An egg white protein loaded nitro radical nitrogen oxide nanocomposite material, a preparation method and application thereof

Abstract

The application discloses an egg white loaded nitro radical nitrogen oxide nanocomposite material and a preparation method and application thereof, and belongs to the technical field of material preparation. The nitro radical nitrogen oxide nanocomposite material is regular in shape, has an average particle size of 100nm-150nm, and has good dispersibility. The preparation method is as follows: chitosan and tetramethylpiperidine-nitrogen oxide are respectively dissolved in water and dichloromethane or dimethyl sulfoxide, the two are mixed after stirring and complete dissolution, dialysis is performed after reaction, the material is washed with ultrapure water, centrifuged, and dispersed in ultrapure water after dialysis. In addition, the egg white is loaded on the nitro radical nitrogen oxide nanocomposite material, so that the nanomaterial is endowed with immunogenicity. The material prepared by the method has a magnetic resonance imaging enhancement effect and potential as an immunoadjuvant.

Description

Technical Field

[0001] This invention relates to a method for preparing nanocomposite materials, specifically a method for preparing ovalbumin-loaded nitro radical oxynitride nanocomposite materials. This method is simple and stable and can be widely applied in the fields of biology and medicine. Background Technology

[0002] Cancer has long been one of the leading causes of death worldwide, claiming millions of lives in the 20th century. To combat this threat, scientists have made countless attempts in recent years, pioneering numerous treatment methods. However, due to the heterogeneity of tumors and certain complications, patient survival rates remain low. Therefore, developing more precise tumor diagnostic techniques and effective tumor treatments remains of great practical significance in saving the lives of cancer patients.

[0003] In recent years, combining nanomedicines with cancer therapy has become a major trend in tumor diagnosis and treatment, and some nanomedicines have already been approved for market entry. Nanoscale therapies have emerged as an alternative model for tumor treatment, offering good targeting and fewer side effects, overcoming the limitations of traditional therapies. Among novel cancer treatments, cancer immunotherapy has become the fifth major mainstream therapy after surgery, radiotherapy, chemotherapy, and targeted therapy, achieving remarkable results. Cancer immunotherapy works by training and activating the body's own immune system, enhancing its immune capacity, and then detecting and killing tumor cells. Nanomedicine-based cancer immunotherapy has three unique advantages: first, it awakens the immune system; second, it triggers antigen-specific immunity; and third, it provides long-term immune memory. Nanomaterials can modulate the host's anti-cancer immune response, providing new insights for developing novel cancer treatments.

[0004] With the development of clinical diagnostic technology and biomedical research, developing safer and more effective disease diagnosis and treatment methods has become a top priority. Since W.R. Röntgen's discovery of X-rays, medical imaging technology has evolved for over 100 years, including magnetic resonance imaging (MRI), computed tomography (CT), gamma-ray imaging, and ultrasound imaging for precise diagnosis. MRI, in particular, is a tomographic imaging technique that uses the spin motion of atomic nuclei, excited by radiofrequency pulses under an external magnetic field, to generate signals, which are then processed by a computer to create images. It is one of the most commonly used diagnostic techniques in clinical practice. MRI also enables high spatial resolution and 3D dynamic monitoring of biological tissues. However, in clinical applications, it has been found that the relaxation times of some normal and diseased tissues overlap, leading to diagnostic difficulties. To address this issue, researchers have introduced MRI contrast agents. Currently, most commercially available MRI contrast agents are gadolinium (Gd)-based, but their high nephrotoxicity and potential brain deposition raise concerns about their safety. Therefore, the development of nuclear magnetic resonance contrast agents that are non-invasive to the human body and have excellent biocompatibility has become an inevitable trend and a research hotspot.

[0005] A novel organic MRI contrast agent based on organic nitrogen oxides (TEMPO) was synthesized and loaded with the model antigen OVA (ovalbumin) to improve its biocompatibility and tissue targeting, enhance its anti-tumor effect and ability to activate the body's immune function, and improve the contrast of MRI imaging. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide an ovalbumin-loaded nitro radical oxynitride nanocomposite material with nuclear magnetic resonance enhancement effect and enhanced immune response, and its preparation method.

