ROS / pH dual-responsive drug-loaded hydrogel and preparation method and application thereof
By preparing a ROS/pH dual-responsive drug-loaded gel and combining it with hyperbaric oxygen therapy, the problems of low drug delivery efficiency, poor targeting, and single treatment strategy in neuroprotection and functional recovery after SCII were solved. Precise drug release and multi-mechanism synergistic effects were achieved, significantly improving the treatment effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JILIN UNIVERSITY
- Filing Date
- 2026-01-21
- Publication Date
- 2026-05-19
AI Technical Summary
Existing treatments for neuroprotection and functional recovery after SCII suffer from problems such as low drug delivery efficiency, poor targeting, unclear treatment time window, single treatment strategy, failure to effectively utilize the dynamic changes of the pathological microenvironment, and lack of multi-mechanism synergistic effects.
A ROS/pH dual-responsive drug-loaded gel was developed by preparing a ROS-responsive monomer and a pH-responsive copolymer containing thioether groups, combined with a miR-22-3p antisense oligonucleotide inhibitor and neurotrophic factor-3, to achieve precise drug release to the spinal cord injury area and form a synergistic effect with hyperbaric oxygen therapy.
This approach achieves a high degree of matching between drug release and disease progression, improving the precision and efficiency of treatment. It is significantly superior to single-therapy approaches and forms a comprehensive treatment system with a clear mechanism and controllable timing.
Abstract
Description
Technical Field
[0001] This invention relates to the field of medical materials technology, and in particular to a ROS / pH dual-responsive drug-loaded gel, its preparation method, and its application. Background Technology
[0002] Spinal cord ischemia-reperfusion injury (SCII) is a common and serious complication following thoracic and abdominal aortic aneurysm repair surgery, spinal trauma surgery, and severe hypotensive shock. Its pathological process is complex. The initial ischemia leads to neuronal energy metabolism failure, while the reperfusion process after the restoration of blood supply triggers a series of more severe secondary injuries, including calcium overload, excitatory amino acid toxicity, inflammatory cell infiltration, and the explosive production of large amounts of reactive oxygen species (ROS). This injury often leads to delayed neurological deficits, placing a heavy burden on patients, their families, and society.
[0003] Currently, clinical treatment options for neuroprotection and functional recovery after SCII are very limited, and the results are unsatisfactory. Existing strategies mainly face the following key technical bottlenecks:
[0004] First, drug delivery efficiency is low and targeting is poor. To maintain effective therapeutic concentrations, repeated invasive administration is often required, which not only increases the risk of surgical trauma and central nervous system infection, but also makes it impossible to match the temporal and spatial distribution of drugs with the complex disease process.
[0005] Secondly, the mechanism of hyperbaric oxygen therapy is unclear, and the therapeutic window is unknown; the optimal treatment time window has never been clearly defined. Clinical treatment is often based on experience, lacking precise guidance, leading to unstable efficacy and significant limitations due to its unpredictable and arbitrary nature.
[0006] Third, the microscopic pathological environment has not been effectively utilized. The SCII region is a dynamic and unique pathological microenvironment, with two most prominent features being high levels of reactive oxygen species (ROS) and acidic pH due to anaerobic metabolism. These should be ideal signals for targeted therapy. However, traditional biomaterials cannot sense and respond to these pathological signals in this environment and can only passively degrade. This causes the drug release curve to become disconnected from the disease development stage, making precision medicine impossible.
[0007] Fourth, treatment strategies are often limited and lack synergistic effects. Existing research primarily focuses on single therapies, either simple drug intervention or simple physical therapy. A major unresolved challenge is how to deeply integrate molecularly targeted therapies that inhibit apoptosis, neurotrophic support that promotes nerve regeneration, and physical therapy that improves the local microenvironment to form a comprehensive treatment system with multi-mechanism synergy and time-matched approach.
[0008] In summary, there is an urgent need in this field for a novel integrated treatment system and method that can intelligently respond to the pathological microenvironment of spinal cord injury, achieve precise and controllable drug release, and produce a temporal synergistic effect with hyperbaric oxygen physical therapy, in order to break through the current treatment bottleneck of delayed paralysis after SCII. Summary of the Invention
[0009] To address the aforementioned technical problems, this invention provides a ROS / pH dual-responsive drug-loaded gel, its preparation method, and its applications.
