Polyethylene glycol modified DNase I-loaded hollow mesoporous cerium dioxide nanoparticles and preparation method thereof

Abstract

The invention provides polyethylene glycol modified DNase I-loaded hollow mesoporous cerium dioxide nanoparticles and a preparation method thereof, and relates to the technical field of biomedicine, and the preparation method is as follows: the preparation method comprises the following steps: preparing hollow mesoporous cerium dioxide by a hydrothermal method; functionalizing the surface of the hollow mesoporous cerium dioxide: realizing surface amino functionalization through APTES modification, further utilizing an EDC / NHS cross-linking technology, loading DNase I and connecting polyethylene glycol in sequence, and preparing nanoparticles with high stability and good biocompatibility. Experimental verification shows that the obtained nanoparticles retain the biological activity of DNase I, and simultaneously show antioxidant and anti-inflammatory effects. According to the preparation method of the DNase I-loaded hollow mesoporous cerium dioxide nano-particles provided by the invention, efficient enzyme loading is realized by optimizing material preparation and functionalization processes, and the DNase I-loaded hollow mesoporous cerium dioxide nano-particles have a remarkable anti-inflammatory effect and good biocompatibility.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to polyethylene glycol-modified DNase I-loaded hollow mesoporous cerium dioxide nanoparticles and a preparation method thereof. Background Art

[0002] In recent years, h-mCeO 2 It has been widely studied for its efficient scavenging ability for reactive oxygen species (ROS) and is used to treat inflammatory diseases (such as inflammatory bowel disease). However, existing preparation technologies often have the following shortcomings: First, the preparation process is complicated and cannot take into account the uniformity, stability and high loading capacity of the material at the same time; second, during the functional modification process, surface modification is difficult to meet the subsequent efficient loading requirements of bioactive molecules; third, the loaded biomolecules (such as DNA enzymes) are easily reduced in activity due to the unsuitable interface environment of the material, affecting the therapeutic effect. Therefore, it is necessary to develop an h-mCeO that can be efficiently prepared, stably functionalized and able to maintain the activity of biomolecules. 2 Nanoparticles have important research value.

[0003] As a nuclease that can degrade extracellular DNA (eDNA), DNA enzyme has shown unique therapeutic potential in the treatment of various inflammatory-related diseases. eDNA is an important component in pathological processes such as extracellular traps (NETs) and is involved in the occurrence of inflammatory responses and tissue damage. By removing eDNA in the pathological environment, DNA enzyme can effectively reduce the inflammatory response and improve the pathological state of the disease. Therefore, delivering DNA enzyme to target and remove eDNA has become an emerging anti-inflammatory treatment strategy.

[0004] However, the application of DNA enzymes in vivo faces many challenges, including its poor stability in complex body fluid environments and easy enzymatic hydrolysis or inactivation. How to improve the loading efficiency of DNA enzymes, protect their activity and achieve controlled release remains a technical difficulty. Traditional carrier materials have low loading efficiency and limited protection for DNA enzymes, resulting in reduced activity and affecting the therapeutic effect. Summary of the invention

[0005] The purpose of the present invention is to solve the technical problems that the loading efficiency of traditional carrier materials is low and the protective effect on DNase I is limited.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing polyethylene glycol-modified hollow mesoporous cerium dioxide nanoparticles loaded with DNase I comprises the following steps:

[0008] S1: Preparation of hollow mesoporous ceria by hydrothermal method;

[0009] S2: Functionalized hollow mesoporous cerium oxide surface: The hollow mesoporous cerium oxide prepared in S1 was added to the APTES solution for reaction; after the reaction, the modified hollow mesoporous cerium oxide was repeatedly washed with ethanol, and finally vacuum dried and stored at 4°C.

