An endoplasmic reticulum-targeted antioxidant functional nanoscale enzyme, and a preparation method and application thereof

Abstract

This invention belongs to the field of biopharmaceuticals, specifically relating to an endoplasmic reticulum-targeting antioxidant nanozyme, its preparation method, and its applications. The novel antioxidant nanozyme (Arg-Mnzyme) provided by this invention can effectively inhibit NOX1 expression, eliminate the accumulation of reactive oxygen species, and inhibit NOX1 activation and oxidative stress after temporomandibular joint arthritis-related pain, thus alleviating pain hypersensitivity. Compared to natural enzymes, nanozymes have stronger modifiability; this invention will construct an endoplasmic reticulum-precise antioxidant nanozyme to achieve precise regulation of oxidative stress.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to an endoplasmic reticulum-targeting antioxidant nanozyme, its preparation method, and its application. Background Technology

[0002] Trigeminal neuralgia (TN) is one of the most common neuropathic pains in the maxillofacial region, typically characterized by sudden, electric shock-like, severe unilateral facial pain, significantly impacting patients' quality of life. Recent studies have found that approximately 30% of trigeminal neuralgia cases are closely related to local inflammatory lesions, also known as trigeminal inflammatory pain (TIP). When inflammatory pain occurs in the temporomandibular joint region, the oxidative stress level in the caudal part of the trigeminal spinal nucleus, the superior center of the pain transmission pathway, is significantly increased. New evidence suggests that NOX1 and central oxidative stress levels play important roles in pain transmission. Oxidative stress-related molecules include hydrogen peroxide (H2O2) and superoxide anion (•O2). − Excessive production of free radicals such as hydroxyl radicals (•OH) and hydroxyl radicals can cause secondary damage and scavenging O2. 2•− And H2O2 (not •OH) to mimic peroxidases including superoxide dismutase (SOD) and catalase. However, its low bioavailability, short half-life, low efficiency in crossing the blood-brain barrier, and side effects on kidney and liver function limit its clinical application. Therefore, there is an urgent need to design and develop drugs with strong reactive oxygen species scavenging activity and ideal physicochemical properties to treat cerebral hemorrhage.

[0003] Natural antioxidants such as catalase and superoxide dismutase are widely used in the treatment of inflammation and in tissue engineering materials, and enzyme immobilization technology is receiving increasing attention. However, problems still exist in the development of natural antioxidants and anti-inflammatory agents in recent years, such as: 1. high cost; 2. easy degradation in vivo; 3. poor alcohol tolerance of enzyme catalysts.

[0004] Compared to natural enzymes, nanozymes have lower development costs, greater stability, and excellent recyclability. They can also catalyze some non-natural biological processes and have been applied in disease diagnosis and treatment, biosensing, environmental remediation, and antibacterial and antifouling applications. Patent 202110706467.8 discloses a transition metal single-atom nanozyme, its preparation method, and its uses. This patent achieves high catalytic performance by mimicking the activity of PODs and is used for antibacterial applications, wastewater treatment, and immunoblotting. This application aims to develop a novel nanozyme with endoplasmic reticulum targeting for relieving central post-stroke pain. Summary of the Invention

[0005] To address the above problems, this invention provides an antioxidant nanoenzyme comprising arginine-chitosan and manganese nanoparticles; arginine-chitosan serves as a bioactive surface decoration, and manganese nanoparticles serve as the functional core.

[0006] This invention also provides a method for preparing the above-mentioned antioxidant nanozyme, comprising the following steps:

[0007] (1) Weigh 80 mg of arginine-modified chitosan and use 80 mL of 0.5 M hydrochloric acid as a solvent to stir and dissolve it to obtain a chitosan hydrochloric acid solution;

[0008] (2) Weigh K3Fe(CN)6 and prepare it into a 1 mM solution, then add chitosan hydrochloric acid solution; slowly add 20 mL of 1 mM K3Fe(CN)6 solution to it and stir for 30 minutes;

[0009] (3) Prepare 20 mL of 1 mM MnCl2 solution and add it dropwise into the solution obtained in the previous step; after stirring for 1 hour, the original solution is obtained, and the antioxidant nanozyme is obtained by dialysis and freeze-drying.

[0010] This invention also provides the application of the above-mentioned antioxidant nanozymes in the preparation of antioxidant drugs.

[0011] The present invention also provides the application of the above-mentioned antioxidant nanozyme in the preparation of drugs for oxidative stress and NOX1 activation in the caudal part of the trigeminal spinal nucleus.

[0012] The present invention also provides the application of the above-mentioned antioxidant nanozyme in the preparation of a drug for relieving joint pain in the temporomandibular joint.