[0010] According to one aspect of the present invention, a method for preparing a ROS / pH dual-responsive drug-loaded gel is provided, comprising: Step one, preparing intermediate 2-(methylthio)ethanol: dissolving sodium hydroxide in anhydrous ethanol, cooling to 0-5°C in an ice bath, adding 2-mercaptoethanol and mixing thoroughly, then adding iodomethane ethanol solution, reacting at room temperature, removing anhydrous ethanol by rotary evaporation under reduced pressure, washing with sodium chloride solution and drying with anhydrous Na2SO4 to obtain intermediate 2-(methylthio)ethanol; Step two, preparing the ROS-responsive monomer 2-(methylthio)ethyl methacrylate containing a thioether group: dissolving the intermediate 2-(methylthio)ethanol obtained in step one in dichloromethane, adding... Triethylamine and 4-dimethylaminopyridine were added, followed by the addition of methacryloyl chloride in an ice bath. The mixture was then heated to room temperature and reacted in the dark. Afterward, it was washed successively with hydrochloric acid, NaHCO3 solution, and saline solution, then dried. Hydroquinone polymerization inhibitor was added, and the mixture was filtered and purified by silica gel column chromatography to obtain the ROS-responsive monomer 2-(methylthio)ethyl methacrylate containing a thioether group. Step three: Preparation of pH-responsive copolymer: Diethylaminoethyl methacrylate, polyethylene glycol methyl ether methacrylate, ethyl α-bromoisobutyrate, and toluene were mixed and stirred until completely dissolved. The mixture was subjected to a preset number of cycles of freezing-vacuuming-thawing with liquid nitrogen to remove oxygen. Under nitrogen protection, CuBr and N2 were added. N,N′,N″,N″-pentamethyldiethylenetriamine was heated in an oil bath. After the reaction was completed, it was cooled to room temperature, and an equal volume of anhydrous tetrahydrofuran was added for dilution. Then, the copper catalyst was adsorbed and removed by chromatography on a neutral alumina column. After further precipitation and filtration, the resulting solid was washed with diethyl ether and dried to constant weight to obtain a white solid pH-responsive copolymer. Step four: Preparation of dual-responsive drug-loaded gel: The pH-responsive copolymer obtained in step three and 2-(methylthio)ethyl methacrylate obtained in step two were dissolved in PBS buffer, and cross-linking was initiated by choosing any of the following methods: Method one, chemical cross-linking: N,N′-methylenediethylenetriamine was added at 4°C. Diacrylamide, after ultrasonic dissolution, is followed by the addition of miR-22-3p antisense oligonucleotide inhibitor and neurotrophic factor-3. After mixing, ammonium persulfate and N,N,N',N'-tetramethylethylenediamine are added sequentially and mixed to obtain a dual-response drug-loaded gel precursor solution. The solution is then reacted at 37°C to obtain a ROS / pH dual-response drug-loaded gel. Alternatively, photocrosslinking is performed: miR-22-3p antisense oligonucleotide inhibitor and neurotrophic factor-3 are added, mixed in the dark at 4°C, and then a photoinitiator is added. After ultrasonic dissolution, a dual-response drug-loaded gel precursor solution is obtained. This solution is then crosslinked and cured using blue light to obtain a ROS / pH dual-response drug-loaded gel.
[0011] Optionally, in step one, the mass of sodium hydroxide is 40g, dissolved in 800mL of anhydrous ethanol; the mass of 2-mercaptoethanol is 100~150g; and the iodomethane ethanol solution is 200mL, wherein the molar amount of iodomethane is 0.5~0.6 times that of 2-mercaptoethanol.
[0012] Optionally, in step one, the reaction time to room temperature is 6 hours; the washing is performed three times with a 3.5 wt% sodium chloride solution.
[0013] Optionally, in step two, the amount of dichloromethane is 1L, the amount of triethylamine is 100mL, the amount of 4-dimethylaminopyridine is 4.9g, the molar amount of methacryloyl chloride is 1.08~1.12 times that of the intermediate 2-(methylthio)ethanol, and the amount of hydroquinone polymerization inhibitor is 1g.