[0010] S3: Loading DNase I: dissolve the functionalized hollow mesoporous ceria, EDC and NHS obtained in S2 in PBS, stir to react, then add DNase I solution, continue to stir to react, after the reaction is completed, wash with PBS, freeze-dry to obtain hollow mesoporous ceria loaded with DNase I;

[0011] S4: Polyethylene glycol modification: The hollow mesoporous ceria loaded with DNase I obtained in S3, EDC and NHS were dissolved in PBS, stirred for reaction, and polyethylene glycol was added after the reaction was completed, and the stirring for reaction was continued. After the reaction was completed, PBS was used for washing, and the hollow mesoporous ceria loaded with DNase I modified with polyethylene glycol was obtained after freeze-drying.

[0012] Preferably, in the hydrothermal method in S1, cerium nitrate hexahydrate, ethylene glycol and acetic acid are added to pure water to obtain a mixed solution, and the mixed solution is subjected to static reaction and calcination to prepare hollow mesoporous cerium dioxide nanoparticles.

[0013] Preferably, the ratio of the added amounts of pure water, acetic acid, ethylene glycol and cerium nitrate hexahydrate in S1 is: (1.0-2.0 mL): (1.0-2.0 mL): (30.0-60.0 mL): (1.0-2.0 g).

[0014] Preferably, the static reaction step in S1 is as follows: placing a mixture of cerium nitrate hexahydrate, ethylene glycol and acetic acid added to pure water in a polytetrafluoroethylene-lined reactor for static reaction, and placing the reactor in a 180° C. blower for 2-4 hours.

[0015] Preferably, the calcination step of S1 is: calcining the reactants obtained by static reaction in a box furnace, raising the temperature to 300° C. at 1° C. / min, and maintaining the temperature for 1 hour.

[0016] Preferably, the concentration of the APTES solution in S2 is 1-5 wt %, and anhydrous ethanol is used as a solvent.

[0017] Preferably, after the hollow mesoporous ceria in S2 is added with the APTES solution, the reaction conditions are: reacting at room temperature or 60° C. for 12-24 hours.

[0018] The present application also provides a polyethylene glycol-modified DNase I-loaded hollow mesoporous cerium dioxide nanoparticle, which is prepared using the above-described preparation method.

[0019] Compared with the prior art, this application has the following beneficial effects:

[0020] In this application, hollow mesoporous cerium dioxide is prepared by a hydrothermal method. The process is simple and controllable. The prepared nanoparticles have a uniform particle size (about 80nm), a high specific surface area and a stable hollow structure, which ensures the high efficiency of the material in subsequent modification and loading processes.

[0021] In addition, the present invention realizes efficient loading of DNase I through surface functionalization technology and EDC / NHS cross-linking strategy, while effectively protecting its activity, avoiding the problem of significant reduction in enzyme activity when traditional carriers load DNase I. The loaded DNase I can stably exist and maintain its ability to degrade extracellular DNA (eDNA) in a complex body fluid environment, thereby significantly improving the biological treatment effect. The biocompatibility, circulation stability and targeting of the nanoparticles are significantly enhanced through polyethylene glycol (PEG) modification.

[0022] In addition, the polyethylene glycol-modified hollow mesoporous ceria loaded with DNase I prepared in this application can significantly inhibit inflammatory response by scavenging reactive oxygen species (ROS) and degrading eDNA. In the model of inflammatory bowel disease, the nanoparticles showed good anti-inflammatory effects and therapeutic potential, providing an efficient solution for the treatment of inflammation-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is characterized by scanning SEM (A), TEM (B), XRD spectrum (C), and EDS-Mapping analysis (D) of the hollow mesoporous cerium dioxide nanospheres.

[0024] Figure 2 1. The zeta potential (A) and particle size distribution diagram (B) of the hollow mesoporous cerium dioxide nanospheres of the present invention. Figure 3 The present invention discloses a N2 adsorption-desorption isotherm (A) and a pore size distribution (B) of the hollow mesoporous ceria nanospheres.

[0025] Figure 4 It is the XPS spectrum of the hollow mesoporous cerium dioxide nanospheres in the present invention.

[0026] Figure 5 The h-mCeO 2 @DNase I EDS-Mapping analysis.

[0027] Figure 6The h-mCeO 2 @DNase I(A) and h-mCeO 2 @DNase I@PEG TEM (B) image, particle size map (C), potential map (D, E), and Fourier analysis (F).

