Oral and injectable sinomenine-gallic acid nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses an oral and injectable sinomenine-gallic acid nano-particle as well as a preparation method and application thereof. A nano-drug is prepared through micromolecule self-assembly. The preparation method adopts a one-step self-assembly process and comprises the following steps: adding 1-10 parts by weight of sinomenine and 1-10 parts by weight of gallic acid into a PBS buffer solution, heating at 60 DEG C for 2 hours, then gradually adding into the PBS buffer solution at 60 DEG C, and continuously stirring and heating for 4 hours. Subsequently, the sample is placed in a dialysis bag having a molecular weight cutoff (MWCO) of 100 Da, and dialyzed with ultrapure water for 12 hours to remove impurities. And finally, the self-assembled sinomenine-gallic acid nanoparticles are successfully obtained. The preparation method disclosed by the invention has the characteristics of simple formula and easy process operation, has oral and injection use modes, and is small in dosage, short in treatment course and remarkable in curative effect.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to sinomenine-gallic acid nanoparticles for oral and injectable nanoparticle medicines for treating sepsis and a preparation method thereof. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection, which is common in patients with severe trauma or infectious diseases. Its causes include infections such as bacteria, fungi, viruses and parasites, which lead to imbalances in the body's inflammatory response and immune regulation. Sepsis can be divided into common sepsis, severe sepsis and septic shock. Globally, more than 19 million people suffer from sepsis each year, of which about 6 million patients die and about 3 million suffer from cognitive dysfunction.

[0003] At present, there are many patent documents that record Chinese medicine compositions for treating or preventing sepsis. For example, Chinese invention patent CN 118697819 A discloses a Chinese medicine composition for treating sepsis. The method uses a variety of Chinese medicines such as Andrographis paniculata, Forsythia suspensa, Anemarrhena asphodeloides, Vaccaria segetalis, Barberry, Scrophularia ningpoensis and Coptis chinensis to prepare a supernatant by mixing, hydrolyzing, filtering and centrifuging, and then freeze-drying to obtain the final drug. Although this method can prepare drugs for treating sepsis, its process is complicated. The mixed drug will lose some effective ingredients during the filtration and centrifugation process, and the freeze-drying process may cause changes in the structure of the active ingredients, thereby reducing the utilization rate of the drug. For example, Chinese invention patent CN 118615352 A discloses a Chinese medicine composition for treating or preventing sepsis. Its preparation method uses a variety of Chinese medicines such as Taraxacum officinale, Ligusticum chuanxiong, Panax notoginseng, Cinnamomum cassia, Aster ternata, Rhizoma scutellariae and Herba Prunellae to prepare decoctions, mixtures, granules, pills, tablets, capsules or powders in different proportions. Although this method can prepare a variety of dosage forms, it has the problems of complex process and inconsistent quality control. In addition, the drug takes a long time to take effect and requires a long course of treatment. Since the above method is only applicable to oral administration, it is particularly important to develop a new drug with simple preparation process, stable efficacy and both oral and injection functions.

[0004] Small molecule self-assembly is an efficient method to construct nanostructures through non-covalent interactions such as hydrogen bonding, electrostatic interactions, π-π stacking, and hydrophobic interactions. The nanoparticles, micelles, or fibers formed have unique physicochemical properties and show broad application prospects in drug delivery, tissue engineering, and biosensing. Compared with traditional methods, small molecule self-assembly has the advantages of simple operation, strong structural controllability, and adjustable functions. In addition, the versatility and responsiveness of the self-assembly system enable it to achieve precise release and targeting in specific environments, becoming a research hotspot in the current fields of materials science and biomedicine. Summary of the invention

[0005] The purpose of the present invention is to provide an oral and injectable sinomenine-gallic acid nanoparticle for treating sepsis, and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution: A method for preparing an oral and injectable nanoparticle drug for treating sepsis comprises the following steps: (1) Weigh 1-10 parts by weight of sinomenine and 1-10 parts by weight of gallic acid and add them into 1-10 parts by weight of PBS buffer to obtain a mixture; (2) Further, the mixture in (1) was heated in a water bath at 60° C. for 2 hours; (3) Further, the mixture solution in (2) is gradually added into 1-10 parts by weight of PBS buffer at 60° C., and the heating and stirring are continued for 4 hours; (4) Further, the mixture solution in (3) is placed in a dialysis bag (Mw = 100 Da); (5) Furthermore, the dialysis bag was placed in ultrapure water for 12 h to obtain an oral or injectable drug for the treatment of sepsis.