[0014] Optionally, in step three, the masses of diethylaminoethyl methacrylate, polyethylene glycol methyl ether methacrylate, and ethyl α-bromoisobutyrate are 50g, 25g, and 1.47g, respectively; the mass of toluene is 200mL; the mass of CuBr is 1.1g; the mass of N,N,N′,N″,N″-pentamethyldiethylenetriamine is 1.9g; the oil bath heating temperature is 70℃; and the reaction time is 24 hours.
[0015] Optionally, in step four, the mass of the pH-responsive copolymer is 2.0 g, the mass of 2-(methylthio)ethyl methacrylate is 0.2 g, the PBS buffer pH is 7.4 and the volume is 8 mL; the miR-22-3p antisense oligonucleotide inhibitor is cholesterol-modified to enhance cell membrane permeability, the dose is 100 nM, 0.1 nmol; and the neurotrophic factor-3 is 20 μg.
[0016] Optionally, in step four, N,N'-methylenebisacrylamide is 0.04 g, ammonium persulfate is 0.01 g, and N,N,N',N'-tetramethylethylenediamine is 10 μL.
[0017] Optionally, in step four, the photoinitiator is 0.01g, and the light is irradiated with 405 nm blue light for 60 seconds.
[0018] According to one aspect of the present invention, a ROS / pH dual-responsive drug-loaded gel is provided, which is prepared by the method for preparing ROS / pH dual-responsive drug-loaded gels as described above.
[0019] According to one aspect of the present invention, the use of the ROS / pH dual-responsive drug-loaded gel as described above in the preparation of a pharmaceutical composition for treating spinal cord injury is provided.
[0020] The beneficial effects of this invention are:
[0021] The hydrogel in this invention has dual ROS and pH responsiveness, enabling it to specifically sense the highly reactive oxygen species and acidic microenvironment unique to the spinal cord injury area and adjust the drug release rate accordingly. Compared with traditional materials, this material enables drug release to be highly matched with the stage-specific needs of disease progression, thereby greatly improving drug utilization efficiency and treatment precision in both time and space.
[0022] This invention does not simply combine hyperbaric oxygen therapy with ROS / pH dual-responsive hydrogel, but rather discovers the synergistic effect between the two. Hyperbaric oxygen not only directly improves local oxygen supply and reduces edema, but the controllable oxidative stress it generates can also serve as an external physical signal to further trigger the responsive degradation of ROS / pH dual-responsive hydrogel. This linkage mechanism of hyperbaric oxygen physical therapy regulating chemical drug release makes the efficacy significantly better than that of single therapy.
[0023] This invention deeply integrates miR-22-3p AMO gene regulation, NT-3 neurotrophic support, ROS / pH dual-responsive hydrogel, and hyperbaric oxygen therapy, forming a complete treatment system with a clear mechanism, controllable timing, and feasible operation. The injectable delivery method of its ROS / pH dual-responsive hydrogel conforms to the concept of minimally invasive surgery and has clinical translational potential and social value. Detailed Implementation
[0024] Example 1: A method for preparing a ROS / pH dual-responsive drug-loaded gel, comprising the following steps:
[0025] Step 1: Dissolve 40g of sodium hydroxide in 800mL of anhydrous ethanol and place it in a 2L reaction vessel. Cool the mixture to 0-5℃ in an ice bath, then slowly add 100-150g of 2-mercaptoethanol dropwise while stirring for 30 minutes. Next, slowly add 200mL of ethanol solution containing 0.5-0.6 times the molar amount of iodomethane (2-mercaptoethanol), controlling the temperature to <5℃. After the addition is complete, raise the temperature to room temperature and react for 6 hours. Finally, remove the anhydrous ethanol by rotary evaporation under reduced pressure. Wash the organic phase three times with 3.5wt% sodium chloride solution and dry it with anhydrous Na2SO4 to obtain the intermediate 2-(methylthio)ethanol.