[0007] To address the technical problem, this invention first provides a method for preparing an ovalbumin-loaded nitro radical oxynitride nanocomposite material, comprising the following steps:

[0008] 1) Dissolve chitosan in ultrapure water, add glacial acetic acid, and mix and stir to obtain solution A;

[0009] 2) Dissolve 4-carboxy-2,2,6,6-tetramethylpiperidine-nitrogen oxide in dichloromethane or dimethyl sulfoxide, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS), and mix and stir at 0℃~5℃ to obtain solution B;

[0010] 3) Add Tween 80 solution to solution A obtained in step 1); then add solution B obtained in step 2), mix to obtain a mixture and stir. After the reaction is complete, dialyze, centrifuge and wash to obtain CS-TEMPO nanoparticles. Disperse the washed CS-TEMPO nanoparticles in ultrapure water to obtain CS-TEMPO nanoparticle dispersion.

[0011] 4) Mix ovalbumin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) at 0℃~5℃ and stir. Then add the CS-TEMPO nanoparticle dispersion obtained in step 3), mix and stir. After the reaction is completed, centrifuge and wash to obtain CS-TEMPO-OVA nanoparticles.

[0012] As a preferred embodiment of the present invention, the degree of deacetylation of the chitosan added to solution A in step 1) is greater than 85%, the molar ratio of chitosan to ultrapure water is 650:1 to 700:1, and the volume ratio of added glacial acetic acid to ultrapure water is 1:100 to 1:200.

[0013] As a preferred embodiment of the present invention, the molar ratio of 4-carboxy-2,2,6,6-tetramethylpiperidine-nitrogen oxide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) added to solution B in step 2) is 1:(0.6-0.7):(0.5-0.7), and the molar ratio of 4-carboxy-2,2,6,6-tetramethylpiperidine-nitrogen oxide and dichloromethane or dimethyl sulfoxide is 4:1-6:1.

[0014] As a preferred embodiment of the present invention, the volume ratio of solution A to solution B in step 3) is 4:3 to 1:1; the volume concentration of the added Tween 80 solution is 1% to 3%; and the volume ratio of the added Tween 80 solution to the mixed solution is 1:35 to 2:35.

[0015] As a preferred embodiment of the present invention, in step 3), the reaction temperature is 5℃~25℃, the reaction conditions are light-protected, the stirring speed is 500-700rpm, and the mixing and stirring time in step 3) is 36h-50h.

[0016] As a preferred embodiment of the present invention, in step 3), the dialysis time is 2 to 4 days and the molecular weight of the dialysis bag is 250 to 300 Da.

[0017] As a preferred embodiment of the present invention, the molar ratio of ovalbumin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl), N-hydroxysuccinimide (NHS) and CS-TEMPO added in step 4) is (100-200):200:(300-400):1.

[0018] As a preferred embodiment of the present invention, the CS-TEMPO-OVA nanoparticles obtained in step 4) are dispersed in PBS and stored at 4°C.

[0019] This invention also provides the application of the above-mentioned ovalbumin-loaded nitro radical oxynitride nanocomposite material in the preparation of nuclear magnetic resonance imaging contrast agents or tumor immunotherapy drugs.

[0020] Compared with the prior art, the beneficial effects of the present invention include:

[0021] (1) The ovalbumin-supported nitro radical oxynitride nanocomposite material (CS-TEMPO-OVA nanoparticles) prepared in this invention has an r1 relaxation rate of 3.1 mM. -1 s -1 It has potential applications in contrast agents for nuclear magnetic resonance imaging;

[0022] (2) The CS-TEMPO nanoparticles loaded with model antigen OVA prepared in this invention also showed an effective effect of improving dendritic cell maturation in vitro, which can indirectly activate T cell immune response, thereby achieving the effect of immunotherapy.

[0023] (3) The preparation method of the present invention has low requirements for experimental instruments, is simple and easy to operate, and produces nanoparticles with uniform shape and size and good dispersibility. Attached Figure Description

[0024] Figure 1 This is a transmission electron microscope (TEM) image of the CS-TEMPO nanoparticles obtained in Example 1 of the present invention.