[0028] Figure 7 The invention relates to ROS detection of hollow mesoporous cerium dioxide nanoparticles with different concentrations.

[0029] Figure 8 The h-mCeO 2 Detection of the NETs clearing ability of @DNase I@PEG. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with specific embodiments.

[0031]

[0032] A method for preparing polyethylene glycol-modified hollow mesoporous cerium dioxide nanoparticles loaded with DNase I comprises the following steps:

[0033] S1: Preparation of hollow mesoporous ceria:

[0034] Cerium nitrate hexahydrate, ethylene glycol and acetic acid are added into pure water to obtain a mixed solution. The mixed solution is subjected to static reaction and calcination to prepare hollow mesoporous cerium dioxide nanoparticles with a particle size of about 80 nm.

[0035] Specifically, in one embodiment, the ratio of the added amounts of pure water, acetic acid, ethylene glycol, and cerium nitrate hexahydrate is: (1.0-2.0 mL): (1.0-2.0 mL): (30.0-60.0 mL): (1.0-2.0 g).

[0036] Static reaction: After obtaining the mixed solution, place the mixed solution in a polytetrafluoroethylene-lined reactor for static reaction, and place the reactor in a blast box at 180°C for 2-4 hours. 。

[0037] Calcination: The reactants obtained by static reaction were calcined in a box furnace, the temperature was increased to 300°C at 1°C / min, and maintained for 1 hour.

[0038] S2: Functionalized hollow mesoporous ceria surface:

[0039] First, an APTES solution is prepared, wherein the concentration of the APTES solution is 1-5 wt %, and anhydrous ethanol is used as a solvent.

[0040] The hollow mesoporous ceria obtained in S1 is added to the APTES solution and reacted at room temperature or 60° C. for 12-24 hours. In one embodiment, the reaction concentration of the hollow mesoporous ceria is 1-10 mg / mL.

[0041] After the reaction, the modified hollow mesoporous ceria was repeatedly washed with ethanol to remove APTES and by-products, and finally vacuum dried and stored at 4 °C.

[0042] S3: Load DNase I

[0043] The functionalized hollow mesoporous cerium dioxide, EDC and NHS prepared by S2 were dissolved in PBS and stirred for 2 hours. The ratio of NHS:EDC was 1:1 and the reaction concentration was 0.01M-0.1M.

[0044] DNase I was added to a reaction concentration of 0.1-1 mg / mL, and stirring was continued at room temperature for 4-24 hours. After the reaction, PBS was used to wash three times, and freeze-dried using a freeze dryer. The obtained DNase I-loaded hollow mesoporous cerium dioxide was stored at 4°C.

[0045] S4: Polyethylene glycol modification

[0046] The DNase I loaded hollow mesoporous ceria obtained in S3, EDC and NHS were dissolved in PBS, the NHS:EDC ratio was 1:1, the reaction concentration was 0.01M-0.1M, and stirred for 2h.

[0047] Polyethylene glycol was added to make the reaction concentration of the polyethylene glycol solution 1-10 mg / mL, and stirring was continued at room temperature for 4-24 hours. After the end, PBS was used to wash 3 times, and freeze-dried in a freeze dryer to obtain polyethylene glycol-modified DNase I-loaded hollow mesoporous ceria. The polyethylene glycol-modified DNase I-loaded hollow mesoporous ceria was stored at 4°C.

[0048] Based on the above preparation method, the present application also provides a polyethylene glycol-modified DNase I-loaded hollow mesoporous ceria.

[0049] The above contents are described below in conjunction with specific embodiments:

[0050] Example 1: Preparation of polyethylene glycol-modified hollow mesoporous ceria loaded with DNase I:

[0051] S1: Preparation of hollow mesoporous ceria by hydrothermal method:

[0052] Weigh cerium nitrate hexahydrate, ethylene glycol, acetic acid and pure water, add cerium nitrate hexahydrate, ethylene glycol and acetic acid to pure water in sequence to obtain a mixed solution, place the mixed solution in a polytetrafluoroethylene-lined reactor, and place the reactor in a blast box at 180° C. to react for 2 hours;

[0053] The product obtained by static reaction was placed in a box furnace for calcination, the temperature was raised to 300° C. at 1° C. / min, and maintained for 1 hour to obtain hollow mesoporous cerium dioxide.