[0026] Step 2: Dissolve the intermediate 2-(methylthio)ethanol obtained in Step 1 in 1L of dichloromethane, then add 100mL of triethylamine and 4.9g of 4-dimethylaminopyridine. Slowly add 1.08~1.12 times the molar amount of methacryloyl chloride (the amount of 2-(methylthio)ethanol) in an ice bath at 0℃. Allow the mixture to rise naturally to room temperature and react in the dark for 12 hours. Wash successively with 5% hydrochloric acid, saturated NaHCO3 solution, and saturated saline solution. Then dry in a vacuum drying oven at 40℃ for 6 hours, and add 1g of hydroquinone polymerization inhibitor. After filtration, purify by silica gel column chromatography (200~300 mesh silica gel, eluent: petroleum ether and ethyl acetate, volume ratio 10:1) to obtain the ROS-responsive monomer 2-(methylthio)ethyl methacrylate containing a thioether group.
[0027] Step 3: In a dry 500mL Schlenk flask, add 50g of diethylaminoethyl methacrylate, 25g of polyethylene glycol methyl ether methacrylate (Mn=500), 1.47g of ethyl α-bromoisobutyrate, and 200mL of toluene sequentially. Stir magnetically at 200rpm until completely dissolved. After removing oxygen by three cycles of liquid nitrogen freezing-vacuuming-thawing, under nitrogen protection, add 1.1g of CuBr and 1.9g of N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA). Heat in an oil bath to 70°C and react for 24 hours. Cool to room temperature and dilute with an equal volume of anhydrous tetrahydrofuran. Pass the diluted solution through a chromatography column packed with sufficient neutral alumina to adsorb and remove the copper catalyst. After further precipitation and filtration, wash the resulting solid three times with diethyl ether and dry under vacuum at 40°C to constant weight to obtain a white solid pH-responsive copolymer.
[0028] Step 4: Dissolve 2.0g of the pH-responsive copolymer obtained in Step 3 and 0.2g of 2-(methylthio)ethyl methacrylate obtained in Step 2 in 8mL of PBS buffer at pH 7.4. Stir overnight at 4°C. Then add 0.04g of N,N'-methylenebisacrylamide, sonicate to dissolve, add 0.1 nmol of miR-22-3p antisense oligonucleotide (AMO) inhibitor and 20 μg of neurotrophic factor-3 (NT-3), gently mix, then add 0.01g of ammonium persulfate and 10μL of N,N,N',N'-tetramethylethylenediamine sequentially and mix rapidly to obtain a biresponsive drug-loaded gel precursor solution. React at 37°C for 30 minutes to form a biresponsive drug-loaded gel.
[0029] Among them, the miR-22-3p AMO inhibitor is modified with cholesterol to enhance cell membrane permeability, at a dose of 100 nM.
[0030] Example 2: Except for step four in Example 1, all other steps are the same as in Example 1.
[0031] In this Example 2, step four is as follows: 2.0 g of pH-responsive copolymer and 0.2 g of 2-(methylthio)ethyl methacrylate are dissolved in 8 mL of PBS buffer at pH 7.4. Then, 0.1 nmol of miR-22-3p antisense oligonucleotide (AMO) inhibitor and 20 μg of neurotrophic factor-3 (NT-3) are added, and the mixture is mixed at 4°C in the dark. Finally, 0.01 g of photoinitiator is added, and the mixture is dissolved by sonication to obtain a dual-responsive drug-loaded gel precursor solution. The solution is then irradiated with 405 nm blue light for 60 seconds to form a dual-responsive drug-loaded gel.
[0032] The photoinitiator is one of the following: lithium phenyl-2,4,6-trimethylbenzoyl phosphite, camphorquinone (0.1-0.3% w / v) and N,N-dimethyl-p-toluidine (0.2-0.8% w / v), or Eosin Y (0.05-0.2 mM) and triethanolamine (0.5-2% v / v).
[0033] Examples 1 and 2 represent two different technical approaches for preparing the ROS / pH dual-responsive drug-loaded gel of the present invention, which can be either chemically crosslinked or photocrosslinked. Example 1 is a chemical crosslinking method, and Example 2 is a photocrosslinking method.
