CN112972392A

CN112972392A - Bilirubin nano-particles and preparation and application thereof

Info

Publication number: CN112972392A
Application number: CN202110259715.9A
Authority: CN
Inventors: 寇龙发; 姚情; 陈瑞杰; 林光勇; 蒋欣宇; 汤莹莹
Original assignee: Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
Current assignee: Second Affiliated Hospital and Yuying Childrens Hospital of Wenzhou Medical University
Priority date: 2021-03-10
Filing date: 2021-03-10
Publication date: 2021-06-18
Anticipated expiration: 2041-03-10
Also published as: CN112972392B

Abstract

The invention discloses bilirubin nanoparticles and preparation and application thereof. The nano-particles are formed by self-assembling bilirubin as a single component according to a specific preparation method; firstly, bilirubin is dissolved in an organic solvent, secondly, the prepared bilirubin solution is slowly dripped into a buffer aqueous solution, and after stirring and mixing, the organic solvent is removed to obtain the bilirubin nano-particles. The bilirubin nanoparticle prepared by the invention has the advantages of 100% drug-loading rate, single component, good stability and excellent clinical application prospect, provides a new mode and way for developing a carrier-free nano delivery drug system, and meets the urgent need of clinical inflammation-related diseases on treatment of high-end preparations.

Description

Bilirubin nano-particles and preparation and application thereof

Technical Field

The invention belongs to a nanoparticle in the technical field of nano-drugs and preparation and application thereof, and particularly relates to a bilirubin nanoparticle and preparation and application thereof.

Background

Bilirubin is a normal catabolite present in heme metabolism. As early as the 80's of the last century, researchers in Stocker et al have proposed that bilirubin is a physiologically significant antioxidant whose antioxidant capacity increases with decreasing oxygen concentration and even exceeds that of alpha-tocopherol under the same experimental conditions. Clinical studies in recent years show that bilirubin levels are negatively associated with various inflammatory diseases, and higher bilirubin levels have a significant gain effect on the occurrence and development of inflammatory diseases. Modern researches show that bilirubin has anti-inflammatory, antioxidant and anti-apoptosis effects, and the therapeutic effect is realized by regulating and controlling NF-kappa B, MAPK signal pathways, endoplasmic reticulum stress and other action mechanisms. Bilirubin can promote repair and regeneration of inflammatory-stimulated damaged cells and significantly reduce serum levels of NO and iNOS. It is worth noting that bilirubin can directly scavenge ROS and be converted into biliverdin, and biliverdin can be converted into bilirubin under the action of biliverdin reductase, so that clinically significant cell protection effect can be achieved under low concentration of bilirubin, and toxic and side effects caused by high concentration are avoided.

Although bilirubin is expected to treat inflammatory diseases by regulating oxidative stress and inflammatory signal pathways, the bilirubin has low solubility, poor stability and half-life period, and the common injection strategy cannot meet the preparation requirements.

The nano-drug delivery system can effectively increase the solubility of the drug, improve the stability, increase the accumulation of the drug at the inflammation part through the size effect and play the role of drug therapy. The bilirubin is chemically combined with polyethylene glycol to form nano particles, and the nano particles are used for intravenous injection drug treatment of colitis. However, PEG chemical modification is complex in preparation and high in cost, the formed nano structure is poor in stability, and the introduction of PEG is easy to induce anaphylactic reaction. In addition, researchers use high molecular polymers, such as poloxamer, polylactic acid-glycolic acid copolymer and the like, to construct nano-carriers of bilirubin nanoparticles, so that the in vivo delivery efficiency of the bilirubin nanoparticles is improved. However, such artificially synthesized polymer materials have problems of poor hydrophilicity, generation of acidic products due to local degradation, and the like. In recent years, natural biomaterials such as silk fibroin and hyaluronic acid are used for drug delivery of bilirubin and have good biocompatibility, but the natural biomaterials also have the potential problems of poor repeatability and unstable batch quality.