[0054] See also Figure 1 , SEM ( Figure 1 A) indicates that hollow mesoporous cerium dioxide nanospheres were successfully synthesized with a particle size of about 80 nm, and transmission electron microscopy ( Figure 1 B) shows that the hollow mesoporous cerium dioxide nanospheres are uniform in size and have a rough porous, hollow spherical structure. Figure 1 C is the XRD spectrum of the prepared hollow mesoporous cerium dioxide, which is used to show the three-dimensional fluorite structure of hollow mesoporous cerium dioxide CeO 2 Characteristic diffraction peaks, Figure 1 D is EDS-Mapping analysis, which is used to show the spatial distribution of Ce and O elements in hollow mesoporous ceria.

[0055] The prepared hollow mesoporous cerium dioxide is as follows Figure 2-Figure 4 As shown, the particle size distribution diagram is 80±10.64nm ( Figure 2 A), the Zeta potential shows a potential of -36.04 ( Figure 2 B);

[0056] N 2 The adsorption-desorption isotherm curve indicates that the specific surface area is 152.58263 m 2 / g( Figure 3 A); The average pore size of hollow mesoporous cerium dioxide nanospheres is 9.99987nm ( Figure 3 B).

[0057] XPS spectrum results show that the e 3+ / (Ce 3+ +Ce 4+ )(%) is 13.75( Figure 4 ).

[0058] S2: Functionalized hollow mesoporous ceria surface:

[0059] The hollow mesoporous cerium dioxide obtained in S1 is added to an APTES solution, wherein the concentration of the APTES solution is 5wt%, and anhydrous ethanol is used as a solvent; the reaction concentration of the hollow mesoporous cerium dioxide is 1 mg / mL, and the reaction is carried out at room temperature for 12 hours to obtain functionalized hollow mesoporous cerium dioxide.

[0060] S3: Hollow mesoporous ceria loaded with DNase I:

[0061] Weigh the functionalized hollow mesoporous ceria, EDC and NHS in S2 and dissolve them in PBS, stirring for 2 h;

[0062] Then, DNase I solution was added and stirring was continued at room temperature for 12 hours. After the stirring was completed, PBS was used to wash three times. After freeze-drying in a freeze dryer, the hollow mesoporous cerium dioxide loaded with DNase I was obtained and stored at 4°C.

[0063] See also Figure 5 , EDS-Mapping analysis shows the spatial distribution of Ce, O, and S elements in the hollow mesopores of ceria loaded with DNase I.

[0064] Also see Figure 6 The obtained DNase I loaded hollow mesoporous ceria transmission electron microscopy is as follows Figure 6 A, and, as Figure 6 C and Figure 6 As shown in D, the particle size of the obtained DNase I-loaded hollow mesoporous cerium dioxide is 108 nm and the potential is -15.74.

[0065] S4: Modification with polyethylene glycol:

[0066] The hollow mesoporous ceria loaded with DNase I in S3, EDC, and NHS were weighed and dissolved in PBS and stirred for 2 h;

[0067] Polyethylene glycol was added and stirring was continued at room temperature for 6 hours. Afterwards, the mixture was washed with PBS and freeze-dried to obtain polyethylene glycol-modified DNase I-loaded hollow mesoporous ceria that was stored at 4°C.

[0068] The obtained polyethylene glycol-modified hollow mesoporous ceria loaded with DNase I is as follows Figure 6 B, and, as Figure 6 C and Figure 6 As shown in E, the particle size of the polyethylene glycol-modified hollow mesoporous ceria loaded with DNase I is 116 nm, and its potential is -17.21.

[0069] See also Figure 6 F, Figure 6 F is h-mCeO2 Fourier analysis of @DNase I@PEG shows that 1637 cm -1 The C=O stretching vibration peak on the protein amide can be seen at 1558 cm -1 The absorption peak at 1109 cm is the NH bending vibration of DNase I. -1 、1035cm -1 The two characteristic peaks are CO stretching vibrations on polyethylene glycol.

