PLGA (poly (lactic-co-glycolic acid)) coated curcumin nano preparation as well as preparation method and application thereof

Abstract

The invention discloses a PLGA (poly (lactic-co-glycolic acid))-coated curcumin nano preparation as well as a preparation method and application thereof.The PLGA and curcumin are dissolved in DMSO (dimethylsulfoxide) solutions respectively to form basic solutions with the mass concentration of 20 mg / mL respectively; the preparation method comprises the following steps: adding a basic solution of PLGA (poly (lactic-co-glycolic acid)) into a basic solution of curcumin in a mass ratio of (1: 2.5)-(1: 10) in a water bath ultrasonic state to obtain a mixed solution A; slowly adding the mixed solution A into a PVA aqueous solution with the concentration of 2% in a water bath ultrasonic state to obtain a mixed solution B; and putting the mixed solution B into an aqueous solution, and dialyzing overnight to obtain the PLGA coated curcumin nano preparation. The curcumin composition effectively overcomes the problems of poor water solubility and low oral bioavailability of curcumin, has a good protection effect on resisting PEDV infection of piglets, effectively reduces the diarrhea rate of the piglets, and can be widely applied to prevention and control of PEDV in livestock and poultry farms.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more particularly to a PLGA-coated curcumin nanoformulation, a preparation method thereof, and an application thereof. Background Art

[0002] During the large-scale pig farming process, important viral diseases of pigs occur frequently, seriously threatening the healthy development of the breeding industry. Porcine epidemic diarrhea (PED) has a wide prevalence in China and is one of the important infectious diseases that seriously endanger the pig industry. PED is an acute and highly contagious intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV). In particular, PEDV causes severe watery diarrhea in young piglets and even 100% death, which is very harmful to suckling piglets. In addition, PEDV also infects medium and large pigs (gilt and breeding sows), and sows can transmit the virus to newborn piglets through vertical transmission. Clinical problems such as continuous virus carriage in the pig herd seriously affect the current safe production of the pig industry, greatly reduce production performance, and cause serious economic losses. According to relevant data, PEDV broke out on a large scale in the pig herd in the United States from 2013 to 2014, directly causing losses of more than 10% of the annual total output value of live pigs in the United States, reaching more than 3 billion US dollars.

[0003] Curcumin is a natural polyphenol compound. Studies have shown that it has significant inhibitory activity against a variety of viruses (such as influenza virus, hepatitis C virus, etc.). Its anti-inflammatory and antioxidant properties can relieve intestinal mucosal damage caused by virus infection. However, curcumin has extremely poor water solubility (<1 μg / mL), low oral bioavailability, and is easily metabolized rapidly in the body (short half-life), which greatly limits its clinical application.

[0004] Nanocarrier technology is an effective means to improve drug delivery. Poly(lactic-co-glycolic acid) (PLGA), as a biodegradable material approved by the FDA, has excellent biocompatibility, controllable degradability, and sustained-release ability, which can protect drugs from degradation and enhance targeting. For example, PLGA-coated doxorubicin significantly increases the drug accumulation at the tumor site. In addition, PLGA nanoparticles can further enhance the cellular uptake efficiency through surface modification (such as folate receptor targeting). However, the current application of PLGA carriers in the antiviral field mostly focuses on hepatitis viruses, and the research on avian and swine coronaviruses has not been fully explored.

[0005] Therefore, providing a PLGA-coated curcumin nanoformulation, a preparation method thereof, and an application thereof can not only overcome the defects of curcumin itself such as low drug loading and poor stability, but also provide an innovative solution for the prevention and treatment of PEDV. Summary of the Invention

[0006] The object of the present invention is to provide a PLGA-coated curcumin nanoformulation, a preparation method and an application, which can effectively solve the technical problems existing in the above-mentioned background art.

[0007] To achieve the above object, the present invention provides the following technical solutions.