[0025] Figure 2 This is a transmission electron microscope (TEM) image of the CS-TEMPO-OVA nanoparticles obtained in Example 1 of the present invention.

[0026] Figure 3 The image shows the Fourier Transform Infrared (FT-IR) spectrum of the CS-TEMPO-OVA nanoparticles obtained in Example 1 of this invention.

[0027] Figure 4 The images show nuclear magnetic resonance imaging (NMR) images and T1 relaxation rate ratio diagrams of the CS-TEMPO-OVA nanoparticles obtained in Example 1 of this invention.

[0028] Figure 5 The figure shows the cytotoxicity results of the product obtained in Example 1 of the present invention co-cultured with (b) BMDC cells and (a) 4T1 cells.

[0029] Figure 6 This is a flow cytometry analysis result of CS-TEMPO-OVA nanoparticles obtained in Example 1 of the present invention after co-culturing with BMDC cells for 24 hours.

[0030] Figure 7 This is a flow cytometry analysis result of CS-TEMPO-OVA nanoparticles obtained in Example 1 of the present invention after co-culturing with BMDC cells for 24 hours.

[0031] Figure 8 The image shows the results of an ELISA (enzyme-linked immunosorbent assay) after co-culturing CS-TEMPO-OVA nanoparticles obtained in Example 1 of this invention with BMDC and T cells for 24 hours.

[0032] Figure 9 This is a transmission electron microscope (TEM) image of the product obtained in the comparative example of this invention. Detailed Implementation

[0033] The present invention will be further described and illustrated below with reference to specific embodiments. The embodiments described are merely examples of the content of this disclosure and do not limit the scope of the invention. The technical features of each embodiment in the present invention can be combined accordingly, provided that there is no mutual conflict.

[0034] Example 1

[0035] 500 mg of chitosan with a degree of deacetylation greater than 85% was dissolved in 20 mL of ultrapure water. To ensure complete dissolution, 200 μL of glacial acetic acid was added, and the mixture was stirred for 2 h until completely dissolved and free of bubbles, to prepare solution A. 250 mg of 4-carboxy-2,2,6,6-tetramethylpiperidine-NOx was dissolved in 15 mL of dichloromethane or dimethyl sulfoxide. 180 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and 100 mg of N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at 0 °C in the dark for 0.5 h to prepare solution B. 1 mL of 2% Tween 80 solution was added to solution A, and after complete dissolution, solutions A and B were mixed and stirred at 500 rpm for 48 h at room temperature. After the reaction was complete, the mixture was dialyzed for 2 days using a dialysis bag with a molecular weight of 300 Da. After the dialysis, the mixture was washed with ultrapure water and centrifuged to obtain an orange intermediate product, which was dispersed in 10 mL of ultrapure water to obtain the CS-TEMPO nanoparticle dispersion. 4 mg of ovalbumin (OVA) was dissolved in 1 mL of ultrapure water, and 1 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and 1 mg of N-hydroxysuccinimide (NHS) were added. 1 mL of the obtained CS-TEMPO nanoparticle dispersion was then added, and the mixture was stirred at 0℃~5℃ for 8 h. Centrifugation was then performed to obtain OVA-loaded CS-TEMPO nanoparticles.

[0036] Transmission electron microscopy images of the product obtained in Example 1 are shown below. Figure 1 and 2 As shown, Figure 1 CS-TEMPO nanoparticles Figure 2 The nanoparticles are CS-TEMPO-OVA. The Fourier Transform Infrared (FTIR) spectrum of the obtained product is shown below. Figure 3 As shown, the nuclear magnetic resonance imaging images and T1 relaxation ratios of the obtained products are, for example... Figure 4 As shown. From Figure 1 and 2 As can be seen, the obtained ovalbumin-supported nitro radical oxynitride nanocomposite material (CS-TEMPO-OVA nanoparticles) exhibits good dispersibility and uniform and stable particle size; from Figure 3 As can be seen, the obtained ovalbumin-loaded nitro radical oxynitride nanocomposite material (CS-TEMPO-OVA nanoparticles) does indeed contain CS and TEMPO groups; from Figure 4 As can be seen, the ovalbumin-loaded nitro radical oxynitride nanocomposite material (CS-TEMPO-OVA nanoparticles) has excellent nuclear magnetic resonance enhancement effect.

