium term, avoiding the risks of inflammatory reactions and secondary surgery that may be caused by long-term retention, and are therefore particularly suitable for use in tissue engineering. In addition, existing degradable polymer CPs, such as conductive polymers blended with polylactide polylactic acid (PLA), polycaprolactone (PCL), etc., although they have certain degradability, these existing conductive polymers must be synthesized in vitro before they can be transplanted into the human body for human tissue engineering. The replaced part of the human engineering tissue must be precisely measured in size, and the transplantation process causes large wounds. At present, there are no reports on the relevant technology of synthesizing degradable conductive polymers in vivo by injecting liquid substances.

[0003] The traditional design trend of biomimetic interfaces is to permanently implant them into living tissues and organs, while conductive polymer materials with good biocompatibility and biodegradability are now shifting towards transient and regenerative biomimetic engineering. Therefore, biodegradable bioelectronic materials have important development and application potential and can be used to monitor physiological functions, temporarily restore disease symptoms, and seamlessly integrate regenerative interfaces from synthetic electronic devices to tissues by reducing inflammatory responses. Currently, there are many methods for preparing bioelectronic materials, however, degradable conductive polymers for biointerfaces are rarely discovered and applied. Therefore, selecting raw materials with good biocompatibility and degradability and optimizing the synthesis process are the key to achieving the synthesis and preparation of green degradable biointerface materials. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the above shortcomings and deficiencies of the prior art, the present invention provides an injectable precursor solution of a degradable conductive polymer and its application, wherein the precursor solution contains a biocompatible and soluble prepolymer monomer, and the precursor solution can undergo oxidative polymerization in a local strong positive active oxygen environment at the bone nerve / heart / brain damaged tissue to obtain a polymer, which has good conductivity and is biodegradable. The present invention realizes the in vivo synthesis of degradable conductive polymers and provides technical support for the non-invasive or minimally invasive implantation of degradable conductive polymers.

[0006] (II) Technical solution

[0007] In a first aspect, the present invention provides an injectable precursor solution of a degradable conductive polymer, comprising: pyrrole monomer and / or pyrrole carboxylic acid monomer, polystyrene sulfonate, hydrogen peroxide and phosphate buffered saline solution; the pyrrole monomer and / or pyrrole carboxylic acid monomer, polystyrene sulfonate and hydrogen peroxide are dissolved in the phosphate buffered saline solution; the total concentration of the pyrrole monomer and / or pyrrole carboxylic acid monomer in the precursor solution is 1-10 mM, and the pH of the precursor solution is 7.0-8.0.

[0008] According to a preferred embodiment of the present invention, the concentration of polystyrene sulfonate PSS in the precursor solution is 0.01-1 μM, such as 0.01-0.05 μM, 0.05-0.1 μM, 0.1-0.2 μM, 0.2-0.4 μM, 0.4-0.6 μM, 0.6-0.8 μM, 0.8-1.0 μM; more preferably 0.1 μM.

[0009] According to a preferred embodiment of the present invention, the concentration of hydrogen peroxide in the precursor solution is 0.1-10mM, such as 0.1-1mM, 1-2mM, 2-3mM, 3-4mM, 4-5mM, 5-6mM, 6-7mM, 7-8mM, 8-9mM or 9-10mM; more preferably 1mM.

[0010] According to a preferred embodiment of the present invention, pyrrole carboxylic acid monomers with better solubility are preferably used.

[0011] According to a preferred embodiment of the present invention, the total concentration of pyrrole monomer and / or pyrrole carboxylic acid monomer in the precursor solution is 1-2mM, 2-3mM, 3-4mM, 4-5mM, 5-6mM, 6-7mM, 7-8mM, 8-9mM or 9-10mM; more preferably 5mM.

[0012] According to a preferred embodiment of the present invention, the pH of the phosphate buffer solution may be 7.0-7.2, 7.2-7.4, 7.4-7.6, 7.6-7.8, 7.8-8.0, preferably 7.4; the concentration of the phosphate buffer solution may be 0.001-0.005M, 0.005-0.01M, 0.01-0.05M, 0.05-0.1M, more preferably 0.01M.