[0013] The present invention has the following beneficial effects:

[0014] 1. This application provides a novel nanomedicine that relieves inflammatory pain in the temporomandibular joint (TMJ), with effects appearing on day 1 after intraperitoneal injection, peaking on days 3-7, and lasting for at least 10-12 days. Systemic nanomedicine delivery eliminates the accumulation of reactive oxygen species, rapidly and effectively inhibiting NOX1 activation in mice with complete Freund's adjuvant intra-articular injection, thus reducing pain hypersensitivity. Using nanozymes within an appropriate time window not only reduces further oxidative stress damage but also produces analgesic effects. In summary, this study, using Arg-CS as a bioactive surface decoration and manganese (Mn) nanoparticles as a functional core, not only provides a novel method for in-situ synthesis of synergistic nanotherapy but also reveals the potential mechanisms underlying the development and progression of inflammatory pain in the temporomandibular joint.

[0015] 2. Using arginine-chitosan (Arg-CS) as a bioactive surface decoration and manganese (Mn) nanoparticles as the functional core, this invention effectively eliminates the accumulation of reactive oxygen species, inhibits the oxidative stress level and NOX1 activation in the upper nerve center of the pain transmission pathway after temporomandibular joint arthritis, and thus reduces pain hypersensitivity. Compared with natural enzymes, nanozymes have stronger modifiability. This invention will construct an endoplasmic reticulum-precision antioxidant nanozyme to achieve precise regulation of oxidative stress. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a transmission electron microscope image of Arg-Mnzyme synthesized in Example 1.

[0018] Figure 2 The image shows the results of the activity assay of the Arg-Mnzyme enzyme synthesized in Example 1.

[0019] Figure 3 After intra-temporomandibular joint injection of complete Freund's adjuvant in mice, hypersensitivity to pain was observed at the injection site, and the expression of ROS and inflammatory factors in the damaged area was increased. However, this trend was reversed in mice injected with nanozymes intraperitoneally. Figure A shows the timeline design for animal model preparation and behavioral testing. Figure B shows the change in mechanical pain threshold at the injection site over time in different groups of mice after intra-temporomandibular joint injection of complete Freund's adjuvant. Figures C and D show the increased expression of ROS (8-OHdG as a marker of oxygen free radicals) (D) and inflammatory factor TNF-α (C) in the caudal part of the ipsilateral trigeminal spinal nucleus after intra-temporomandibular joint injection of complete Freund's adjuvant in mice. This trend was reversed in mice injected with nanozymes intraperitoneally.

[0020] Figure 4 West Blotting (A, B), QT-PCR (C), and immunofluorescence (D) all indicated that intra-temporal injection of complete Freund's adjuvant into the temporomandibular joint of mice led to increased expression of NOX1, an inflammatory pain-related protein in the caudal part of the ipsilateral trigeminal spinal nucleus, while this trend was reversed in mice injected intraperitoneally with nanozymes.

[0021] Figure 5The effect of nanozyme administration on reducing temporomandibular joint pain after intra-articular injection of complete Freund's adjuvant without obvious toxic side effects is shown in Figure A: Nanozyme administration reduces hyperalgesia at the temporomandibular joint in mice after intra-articular injection of complete Freund's adjuvant without causing substantial damage to the heart, liver, spleen, lungs, and kidneys of mice. Figure B: Nanozyme (8 mg / kg) does not affect liver and kidney function in both groups of mice and does not show in vivo toxicity. Detailed Implementation

[0022] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Example 1

[0028] The preparation method of the antioxidant nanozyme that targets and inhibits nitric oxide synthase activity in this embodiment includes the following steps:

[0029] Weigh 80 mg of arginine-modified chitosan and dissolve it completely in 80 mL of 0.5 M hydrochloric acid using 0.5 M hydrochloric acid. Weigh K3Fe(CN)6 and prepare a 1 mM solution in a round-bottom flask, then add the chitosan hydrochloric acid solution obtained in the previous step. Slowly add 20 mL of the 1 mM K3Fe(CN)6 solution dropwise while stirring for 30 minutes. The solution color changes from colorless to light green, and finally to indigo blue. Prepare 20 mL of 1 mM MnCl2 solution separately, then add the mixed solution dropwise to the solution obtained in the previous step. After stirring for 1 hour, the Arg-Mnzyme stock solution is obtained, which is dialyzed and lyophilized. The Arg-Mnzyme product is analyzed by transmission electron microscopy, and the results are as follows: Figure 1 As shown, by Figure 1 It is known that Arg-Mnzyme has a uniform cubic structure with a size of about 80 nm, indicating that the present invention has the potential to effectively penetrate the blood-brain barrier.

