An injectable degradable conductive polymer precursor solution and its use

Abstract

This invention relates to a precursor solution for an injectable, biodegradable conductive polymer, comprising: pyrrole monomers and / or pyrrole carboxylic acid monomers, polystyrene sulfonate, hydrogen peroxide, and a phosphate buffer solution; the pyrrole monomers and / or pyrrole carboxylic acid monomers, polystyrene sulfonate, and hydrogen peroxide are dissolved in the phosphate buffer solution; the total concentration of the pyrrole monomers and / or pyrrole carboxylic acid monomers in the precursor solution is 1-10 mM, and the pH of the precursor solution is 7.0-8.0. This invention uses raw materials with good biocompatibility and solubility to prepare the precursor solution. Under endogenous polymerization conditions in damaged tissue, utilizing the strong ROS-positive expression and the reactivity of CAT enzymes (or HRP enzymes) at the damaged site, a biodegradable and biocompatible conductive polymer can be synthesized in situ at the biological tissue interface. Testing has shown that this conductive polymer exhibits good conductivity, overcoming the problem of existing conductive polymers being non-degradable in vivo.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering materials, specifically relating to an injectable precursor solution of a biodegradable conductive polymer and its application. Background Technology

[0002] Conductive polymers (CPs) have been widely used in biomedical fields such as biosensors and tissue engineering; however, their non-degradability limits their applications. 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 mechanical properties and electrode impedance optimization of these materials can improve tissue-electrode interactions, but their non-degradability limits their application in tissue engineering and regenerative medicine. Degradable CPs can degrade in the short to medium term, avoiding the risks of inflammatory reactions and secondary surgeries that may be caused by long-term placement, making them particularly suitable for tissue engineering. Furthermore, existing degradable polymer CPs, such as conductive polymers blended with polylactic acid (PLA) and polycaprolactone (PCL), while possessing some degradability, must be synthesized in vitro before being transplanted into the human body for use in human tissue engineering. The replaced human tissue must undergo precise dimensional measurements, and the transplantation process involves a large wound. Currently, there are no reports of technologies for synthesizing degradable conductive polymers in vivo by injecting liquid substances.

[0003] Traditional biomimetic interface design trends focus on permanent implantation into living tissues and organs, while biocompatible and biodegradable conductive polymer materials are now shifting towards transient and regenerative biomimetic engineering. Therefore, biodegradable bioelectronic materials hold significant development and application potential, enabling the monitoring of physiological functions, temporary relief of disease symptoms, and seamless integration from synthetic electronics to tissue regeneration interfaces by reducing inflammatory responses. While various methods exist for preparing bioelectronic materials, biodegradable conductive polymers for biointerface applications are rarely discovered and utilized. Therefore, selecting biocompatible and biodegradable raw materials and optimizing synthesis processes are crucial for the successful synthesis and preparation of green and biodegradable biointerface materials. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides an injectable precursor solution of a biodegradable conductive polymer and its application. The precursor solution contains a biocompatible and soluble prepolymer monomer. The precursor solution undergoes oxidative polymerization in a localized, strongly positive reactive oxygen species environment at the site of bone, nerve, heart, or brain injury tissue to obtain the polymer. This polymer exhibits good conductivity and is biodegradable. The present invention realizes the in vivo synthesis of biodegradable conductive polymers, providing technical support for the non-invasive or minimally invasive implantation of biodegradable conductive polymers.

[0006] (II) Technical Solution

[0007] In a first aspect, the present invention provides an injectable precursor solution for a biodegradable conductive polymer, comprising: pyrrole monomer and / or pyrrole carboxylic acid monomer, polystyrene sulfonate, hydrogen peroxide, and a phosphate buffer solution; wherein the pyrrole monomer and / or pyrrole carboxylic acid monomer, polystyrene sulfonate, and hydrogen peroxide are dissolved in the phosphate buffer 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, or 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 0.1-10 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.

[0010] According to a preferred embodiment of the present invention, a pyrrole carboxylic acid monomer with better solubility is 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 1-2 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 5 mM.