[0034] The specific principle of the responsive degradation of the ROS / pH dual-responsive drug-loaded gel of this invention is as follows:
[0035] When the ROS / pH dual-responsive gel (i.e., the ROS / pH dual-responsive drug-loaded gel of this invention) is placed in a high-pressure oxygen environment, the introduced thioether groups (-S-) are oxidized. First, the sulfur atoms lose electrons and are oxidized from the thioether (-S-) to sulfoxide (-SO-). This is a relatively rapid process. Under continuous high-pressure oxygen, the sulfoxide can be further oxidized to sulfone (-SO2-).
[0036] The introduction of sulfoxide and sulfone groups brought about two key changes:
[0037] 1. CS bonds become unstable after oxidation and are more likely to undergo hydrolysis and breakage under physiological conditions (such as the action of water molecules).
[0038] 2. Thioethers are hydrophobic, while sulfoxides and sulfones are strongly hydrophilic. This dramatic change in polarity leads to a surge in the interaction forces between polymer segments and water molecules, generating intense osmotic pressure. This causes the gel network to swell dramatically, its structure to loosen, and the segments between crosslinking points to break more easily. Under high-pressure oxygen stimulation, the ROS / pH dual-responsive gel experiences accelerated mass loss, a sharp increase in swelling rate, a rapid decrease in modulus, and ultimately, overall network dissociation, resulting in the release of a large amount of the encapsulated drug.
[0039] pH-responsive degradation process and principle: Under normal physiological pH (~7.4), the tertiary amine groups in the ROS / pH dual-responsive gel are in a neutral, hydrophobic state. When the ROS / pH dual-responsive gel is in an acidic microenvironment (such as the SCII region, where the pH can drop to 6.5~6.8), H... + As the concentration increases, the tertiary amine group undergoes protonation, transforming into a positively charged quaternary ammonium salt cation (-N). + H(CH2CH3)2). A large number of positively charged amino ions generate strong electrostatic repulsion. This repulsion overcomes the hydrophobic interactions and physical entanglement between polymer chains, expanding the originally compact network. At the same time, in order to balance the concentration difference of ions inside and outside the polymer network, a large number of water molecules and anions rush into the gel, generating huge osmotic pressure, causing macroscopic and dramatic swelling of the ROS / pH dual-responsive gel.
[0040] The highly swollen state allows water to more easily penetrate into the ROS / pH dual-responsive gel. Furthermore, the polymer chains are more extended, making them more susceptible to attack by hydrolytic enzymes, and hydrolyzable chemical bonds (such as ester bonds) are more fully exposed. These factors combined result in a significantly accelerated hydrolytic degradation rate of the ROS / pH dual-responsive gel backbone or cross-linking points.
[0041] Macroscopically, under acidic pH conditions, the ROS / pH dual-responsive gel initially exhibits rapid volume expansion and softening, followed by a rapid mass loss phase, with the drug released through a synergistic mechanism of diffusion and skeleton erosion.
[0042] When using the ROS / pH dual-responsive drug-loaded gel of this invention:
[0043] During the acute phase of injury (0–72 hours): a burst of hyperbaric oxygen occurs, and pH drops significantly. The dual stimulation is most potent, and the hydrogel responds rapidly, accelerating drug release, designed to deliver miR-22-3p AMO with maximum efficiency to inhibit early apoptosis storm.
[0044] Subacute phase of injury (days to weeks): With hyperbaric oxygen therapy and self-repair, ROS and acidity levels decrease. The hydrogel response diminishes, entering a stable, long-lasting sustained-release phase, continuously providing NT-3 to support nerve regeneration and remodeling. This perfectly matches the different treatment needs at different stages of the disease.
[0045] Application Example: The following experiment verifies the effectiveness of the ROS / pH dual-responsive drug-loaded gel (combined with hyperbaric oxygen therapy) of the present invention in spinal cord injury repair.
[0046] Animals and grouping: Healthy SD rats (220~250g) were divided into healthy rat group (G1), SCII group (G2), single drug-loaded gel group (G3), single hyperbaric oxygen group (G4), and combined treatment group (G5).
[0047] G2: SCII Group:
[0048] The SCII rat model was obtained by using the abdominal aortic clamping method. After anesthesia, the abdominal aorta below the kidney was exposed via an abdominal approach and clamped with a non-invasive arterial clamp for 14 minutes to induce spinal cord ischemia. Reperfusion was achieved after the clamp was released.