[0007] (6) The present invention uses a sepsis mouse model induced by intraperitoneal injection of lipopolysaccharide (LPS) into C57BL / 6 mice, and finds that the drug of the present invention can significantly reduce the degree of damage to the lung tissue of sepsis mice. These results indicate that the drug of the present invention has a good effect in treating and / or preventing sepsis and can be used to develop drugs for sepsis diseases.

[0008] The present invention provides sinomenine gallic acid nanoparticles (abbreviated as SGNPs) which can be taken orally or injected for treating sepsis and are prepared by the above method.

[0009] Preferably, the oral administration and injectable administration include oral gavage, intraperitoneal injection and tail vein injection.

[0010] Preferably, the sepsis is an acute inflammatory disease such as acute pulmonary dysfunction, cardiac dysfunction and renal dysfunction.

[0011] Preferably, the dosage for intraperitoneal injection is 80-100 mg / kg; the dosage for tail vein injection is 80 mg / kg.

[0012] The present invention has the following beneficial effects: (1) The present invention improves the insolubility of sinomenine by small molecule self-assembly. This method has the advantages of no carrier, simple synthesis and good biocompatibility. This study provides a new direction for the high-value utilization of insoluble small molecules.

[0013] (2) The raw materials selected in the present invention are sinomenine and gallic acid monomers, which have simple ingredients and stable efficacy.

[0014] (3) The raw material sinomenine selected in the present invention has good anti-inflammatory effect and a wide range of applications, and can have a good therapeutic effect on inflammatory diseases such as rheumatoid arthritis.

[0015] (4) The SGNPs provided by the present invention have good stability at room temperature or in solution state and will not decompose for at least one week.

[0016] (5) The SGNPs provided by the present invention can prevent and reduce LPS-induced septic mouse lung inflammation, reduce pulmonary edema, and reduce the content of inflammatory factors in blood vessels through three administration methods: pre-oral gavage, intraperitoneal injection, and tail vein injection.

[0017] (6) Gallic acid has multiple effects such as anti-oxidation, anti-inflammatory, immunomodulatory, antibacterial and multi-organ protection. The sinomenine-gallic acid nanoparticles based on nanotechnology of the present invention significantly improve the stability, solubility and bioavailability of the drug, while enhancing the therapeutic effect and reducing toxic side effects. However, the existing sinomenine hydrochloride injection, as the hydrochloride form of sinomenine, has good therapeutic effects but still has certain toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is the full-wavelength ultraviolet scanning image of the SGNPs prepared in Example 1; Figure 2 The infrared spectrum of SGNPs prepared in Example 1; Figure 3 1HNMR spectrum of SGNPs prepared in Example 1; Figure 4 Transmission electron microscopy image (left) and particle size image (right) of SGNPs prepared in Example 1; Figure 5 Hemolysis experiment of SGNPs prepared in Example 1; Figure 6 Stability determination of SGNPs prepared in Example 1; Figure 7HE staining diagram of the protective effect of tail vein injection of SGNPs prepared in Example 1 on the LPS-induced sepsis mouse model; Figure 8 Determination of lung tissue mRNA for the protective effect of tail vein injection of SGNPs prepared in Example 1 on LPS-induced sepsis mouse model; Fig. 9 HE staining diagram of the protective effect of intragastric administration of SGNPs prepared in Example 1 on the LPS-induced sepsis mouse model; Fig.10 Determination of lung tissue mRNA for the protective effect of oral administration of SGNPs prepared in Example 1 on the LPS-induced sepsis mouse model; Fig.11 HE staining of the therapeutic effect of the SGNPs prepared in Example 1 on the LPS-induced sepsis mouse model via tail vein injection; Fig.12 Determination of lung tissue mRNA for the therapeutic effect of tail vein injection of SGNPs prepared in Example 1 on the LPS-induced sepsis mouse model. DETAILED DESCRIPTION

[0020] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. The described examples are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] The present invention provides a method for preparing an oral and injectable nanoparticle drug for treating sepsis, comprising the following steps: (1) Weigh 2 parts by weight of sinomenine and 1 part by weight of gallic acid, add them into 3 parts by weight of PBS buffer (0.01 M, pH = 7.2-7.4), stir vigorously to obtain a mixture; (2) Further, the mixture in (1) was heated in a water bath at 60°C for 2 hours; (3) Further, the mixture solution in (2) was gradually added into 10 parts by weight of PBS buffer (0.01 M, pH = 7.2-7.4) at 60° C., and the mixture was heated and stirred for 4 hours; (4) Further, the mixture solution in (3) is placed in a dialysis bag (MWCO = 100 Da); (5) Furthermore, the dialysis bag was placed in ultrapure water for 12 h to obtain an oral or injectable drug for the treatment of sepsis.

