Preparation method of multifunctional HAMA hydrogel system and application of multifunctional HAMA hydrogel system in treatment of psoriasis skin lesion

Abstract

The invention provides a preparation method of a multifunctional HAMA hydrogel system and application of the multifunctional HAMA hydrogel system in treatment of psoriasis skin lesion, relates to the technical field of biomedicine, prepares the multifunctional HAMA hydrogel system, and further discloses application of the multifunctional HAMA hydrogel system in treatment of psoriasis. The multifunctional HAMA hydrogel system comprises cerium dioxide particles, and hyaluronic acid wraps cerium dioxide and carries DNase I. Experiments include preparation of cerium dioxide, wrapping of cerium dioxide with hyaluronic acid and carrying of DNase I. A series of characterization experiments prove that the material is successfully synthesized, and in-vitro experiments prove that the HAMA hydrogel system has good enzymatic activity and biological safety, and can successfully remove DNA skeletons of neutrophil extracellular traps (NETs) and reduce the free DNA level. According to the application, the application of the multifunctional hydrogel in treatment of psoriasis is explored, and research shows that the HAMA hydrogel as an efficient and safe drug delivery carrier has a relatively good application prospect in treatment of psoriasis.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to a preparation method of a multifunctional HAMA hydrogel system and application of the system in treating psoriasis skin lesions. Background Art

[0002] Psoriasis is a chronic, cyclical systemic autoimmune disease characterized by the appearance of red papules or plaques on the skin, covered with multiple layers of silvery-white scales. These lesions can be confined to specific areas or widely distributed throughout the body. The disease has a polygenic genetic tendency, affecting approximately 2-4% of the world's population, of which 5% to 20% may further develop psoriatic arthritis, manifested as joint pain and stiffness, which may lead to movement disorders or even disability in severe cases. The pathogenesis of psoriasis involves multiple cell types, among which neutrophils play a key role. Their role in the onset and development of psoriasis is mainly reflected in the following aspects: enhanced cell survival and migration ability, abnormal inflammatory activity, increased oxidative stress levels, and increased release of neutrophil extracellular traps (NETs). These mechanisms work together to activate other immune cells, leading to a persistent inflammatory response, which in turn triggers excessive skin proliferation and tissue damage in psoriasis.

[0003] Reactive oxygen species (ROS) are intermediate products produced during cell metabolism, including superoxide anions (O2 - ), hydroxyl radicals (OH) and hydrogen peroxide (H2O2), etc. Under normal physiological conditions, the generation and clearance of ROS in the body maintain a dynamic balance. Appropriate amounts of ROS participate in signal transduction, promote cell proliferation, migration and differentiation, while excessive ROS destroy the redox balance, induce oxidative stress, and damage the structure and function of cellular macromolecules such as proteins, lipids and nucleic acids. In long-term inflammatory responses, the body will produce a large amount of ROS and reactive nitrogen. When the body's clearance capacity is exceeded, excessive ROS can cause further damage to the body through oxidative damage, initiation of inflammatory cascades and systemic disorders. Neutrophils, as terminally differentiated cells, are the main source of ROS. ROS are essential for the formation of neutrophil extracellular traps (NETs) or the NETosis process. In patients with psoriasis, neutrophils in skin lesions and peripheral blood show enhanced NETs formation ability. The formation of NETs may be one of the key processes leading to the citrullination of autoantigens. NETs play an important role in the pathogenesis of psoriasis; therefore, inhibiting the production of NETs may have potential benefits in the treatment of psoriasis. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problem of lack of drugs for treating psoriasis in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A multifunctional HAMA hydrogel system comprises cerium dioxide, a hyaluronic acid layer is wrapped around the periphery of the cerium dioxide, and the hyaluronic acid layer wrapped around the periphery of the cerium dioxide is also loaded with DNase I.