How to construct a bilirubin preparation can exert the advantages of nano-drugs to the maximum extent, avoid potential side effects brought by carrier materials and have great innovation value and application prospect.

Disclosure of Invention

In order to solve the problems existing in the background technology, the invention provides bilirubin single-component nano-particles and a preparation method thereof, which are used for preparing medicaments for treating inflammatory diseases through the technical advantages of nano-carriers. The bilirubin nano-particles contain a single component, and have the advantages of 100% of drug loading, stable property, strong operability, low cost and the like.

The technical scheme adopted by the invention is as follows:

a bilirubin nanoparticle:

the nanoparticles are formed by self-assembly of bilirubin as a single component only.

Secondly, a preparation method of the bilirubin nano-particles comprises the following steps:

1) dissolving a certain amount of bilirubin into a suitable organic solvent to form an organic phase;

2) preparing a buffer aqueous solution with a certain pH range as a water phase;

3) slowly dripping the organic phase into the water phase, and stirring and mixing to obtain a mixed solution;

4) and removing the organic solvent in the mixed solution to obtain the nano-particle solution.

In the step 1), the organic solvent is one or a mixture of two of dimethyl sulfoxide and ethanol.

In the step 1), the concentration of bilirubin in the organic phase is 0.2-2.0 mg/mL.

The pH range in the step 2) is 6.8-10.6.

The buffer aqueous solution in the step 2) is one or a mixture of more of phosphate buffer solution, borate buffer solution, carbonate buffer solution, glycinate buffer solution and Tris buffer solution.

The volume ratio of the organic phase to the aqueous phase in the step 3) is 3: 1-1: 1.

The bilirubin nano-particles prepared by the preparation method of the invention are applied to the preparation of a drug delivery system.

The application in preparing the medicine for treating the inflammatory diseases, wherein the inflammatory diseases are specifically acute pancreatitis, rheumatoid arthritis, diabetic wound repair and the like, but not limited to the applications.

The use in the manufacture of an injectable, oral or topical delivery system.

The advantages and the beneficial effects of the invention include:

1) the single component is used for constructing the nano particles, so that the introduction of other components is avoided, and the drug loading of bilirubin can be improved;

2) the nano-particles utilize the nano-size of the nano-particles to improve the half-life period in vivo, thereby being beneficial to improving the bioavailability of bilirubin;

3) the bilirubin nano-particles have inflammation targeting properties, and the delivery efficiency is improved;

4) the preparation process of the nano particles is simple, the operability is strong, the reproducibility is good, and the industrialization prospect is good;

5) the bilirubin nano-particles can be used for directly treating inflammatory diseases, can also be used as nano-carriers of other active substances, and have wide application prospects.

Drawings

FIG. 1 is a transmission electron micrograph of a bilirubin nanoparticle sample 1 prepared in example 1 of the present invention;

FIG. 2 is a graph showing the anti-inflammatory effect of bilirubin nanoparticles prepared in accordance with example 1 of the present invention on macrophages;

FIG. 2 (a) shows the mRNA expression level of TNF-. alpha.and FIG. 2 (b) shows the mRNA expression level of IL-1. beta., and FIG. 2 (c) shows the IL-6 expression level of TNF-. alpha.and FIG. 3 is a graph showing the results of arthritis index values after the treatment of groups of drugs on rheumatoid arthritis model mice;

FIG. 4 is a graph showing the results of the sole thickness values of the groups of drugs after treatment of rheumatoid arthritis model mice;

FIG. 5 is a graph showing the results of the healing rate of each group of drugs on the wound repair treatment of diabetic mice.

Detailed Description

The invention is further described below with reference to the figures and examples.

The invention is implemented as follows:

example 1 preparation of bilirubin nanoparticles

According to Table 1, an amount of bilirubin was dissolved in 2mL of dimethyl sulfoxide to form an organic phase; using a buffer aqueous solution as a water phase; slowly dripping the organic phase into the water phase, and continuously stirring to obtain a mixed solution; dialyzing to remove the organic solvent to obtain the bilirubin nano-particle solution.