[0037] like Figure 5As shown, ovalbumin-loaded nitro radical oxynitride nanocomposite materials (CS-TEMPO-OVA nanoparticles) of different concentrations were co-cultured with 4T1 (mouse breast cancer cells) and BMDC (mouse bone marrow-derived dendritic cells) cells for 24 h. Cell viability was detected by CCK-8 assay. Ovalbumin-loaded nitro radical oxynitride nanocomposite materials (CS-TEMPO-OVA nanoparticles) showed good biocompatibility.

[0038] like Figure 6 and Figure 7 As shown, a certain concentration of ovalbumin-loaded nitro radical oxynitride nanocomposite material (CS-TEMPO-OVA nanoparticles) and BMDC (mouse bone marrow-derived dendritic cells) cells were co-cultured for 24 h. After staining with flow cytometry antibodies CD11c, CD80 and CD86, the results were detected by flow cytometry. It was found that CS-TEMPO-OVA nanoparticles can efficiently promote the maturation of dendritic cells.

[0039] like Figure 8 As shown, a certain concentration of ovalbumin-loaded nitro radical oxynitride nanocomposite material (CS-TEMPO-OVA nanoparticles) was co-cultured with BMDC (mice bone marrow-derived dendritic cells) cells and T (mouse spleen T cells) cells for 24 h. The concentration of cytokines in the culture supernatant was then detected by an ELISA enzyme-linked immunosorbent assay kit. It was found that CS-TEMPO-OVA nanoparticles can activate the T cell immune response and promote the production of cytokines that kill cancer cells by T cells, thus proving that this nanocomposite material has a good anti-tumor effect.

[0040] Example 2

[0041] 500 mg of chitosan with a degree of deacetylation greater than 85% was dissolved in 20 mL of ultrapure water. To ensure complete dissolution, 200 μL of glacial acetic acid was added, and the mixture was stirred for 2 h until completely dissolved and free of bubbles, to prepare solution A. 250 mg of 4-carboxy-2,2,6,6-tetramethylpiperidine-NOx was dissolved in 20 mL of dimethyl sulfoxide, and 180 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and 100 mg of N-hydroxysuccinimide (NHS) were added. The mixture was stirred at 0 °C in the dark for 0.5 h to prepare solution B. 3 mL of 2% Tween 80 solution was added to solution A. Solutions A and B were then mixed and stirred at 500 rpm for 48 h at room temperature. Afterwards, dialysis was performed using a dialysis bag with a molecular weight of 300 Da for 2 days. Afterwards, the sample was washed with ultrapure water and centrifuged to obtain an orange-colored product, which was further dispersed in 10 mL of ultrapure water to obtain the CS-TEMPO nanoparticle dispersion. 5 mg of ovalbumin (OVA) was dissolved in 1 mL of ultrapure water, and 1 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and 1 mg of N-hydroxysuccinimide (NHS) were added. 1 mL of the obtained CS-TEMPO nanoparticle dispersion was then added, and the mixture was stirred at 0℃~5℃ for 8 h. Centrifugation was then performed to obtain CS-TEMPO-OVA nanoparticles.