[0007] Preferably, the preparation method of the multifunctional HAMA hydrogel system is as follows:

[0008] S1: Preparation of cerium dioxide nanoparticles;

[0009] S2: Preparation of functionalized cerium dioxide solution:

[0010] DSPE-MPEG2000 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] were dissolved in chloroform, and the cerium dioxide nanoparticles prepared by S1 were added;

[0011] The mixture was sonicated, the chloroform was removed, and the mixture was dried;

[0012] Deionized water was added to the sample obtained in (2), followed by ultrasonic treatment to obtain a transparent colloidal suspension;

[0013] The colloidal suspension is washed by centrifugation and then purified by a filter to obtain a functionalized cerium dioxide solution;

[0014] S3: Preparation of HAMA hydrogel;

[0015] S4: Preparation of hydrogel system:

[0016] Weigh HAMA hydrogel and dissolve it in PBS solution, stirring;

[0017] After complete dissolution, add NHS and EDC and stir;

[0018] Add DNase I and the functionalized ceria solution obtained in S2 and stir to react overnight;

[0019] The solution is dialyzed against pure water for 2-3 days, and the pure water is replaced once a day;

[0020] Freeze-dry for 2-4 days to obtain the multifunctional HAMA hydrogel material.

[0021] Preferably, in the step S2 (1), the mass ratio of DSPE-MPEG2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] and cerium dioxide nanoparticles is 2:1:2.

[0022] Preferably, the ultrasonic treatment in step (2) of S2 is performed for 5 minutes, and after the ultrasonic treatment is completed, chloroform is removed from the sample using a rotary evaporator, and then further dried in a vacuum oven at 60° C. for 3 hours to ensure that the chloroform is completely removed.

[0023] Preferably, in step (4), the excess polyethylene glycol is removed by washing at 5000G centrifugation, and then purified by a 0.22 μm filter to obtain a functionalized cerium dioxide solution.

[0024] Preferably, the cerium dioxide nanoparticles are prepared as follows:

[0025] (1) Weighing cerium nitrate hexahydrate and dissolving it in ddH2O to form liquid A; weighing disodium citrate and dissolving it in ddH2O to form liquid B; mixing A and B evenly;

[0026] (2) The mixture in (1) was quickly added into 0.4 M ammonia water and stirred at room temperature for 24 h to obtain a yellow solution;

[0027] (3) Centrifuge at 2600 g for 30 min to remove aggregated particles and collect the supernatant;

[0028] (4) dialyze the supernatant against a 3 kDa dialysis bag for 24 h;

[0029] (5) Centrifuge at 15,000 g for 10 min and filter through a 0.22 μm filter;

[0030] The mixture was freeze-dried and stored at 4°C to obtain cerium dioxide nanoparticles.

[0031] Preferably, the HAMA preparation method is as follows:

[0032] Weigh HA and add it to DPBS, stir at 50°C for 3 h;

[0033] After the hyaluronic acid is completely dissolved, adjust the pH value of the HA solution to 8.5, add 12 mL of MA dropwise into the bottle, place in an ice-water bath, stir for 4 hours, and monitor the pH every half an hour to maintain the pH at 7.5-8.5;

[0034] The reacted solution was poured into a 50 mL centrifuge tube and the centrifuge was set to the centrifugal force mode at 3500 g for 3 min to remove the unreacted MA;

[0035] The HAMA supernatant was poured into a large beaker, diluted with 200 mL of 40°C pure water, and stirred at 600 rpm overnight;

[0036] The HAMA solution was poured into a 12-14 kDa dialysis bag and dialyzed in 40°C pure water for 7 days, with the pure water being changed at least once a day;

[0037] After dialysis, the pH of the HAMA solution was adjusted to about 7.2;

[0038] HAMA was obtained by freeze-drying.

[0039] The present application also provides the use of a multifunctional HAMA hydrogel material in the preparation of a drug for treating psoriasis lesions, wherein the multifunctional HAMA hydrogel material is the multifunctional HAMA hydrogel material described above.

[0040] The present application provides a preparation of a multifunctional HAMA hydrogel system and its application in the treatment of psoriasis. The multifunctional hydrogel system of the present application is a cerium dioxide nanoparticle encapsulated by hyaluronic acid carrying DNase I. The cerium dioxide nanosystem has good enzyme activity and biosafety, and can successfully remove the DNA skeleton of neutrophil extracellular traps (NETs), and alleviate psoriasis skin inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 FIG. 5 is a scanning electron micrograph of HAMA hydrogel according to one embodiment of the present invention.