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

[0013] The pyrrole monomers or pyrrole carboxylic acid monomers in the precursor solution are the monomers for synthesizing the conductive polymer, while polystyrene sulfonate (PSS) acts as a dopant. Hydrogen peroxide in the precursor solution acts as a trigger for initiating oxidative polymerization. PSS is an anionic polymer; by doping with PSS, several properties of the polymer can be improved, such as: increasing conductivity (making biopolymers better materials for biosensors, biobatteries, or biocompatible electronic components), enhancing the mechanical strength and toughness of the polymer (making it more durable and suitable for biomedical applications requiring mechanical stress, such as tissue engineering scaffolds or wearable medical devices), improving biocompatibility and degradability (reducing immune responses or promoting material biodegradation), improving polymer properties (such as higher water solubility, better optical transparency, or specific hydrophilic / hydrophobic properties), and promoting cell adhesion (PSS doping can improve the surface properties of biopolymers, making them more conducive to cell adhesion, proliferation, and differentiation, thereby promoting tissue regeneration).

[0014] In preparing the precursor solution, an appropriate amount of pyrrole monomer (Py) and / or pyrrole carboxylic acid monomer (PyCA) is dissolved in a phosphate buffer solution. Then, a polystyrene sulfonate solution (PSS) and hydrogen peroxide (H2O2) solution are added, and the mixture is ultrasonically mixed to form a transparent and fluid prepolymer solution, which is then sterilized. The ultrasonic treatment time is 2-10 s, 10-20 s, or 20-30 s. In a preferred embodiment of the invention, the ultrasonic treatment time is 10 s.

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

[0016] Thirdly, the present invention provides the application of the precursor solution of the above embodiments in the preparation of regenerative biological interfaces or conductive bioelectronic materials for biological tissues.

[0017] The regenerated biointerface or conductive bioelectronic material has good conductivity and biodegradability, and can be catalytically degraded in alkaline environments and H2O2 solutions.

[0018] (III) Beneficial Effects

[0019] 1. This 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 the application of tissue engineering and regenerative medicine because they cannot be degraded.

[0020] 2. Since the precursor solution provided by this invention can generate a polymerization reaction to synthesize functionalized degradable conductive polymers under the trigger of an endogenous oxidative environment, and ROS and peroxidase (CAT or HRP) will naturally occur at the site of injury to the sciatic nerve, heart or brain, the precursor solution provided by this 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 damaged 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 has the potential to be applied in the fields of regenerative medicine, neural interfaces, wearable medical devices and biosensors. Attached Figure Description

[0022] Figure 1 The toxicity of different concentrations of Py or PyCA monomers to DRG neurons was investigated.

[0023] Figure 2 A comparison of DHE indexes for local reactive oxygen species production in normal and damaged areas.

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

[0025] Figure 4 This is a schematic diagram of the electrochemical testing of PPy and PPyCA CMVs generated 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 concentrations of H2O2 and pH conditions from 0 to 30 days is shown. Detailed Implementation

[0028] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0029] This invention provides an injectable precursor solution for a biodegradable conductive polymer, prepared as follows: Appropriate amounts of pyrrole monomer (Py) and / or pyrrole carboxylic acid monomer (PyCA) are dissolved in a phosphate buffer solution. Then, a polystyrene sulfonate solution (PSS) and hydrogen peroxide solution (H2O2) are added. After ultrasonic mixing, a fluid and transparent prepolymer monomer solution is formed. After sterilization, this solution can be delivered to damaged tissues in an animal via a syringe. Triggered by endogenous synthesis conditions (reactive oxygen species (ROS), enzyme reactivity (CAT), etc.) at the tissue damage site, the precursor solution undergoes oxidative polymerization at the tissue damage site, yielding a biodegradable 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 both be used as monomers for conductive polymers. In some preferred embodiments, the pyrrole carboxylic acid monomer PyCA, which has better solubility, is preferred. The structural formula of the pyrrole carboxylic acid monomer PyCA can be as follows:

[0031]

[0032] The concentration of pyrrole monomer (Py) and / or pyrrole carboxylic acid monomer (PyCA) in the precursor solution can be 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. In some preferred embodiments, the total concentration of Py and / or PyCA is 5 mM.

[0033] The phosphate buffer solution has a pH of 7.0-7.2, 7.2-7.4, 7.4-7.6, 7.6-7.8, or 7.8-8.0, more preferably 7.4; and the concentration of the phosphate buffer solution can be 0.001-0.005M, 0.005-0.01M, 0.01-0.05M, or 0.05-0.1M, more preferably 0.01M.

[0034] The ultrasound duration can be 2-10s, 10-20s, or 20-30s, with 10s being the preferred duration.

[0035] 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, or 0.8-1.0 μM; more preferably 0.1 μM.

[0036] 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.