[0049] G3: Single-loaded drug-eluting gel group:
[0050] Dual-responsive drug-loaded gel implantation surgery: 36±2 hours after SCII, rats were re-anesthetized and placed in a prone position. The T8-T10 segment of the spine was exposed through the original incision, and laminectomy was performed to clearly expose the injured segment of the spinal cord. Using a Hamilton microsyringe equipped with a 33G needle, 10 μL of the dual-responsive drug-loaded gel precursor solution obtained in Example 1 or Example 2 was drawn up. Under a surgical microscope, injections were made at three sites: the center of the injury and 2 mm from the head and tail. The needle tip was beveled dorsally and slowly inserted into the spinal cord parenchyma to a depth of 1.0 mm. The injection volume at each site was 3.3 μL, and the injection rate was 0.25 μL / min. After injection, the needle was left in place for 3 minutes and then slowly withdrawn. The solution was observed to remain in the tissue and transform from a liquid state to a non-diffusion gel state with body temperature (37℃) for 30 min or irradiation with 405 nm blue light for 60 s. Finally, the needle holes were gently pressed with gelatin sponge and sutured layer by layer.
[0051] G4: Single Hyperbaric Oxygen Group:
[0052] Rats after SCII were placed in an animal-specific hyperbaric oxygen chamber. The pressure was increased to 0.2 MPa (2 ATA) at a rate of 1.0 MPa / min, maintaining an oxygen concentration >95% for 40 minutes. Then, the pressure was slowly reduced at a rate of 0.01 MPa / min for the entire treatment, which lasted approximately 70 minutes. Treatment was administered once daily for 7 consecutive days.
[0053] G5: Combined Treatment Group:
[0054] Using the G3 single-drug-loaded gel method, the first hyperbaric oxygen therapy was initiated 2 hours after implantation of the dual-response drug-loaded gel. Using the G4 single-hyperbaric oxygen therapy method, the hydrogel had already formed in the rats after SCII at the time of the first hyperbaric oxygen therapy. The physiological oxidative stress generated by hyperbaric oxygen served as a trigger signal, slightly accelerating hydrogel degradation and drug release, thus synchronizing the initiation of treatment.
[0055] BBB motor scoring, inclined plane test, and motor evoked potential testing were performed on the SCII group, the single drug-loaded gel group, the single hyperbaric oxygen group, and the combined treatment group. The specific methods are as follows:
[0056] BBB motor assessment: was performed preoperatively and on days 1, 3, 7, 14, 21, and 28 postoperatively. Rats were placed in a circular, opaque test chamber with a diameter of 90 cm. Two researchers, whose groups were unknown, independently observed the rats' free movement for 4 minutes. The rats' hind limb joint movement, gait, trunk stability, and coordination were scored using the 21-point BBB scale, and the average score was taken as the final score.
[0057] Inclined board test: Performed on days 7, 14, 21, and 28 post-surgery. An adjustable-angle inclined board apparatus with a rough rubber pad on the surface was used. The rat was placed head-up in the center of the inclined board, and the board was raised at a constant speed to the starting angle (30°). The rat's ability to maintain the angle for 5 seconds was observed. If successful, the angle was increased by 5° and the test was continued; if unsuccessful, the test was repeated once. The maximum angle that the rat could successfully maintain for 5 seconds was recorded. Data for each group are expressed as mean ± standard deviation, and one-way ANOVA was used for inter-group comparisons.
[0058] Motor evoked potential testing: Performed on day 28 post-surgery. Rats were deeply anesthetized with urethane (1.2 g / kg, intraperitoneal injection), and their body temperature was maintained at 37.0℃. The skull was exposed, and the anode of the stimulating electrode was placed 1.0 mm anterior to the anterior fontanelle and 1 mm to the left of the sagittal suture, while the cathode was placed superior to the nasal bone. The recording electrode was inserted into the belly of the right tibialis anterior muscle. Monophasic square wave stimulation (0.2 ms duration, 0.5 Hz frequency, and 120% intensity of tympany) was used to record the motor evoked potential waveforms. Analytical parameters included latency and peak-to-peak amplitude, and the amplitude recovery rate was calculated based on the mean of the healthy rat group.