[0042] Figure 2 : is a characterization diagram of cerium dioxide nanoparticles in one embodiment of the present invention, wherein A is the particle size of the cerium dioxide nanoparticles, and B is the potential diagram of the cerium dioxide nanoparticles.

[0043] Figure 3 It is a schematic diagram demonstrating the enzyme activity of cerium dioxide nanoparticles in one embodiment of the present invention, which includes hydroxyl radical scavenging rate and SOD activity.

[0044] Figure 4 Schematic diagram demonstrating the ROS scavenging ability of cerium dioxide nanoparticles in one embodiment of the present invention.

[0045] Figure 5 Schematic diagram demonstrating the ability of the multifunctional hydrogel system to clear NETs in one embodiment of the present invention. DETAILED DESCRIPTION

[0046] Some English interpretations of the text:

[0047]

[0048]

[0049] The present application provides a multifunctional HAMA hydrogel system, wherein the hydrogel system comprises hyaluronic acid carrying DNase I and a cerium dioxide nanosystem, wherein the hyaluronic acid carrying DNase I encapsulates cerium dioxide nanoparticles.

[0050] The present application also provides a method for preparing a multifunctional HAMA hydrogel system, which comprises the following steps:

[0051] S1: Preparation of CeO2 Nanoparticles:

[0052] (1) Weigh 108.5 mg of cerium nitrate hexahydrate and dissolve it in 2 mL of ddH2O to form solution A; weigh 100 mg of disodium citrate and dissolve it in 1 mL of ddH2O to form solution B; mix A and B evenly.

[0053] (2) The mixture in (1) was quickly added into 50 mL of 0.4 M aqueous ammonia and stirred at room temperature for 24 h to obtain a yellow solution.

[0054] (3) Centrifuge at 2600 g for 30 min to remove aggregated particles and collect the supernatant.

[0055] (4) Dialyze the supernatant using a 3 kDa dialysis bag for 24 h.

[0056] (5) Centrifuge at 15000 g for 10 min and filter using a 0.22 μm filter.

[0057] (6) freeze-drying and storing at 4°C to obtain cerium dioxide nanoparticles.

[0058] S2: Obtaining functionalized cerium dioxide solution:

[0059] (1) 20 mg of DSPE-MPEG2000 and 10 mg of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] were dissolved in 10 mL of chloroform, and 20 mg of the cerium dioxide nanoparticles obtained in S1 were added.

[0060] (2) The mixture was sonicated for 5 min, and the chloroform was removed from the sample using a rotary evaporator and further dried in a vacuum oven at 60 °C for 3 h to ensure complete removal of chloroform.

[0061] (3) Add 4 mL of deionized water to the sample and treat it with ultrasound to obtain a transparent colloidal suspension.

[0062] (4) Washing by centrifugation at 5000 G to remove excess polyethylene glycol, and purifying with a 0.22 μm filter to obtain a functionalized cerium dioxide solution.

[0063] S3: Preparation of HAMA:

[0064] (1) 1.4 g HA was added to 200 mL DPBS and stirred at 50 °C for 3 h.

[0065] (2) After the hyaluronic acid is completely dissolved, adjust the pH value of the HA solution to 8.5 with 1 M NaHCO3 (or 1 M NaOH), add 12 mL MA dropwise into the bottle, place in an ice water bath, stir for 4 h, and monitor the pH every half an hour to maintain the pH between 7.5 and 8.5.

[0066] (3) The solution after the reaction was poured into a 50 mL centrifuge tube and the centrifuge was set to the centrifugal force mode at 3500 g for 3 min to remove the unreacted MA.

[0067] (4) Pour the HAMA supernatant into a large beaker, add 200 mL of 40°C pure water to dilute, and stir at 600 rpm overnight.

[0068] (5) The HAMA solution was poured into a 12-14 kDa dialysis bag and dialyzed in 40°C pure water for 7 days, with the pure water being changed at least once a day.

[0069] (6) After dialysis, adjust the pH of the HAMA solution to about 7.2 using 1 M NaHCO3 (1 M NaOH can also be used).