[0037] The aforementioned precursor solution is injected into the sciatic nerve, heart, or brain injury tissue. Through the catalytic action of endogenous synthetic conditions in these tissues (reactive oxygen species (ROS), enzyme-reactive CAT, or HRP, etc.), the pyrrole monomers and / or pyrrole carboxylic acid monomers (PyCA) in the precursor solution polymerize, forming a biodegradable and biocompatible conductive polymer at the damaged tissue interface. The reaction process is described below:

[0038]

[0039] Where R is H or COOH; when R is H, it is a pyrrole monomer, denoted as Py; when R is COOH, it is a pyrrole carboxylic acid monomer, denoted as PyCA.

[0040] The present invention will now be described in more detail with reference to specific embodiments.

[0041] Example 1

[0042] This study investigated the biocompatibility of pyrrole monomers (Py) and pyrrole carboxylic acid monomers (PyCA). The method involved culturing primary DRG neurons for 5 days, followed by incubation with either Py or PyCA monomers (0.5 mM to 10 mM, PBS solution) for 24 hours. 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.

[0043] Experimental results are as follows Figure 1 As shown, at monomeric concentrations of Py or PyCA up to 1 mM, no decrease in cell viability was observed due to Py or PyCA (e.g., Figure 1 Cases A and B indicate that at low concentrations (1 mM), neither Py nor PyCA is toxic to DRG neurons; at monomeric PyCA concentrations as high as 5 mM, only a slight decrease in cell viability was observed, and at high concentrations (2-5 mM), PyCA showed significantly better biocompatibility than Py (see [reference]). Figure 1 (C, D).

[0044] Example 2

[0045] In this embodiment, SD rats were used to establish a sciatic nerve injury model. The modeling method was as follows: 90 adult male SD rats (weighing between 200-230g) were randomly divided into three groups. Under anesthesia, the sciatic nerve of the left leg was exposed, and it was squeezed with thin forceps for 30 seconds to cause sciatic nerve injury.

[0046] ROS levels at the sciatic nerve injury site were measured using a DHE probe. Freshly collected tissue was immediately sectioned on a cryostat and then incubated with DHE (ethidium dihydrogen phosphate, 100 μM, dissolved in PBS) in the dark at 37°C for 30 min. Images of the samples were captured using a fluorescence microscope.

[0047] like Figure 2 As shown, 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 entire repair process. DHE is a typical indicator of local reactive oxygen species (ROS) production at the injury site (e.g., DHE). Figure 2 As part of the feedback mechanism for ROS scavenging, antioxidant enzymes such as catalase (CAT) are overexpressed. In the following experiments, it is hoped that the environment at the injury site will be used as a polymerization triggering condition, allowing the prepolymer solution to polymerize at the tissue injury site to obtain the polymer.

[0048] Example 3

[0049] This embodiment relates to a precursor solution for an injectable, biodegradable conductive polymer, prepared as follows: pyrrole monomer (Py) or pyrrole carboxylic acid monomer (PyCA) is dissolved in a 0.01 mol / L PBS solution at pH 7.4. Polystyrene sulfonate (PSS) and hydrogen peroxide are added, and the mixture is ultrasonically mixed (400 W, 50 kHz) for 10 seconds to form a transparent prepolymer solution. The prepolymer solution is sterilized and then used for later use, yielding Py prepolymer solution and PyCA prepolymer solution respectively. The concentrations of Py or PyCA in the prepolymer solution are 5 mM, the PSS concentration is 0.1 μM, and the H2O2 concentration is 1 mM.

[0050] Example 4

[0051] In this embodiment, the Py prepolymer solution and PyCA prepolymer solution prepared in Example 3 were injected into the sciatic nerve injury area of ​​the rat model constructed in Example 2 using a syringe. Specifically, using a 0.3×8mm² insulin injection needle, the Py prepolymer solution and PyCA prepolymer solution were injected into the sciatic nerve injury site of the rat model as the experimental group, while an equal volume of PBS solution was injected into the sciatic nerve injury site of the rat model as the control group.

[0052] Each group of model rats was individually housed in cages and fed under a 12-hour light / dark cycle. During feeding, the rats had free access to food and water. After 24 hours of feeding, the sciatic nerve injury site was incised, and scanning electron microscopy (SEM) revealed that in the experimental group, polymers PPy and PPyCA were polymerized at the sciatic nerve injury site. Their microstructures are shown in [reference needed]. Figure 3 In contrast, no polymer formation was observed in the control group. Furthermore, during the experiment, both Py prepolymer solution and PyCA prepolymer solution were injected into the sciatic nerve region of healthy rats. After 24 hours of feeding, no polymer formation was observed by SEM scanning.