[0022] The SGNPs provided by the present invention have been experimentally verified to be able to effectively inhibit the inflammatory expression of mice induced by sepsis and alleviate organ dysfunction, thereby providing a new medical means for the clinical treatment of sepsis animals, and having great research significance and application prospects.

[0023] In the above preferred embodiment, the increase in sepsis-induced inflammation includes: high expression of IL-6 and IL-1β in blood serum; high expression of IL-6, IL-1β, iNOS, PTGS 2 and IL-10 in tissues.

[0024] In the above preferred embodiment, the sepsis-induced multiple organ dysfunction includes: pulmonary dysfunction, cardiac dysfunction, liver dysfunction and renal dysfunction. In the present invention, pulmonary dysfunction will be selected for research.

[0025] The drug for treating and / or preventing sepsis provided by the present invention can effectively alleviate sepsis-induced inflammation and pulmonary organ dysfunction.

[0026] In a preferred embodiment of the present invention, the dosage form of the drug is oral and injection.

[0027] In a preferred embodiment of the present invention, the diluent for the drug injection is physiological saline.

[0028] In the above preferred embodiment, the administration route of the injection includes one of intravenous injection and intraperitoneal injection.

[0029] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0030] Example 1 (1) Establishing experimental animal models: Male C57BL / 6 mice aged 8 weeks and weighing between 20-25 g were used to establish a mouse sepsis model by intraperitoneal injection of lipopolysaccharide (LPS). The specific modeling method is as follows: 1 mg / mL LPS was prepared with normal saline and injected intraperitoneally into C57BL / 6 mice at a dose of 10 mg / kg.

[0031] The experimental animals were randomly grouped as follows: intraperitoneal injection of normal saline served as the control group (n=6); Intraperitoneal injection of LPS was used as the sepsis model group (n=6); The patients were injected with SGNPs (low dose, 20 mg / kg) through the tail vein for three days and LPS was injected on the third day as the low-dose protection group (n=6); The patients were injected with SGNPs (high dose, 80 mg / kg) through the tail vein for three days and LPS was injected on the third day as the high-dose protection group (n=6); Oral gavage of SGNPs (low dose, 25 mg / kg) for three days, and injection of LPS on the third day served as the low-dose protection group (n=6); Oral administration of SGNPs (high dose, 100 mg / kg) for three days, and injection of LPS on the third day served as a low-dose protection group (n=6); LPS was injected intraperitoneally, and SGNPs (low dose, 20 mg / kg) were injected into the tail vein 1 hour later as the low-dose treatment group (n=6); LPS was injected intraperitoneally, and SGNPs (high dose, 80 mg / kg) were injected into the tail vein 1 hour later as the low-dose treatment group (n=6); In the above preferred embodiments, the dosage of LPS is 10 mg / kg, injected intraperitoneally.

[0032] In the above preferred embodiment, when studying the preventive and protective effect of the drug, the experimental scheme is that SGNPs are gavage / injected three days before gavage / tail vein injection / or intraperitoneal injection of the drug, and LPS is injected one hour after the SGNPs drug is given on the third day. The mice are euthanized 24 hours after the LPS injection, and the mouse serum and tissues are collected for subsequent testing.

[0033] In the above preferred embodiment, when studying the therapeutic effect of the drug, a single administration method is used, that is, LPS is injected intraperitoneally to establish a mouse sepsis model. After 1 hour, SGNPs are injected into the tail vein. The mice are euthanized 24 hours after the LPS injection, and the mouse serum and tissues are collected for subsequent testing.

[0034] (2) Detection of mouse serum inflammatory factors and lung tissue damage markers. The specific methods are as follows: 24 hours after intraperitoneal injection of LPS, blood was collected from the eyeball of mice, and after standing at room temperature for 30 minutes, the blood was centrifuged at 3000 rpm for 20 minutes to separate serum and plasma. The collected serum was used to detect mouse interleukin-6 (IL-6) and mouse interleukin-1β (IL-1β) using ELISA kits.

[0035] 24 hours after the intraperitoneal injection of LPS, blood was collected through the eyeballs of the mice, and then the mice were euthanized. The chest cavity of the mice was carefully cut open, and the lung tissue was fully irrigated. The lungs were removed and immersed in PBS for cleaning, and then the water was absorbed and used for subsequent experiments such as the determination of inflammatory factor mRNA.