[0070] (7) HAMA is obtained by freeze-drying and stored at -80°C for at least one year.

[0071] S4: Preparation of hydrogel system:

[0072] (1) Weigh 200 mg of HAMA and dissolve it in 4 mL of PBS solution and stir.

[0073] (2) After complete dissolution, add 115.09 mg NHS and 218.5 μL LEDC and stir for 30 min.

[0074] (3) Add 400 μg of DNase I and an excess of the functionalized cerium dioxide solution obtained in S2 and stir overnight to react.

[0075] (4) Pour the solution into a dialysis bag (make sure that the small molecular weight molecules of NHS and EDC are dialyzed out, and the molecular weight cut-off is greater than 1 kDa), and dialyze in pure water for 2-3 days, changing the pure water every half a day.

[0076] (5) Freeze-drying for 2-4 days to obtain a multifunctional HAMA hydrogel material.

[0077] The present application also provides the use of the multifunctional HAMA hydrogel material in the preparation of a drug for treating psoriasis lesions.

[0078] The above contents are described below in conjunction with specific embodiments.

[0079] Example 1: Properties of the obtained multifunctional HAMA hydrogel material:

[0080] See also Figure 1 After sampling, fixation, dehydration, drying, and sample conductive treatment, the HAMA hydrogel structure was observed under a microscope. Figure 1 shown.

[0081] Take 20 mg of multifunctional HAMA hydrogel sample and add it to pure water for dispersion, ultrasonicate for 1 min, and then drop it into the test chamber. Wait for the intensity to be moderate, repeat the above three times, and obtain the average result and curve spectrum of the sample. The potential and particle size of the sample are as follows Figure 2 shown.

[0082] Example 2: Verification of enzyme activity of cerium dioxide nanoparticles:

[0083] Experimental steps:

[0084] 1. Prepare instruments: any specification of microplate reader (450±10nm wavelength), ordinary disposable 96-well microplate, micropipette (single-channel pipette, multi-channel pipette), 37℃ constant temperature incubator (or water bath), ordinary low-speed centrifuge

[0085] 2. According to the operation table of Nanjing Jiancheng kit, add the sample to be tested (cerium dioxide nanoparticles), distilled water, enzyme working solution, enzyme diluent, and substrate application solution, gently shake the well plate to mix, incubate at 37℃ for 20 minutes, take out the microplate reader reading at 450nm, and calculate according to the formula described in the manual

[0086] Experimental results:

[0087] The results are as follows Figure 3 As shown, the sample prepared in the present application has enzyme activity, including hydroxyl radical scavenging rate, SOD activity, and has anti-inflammatory properties.

[0088] Example 3: ROS scavenging ability of cerium dioxide nanoparticles

[0089] Experimental steps:

[0090] 1. Prepare cells: Evenly spread the target cells (Raw264.7 cells) in a six-well plate, perform corresponding treatments on the experimental group and the control group, and preset a blank tube (i.e., no probe loaded) until the cell density reaches 80%-95%.

[0091] 2. Washing: Wash the cells in the six-well plate 1-2 times with PBS and discard the PBS.

[0092] 3. Digestion: Use trypsin to digest the cells in the six-well plate, transfer them to a 1.5 mL EP tube, and mark it.

[0093] 4. Centrifugation: 1200 rpm, room temperature, centrifugation for 5 minutes.

[0094] 5. Probe preparation: Prepare 2 μL DCFH-DA stock solution: 3 mL serum-free culture medium, mix by inverting (keep away from light).

[0095] 6. Add probe: discard 4 culture media, add 1mL serum-free culture medium to the blank tube, add 1mL prepared probe solution to the rest, and resuspend the cells (be careful to avoid light).

[0096] 7. Incubation: Incubate at 37°C in dark for 20 min.

[0097] 8. Centrifugation: 1200 rpm, room temperature, centrifuge for 5 minutes, discard the supernatant.

[0098] 9. Washing: Wash with 1 mL of cold PBS, centrifuge at 1200 rpm for 5 min at room temperature, discard the supernatant, and repeat twice (keep away from light).