[0013] The pyrrole monomer or pyrrole carboxylic acid monomer in the precursor solution is a monomer for synthesizing a conductive polymer, and the polystyrene sulfonate PSS plays a doping role; hydrogen peroxide is used as a trigger to start oxidative polymerization in the precursor solution. Polystyrene sulfonate PSS is an anionic polymer. By doping some PSS, some properties of the polymer can be improved, such as: improving conductivity (making biopolymers better biosensors, biobatteries or biocompatible electronic component materials), enhancing the mechanical strength and toughness of the polymer (making it more durable and suitable for biomedical applications that need to withstand certain mechanical stresses, such as tissue engineering scaffolds or wearable medical devices), improving biocompatibility and degradability (reducing immune response or promoting biodegradation of materials), improving polymer performance (such as higher water solubility, better optical transparency or specific hydrophilic / hydrophobic properties), promoting cell adhesion (doping with PSS can improve the surface properties of biopolymers, making them more conducive to cell adhesion, proliferation and differentiation, thereby promoting tissue regeneration), etc.

[0014] When preparing the precursor solution, take an appropriate amount of pyrrole monomer (Py) or / and pyrrole carboxylic acid monomer (PyCA) and dissolve it in a phosphate buffer solution, then add polystyrene sulfonate solution (PSS) and hydrogen peroxide (H2O2) solution thereto, ultrasonically mix to form a transparent and fluid prepolymer solution, and then sterilize. The ultrasonic treatment time is 2-10s, 10-20s, 20-30s. In a preferred embodiment of the present invention, the ultrasonic time is 10s.

[0015] In a second aspect, the present invention provides a method for forming a functionalized degradable conductive polymer, which comprises: contacting the above-mentioned precursor solution with reactive oxygen species ROS and peroxidase, so that the precursor solution undergoes oxidative polymerization to generate a degradable conductive polymer; the peroxidase is catalase (CAT) and / or horseradish peroxidase (HRP).

[0016] In a third aspect, the present invention provides the use of the precursor solution of the above embodiment in preparing a regenerative biological interface of biological tissue or a conductive bioelectronic material.

[0017] The regenerative biointerface or conductive bioelectronic material has good conductivity and biodegradability, and can be degraded in an alkaline environment and under the catalysis of H2O2 solution.

[0018] (III) Beneficial effects

[0019] 1. The present invention prepares a precursor solution by selecting raw materials with good biocompatibility and solubility. The precursor solution can be polymerized to obtain a conductive polymer with good biocompatibility and degradability, which solves the problem that existing bioconductive materials are limited in tissue engineering and regenerative medicine applications due to their non-degradability.

[0020] 2. Since the precursor solution provided by the present invention can produce a polymerization reaction under the triggering of an endogenous oxidative environment to synthesize a functionalized degradable conductive polymer, and ROS and peroxidase (CAT or HRP) naturally occur at the damaged site of the sciatic nerve, heart or brain, the precursor solution provided by the present invention can achieve the repair of damaged tissues such as the sciatic nerve, heart or brain.

[0021] 3. The precursor solution of the present invention can be used to form biodegradable bioelectronic materials for monitoring physiological functions, temporarily restoring symptoms of damaging diseases, and seamlessly integrating synthetic electronic devices with the regenerative interface of biological tissues by reducing inflammatory responses, so as to achieve the purpose of monitoring, treating or enhancing biological functions. Therefore, the precursor solution can exert application potential in the fields of regenerative medicine, neural interfaces, wearable medical devices and biosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The toxicity of different concentrations of Py or PyCA monomers on DRG neuronal cells.

[0023] Figure 2 Comparison of DHE index of local reactive oxygen species produced in normal and injured areas.

[0024] Figure 3 The polymer formation was observed 24 hours after the injection of Py and PyCA prepolymer solutions and PBS solutions at the sciatic nerve injury site in model rats.

[0025] Figure 4 Schematic diagram of the operation of electrochemical testing of PPy and PPyCA CMVs produced in Example 4 in Example 5.

[0026] Figure 5 Electrochemical test results of PPy and PPyCA CMVs generated at the sciatic nerve injury site in model rats.

[0027] Figure 6 The degradation of PPy and PPyCA under different H2O2 concentrations and pH conditions from 0 to 30 days. DETAILED DESCRIPTION

[0028] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below through specific implementation modes in conjunction with the accompanying drawings.