[0036] For tissue fixation, 24 hours after intraperitoneal injection of LPS, blood was collected through the eyeballs of mice, and then the mice were euthanized. The chest cavity of the mice was carefully cut open, and the lung tissue was fully lavaged and then injected with 4% paraformaldehyde, and then immersed in 4% paraformaldehyde for subsequent section staining.

[0037] The specific experimental results are as follows: 1. The samples were tested using different spectroscopic methods and compared with the KP / PI physical mixture and individual drugs: first Figure 1 The results of the full-wavelength UV scan showed that SIN (sinomenine) and GA (gallic acid) showed maximum absorbance (λmax) at 260 and 220 nm, respectively. The SGNPs (sinomenine-gallic acid nanoparticles) sample showed a combination of two peaks corresponding to the constituent drugs, with no red shift or blue shift, indicating that the structures of the two drugs had not changed. However, the intensity of these two peaks was significantly lower than that of a single drug, although the concentration of each drug was comparable. This indicates that the interaction between the two drugs is significant.

[0038] 2. From FTIR spectrum data ( Figure 2 ), the mixture spectrum shows all the main peaks of SIN (sinomenine) and GA (gallic acid), while SGNPs (sinomenine-gallic acid nanoparticles) have a peak at 3427 cm −1 The broad peak at shows that hydrogen bonds were formed between the carboxyl OH group of GA (gallic acid) and the carbon group of SIN (sinomenine).

[0039] 3. 1 H NMR spectra of different samples Figure 3 To further determine the type and location of the weak interaction between SIN and GA, we measured the 1 H NMR and compared with monomers SIN and GA under the same conditions. 1 H NMR spectrum showed that the hydroxyl signal of GA in SGNPs disappeared, indicating the possible formation of hydrogen bonds between SGNPs.

[0040] 4. Figure 4 The transmission electron microscopy image of the SGNPs provided in this embodiment was measured, and as shown, the SGNPs sample was monodispersed, spherical, and had a uniform size distribution, with a particle size of about 63 nm.

[0041] 5. Figure 5 The hemolytic activity of the SGNPs sample was evaluated by direct contact method. The hemolytic activity of the SGNPs sample was greatly reduced. As shown in the figure, the supernatant is transparent, the precipitated red blood cells are shown in the figure, and green is the sample color of the SGNPs.

[0042] 6. Figure 6 The stability of the SGNPs provided in this example was determined, and the particle size of the SGNPs was stable for at least one week.

[0043] 7. Figure 7 The SGNPs provided in this embodiment can prevent and protect pulmonary edema, alveolar wall thickening, and pulmonary congestion in the lung tissue of septic mice by tail vein injection. Representative images of HE staining. The alveolar epithelial cells of the lung tissue of the control group were arranged in an orderly manner, with a neat structure, and no alveolar interstitial edema or alveolar wall thickening. Compared with the control group, the alveolar wall of the model group was significantly thickened, with infiltration of neutrophils and monocytes, accompanied by increased alveolar interstitial exudate, alveolar edema, and congestion. This shows that the mouse acute lung injury model was successfully constructed. Compared with the model group, SGNPs can inhibit extensive alveolar wall thickening, inflammatory cell infiltration, and interstitial edema to varying degrees, and are concentration-dependent.

[0044] 8. Figure 8 The SGNPs provided in this embodiment can reduce the content of inflammatory factor mRNA in the lung tissue of septic mice by tail vein injection. The mRNA content was determined by qPCR. As shown in the figure, IL-6 and IL-1β in the septic mouse group were significantly increased by 286.07±123.59 and 17.37±2.47 times, while compared with the model group, the inflammatory factors in the SGNPs prevention and protection group were significantly decreased, among which IL-6 and IL-1β in the low-dose administration group decreased to 109.54±89.31 and 13.56±3.5 times, respectively, and IL-6 and IL-1β in the high-dose administration group decreased to 8.85±5.39 and 8.60±2.59 times, respectively, and were concentration-dependent.

[0045] 9. Fig. 9 The SGNPs provided in this embodiment can prevent and protect pulmonary edema, alveolar wall thickening, and pulmonary congestion in the lung tissue of septic mice by oral gavage. Representative images of HE staining. The alveolar epithelial cells of the lung tissue of the control group were arranged in an orderly manner, with a neat structure, and no alveolar interstitial edema or alveolar wall thickening. Compared with the control group, the alveolar wall of the model group was significantly thickened, with infiltration of neutrophils and monocytes, and accompanied by increased alveolar interstitial exudate, alveolar edema, and congestion. This shows that the mouse acute lung injury model was successfully constructed. Compared with the model group, the low-dose and high-dose SGNPs in the experimental group can inhibit extensive alveolar wall thickening, inflammatory cell infiltration and interstitial edema to varying degrees, and are concentration-dependent. SIN is used as a positive control group.

