Application of selenoprotein nanoparticles in preparation of medicine for preventing and treating diabetic macrovascular complications
By coating selenoprotein onto the surface of inorganic nanoparticles, selenoprotein nanoparticles were prepared, solving the problem of poor in vivo delivery stability of selenoprotein S and achieving a highly efficient and safe treatment effect for diabetic macrovascular complications.
Patent Information
- Application Number
- CN202511342371.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-19
- Publication Date
- 2025-12-26
AI Technical Summary
In existing technologies, selenoprotein S has poor delivery stability in vivo and limited targeting effects, making it difficult to effectively prevent and treat macrovascular complications of diabetes.
Inorganic nanoparticles were used as carriers to coat selenoproteins onto their surfaces. Selenoprotein nanoparticles were prepared by incubation, washing, and centrifugation, maintaining the exposure of their active structural domains and improving stability and biocompatibility.
This study achieves the stability of selenoproteins in vivo and the effective exposure of their active structural domains, improving the prevention and treatment of macrovascular complications of diabetes. Furthermore, the preparation process is simple and the materials are highly safe.
Smart Images
Figure CN121197355A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application relates to the technical field of biological medicine, and particularly relates to application of a selenoprotein nanoparticle in preparation of a medicine for preventing and treating diabetic macrovascular complications. BACKGROUND
[0002] With the aggravation of global aging, the incidence of diabetes is increasing year by year. Diabetic complications are numerous, and diabetic cardiovascular lesions are one of the main and dangerous complications of diabetes, which are often manifested as atherosclerosis. Endothelial dysfunction occurs in the important stage of the occurrence and development of atherosclerosis and cardiovascular diseases. Therefore, the development of a medicine capable of protecting diabetic vascular endothelial dysfunction will greatly promote the prevention and treatment process of diabetic cardiovascular diseases.
[0003] Selenoprotein is a kind of protein containing selenocysteine, which widely exists in organisms. Studies have found that in vivo regulation of selenoprotein S shows specificity in the prevention and treatment of diabetic macrovascular complications. However, the prevention and treatment of diabetic macrovascular complications by in vivo regulation of selenoprotein S is indirect regulation, the method is complex, the content of selenoprotein S in vivo is low, and the targeting effect is limited. At the same time, protein drugs have poor in vivo delivery stability and are easily removed by the circulatory system. Therefore, how to stabilize the in vivo delivery stability of in vitro expressed selenoprotein S and still exhibit its specificity is crucial.
[0004] Nanoprotein crown is a drug-loaded form in which protein is adsorbed on the surface of nanoparticles. How to adsorb selenoprotein on the surface of nanoparticles while still maintaining its drug activity is still worth breaking through and innovating. SUMMARY
[0005] The application aims to provide application of a selenoprotein nanoparticle in preparation of a medicine for preventing and treating diabetic macrovascular complications.
[0006] In order to achieve the above-mentioned purpose of the application, the technical scheme adopted by the application is as follows:
[0007] Application of a selenoprotein nanoparticle in preparation of a medicine for preventing and treating diabetic macrovascular complications.
[0008] The selenoprotein nanoparticle is a mixture of inorganic nanosphere particles and selenoprotein with biological activity, so that the selenoprotein is coated on the surface of the inorganic nanosphere particles.
[0009] Further, the selenoprotein nanoparticle is:
[0010] (1) a selenoprotein plasmid with a label is transferred into an expression host, the host expresses and amplifies the protein, and then total protein obtained is purified to obtain selenoprotein;
[0011] (2) dissolving the selenoprotein obtained in step (1) and slowly dropping into the well-dispersed inorganic nanoparticle solution; wherein the selenoprotein and inorganic nanoparticle have a molar ratio of (10000-1:1);
[0012] (3) after mixing, the purified selenoprotein nanoparticle is prepared by incubation, washing, and centrifugation.
[0013] The selenoprotein is human and / or non-human selenoprotein S.
[0014] The label is selected from one of the recognition labels that can obtain the selenoprotein from the total protein in the purification process, not limited to histone tag, maltose binding protein tag, etc.
[0015] The expression host is selected from one of the hosts that can express selenoprotein, not limited to Escherichia coli.
[0016] The inorganic nanoparticle is a soluble, surface negatively charged, highly biocompatible nanoparticle with a particle size of 1-100 nm. For example: gold, silicon dioxide, titanium dioxide nanoparticles.
[0017] In step (3), the incubation is carried out at 25-37℃ for 30min-24h. The washing is carried out for more than or equal to 3 times. The centrifugal speed is 2000-20000rpm.
[0018] Compared with the prior art, the present application has the following beneficial effects:
[0019] The present application combines the protein drug selenoprotein S stably on the surface of the nanoparticle, and the binding domain and the nanoparticle stably combined can stabilize the stability of the protein drug, and the active domain exposed on the surface of the selenoprotein nanoparticle still maintains the activity of the protein drug. Further:
[0020] (1) The present application uses inorganic nanoparticles as the drug carrier of selenoprotein, which can improve the stability of selenoprotein by binding to the binding region of selenoprotein, and is not easily degraded by various enzymes in the body.