The observation result of the bilirubin nanoparticle sample under a transmission electron microscope is shown in FIG. 1.

TABLE 1 preparation points of bilirubin nanoparticles

Example 2 characterization of bilirubin nanoparticles

The samples prepared in example 1 were used for visual observation, and the particle size of the sample having the tyndall effect was measured. The results are reported in Table 2. And the morphology of the sample 1 was examined by a transmission electron microscope, specifically, bilirubin nanoparticles were diluted to an appropriate concentration, dropped onto a copper mesh coated with a carbon film to prepare a sample, and then the morphology of the sample was observed under a transmission electron microscope. The bilirubin nanoparticles (samples 1 to 6) prepared in example 1 were collected and stored at room temperature for 3 months, and the particle size and the encapsulation efficiency were measured, respectively, and the results are shown in Table 2.

TABLE 2 characterization of bilirubin nanoparticles

The bilirubin nano-particles prepared by the method are spherical, the edges are flat and smooth, the particle size range is 160-200 nm, and the indication shows that under appropriate preparation conditions, bilirubin can successfully prepare single-component nano-particles, and the experimental preparation method is reasonable and simple. When the concentration of bilirubin is too high or too low, bilirubin nanoparticles may not be formed or may not be stably present. When an inappropriate organic solvent is selected, the drug cannot be dissolved, the organic phase and the aqueous phase cannot be mixed, and the like, so that the experiment fails. When the pH system of the buffer solution in the water phase is too low or too high, bilirubin cannot self-assemble to form a nano-result. Therefore, bilirubin single-component nanoparticles between 160 nm and 200nm can be successfully prepared according to the provided preparation method (reference samples 1-6).

The bilirubin nano crinite prepared by the method has good storage stability, and still keeps a good nano structure after being placed at room temperature for 1 month.

Example 3 anti-inflammatory action of bilirubin nanoparticles on macrophages

Bilirubin nanoparticle samples 1-6 or free drug prepared in example 1 were co-cultured with macrophages activated by lipopolysaccharide. And collecting cells after 48 hours, extracting cell RNA, and detecting the expression of three key inflammatory factors TNF-alpha, IL-1 beta and IL-6 by a real-time quantitative PCR method.

As can be seen from fig. 2, the bilirubin nanoparticles prepared according to the present invention can achieve highly effective anti-inflammatory effects at the cell level, as compared to free drugs.

Example 4 anti-inflammatory therapeutic Effect of bilirubin nanoparticles on acute pancreatitis

After a rat model for acute pancreatitis is constructed by adopting an abdominal arginine injection method, physiological saline, a bilirubin solution and bilirubin nanoparticles are respectively injected into the abdomen. After 24 hours, serum was collected for amylase, lipase and inflammatory factor levels (TNF-. alpha., IL-6), and pancreatic tissue was collected for double-blind histopathological scoring after routine H & E staining. The scoring standard refers to Schmidt pancreatic histopathology scoring standard (0-4 points), and the higher score indicates the higher pathological malignancy degree.

TABLE 3 anti-inflammatory therapeutic effect of bilirubin nanoparticles on acute pancreatitis

The results of the experiment are shown in table 3. The levels of amylase and lipase of the bilirubin nanoparticles are obviously lower than those of a pure die set and other administration intervention groups, and compared with a bilirubin solution group, the bilirubin nanoparticle injection has the advantage of relieving the condition of pancreatitis better. The result shows that the bilirubin nanoparticles can effectively reduce amylase and lipase in rats with acute pancreatitis and relieve inflammation of pancreatic tissues, thereby prompting that the occurrence and development of the acute pancreatitis are improved. The bilirubin nanoparticles have a better anti-inflammatory effect than bilirubin solutions.