[0053] Example 5

[0054] This embodiment describes the electrochemical testing of PPy and PPyCA CMVs (conductive microvesicles) generated at the sciatic nerve injury site in the model rats of Example 4. The testing method involved harvesting the sciatic nerve, cutting it into samples of appropriate length and thickness, placing the samples on a self-made microelectrode chip, and performing the test on an electrochemical workstation. The width of the gold electrode and the electrode spacing were both 100 μm. The CV curve scan rate was 10 mV. −1 The potential window is 0-2.0V. At 10 0 -10 5 Tests were conducted at Hz frequency and 10mV amplitude (see test diagram). Figure 4 ).

[0055] Cyclic voltammetry curves show that, compared to PPy, PPyCA undergoes redox processes more readily, thereby promoting electron transfer ( Figure 5 Electrochemical impedance spectroscopy showed that the conductivity of PPyCA CMVs in tissue was significantly higher than that of PPy CMVs. The calculated results were: the charge transfer impedance of PPy CMVs was 2869 Ω, while that of PPyCA CMVs was only 16.7 Ω. Figure 5 (B). Therefore, compared with CMVs formed by Py polymerization, CMVs formed by PyCA polymerization have better biocompatibility, tissue affinity and conductivity, and can provide a more seamless interface for intercellular signal transduction.

[0056] Example 6

[0057] This embodiment examines the degradability of the PyCA polymer PPyCA. The experimental method is as follows:

[0058] Fe 2+Ammonium persulfate provides an oxidizing environment, enabling the oxidative polymerization of PyCA monomer solution to synthesize PPyCA. 600 μg of PPyCA was dispersed in PBS solutions at 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.

[0059] Test results are as follows Figure 6 As shown, PPyCA degradation in PBS solution is not significant in the absence of H2O2. With the addition of H2O2, PPyCA gradually degrades, and the higher the H2O2 concentration, the more pronounced the degradation. When the H2O2 concentration reaches 5 mM, at pH 7.4, the PPyCA solution changes from a brownish-green color on day 1 to a light yellow color on day 5, and becomes almost completely clear on day 30, at which point PPyCA is completely degraded. Degradation is even slower at an H2O2 concentration of 1 mM. Furthermore, PPyCA degradation is also slower at pH 6.5 compared to pH 7.4.

[0060] like Figure 6 As shown, regardless of whether the pH is 6.5 or 7.4, PPy only begins to degrade after 15 days. With increasing degradation time and H2O2 concentration, the degradation rate of PPy is consistently slower than that of PPyCA. This indicates that PPyCA is more easily degraded, while PPy requires strong oxidizing conditions to degrade.

[0061] In summary, the precursor solution provided by this invention can, under the action of endogenous polymerization conditions in damaged tissue, utilize the strong positive expression of ROS and the reactivity of CAT enzyme (or HRP enzyme) at the damaged site to synthesize a degradable and biocompatible conductive polymer in situ at the biological tissue interface. The conductive polymer has been tested and found to have good conductivity, overcoming the problem that existing conductive polymers such as PEDOT cannot be degraded in vivo.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 for in vivo synthesis of a functionalized degradable conductive polymer, characterized in that, It is prepared from pyrrole monomers and / or pyrrole carboxylic acid monomers, polystyrene sulfonate, hydrogen peroxide, and phosphate buffer solution; pyrrole monomers and / or pyrrole carboxylic acid monomers, polystyrene sulfonate, and hydrogen peroxide are dissolved in phosphate buffer solution; the total concentration of pyrrole monomers and / or pyrrole carboxylic acid monomers in the precursor solution is 1-10 mM, and the pH of the precursor solution is 7.0-8.0; the concentration of polystyrene sulfonate PSS in the precursor solution is 0.01-1 μM; the concentration of hydrogen peroxide in the precursor solution is 0.1-10 mM; the provided precursor solution can undergo polymerization reaction triggered by an endogenous oxidative environment to synthesize functionalized degradable conductive polymers.

2. The precursor solution of claim 1, wherein, The monomer in the precursor solution is a pyrrolic acid monomer.

3. The precursor solution of claim 1, wherein, 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.

4. The precursor solution of claim 1, wherein, Phosphate buffer solutions can have pH values ​​of 7.0-7.2, 7.2-7.4, 7.4-7.6, 7.6-7.8, or 7.8-8.

0.

5. The use of the precursor solution of the injectable in vivo synthetic functionalized degradable conductive polymer according to any one of claims 1-4 in the preparation of materials for repairing sciatic nerve, heart or brain injury tissues.

Images