[0021] (2) The present application uses inorganic nanoparticles as the drug carrier of selenoprotein, which can fully and effectively expose the active region of selenoprotein, and improve the effect of selenoprotein.
[0022] (3) The selenoprotein nanoparticle prepared by the present application still has high biocompatibility and good biological activity; the nanoparticle preparation process is simple, the materials used are all highly biocompatible, the composition is simple, and the safety is high.
[0023] (4) The selenoprotein nanoparticles prepared by the application still have the prevention and treatment value of selenoprotein for diabetic macroangiopathy, and are more stable and efficient, and have a longer action time. BRIEF DESCRIPTION OF DRAWINGS
[0024] Figure 1 is the Western Blot result of selenoprotein obtained after expression and purification.
[0025] Figure 2 is the transmission electron microscopy characterization result of the inorganic nanoparticles prepared.
[0026] Figure 3 is the activity identification result of selenoprotein and selenoprotein nanoparticles.
[0027] Figure 4 is the comparison result of the protective effect of selenoprotein, inorganic nanoparticles and selenoprotein nanoparticles on diabetic vascular endothelial injury. DETAILED DESCRIPTION
[0028] The application is further described below in combination with specific examples, but should not be understood as limiting the application. Based on the examples in the application, all other examples obtained by modifying or replacing the application by those skilled in the art without creative labor shall fall within the scope of protection of the application.
[0029] Unless otherwise defined in the application, the technical and scientific terms used in the examples are the conventional technical and scientific terms known to those skilled in the art. Unless otherwise stated in the application, the reagents used in the examples are analytical pure or above.
[0030] The selenoprotein nanoparticles of the application are prepared by gold nanoparticles with chemical groups on the surface, so that selenoprotein is combined with inorganic nanoparticles on the surface by physical or chemical methods. The obtained selenoprotein nanoparticles have high stability and can prolong the half-life of selenoprotein. At the same time, the selenoprotein nanoparticles prepared by the application have excellent biocompatibility, good activity, stability and high efficiency, simple preparation process, and can be used as a new type of nanoprotein drug for the prevention and treatment of diabetic macrovascular complications and even more metabolic diseases and their complications, providing a very promising protein drug optimization model.
[0031] Moreover, the application adopts a nano-protein crown drug model, coats selenoprotein on the surface of inorganic nanoparticles with high biocompatibility, so that the selenoprotein nano-protein crown in the particles can still maintain the structure and activity of selenoprotein; and the selenoprotein nano-protein crown can prolong the action time of selenoprotein in the body.
[0032] The selenoprotein S used in the following examples is human selenoprotein S.
[0033] Expression and purification of selenoprotein
[0034] First, the constructed selenoprotein S plasmid was transfected into E. coli in vivo for selenoprotein expression, which specifically included adding 1 uL of selenoprotein plasmid with histone tag (i.e., the plasmid carried selenoprotein sequence and tag, the plasmid was pET-28a(+), and the selenoprotein sequence was referred to NCBI, Gene ID: 109815, NM_024439.3) into the BL21 E. coli solution and mixing uniformly, ice bath for 30 minutes; then, after ice bath for 2 minutes at 42℃ for 60 seconds, the suspension was cultured in a conventional LB medium for 1 hour (37℃); the suspension was added to the conventional LB solid medium for plating, and cultured at 37℃ overnight; finally, a single colony was picked up and amplified in a liquid medium (37℃); when the OD value of the medium reached 0.6, IPTG (isopropyl-β-D-thiogalactoside) was added for protein induction expression. Finally, the E. coli after expressing the protein was lysed to obtain whole protein (see Figure 1 ), and then the selenoprotein was grabbed using a nickel-containing magnetic bead, and the selenoprotein solution was replaced by a purification column to finally obtain a simple and easily preserved selenoprotein solution.
[0035] By Figure 1 The Western Blot identification result of the selenoprotein after expression and purification showed that a single selenoprotein product could be obtained by the above conditions.
[0036] Example 2 Preparation of inorganic nanoparticles
[0037] Gold nanoparticles were synthesized by sodium citrate reduction method, and the specific preparation steps included: first, 100 mL of 0.5 mmol / L chloroauric acid solution was heated to 97℃ in a microwave reactor; after stabilizing for 5 minutes, 2.5 mL of 0.1 mol / L sodium citrate solution was injected into the chloroauric acid solution with vigorous stirring, and the whole reaction was kept at 97℃ for 20 minutes; finally, the suspension was cooled to room temperature, and the nanoparticles were obtained by centrifugation under the conditions of 12000g, 25 minutes, and 4℃ (see Figure 2 ).
[0038] By Figure 2 The electron microscope characterization graph of the prepared inorganic gold nanoparticles showed that spherical nanoparticles with a particle size of 30 nm were finally obtained.