[0029] The present invention provides an injectable precursor solution of a degradable conductive polymer. The preparation method thereof is as follows: an appropriate amount of pyrrole monomer (Py) or / and pyrrole carboxylic acid monomer (PyCA) is weighed and dissolved in a phosphate buffer solution, and then a polystyrene sulfonate solution (PSS) and a hydrogen peroxide solution (H2O2) are added thereto, and after ultrasonic mixing, a fluid and transparent prepolymer monomer solution is formed. After sterilization, the prepolymer solution can be delivered to a damaged tissue in an animal body through a syringe or the like, and the precursor solution is oxidatively polymerized at the damaged tissue site through the triggering effect of endogenous synthetic conditions (active oxygen species ROS, enzyme reactive CAT, etc.) formed at the damaged tissue site, so as to obtain a degradable polymer with good biocompatibility and conductivity.

[0030] Among them, pyrrole monomer (Py) and / or pyrrole carboxylic acid monomer (PyCA) have good solubility and biocompatibility and can be used as monomers of conductive polymers. In some preferred embodiments, pyrrole carboxylic acid monomer PyCA with better solubility is more preferably used. The structural formula of pyrrole carboxylic acid monomer PyCA can be as follows:

[0031] The concentration of pyrrole monomer (Py) or / and pyrrole carboxylic acid monomer (PyCA) in the precursor solution may be 1-2mM, 2-3mM, 3-4mM, 4-5mM, 5-6mM, 6-7mM, 7-8mM, 8-9mM or 9-10mM. In some preferred embodiments, the total concentration of Py or / and PyCA is 5mM.

[0032] The pH of the phosphate buffer solution may be 7.0-7.2, 7.2-7.4, 7.4-7.6, 7.6-7.8, 7.8-8.0, and more preferably 7.4; the concentration of the phosphate buffer solution may be 0.001-0.005M, 0.005-0.01M, 0.01-0.05M or 0.05-0.1M, and more preferably 0.01M.

[0033] The ultrasonic time may be 2-10s, 10-20s and 20-30s, preferably 10s.

[0034] The concentration of polystyrene sulfonate PSS in the precursor solution is 0.01-1 μM, such as 0.01-0.05 μM, 0.05-0.1 μM, 0.1-0.2 μM, 0.2-0.4 μM, 0.4-0.6 μM, 0.6-0.8 μM, 0.8-1.0 μM; more preferably 0.1 μM.

[0035] The concentration of hydrogen peroxide in the precursor solution is 0.1-10 mM, such as 0.1-1 mM, 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM or 9-10 mM; more preferably 1 mM.

[0036] The precursor solution is injected into the sciatic nerve, heart or brain damaged tissues, and the pyrrole monomer or / and pyrrole carboxylic acid monomer (PyCA) in the precursor solution are polymerized through the catalytic action of the endogenous synthetic conditions of these damaged tissues (active oxygen ROS, enzyme reactive CAT or HRP, etc.), and a biodegradable and biocompatible conductive polymer is formed at the damaged tissue interface. The reaction process is shown as follows:

[0037]

[0038] Wherein, R is H or COOH; when R is H, it is a pyrrole monomer, recorded as Py; when R is COOH, it is a pyrrole carboxylic acid monomer, recorded as PyCA.

[0039] The present invention will be described in more detail below with reference to specific embodiments.

[0040] Example 1

[0041] This example investigates the biocompatibility of pyrrole monomer (Py) and pyrrole carboxylic acid monomer (PyCA), and the investigation method is as follows: primary DRG neuron cells were cultured for 5 days, and then incubated with Py or PyCA monomer (0.5mM to 10mM, PBS solution) for 24h. After washing three times with PBS solution (pH 7.4), the cells were stained with a Live / Dead staining kit and observed under a fluorescence microscope.

[0042] The experimental results are as follows Figure 1 As shown, no decrease in cell viability was observed with Py or PyCA at Py or PyCA monomer concentrations up to 1 mM (e.g. Figure 1 A, B), indicating that at low concentrations (1 mM), Py or PyCA were non-toxic to DRG neuronal cells; when the PyCA monomer concentration was as high as 5 mM, only a slight reduction in cell viability was observed, and at high concentrations (2-5 mM), PyCA showed significantly better biocompatibility than Py (see Figure 1 C, D).

[0043] Example 2

[0044] In this example, SD rats were used to establish a sciatic nerve injury model, and the modeling method was as follows: 90 adult male SD rats (weight distribution ranged from 200 to 230 g) were randomly divided into three groups. Under anesthesia, the sciatic nerve of the left leg was exposed, and fine forceps were used to squeeze for 30 seconds to cause sciatic nerve injury.