[0046] 10. Fig.10The SGNPs provided in this embodiment can reduce the content of inflammatory factor mRNA in the lung tissue of septic mice by oral gavage. The content of mRNA is determined by qPCR. As shown in the figure, IL-6 and IL-1β in the septic mouse group are significantly increased by 38.92±28.23 times and 24.12±8.23, while compared with the model group, the inflammatory factors in the SGNPs prevention and protection group are significantly decreased, among which IL-6 and IL-1β in the low-dose administration group are decreased to 13.98±12.41 and 16.84±6.95 times, respectively, and IL-6 and IL-1β in the high-dose administration group are decreased to 4.62±5.11 and 7.02±4.31 times, respectively, and are concentration-dependent. SIN as a positive control group is decreased by 6.43±6.08 and 17.65±10.97 times, respectively. From the above results, it can be seen that SGNPs have better efficacy in septic mice.

[0047] 11. Fig.11 The SGNPs provided in this embodiment can treat pulmonary edema, alveolar wall thickening, and pulmonary congestion in the lung tissue of septic mice by a single tail vein injection. Representative images of HE staining. The alveolar epithelial cells of the lung tissue of the control group were arranged in an orderly manner, with a neat structure, and no alveolar interstitial edema or alveolar wall thickening. Compared with the control group, the alveolar wall of the model group was significantly thickened, with infiltration of neutrophils and monocytes, accompanied by increased alveolar interstitial exudate, alveolar edema, and congestion. This shows that the mouse acute lung injury model was successfully constructed. Compared with the model group, SGNPs can inhibit extensive alveolar wall thickening, inflammatory cell infiltration, and interstitial edema to varying degrees, and are concentration-dependent.

[0048] 12. Fig.12 The SGNPs provided in this embodiment can reduce the content of mRNA of inflammatory factors in the lung tissue of septic mice through a single tail vein injection. The mRNA content was determined by qPCR. As shown in the figure, IL-6 and IL-1β in the septic mouse group were significantly increased by 309.61±13.12 and 13.75±2.85 times, while compared with the model group, the inflammatory factors in the SGNPs prevention and protection group were significantly decreased, among which IL-6 and IL-1β in the low-dose administration group decreased to 138.35±97.1 and 12.45±2.85 times, respectively, and IL-6 and IL-1β in the high-dose administration group decreased to 10.35±5.17 and 8.56±4.27 times, respectively, and were concentration-dependent.

[0049] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing orally administrable and injectable sinomenine-gallic acid nanoparticles, characterized in that: The following steps are involved: (1) adding 1-10 parts by weight of sinomenine and 1-10 parts by weight of gallic acid into PBS buffer to obtain a mixture; (2) heating the mixture obtained in step (1) at 60° C. for 2 hours; (3) gradually adding the mixture solution obtained in step (2) into 60°C PBS buffer, and continuing heating and stirring for 4 hours; (4) The mixture solution was then dialyzed for 12 h to obtain orally and injectably available sinomenine-gallic acid nanoparticles.

2. The preparation method according to claim 1, characterized in that: The amount of PBS buffer used in step (1) is 1-10 parts by weight.

3. The preparation method according to claim 1, characterized in that: The concentration of PBS buffer in step (1) is 0.01 M and the pH is 7.2-7.

4.

4. The preparation method according to claim 1, characterized in that: The amount of PBS buffer used in step (3) is 1-10 parts by weight.

5. The preparation method according to claim 1, characterized in that: The concentration of PBS buffer in step (3) is 0.01 M and the pH is 7.2-7.

4.

6. The preparation method according to claim 1, characterized in that: The heating method in step (2) is water bath heating.

7. The preparation method according to claim 1, characterized in that: The water used for dialysis in step (4) is distilled water or ultrapure water.

8. The preparation method according to claim 1, characterized in that: The molecular weight cutoff MWCO of the dialysis bag used for dialysis in step (4) is 100 Da.

9. Orally administrable and injectable sinomenine-gallic acid nanoparticles prepared according to the preparation method according to any one of claims 1 to 8.

10. Use of the orally administrable and injectable sinomenine-gallic acid nanoparticles according to claim 9 in the preparation of a drug for treating sepsis.

Images