[0099] 10. Loading: Resuspend the cells in 500 μL cold PBS, transfer to flow cytometry tube, and load onto the machine.

[0100] Experimental results:

[0101] See also Figure 4 The antioxidant properties of the HAMA hydrogel provided in the present invention help neutralize ROS, which is a key factor in the development of psoriasis. The flow cytometry results demonstrate the ability to reduce ROS at different concentrations, thereby reflecting the efficacy of psoriasis.

[0102] Example 4: Multifunctional hydrogel system's ability to remove NETs:

[0103] Experimental steps:

[0104] 1. Peripheral blood (normal human peripheral blood) was centrifuged at 500 g for 10 minutes to obtain blood cells;

[0105] 2. Mix blood cells with saline in a ratio of 1:1, add to a 15 mL centrifuge tube containing 3 mL of ficol and centrifuge at 700 g for 30 minutes;

[0106] 3. Take the white film layer between blood cells and PBMC, add red blood cell lysis solution to split the red blood cells 2-3 times to obtain neutrophil precipitate;

[0107] 4. Count 2*10 6 Each well of secondary neutrophils (24-well plate) was added with 500 μL of 1640 complete culture and 5 μL of PMA stimulator, and the corresponding grouped wells were added to the HAMA hydrogel system incubator and incubated for 4 h;

[0108] 5. DAPI staining and observation under fluorescence microscope.

[0109] Experimental results:

[0110] See also Figure 5 Microscopic observation showed that the synthesized HAMA hydrogel system could successfully remove the DNA skeleton of neutrophil extracellular traps (NETs), thereby eliminating neutrophil extracellular traps in the psoriasis skin microenvironment and alleviating psoriasis skin inflammation.

[0111] Experimental materials and sources of the above embodiments:

[0112] Total Superoxide Dismutase (SOD) Assay Kit Nanjing completed Hydroxyl Radical Test Kit Nanjing completed Reactive oxygen species detection kit Solebao PMA Sigma DAPI Blue Sky

[0113] CeO2 nanoparticles exhibit biological activities similar to superoxide dismutase (SOD), catalase (CAT) and oxidase, and can effectively scavenge hydroxyl radicals and nitric oxide free radicals. 3+ / Ce 4+ When the ratio is high, CeO2 nanoparticles mainly show SOD-like enzyme activity; 3+ / Ce 4+ When the ratio is low, it mainly manifests as CAT-like enzyme activity. This dual characteristic makes CeO2 nanoparticles show great potential in the treatment of various diseases. Compared with other materials, CeO2 nanoparticles have low toxicity to animal cells, simple preparation process, high biocompatibility, and catalytic performance that can be recycled, which has significant application advantages in the biomedical field.

[0114] In view of the enzyme-like activity of CeO2 nanoparticles, in order to eliminate neutrophil extracellular traps (NETs) in the psoriasis skin microenvironment, this application has developed a system of HAMA hydrogel-encapsulated cerium oxide carrying DNase I to degrade NETs, ​​thereby reducing the inflammatory response. This strategy not only targets the removal of NETs, ​​but also achieves local delivery of DNase I to the lesion site through the sustained-release properties of HAMA hydrogel, thereby improving the therapeutic effect and reducing systemic side effects. Through this innovative treatment method, researchers have provided a new perspective for the treatment of psoriasis, which is expected to achieve more effective inflammation control and skin lesion repair.

[0115] In summary, the present invention verifies the successful synthesis of the material through characterization experiments, and draws conclusions through in vitro experiments that the multifunctional hydrogel system has good enzyme activity and biosafety, and can successfully remove the DNA backbone of neutrophil extracellular traps (NETs). This application explores the application of the multifunctional hydrogel system in the treatment of psoriasis lesions, and through research, it is shown that HAMA hydrogel, as an efficient and safe drug delivery carrier, has good application prospects for the treatment of psoriasis.

Claims

1. A multifunctional HAMA hydrogel system, characterized in that: The multifunctional HAMA hydrogel system comprises cerium dioxide, a hyaluronic acid layer is wrapped around the periphery of the cerium dioxide, and the hyaluronic acid layer wrapped around the periphery of the cerium dioxide is also loaded with DNase I.