[0070] Example 2: ROS scavenging ability of hollow mesoporous ceria

[0071] 1. Prepare cells: Raw264.7 cells were evenly plated in six-well plates and divided into control, H 2 O 2 group, 10μg / mL, 50μg / mL, 100μg / mL intervention group (hollow mesoporous cerium dioxide), density 5×10 5 cells / well and incubate overnight.

[0072] 2. Dosing: Except for the control group and H 2 O 2 After 12 hours of intervention with hollow mesoporous ceria, H 2 O 2 (400 μM) for 4 hours.

[0073] 3. Washing: Wash the cells in the six-well plate 1-2 times with culture medium and discard.

[0074] 4. Probe preparation: 1 μL DCFH-DA stock solution: 1 mL serum-free culture medium, pipette and mix.

[0075] 5. Add the probe: prepare 1 μL DCFH-DA stock solution: 1 mL serum-free culture medium, pipette to mix well, and add to a 6-well plate, 1 mL per well.

[0076] 6. Incubation: Incubate at 37°C in dark for 20 min.

[0077] 7. Washing: Wash the cells in the six-well plate three times with culture medium, and transfer to a 1 mL centrifuge tube after pipetting.

[0078] 8. Centrifugation: 700g / min, room temperature, centrifuge for 5 minutes, discard the supernatant.

[0079] 9. On-line: Resuspend the cells in 400 μL cold PBS, transfer to a flow cytometer, and analyze using an LSR Fortessa flow cytometer.

[0080] The results are as follows Figure 7As shown, hollow mesoporous cerium oxide has the ability to scavenge ROS, and the scavenging activity increases with the increase of concentration.

[0081] Example 3: Verification of h-mCeO 2 Detection of the NETs clearing ability of @DNase I@PEG.

[0082] 1. Take 2 mL of normal human peripheral blood and centrifuge at 800 g for 5 minutes, discard the serum;

[0083] 2. Mix the remaining blood cells with saline in a ratio of 1:1, then add equal proportions of ficol (blood: PBS: ficol = 1:1:1) at 45°C, centrifuge at 800g for 20 minutes, discard the supernatant and PBMC, and keep the buffy coat and red blood cells;

[0084] 3. Add red blood cell lysis buffer, pipette and mix, centrifuge at 500g for 20 minutes, and discard the supernatant. Repeat the above steps 1-2 times. Add 8mL PBS to resuspend, centrifuge at 500g for 5 minutes, discard the supernatant, obtain the neutrophil precipitate, and add 1mL PBS to count;

[0085] 4. The cells were divided into control group, PMA stimulation group, DNase I group, h-mCeO 2 @DNase I@PEG, cells were added to a 24-well plate, with 5*10 cells per well. 5 (24-well plate), incubated in an incubator for 4 h;

[0086] 5. DAPI staining and observation under fluorescence microscope.

[0087] The results showed that h-mCeO 2 @DNase I@PEG can effectively remove NETs.

[0088] Materials and sources in the above embodiments of this application:

[0089] Material source Cerium nitrate Aladdin Acetic acid Sinopharm Ethylene glycol Sinopharm DNaseI Roche Polyethylene glycol Maclean ROS Detection Kit Blue Sky EDC Shanghai Yuanye NHS Sigma

[0090] In summary, the present application prepares hollow mesoporous cerium dioxide by a hydrothermal method. The process is simple and controllable. The prepared nanoparticles have uniform particle size (about 80 nm), high specific surface area and stable hollow structure, ensuring the high efficiency of the material in subsequent modification and loading processes.

[0091] In addition, the present invention realizes efficient loading of DNase I through surface functionalization technology and EDC / NHS cross-linking strategy, while effectively protecting its activity, avoiding the problem of significant reduction in enzyme activity when traditional carriers load DNase I. The loaded DNase I can stably exist and maintain its ability to degrade extracellular DNA (eDNA) in a complex body fluid environment, thereby significantly improving the biological treatment effect. The biocompatibility, circulation stability and targeting of the nanoparticles are significantly enhanced through polyethylene glycol (PEG) modification.