[0039] Example 3 Preparation of selenoprotein nanoparticles
[0040] The preparation steps include: firstly, rinsing the gold nanoparticles obtained in Example 2 above with 20 mmol / L 4-hydroxyethylpiperazine ethanesulfonic acid solution for at least 3 times, centrifuging to remove the supernatant, and then using a phosphoric acid buffer to dissolve the nanoparticles by using an ultrasonic disperser to prevent aggregation of the nanoparticles; then dissolving the selenoprotein S obtained in Example 1 above with 4-hydroxyethylpiperazine ethanesulfonic acid solution; according to the mass / volume ratio, the ratio of 0.5 mg / mL gold nanoparticles to 1 mg / mL selenoprotein, slowly adding the protein solution to the nanoparticle solution, and incubating together at 37°C for 1 hour; finally, removing the supernatant by high-speed centrifugation, repeating the rinsing 3 times to remove free proteins, and finally obtaining selenoprotein nanoparticles in which the selenoprotein is adsorbed to the surface of the nanoparticles.
[0041] Example 4: Activity verification of selenoprotein nanoparticles
[0042] In order to maintain the activity of the selenoprotein in the protein crown, the entire process of expressing the protein and constructing the protein crown needs to be maintained in a 4°C environment. The activity verification steps include: firstly, adding 10 μL of a phosphoric acid buffer, the selenoprotein purified in Example 1, and the selenoprotein nanoparticle sample obtained in Example 3 in sequence and separately in a 96-well plate, with three repeats; then adding 190 μL of a hydrogen peroxidase activity detection working solution in sequence, and detecting the absorbance value using an enzyme marker at a fixed absorbance. The enzyme activity value is obtained according to the conversion formula, with the unit defined as 1 μmol of H2O2 degraded per minute in the reaction system per mg of protein, and defined as one enzyme activity unit (see Figure 3 ).
[0043] From the comparison of the activities of the selenoprotein and the selenoprotein nanoparticles, it can be seen that the selenoprotein combined to the surface of the inorganic nanoparticles still has good selenoprotein activity. Figure 3
[0044] Example 5: Comparison of the effects of selenoprotein S and selenoprotein S nanoparticles on resisting high-sugar-induced vascular endothelial injury
[0045] In this example, a diabetic vascular endothelial injury model is constructed by stimulating human aortic endothelial cells with high sugar. The control glucose concentration is 5.5 mmol / L, and the high-sugar concentration is 30 mmol / L. Selenoprotein, inorganic nanoparticles, and selenoprotein nanoparticles are added before high-sugar stimulation for protection, and a total of five groups are set up, with three repeats for each group. The stimulation time is set to 0 h, 8 h, 24 h, and 48 h. The endothelial injury factor is determined for each group, and the sample is added and the absorbance is determined by an enzyme marker for the above groups at the same time.
[0046] Figure 4 The column chart of protective effects of selenoprotein, inorganic nanoparticles and selenoprotein nanoparticles on endothelial injury. The results show that human aortic endothelial cells are damaged when stimulated by high glucose for 24 h; at 24 h, the protective effect of selenoprotein on endothelial injury is equivalent to that of selenoprotein nanoparticles, but at 48 h, the protective effect of selenoprotein nanoparticles on endothelial injury is superior to that of selenoprotein. It is suggested that the stability of selenoprotein nanoparticles is superior to that of selenoprotein, and the time effectiveness of selenoprotein nanoparticles is superior to that of selenoprotein.
Claims
1. Use of selenoprotein nanoparticles in the preparation of a medicament for preventing and treating diabetic macrovascular complications.
2. Use according to claim 1, characterized in that: The selenoprotein nanoparticles are obtained by mixing inorganic nanospheres with bioactive selenoprotein, so that the selenoprotein is coated on the surface of the inorganic nanospheres.
3. Use according to claim 2, characterized in that: The selenoprotein nanoparticles are (1) introducing a selenoprotein plasmid with a label into an expression host, expressing and amplifying the protein by the host, purifying the total protein obtained to obtain selenoprotein; (2) slowly dropping the selenoprotein obtained in step (1) into a solution of inorganic nanoparticles which is fully dispersed, wherein the molar ratio of selenoprotein to inorganic nanoparticles is (10000-1:1); (3) obtaining the purified selenoprotein nanoparticles by incubation, washing and centrifugation after mixing.
4. Use according to claim 2 or 3, characterized in that: The selenoprotein is human and / or non-human selenoprotein S.
5. Use according to claim 2 or 3, characterized in that: The inorganic nanoparticles are soluble, surface negatively charged, high biocompatibility nanoparticles with a particle size of 1-100 nm.
6. Use according to claim 3, characterized in that: In step (3), the incubation is carried out at 25-37℃ for 30 min-24 h, the washing is carried out for no less than 3 times, and the centrifugal speed is 2000-20000 rpm.