2. The multifunctional HAMA hydrogel system according to claim 1, characterized in that: The preparation method of the multifunctional HAMA hydrogel system is as follows: S1: Preparation of cerium dioxide nanoparticles; S2: Preparation of functionalized cerium dioxide solution: (1) DSPE-MPEG2000 and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] were dissolved in chloroform, and the cerium dioxide nanoparticles prepared in S1 were added; (2) treating the mixture by ultrasonic treatment, removing the chloroform, and then drying the mixture; (3) adding deionized water to the sample obtained in (2), and then subjecting the sample to ultrasonic treatment to obtain a transparent colloidal suspension; (4) washing the colloidal suspension by centrifugation, and then purifying it by filtering to obtain a functionalized cerium dioxide solution; S3: Preparation of HAMA hydrogel; S4: Preparation of hydrogel system: Weigh HAMA hydrogel and dissolve it in PBS solution, stirring; After complete dissolution, add NHS and EDC and stir; Add DNase I and the functionalized ceria solution obtained in S2 and stir to react overnight; The solution is dialyzed against pure water for 2-3 days, and the pure water is replaced once a day; Freeze-dry for 2-4 days to obtain the multifunctional HAMA hydrogel material.

3. The multifunctional HAMA hydrogel system according to claim 2, characterized in that: In the step S2 (1), the mass ratio of DSPE-MPEG2000, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] and cerium dioxide nanoparticles is 2:1:

2.

4. The multifunctional HAMA hydrogel system according to claim 2, characterized in that: In step (2) of S2, the ultrasonic treatment is performed for 5 minutes, and after the ultrasonic treatment is completed, chloroform is removed from the sample using a rotary evaporator, and then further dried in a vacuum oven at 60° C. for 3 hours to ensure that the chloroform is completely removed.

5. The multifunctional HAMA hydrogel system according to claim 2, characterized in that: In the step (4), the excess polyethylene glycol is removed by washing at 5000G centrifugation, and then purified by a 0.22 μm filter to obtain a functionalized cerium dioxide solution.

6. The multifunctional HAMA hydrogel system according to claim 2, characterized in that: The cerium dioxide nanoparticles are prepared as follows: (1) Weighing cerium nitrate hexahydrate and dissolving it in ddH2O to form liquid A; weighing disodium citrate and dissolving it in ddH2O to form liquid B; mixing A and B evenly; (2) The mixture in (1) was quickly added into 0.4 M ammonia water and stirred at room temperature for 24 h to obtain a yellow solution; (3) Centrifuge at 2600 g for 30 min to remove aggregated particles and collect the supernatant; (4) dialyze the supernatant against a 3 kDa dialysis bag for 24 h; (5) Centrifuge at 15,000 g for 10 min and filter through a 0.22 μm filter; (6) freeze-drying and storing at 4°C to obtain cerium dioxide nanoparticles.

7. The multifunctional HAMA hydrogel system according to claim 3, characterized in that: The HAMA preparation method is as follows: (1) Weigh HA and add it to DPBS, stir at 50°C for 3 h; (2) After the hyaluronic acid is completely dissolved, adjust the pH value of the HA solution to 8.5, add 12 mL of MA dropwise into the bottle, place in an ice water bath, stir for 4 h, and monitor the pH every half an hour to maintain the pH between 7.5 and 8.5; (3) The reacted solution was poured into a 50 mL centrifuge tube and the centrifuge was set to the centrifugal force mode at 3500 g for 3 min to remove the unreacted MA; (4) Pour the HAMA supernatant into a large beaker, add 200 mL of 40°C pure water to dilute, and stir at 600 rpm overnight; (5) HAMA solution was poured into a 12-14 kDa dialysis bag and dialyzed against 40°C pure water for 7 days, with the pure water being changed at least once a day; (6) After dialysis, adjust the pH of the HAMA solution to about 7.2; (7) Freeze-drying to obtain HAMA.

8. Application of multifunctional HAMA hydrogel material in the preparation of a drug for treating psoriasis lesions, characterized in that: The multifunctional HAMA hydrogel material is the multifunctional HAMA hydrogel material according to any one of claims 1 to 7.

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