[0030] Implementation Results Example

[0031] 1. Laboratory animals and procedures:

[0032] We used male C57 mice (7-8 weeks old, 20-25g) purchased from Liaoning Changsheng Biotechnology Co., Ltd. Five mice were housed in each standardized cage, kept in a standard 12-hour light / dark cycle, and had free access to clean water and food. The animal experiments were approved by the Medical Ethics Committee of the Second Affiliated Hospital of Zhengzhou University (ethics approval number: KY2024186). To minimize individual variability in behavioral tests, all animals underwent one week of environmental acclimatization before the tests. During the behavioral tests, to ensure objectivity and authenticity, the experimenters were unaware of the mice's treatment conditions.

[0033] 2. Establishment of a temporomandibular joint arthritis pain model

[0034] The method for establishing a mouse model of temporomandibular joint arthritis pain was based on previous literature, with some modifications made to our preliminary experiments. Mice were anesthetized by intraperitoneal injection of tribromoethanol (10 ml / kg). The mice were placed in a lateral decubitus position, and the temporomandibular joint was located using forceps. Using a microsyringe, a needle was inserted perpendicularly to the skin at the location of the left temporomandibular joint projection on the body surface to a depth of about 3 mm. 15 μL of complete Freund's adjuvant was slowly injected for more than 30 seconds. The microsyringe was removed 30 seconds after injection.

[0035] 3. Behavioral Science

[0036] The head retraction threshold under mechanical stimulation was measured using methods similar to those used in previous studies. Mice were placed in a single plexiglass restraint, with only their heads protruding from the restraint, and allowed to adapt to the environment for half an hour. An electronic von Frey wire was then attached to the surface projection of the left temporomandibular joint of the mouse. Stimulation was stopped immediately after the mouse's head retracted, and this was repeated three times. Rapid head retraction by the mouse is a positive response, and the average weight of the electronic von Frey wire measured in the three head retractions was taken as the final measurement result.

[0037] 4. Serum biochemical index monitoring

[0038] Mice were injected intraperitoneally with physiological saline or Arg@Mn solution (dissolved in physiological saline, 15 mg / kg). Three days later, the mice were deeply anesthetized, and blood was collected by puncturing the orbital fossa. The blood was placed in a 1.5 ml centrifuge tube and allowed to stand at room temperature for 1 hour. Then, the tube was centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. The corresponding serum indicators were detected according to the biochemical reagent kit (Seville, Wuhan).

[0039] 5. Western blotting

[0040] After deep anesthesia, mice were harvested from the brain and processed on ice. Protein extraction was performed from the caudal portion of the trigeminal spinal nucleus. The protein sample was mixed with loading buffer and heated at 100°C for 5 minutes. It was then loaded onto a 10% polyacrylamide gel, electrophoresed, and subsequently transferred to a PVDF membrane (Millibor, USA). The membrane was placed in 3% skim milk and blocked on a shaker at room temperature for two hours. The PVDF membrane was then incubated overnight at 4°C with primary antibodies, including NOX1 (ABCAM, ab131088) and GAPDH (Wuhan Sanying 10494-1-AP). The following day, the PVDF membrane was washed three times with TBST for 10 minutes each time, followed by co-incubation with secondary antibodies for 2 hours, including goat anti-rabbit secondary antibody (Wuhan Sanying, SA00001-2). This was followed by three more TBST washes for 10 minutes each. Development was performed using ECL chemiluminescence solution (Abkine, BMU102) and the Invitrogen iBright system. Blot intensity was measured using Image software.

[0041] 6. QT-PCR

[0042] After euthanizing mice with tribromoethanol, the caudal portion of the trigeminal spinal nucleus was removed and placed in a 1.5 ml sterile, enzyme-free tube, flash-frozen in liquid nitrogen, and then stored at -80°C. RNA was extracted using an RNA column extraction kit (Aikerui, Changsha). Reverse transcription was performed to obtain cDNA. The primer sequences were: NOX1 upstream 5'-TGTTCCCATTCTTTTCACGAGTG-3', downstream 5'-CTIGGTAATCACGACCTICIGC-3'; TNF-α upstream 5'-CTTTGAGATCCATGCCGTTG-3', downstream 5'-CTTTGAGATCCATGCCGTTG-3'; GAPDH upstream 5'-CCTCGTCCCGTAGACAAAATG-3', downstream 5'-TGAGGTCAATGAAGGGGTCGT-3'. QT-PCR was then performed according to the manufacturer's instructions.