Example 5 anti-inflammatory therapeutic Effect of bilirubin nanoparticles on rheumatoid arthritis

A mouse model of rheumatoid arthritis is constructed by adopting classical collagen-induced arthritis. After 5 days of immunization, a peak of inflammation appears, which is marked by the obvious swelling of the two paw sides of the mouse and can affect the forepaw. Physiological saline, bilirubin solution, and bilirubin nanoparticles (bilirubin nanoparticle sample 1 prepared in example 1) were injected into the joint cavity, respectively. The anti-inflammatory treatment effect of the bilirubin nanoparticles on rheumatoid arthritis was evaluated by means of the arthritis index and the sole thickness values. A lower value of the arthritis index indicates a weaker degree of inflammation of the joint. The smaller the thickness value of the sole, the lower the swelling degree of arthritis is also indicated.

The results are shown in FIGS. 3 and 4. The result shows that the bilirubin nano-particles prepared by the invention have lower joint index values and smaller sole thickness values after being used for treating rheumatoid arthritis model mice. The bilirubin nano-particles have the best treatment effect on rheumatoid arthritis, and the treatment effect is superior to that of free medicines.

Example 5 therapeutic Effect of bilirubin nanoparticles on diabetic mouse wounds

A diabetic mouse model (blood glucose concentration continuously exceeds 250mg/dL) is constructed by injecting streptozotocin into the abdominal cavity. After two weeks, a full-thickness circular skin with a diameter of 6mm was cut from the back of the diabetic mouse, and a full-thickness skin defect wound model was prepared. The wound surface was injected with physiological saline, bilirubin solution and bilirubin nanoparticles (bilirubin nanoparticle sample 1 prepared in example 1), respectively. And then changing the wound dressing to inject the medicine and photographing and recording the area of the wound on the 4 th, 7 th, 9 th and 15 th days after the operation. Wound healing rate (%) — original wound area-residual wound area)/original wound area × 100%. The therapeutic effect of bilirubin nanoparticles on diabetic wound repair was evaluated by wound healing rate (%).

The results are shown in FIG. 5. The result shows that the bilirubin nano-particles prepared by the invention can effectively improve the healing rate of the diabetic wound and shorten the healing time of the wound.

While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

1. A bilirubin nanoparticle, wherein the nanoparticle is formed by self-assembly of bilirubin as a single component.

2. A method of preparation of bilirubin nanoparticles as described in claim 1, wherein the method comprises:

1) dissolving a certain amount of bilirubin into an organic solvent to obtain an organic phase;

3) dripping the organic phase into the water phase, and stirring and mixing to obtain a mixed solution;

3. The method of producing bilirubin nanoparticles as in claim 2, wherein in step 1) the organic solvent is one or a mixture of two of dimethylsulfoxide and ethanol.

4. The method of producing bilirubin nanoparticles as in claim 2, wherein in step 1), the bilirubin concentration in the organic phase is 0.2-2.0 mg/mL.

5. The method for producing bilirubin nanoparticles as in claim 2, wherein the pH in step 2) is in the range of 6.8 to 10.6.

6. The method of claim 2, wherein the aqueous buffer solution in step 2) is one or more selected from the group consisting of phosphate buffer, borate buffer, carbonate buffer, glycinate buffer, and Tris buffer.

7. The method for producing bilirubin nanoparticles as in claim 2, wherein the volume ratio of the organic phase to the aqueous phase in step 3) is from 3:1 to 1: 1.

8. Use of bilirubin nanoparticles as described in any one of claims 1 to 3 or produced by a process as described in any one of claims 1 to 3 in the preparation of a drug delivery system.

9. Use of bilirubin nanoparticles as claimed in claim 8, wherein: the application in preparing the medicine for treating the inflammatory diseases, wherein the inflammatory diseases are specifically acute pancreatitis, rheumatoid arthritis, diabetic wound repair and the like, but not limited to the applications.

10. Use of bilirubin nanoparticles as claimed in claim 8, wherein: the use in the manufacture of an injectable, oral or topical delivery system.