[0045] ROS detection was performed on the injured sciatic nerve by using a DHE probe to detect ROS levels. Freshly collected tissues were immediately sliced ​​on a freezer and then incubated with DHE (dihydroethidium oxide, 100 μM, dissolved in PBS) at 37°C in the dark for 30 min, and images of the samples were captured using a fluorescence microscope.

[0046] like Figure 2 As shown in , when nerves are injured or damaged, local tissues respond by recruiting macrophages and undergoing Wallerian degeneration, resulting in complex changes in the chemical and biological environment. These dynamic changes occur throughout the repair process. DHE is a typical indicator of local reactive oxygen species (ROS) production at the site of injury (e.g. Figure 2 As part of the feedback mechanism for ROS removal, antioxidant enzymes such as catalase (CAT) are overexpressed. In the next experiment, it is expected that the environment of the damaged site will be used as a polymerization trigger to polymerize the prepolymer solution at the site of tissue damage to obtain polymers.

[0047] Example 3

[0048] This embodiment relates to an injectable precursor solution of a degradable conductive polymer, the preparation method of which is as follows: dissolving pyrrole monomer (Py) or pyrrole carboxylic acid monomer (PyCA) in a 0.01 mol / L PBS solution with a pH of 7.4, adding polystyrene sulfonate PSS and hydrogen peroxide, mixing under ultrasonication (power 400W, frequency 50kHz) for 10s to form a transparent prepolymer solution, sterilizing the prepolymer solution for standby use, and obtaining a Py prepolymer solution and a PyCA prepolymer solution, respectively. The concentration of Py or PyCA in the prepolymer solution is 5mM, the concentration of PSS is 0.1μM, and the concentration of H2O2 is 1mM.

[0049] Example 4

[0050] In this example, the Py prepolymer solution and the PyCA prepolymer solution prepared in Example 3 were injected into the sciatic nerve injury area of ​​the model rat constructed in Example 2 using a syringe. The specific method is: using a 0.3×8 mm 2 The Py prepolymer solution and the PyCA prepolymer solution were injected into the sciatic nerve injury site of the model rats as the experimental group, and the same amount of PBS solution was injected into the sciatic nerve injury site of the model rats as the control group.

[0051] Each group of model rats was individually caged and fed with a 12-hour light / dark cycle. During the feeding period, each model rat had free access to food and water. After 24 hours of feeding, the sciatic nerve injury site was cut open and scanning electron microscopy (SEM) was used to find that in the experimental group, the sciatic nerve injury site of the model rats was polymerized to produce polymers PPy and PPyCA. Their microscopic morphology can be found in Figure 3 In addition, during the experiment, the Py prepolymer solution and the PyCA prepolymer solution were injected into the sciatic nerve area of ​​healthy rats. After 24 hours of feeding, no polymer was observed by SEM scanning.

[0052] Example 5

[0053] In this example, the PPy and PPyCA CMVs (conductive microvesicles) generated at the sciatic nerve injury site of the model rat in Example 4 were subjected to electrochemical testing. The testing method was as follows: the sciatic nerve was taken, cut into samples of appropriate length and thickness, the samples were placed on a homemade microelectrode chip, and the test was performed on an electrochemical workstation. The width and electrode spacing of the gold electrode were each 100 μm. The CV curve scanning rate was 10 mV -1 , the potential window is 0-2.0V. 0 -10 5 Hz frequency and 10mV amplitude (see the test diagram for details) Figure 4 ).

[0054] The cyclic voltammetry curves show that compared with PPy, PPyCA is more susceptible to redox processes, thereby promoting electron transfer ( Figure 5 A). Electrochemical impedance spectroscopy showed that the conductivity of PPyCA CMVs in tissues was significantly higher than that of PPy CMVs. The calculated charge transfer impedance of Ppy CMVs was 2869Ω, while that of PPyCA CMVs was only 16.7Ω ( Figure 5 B) and the impedance of PPyCA CMVs 10 4 Up to 10 5 Hz, compared to the PPy CMVs which are relatively stable in the range of 10 3 Up to 10 4 Hz impedance increase ( Figure 5 A similar trend can be found in the phase diagram, where the PPy frequency range is smaller when the phase angle is close to 0° ( Figure 5 D). Therefore, compared with CMVs formed by Py polymerization, CMVs formed by PyCA polymerization have better biocompatibility, tissue affinity and electrical conductivity, and can provide a more seamless interface for intercellular signaling.