[0092] In addition, the polyethylene glycol-modified hollow mesoporous ceria loaded with DNase I prepared in this application can significantly inhibit inflammatory response by scavenging reactive oxygen species (ROS) and degrading eDNA. In the model of inflammatory bowel disease, the nanoparticles showed good anti-inflammatory effects and therapeutic potential, providing an efficient solution for the treatment of inflammation-related diseases.

Claims

1. A method for preparing polyethylene glycol-modified hollow mesoporous cerium dioxide nanoparticles loaded with DNase I, characterized in that: The following steps are involved: S1: Preparation of hollow mesoporous ceria by hydrothermal method; S2: Functionalized hollow mesoporous ceria surface: The hollow mesoporous ceria prepared in S1 was added to the APTES solution for reaction; after the reaction, the modified hollow mesoporous ceria was repeatedly washed with ethanol, and finally vacuum dried and stored at 4°C; S3: Loading DNase I: dissolve the functionalized hollow mesoporous ceria, EDC and NHS obtained in S2 in PBS, stir to react, then add DNase I solution, continue to stir to react, after the reaction is completed, wash with PBS, freeze-dry to obtain DNase I loaded hollow mesoporous ceria; S4: Polyethylene glycol modification: The hollow mesoporous ceria loaded with DNase I obtained in S3, EDC and NHS were dissolved in PBS, stirred for reaction, and polyethylene glycol was added after the reaction was completed, and the stirring for reaction was continued. After the reaction was completed, PBS was used for washing, and the hollow mesoporous ceria loaded with DNase I modified with polyethylene glycol was obtained after freeze-drying.

2. The method for preparing a polyethylene glycol-modified DNase I-loaded hollow mesoporous ceria nanoparticle according to claim 1, characterized in that: The hydrothermal method in S1 is as follows: adding cerium nitrate hexahydrate, ethylene glycol and acetic acid into pure water to obtain a mixed solution, and the mixed solution is subjected to static reaction and calcination to prepare hollow mesoporous cerium dioxide nanoparticles.

3. The method for preparing a polyethylene glycol-modified DNase I-loaded hollow mesoporous ceria nanoparticle according to claim 2, characterized in that: The ratio of the added amounts of pure water, acetic acid, ethylene glycol and cerium nitrate hexahydrate in the S1 is: (1.0-2.0 mL): (1.0-2.0 mL): (30.0-60.0 mL): (1.0-2.0 g).

4. The method for preparing a polyethylene glycol-modified DNase I-loaded hollow mesoporous cerium dioxide nanoparticle according to claim 3, characterized in that: The static reaction step in S1 is as follows: a mixture of cerium nitrate hexahydrate, ethylene glycol and acetic acid added to pure water is placed in a polytetrafluoroethylene-lined reactor for static reaction, and the reactor is placed in a 180° C. blower for 2-4 hours.

5. The method for preparing a polyethylene glycol-modified DNase I-loaded hollow mesoporous ceria nanoparticle according to claim 4, characterized in that: The calcination step of S1 is: calcining the reactants obtained by static reaction in a box furnace, raising the temperature to 300° C. at 1° C. / min, and maintaining for 1 hour.

6. The method for preparing a polyethylene glycol-modified DNase I-loaded hollow mesoporous ceria nanoparticle according to claim 5, characterized in that: The concentration of the APTES solution in S2 is 1-5 wt %, and anhydrous ethanol is used as a solvent.

7. The method for preparing a polyethylene glycol-modified DNase I-loaded hollow mesoporous ceria nanoparticle according to claim 6, characterized in that: After the hollow mesoporous cerium dioxide in S2 is added with the APTES solution, the reaction conditions are: reacting at room temperature or 60° C. for 12-24 hours.

8. A polyethylene glycol-modified DNase I-loaded hollow mesoporous ceria nanoparticle, characterized in that: The method is prepared by any one of claims 1 to 7.

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