[0059] The specific indicators for different rat groups are shown in the table below:
[0060] Group BBB Sports Rating Maximum maintenance angle Motion evoked potentials G1 21.0 ± 1.3 >75° 98%± 1% G2 9.8 ± 1.5 38.5° ± 5.0° 25% ± 10% G3 13.5 ± 1.8 52.4° ± 5.8° 55% ± 12% G4 12.1 ± 2.0 48.7° ± 6.2° 48% ± 15% G5 17.2 ± 1.5 65.8° ± 6.5° 82% ± 10%
[0061] As can be seen from the table, the G3 group (compared to the G2 and G4 groups) showed an improvement in BBB motor score, indicating that the rats' motor function was effectively improved. The maximum sustained angle indicated that their joint movement and control ability was enhanced, and the motor evoked potential showed that the nerve conduction function was restored. The results of the above three indicators prove that the ROS / pH dual-response drug-loaded gel in this invention can effectively promote the repair of nerve structure and function.
[0062] Furthermore, the combination of hyperbaric oxygen therapy with the ROS / pH dual-response drug-loaded gel (i.e., group G5) showed a greater degree of improvement in all indicators than group G3, which used the single drug-loaded gel. This indicates that the synergistic effect of hyperbaric oxygen therapy and the ROS / pH dual-response drug-loaded gel demonstrates a significant additive gain effect in functional recovery.
[0063] The above results demonstrate that the ROS / pH dual-response drug-loaded gel proposed in this invention has a good repair effect. When combined with hyperbaric oxygen therapy, it can achieve optimal spinal cord injury repair through deep synergy between intelligent drug release and physical therapy regulation, fully demonstrating its innovation and clinical translation potential.
[0064] The sequence numbers of the above embodiments are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments. The descriptions of each embodiment in the above embodiments have different emphases; for parts not described in detail in a certain embodiment, please refer to the relevant descriptions of other embodiments.
[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing a ROS / pH dual-responsive drug-loaded gel, characterized in that, include: Step 1: Preparation of intermediate 2-(methylthio)ethanol: Sodium hydroxide was dissolved in anhydrous ethanol, cooled to 0-5°C in an ice bath, 2-mercaptoethanol was added and mixed evenly, then iodomethane ethanol solution was added, the mixture was heated to room temperature and reacted, anhydrous ethanol was removed by rotary evaporation under reduced pressure, and the product was washed with sodium chloride solution and dried with anhydrous Na2SO4 to obtain the intermediate 2-(methylthio)ethanol. Step 2: Preparation of ROS-responsive monomer 2-(methylthio)ethyl methacrylate containing thioether groups: The intermediate 2-(methylthio)ethanol obtained in step one was dissolved in dichloromethane, and triethylamine and 4-dimethylaminopyridine were added. Methacryl chloride was added under ice bath conditions, and the mixture was heated to room temperature and reacted in the dark. After that, it was washed successively with hydrochloric acid, NaHCO3 solution, and saline solution, and then dried. Hydroquinone polymerization inhibitor was added, and the mixture was filtered and purified by silica gel column chromatography to obtain the ROS-responsive monomer 2-(methylthio)ethyl methacrylate containing a thioether group. Step 3, Preparation of pH-responsive copolymer: Diethylaminoethyl methacrylate, polyethylene glycol methyl ether methacrylate, ethyl α-bromoisobutyrate, and toluene were mixed and stirred until completely dissolved. The mixture was subjected to a predetermined number of cycles of freezing-vacuuming-thawing in liquid nitrogen to remove oxygen. Under nitrogen protection, CuBr and N,N,N′,N″,N″-pentamethyldiethylenetriamine were added. The mixture was heated in an oil bath. After the reaction was completed, the mixture was cooled to room temperature and diluted with an equal volume of anhydrous tetrahydrofuran. The copper catalyst was then adsorbed and removed by chromatography on a neutral alumina column. The solid obtained after further precipitation and filtration was washed with diethyl ether and dried to constant weight to obtain a white solid pH-responsive copolymer. Step 4: Preparation of dual-response drug-loaded gel: The pH-responsive copolymer obtained in step three and the 2-(methylthio)ethyl methacrylate obtained in step two are dissolved in PBS buffer, and cross-linking is initiated by choosing any of the following methods: Method 1, chemical cross-linking: N,N'-methylenebisacrylamide was added at 4°C and dissolved by sonication. Then, miR-22-3p antisense oligonucleotide inhibitor and neurotrophic factor-3 were added and mixed. Ammonium persulfate and N,N,N',N'-tetramethylethylenediamine were added sequentially and mixed to obtain a dual-response drug-loaded gel precursor solution. The solution was placed at 37°C for reaction. After the reaction was completed, ROS / pH dual-response drug-loaded gel was obtained. Method 2, photocrosslinking: Add miR-22-3p antisense oligonucleotide inhibitor and neurotrophic factor-3, mix at 4°C in the dark, then add photoinitiator, and sonicate to dissolve to obtain a dual-response drug-loaded gel precursor solution. Use blue light to irradiate and crosslink to cure to obtain ROS / pH dual-response drug-loaded gel.