[0055] Example 6

[0056] This example investigates the degradability of the polymer PPyCA of PyCA, and the experimental method is as follows:

[0057] With Fe 2+ The oxidative environment was provided by ammonium persulfate to oxidize and polymerize the PyCA monomer solution to synthesize PPyCA. 600 μg of PPyCA was dispersed in PBS solutions with different pH values ​​(7.4 and 6.5), and different concentrations of H2O2 (0, 1 mM, and 5 mM) were added to test the degradation time of PPyCA. Low concentrations of H2O2 and pH values ​​(7.4 and 6.5) simulated the oxidative environment in animals.

[0058] The test results are as follows Figure 6 As shown in the figure, in the absence of H2O2, the degradation of PPyCA in PBS solution is not obvious. When H2O2 is added, PPyCA gradually degrades, and the higher the H2O2 concentration, the more obvious the degradation of PPyCA. When the H2O2 concentration reaches 5mM, at pH 7.4, the PPyCA solution changes from brown-green on the first day to light yellow on the 5th day, and almost completely becomes a clear solution on the 30th day, at which time PPyCA is completely degraded. When the H2O2 concentration is 1mM, PPyCA degrades more slowly. In addition, in an environment of pH 6.5, PPyCA degradation is also slower than when the pH is 7.4.

[0059] like Figure 6 As shown in the figure, whether the pH is 6.5 or 7.4, PPy starts to degrade from the 15th day. With the extension of degradation time and the increase of H2O2 concentration, it is obvious that the degradation rate of PPy is always slower than that of PPyCA. It can be seen that PPyCA is easier to degrade, and compared with PPyCA, PPy can be degraded under strong oxidative conditions.

[0060] In summary, the precursor solution provided by the present invention can synthesize in situ a degradable and biocompatible conductive polymer at the interface of biological tissue under the endogenous polymerization conditions of damaged tissue by utilizing the strong positive expression of ROS and the reactivity of CAT enzyme (or HRP enzyme) at the damaged site. The conductive polymer has good conductivity after testing, which overcomes the problem that existing conductive polymers such as PEDOT cannot be degraded in vivo.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An injectable precursor solution of a degradable conductive polymer, characterized in that: These include: Pyrrole Monomer and / or pyrrole carboxylic acid monomer, polystyrene sulfonate, hydrogen peroxide and phosphate buffered saline solution; pyrrole monomer and / or pyrrole carboxylic acid monomer, polystyrene sulfonate, hydrogen peroxide are dissolved in phosphate buffered saline solution; the total concentration of pyrrole monomer and / or pyrrole carboxylic acid monomer in the precursor solution is 1-10mM, and the pH of the precursor solution is 7.0-8.

0.

2. The precursor solution according to claim 1, characterized in that The concentration of polystyrene sulfonate PSS in the precursor solution is 0.01-1 μM.

3. The precursor solution according to claim 1, characterized in that: The concentration of hydrogen peroxide in the precursor solution is 0.1-10 mM.

4. The precursor solution according to claim 1, characterized in that: The units in the precursor solution are pyrrole carboxylic acid monomers.

5. The precursor solution according to claim 1, characterized in that: The total concentration of pyrrole monomer and / or pyrrole carboxylic acid monomer in the precursor solution is 1-2 mM, 2-3 mM, 3-4 mM, 4-5 mM, 5-6 mM, 6-7 mM, 7-8 mM, 8-9 mM or 9-10 mM.

6. The precursor solution according to claim 1, characterized in that: The pH of the phosphate buffer solution may be 7.0-7.2, 7.2-7.4, 7.4-7.6, 7.6-7.8, or 7.8-8.

0.

7. A method for forming a functionalized degradable conductive polymer, comprising: contacting the precursor solution according to any one of claims 1 to 6 with reactive oxygen species (ROS) and peroxidase, so that the precursor solution undergoes oxidative polymerization to generate a degradable conductive polymer; The peroxidase is catalase CAT and / or horseradish peroxidase HRP.

8. Use of the injectable precursor solution for in vivo synthesis of functionalized degradable conductive polymers according to any one of claims 1 to 6 in the preparation of regenerative biological interfaces of biological tissues or conductive bioelectronic materials.

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