[0042] Comparative Example

[0043] 500 mg of chitosan with a degree of deacetylation greater than 85% was dissolved in 20 mL of ultrapure water. To ensure complete dissolution, 200 μL of glacial acetic acid was added, and the mixture was stirred for 2 h until completely dissolved and free of bubbles, to prepare solution A. 250 mg of 4-carboxy-2,2,6,6-tetramethylpiperidine-nitride was dissolved in 20 mL of dichloromethane or dimethyl sulfoxide. 200 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and 100 mg of N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at room temperature in the dark for 1 h to prepare solution B. After complete dissolution, solutions A and B were mixed and stirred at 500 rpm for 48 h at room temperature. After the reaction was complete, the mixture was dialyzed using a dialysis bag with a molecular weight of 300 Da for 2 days. After the dialyzed reaction, the mixture was washed with ultrapure water, centrifuged, and dispersed in 10 mL of ultrapure water to obtain the CS-TEMPO nanoparticle dispersion. 5 mg of ovalbumin (OVA) was dissolved in 1 mL of ultrapure water, 1 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and 1 mg of N-hydroxysuccinimide (NHS) were added, and 1 mL of the obtained CS-TEMPO nanoparticle dispersion was added. The mixture was stirred at room temperature for 8 h and centrifuged to obtain CS-TEMPO-OVA nanoparticles.

[0044] like Figure 9 The image shown is a transmission electron microscope (TEM) image of the product obtained in the comparative example of this invention; from Figure 9 As can be seen, the ovalbumin-loaded nitro radical oxynitride nanocomposite material (CS-TEMPO-OVA nanoparticles) obtained through this comparative example has an uneven particle size and poor dispersion.

[0045] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for preparing an ovalbumin-supported nitro radical oxynitride nanocomposite material, characterized in that, Includes the following steps: 1) Dissolve chitosan in ultrapure water, add glacial acetic acid, and mix and stir to obtain solution A; 2) Dissolve 4-carboxy-2,2,6,6-tetramethylpiperidine-nitrogen oxide in dichloromethane or dimethyl sulfoxide, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC.HCl and N-hydroxysuccinimide NHS, and mix and stir at 0℃~5℃ to obtain solution B; 3) Add Tween 80 solution to solution A obtained in step 1); then add solution B obtained in step 2), mix to obtain a mixture and stir. After the reaction is complete, dialyze, centrifuge and wash to obtain CS-TEMPO nanoparticles. Disperse the washed CS-TEMPO nanoparticles in ultrapure water to obtain CS-TEMPO nanoparticle dispersion. 4) Mix ovalbumin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS) at 0℃~5℃, then add the CS-TEMPO nanoparticle dispersion obtained in step 3), mix and stir, and centrifuge and wash after the reaction to obtain CS-TEMPO-OVA nanoparticles.

2. The method for preparing the ovalbumin-supported nitro radical oxynitride nanocomposite material according to claim 1, characterized in that, The chitosan added to solution A in step 1) has a deacetylation degree greater than 85%. 500 mg of chitosan with a deacetylation degree greater than 85% is dissolved in 20 mL of ultrapure water. The volume ratio of glacial acetic acid to ultrapure water is 1:100 to 1:

200.

3. The method for preparing the ovalbumin-supported nitro radical oxynitride nanocomposite material according to claim 1, characterized in that, In step 3), the volume ratio of solution A to solution B is 4:3 to 1:1; the volume concentration of the added Tween 80 solution is 1% to 3%, and the volume ratio of the added Tween 80 solution to the mixed solution is 1:35 to 2:

35.

4. The ovalbumin-supported nitro radical oxynitride nanocomposite material according to claim 1, characterized in that, In step 3), the reaction temperature is 5℃~25℃, the reaction conditions are to avoid light, the stirring speed is 500-700 rpm, and the stirring time is 36 h-50 h.

5. The method for preparing the ovalbumin-supported nitro radical oxynitride nanocomposite material according to claim 1, characterized in that, In step 3), the dialysis time is 2-4 days, and the molecular weight of the dialysis bag is 250-300 Da.

6. The method for preparing the ovalbumin-supported nitro radical oxynitride nanocomposite material according to claim 1, characterized in that, The CS-TEMPO-OVA nanoparticles obtained in step 4) were dispersed in PBS and stored at 4 °C.

7. An ovalbumin-loaded nitro radical oxynitride nanocomposite material prepared by the preparation method according to any one of claims 1-6.

8. The use of the ovalbumin-loaded nitro radical oxynitride nanocomposite material according to claim 7 in the preparation of nuclear magnetic resonance imaging contrast agents or tumor immunotherapy drugs.

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