2. The method for preparing the ROS / pH dual-responsive drug-loaded gel according to claim 1, characterized in that, In step one, the mass of sodium hydroxide is 40g, dissolved in 800mL of anhydrous ethanol; the mass of 2-mercaptoethanol is 100~150g; the mass of iodomethane ethanol solution is 200mL, wherein the molar amount of iodomethane is 0.5~0.6 times that of 2-mercaptoethanol.
3. The method for preparing the ROS / pH dual-responsive drug-loaded gel according to claim 1, characterized in that, In step one, the reaction time to room temperature is 6 hours; the washing is performed by washing three times with a 3.5 wt% sodium chloride solution.
4. The method for preparing the ROS / pH dual-responsive drug-loaded gel according to claim 1, characterized in that, In step two, the amount of dichloromethane is 1L, triethylamine is 100mL, and 4-dimethylaminopyridine is 4.9g; the molar amount of methacryloyl chloride is 1.08~1.12 times that of the intermediate 2-(methylthio)ethanol; the amount of hydroquinone polymerization inhibitor is 1g; the silica gel used for silica gel column chromatography purification is 200~300 mesh; and the eluent is petroleum ether and ethyl acetate in a volume ratio of 10:
1.
5. The method for preparing the ROS / pH dual-responsive drug-loaded gel according to claim 1, characterized in that, In step three, the masses of diethylaminoethyl methacrylate, polyethylene glycol methyl ether methacrylate, and ethyl α-bromoisobutyrate are 50g, 25g, and 1.47g, respectively; the mass of toluene is 200mL; the mass of CuBr is 1.1g; the mass of N,N,N′,N″,N″-pentamethyldiethylenetriamine is 1.9g; the oil bath heating temperature is 70℃; and the reaction time is 24 hours.
6. The method for preparing the ROS / pH dual-responsive drug-loaded gel according to claim 1, characterized in that, In step four, the mass of the pH-responsive copolymer is 2.0 g, the mass of 2-(methylthio)ethyl methacrylate is 0.2 g, the PBS buffer pH is 7.4 and the volume is 8 mL; the miR-22-3p antisense oligonucleotide inhibitor is cholesterol-modified to enhance cell membrane permeability, and the dosage is 100 nM, 0.1 nmol. Neurotrophic factor-3 was 20 μg.
7. The method for preparing the ROS / pH dual-responsive drug-loaded gel according to claim 1, characterized in that, In step four, N,N'-methylenebisacrylamide is 0.04 g, ammonium persulfate is 0.01 g, and N,N,N',N'-tetramethylethylenediamine is 10 μL.
8. The method for preparing the ROS / pH dual-responsive drug-loaded gel according to claim 1, characterized in that, In step four, the photoinitiator is 0.01g, and the light is irradiated with 405 nm blue light for 60 seconds.
9. A ROS / pH dual-responsive drug-loaded gel, characterized in that, It was prepared using the method described in any one of claims 1 to 8 for preparing the ROS / pH dual-responsive drug-loaded gel.
10. The use of the ROS / pH dual-responsive drug-loaded gel as described in claim 9 in the preparation of a pharmaceutical composition for treating spinal cord injury.