[0043] 7. Statistical Analysis

[0044] Statistical analysis was performed using GraphPad Prism 8 software, and the results are shown in the figure. One-way or two-way ANOVA was used to compare the data from the three independent categorical groups. Unpaired t-tests were used for comparisons between two groups. A p-value less than 0.05 was considered significant in all statistical tests. Data are expressed as mean ± SEM.

[0045] in conclusion:

[0046] Enzyme activity was determined using the product Arg-Mnzyme prepared in Example 1. The specific detection method is as follows: Test conditions for absorbance ΔA1 of the control group:

[0047]

[0048] The enzyme-linked immunosorbent assay (ELISA) was used to measure at 550 nm for 1 min, and the xanthine oxidase concentration was adjusted to stabilize ΔA1 at around 0.0225.

[0049] Test conditions for absorbance ΔA2 in the experimental group:

[0050]

[0051] Use an ELISA reader to measure at 550 nm for 1 min, and serially dilute the sample, for example: 1, 1 / 5, 1 / 5^2, 1 / 5^3, 1 / 5^4, 1 / 5^5, 1 / 5^6; use a UV spectrophotometer to measure at 550 nm for 1 min ΔA2.

[0052] The results are as follows Figure 2 As shown, by Figure 2As can be seen from the analysis, Arg-Mnzyme has good superoxide dismutase (SOD) activity. Endoplasmic reticulum-targeted liposome encapsulation does not affect the enzyme-like activity of the nanozyme, indicating that the present invention can effectively scavenge free radicals.

[0053] Mice injected intra-temporal joint with complete Freund's adjuvant exhibited pain hypersensitivity and increased NOX1 expression and oxidative stress levels in the affected area.

[0054] like Figure 3 , 4 As shown, compared with the unilateral injection of saline, unilateral microinjection of complete Freund's adjuvant (…) Figure 3 B) led to mechanical hyperalgesia. This hyperalgesia occurred 2 days after the lesion and lasted at least 10 days compared to the control group, with NOX1 ( ) in the lesion area. Figure 4 AC) and inflammatory factor TNF-α Figure 3 C), and the oxidative stress marker 8-OHdG ( Figure 3 D) Increased expression.

[0055] Nanozyme administration can alleviate oxidative stress levels and reduce the expression of NOX1 and TNF-α in the damaged area:

[0056] Arg@Mn (15 mg / kg) and its carrier (saline) were administered intraperitoneally 30 minutes after intra-articular injection of either Freund's adjuvant or saline into the temporomandibular joint. Behavioral testing was performed on days 0, 1, 2, 3, 5, 7, 9, 11, and 13 post-modeling. Results are as follows: Figure 3 B confirmed that intraperitoneal injection of Arg@Mn could alleviate mechanoresonance at the temporomandibular joint in mice after intra-articular injection of complete Freund's adjuvant, and reduced NOX1 expression in the caudal part of the trigeminal spinal nucleus, an upstream nerve center. Figure 3 It can be seen that after Arg@Mn administration, ROS (8-OHdG oxygen free radical markers) in the brain injury area ( Figure 3 D), inflammatory factor TNFα ( Figure 3 The expression of C) was significantly reduced. (By...) Figure 4 It can be seen that after nanozyme administration, pain behavior tests ( Figure 3 B) showed that the pain hypersensitivity phenomenon in mice in the nanozyme administration group was improved, and QT-PCR ( Figure 4 C), West Blotting Figure 4 B) Both indicate decreased NOX1 expression.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of an antioxidant functional nanoscale enzyme in the preparation of a medicament for alleviating temporomandibular joint inflammatory pain, characterized in that, The antioxidant nanoenzyme comprises arginine-chitosan and manganese nanoparticles; arginine-chitosan is used as a bioactive surface decoration, and manganese nanoparticles are used as a functional core; the preparation method of the antioxidant nanoenzyme includes the following steps: (1) Weigh 80 mg of arginine-modified chitosan and use 80 mL of 0.5 M hydrochloric acid as a solvent to stir and dissolve it to obtain a chitosan hydrochloric acid solution; (2) Weigh K3Fe(CN)6 and prepare it into a 1 mM solution, then add chitosan hydrochloric acid solution; slowly add 20 mL of 1 mM K3Fe(CN)6 solution to it and stir for 30 minutes; (3) Prepare 20 mL of 1 mM MnCl2 solution and add it dropwise into the solution obtained in the previous step; after stirring for 1 hour, the original solution is obtained, and the antioxidant nanozyme is obtained by dialysis and freeze-drying.

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