[0008] A PLGA-coated curcumin nanoformulation and a preparation method thereof, characterized in that: the PLGA-coated curcumin nanoformulation is spherical in shape, with a particle size of 200-235 nm, an encapsulation efficiency of 40% to 80%, and the particle size is stable within 7 days in phosphate buffer solution, aqueous solution and 10% fetal bovine serum;

[0009] The preparation method of the PLGA-coated curcumin nanoformulation is as follows:

[0010] S1. Dissolve PLGA and curcumin in DMSO solution respectively to form basic solutions with a mass concentration of 20 mg / mL;

[0011] S2. Based on S1, under the state of water bath ultrasound, slowly add the basic solution containing PLGA to the basic solution containing curcumin to obtain a mixed solution A, wherein the mass ratio of curcumin to PLGA is 1:2.5 to 1:10;

[0012] S3. Based on S2, slowly add the mixed solution A in step S2 to a 2% PVA aqueous solution under the state of water bath ultrasound to obtain a mixed solution B, wherein the volume ratio of the mixed solution A to the mixed solution B is 1:5 to 1:5.5;

[0013] S4. Based on S3, dialyze the mixed solution B in an aqueous solution overnight to obtain a PLGA-coated curcumin nanoformulation.

[0014] Application of the PLGA-coated curcumin nanoformulation, the application of the PLGA-coated curcumin nanoformulation in the preparation of anti-porcine epidemic diarrhea virus drugs.

[0015] Preferably, when the concentration of the PLGA-coated curcumin nanoformulation is 60-100 μM, it can inhibit 3.84-4.33 Lg TCID 50 / mL PEDV virus titer.

[0016] Preferably, the PLGA-coated curcumin nanoformulation is administered by the oral route, with a dose of 1 mg / kg body weight, used twice a day, and continuously orally administered for 4 days.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) In the present invention, PLGA and curcumin are separately dissolved in DMSO solution to form basic solutions with a mass concentration of 20 mg / mL respectively. Under the condition of water bath ultrasonic treatment, the basic solution containing curcumin is slowly added to the basic solution containing PLGA to obtain a mixed solution A, where the mass ratio of curcumin to PLGA is 1:2.5 - 1:10. The mixed solution A is slowly added to a 2% PVA aqueous solution under the condition of water bath ultrasonic treatment to obtain a mixed solution B, where the volume ratio of the mixed solution A to the mixed solution B is 1:5 - 1:5.5. The mixed solution B is dialyzed overnight in an aqueous solution to obtain a PLGA-coated curcumin nano-preparation. The PLGA-coated curcumin nano-preparation prepared by this method is spherical in shape, with a particle size of 200 - 235 nm, an encapsulation efficiency of 40% - 80%, and the particle size is stable within 7 days in phosphate buffer solution, aqueous solution and 10% fetal bovine serum. It effectively overcomes the problems of poor water solubility and low oral bioavailability of curcumin, effectively protects piglets from PEDV infection, significantly reduces the diarrhea rate of piglets, and its protection rate reaches 80%. It shows that the PLGA-coated curcumin nano-preparation has a good protective effect on piglets against PEDV infection and can be widely used for the prevention and control of PEDV in livestock and poultry farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a transmission electron microscope result diagram of the PLGA-coated curcumin nano-preparation of the present invention.

[0020] Figure 2 It is a particle size result diagram of the PLGA-coated curcumin nano-preparation of the present invention at different time points.

[0021] Figure 3 It is a hemolysis result diagram of the PLGA-coated curcumin nano-preparation of the present invention.

[0022] Figure 4 It is a schematic diagram of the results of inhibiting PEDV virus TCID by PLGA-coated curcumin nano-preparations with different concentrations of the present invention. 50 RESULT SCHEMATIC DIAGRAM DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention will be further described below in conjunction with the drawings and specific embodiments.

[0024] In the description of the present invention, unless otherwise specified, "a plurality of" means two or more; the orientation or positional relationship indicated by terms such as "upper", "lower", "left", "right", "inner", "outer", "front end", "rear end", "head", "tail", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation on the present invention. In addition, terms such as "first", "second", "third", etc. are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0025] In the description of the present invention, it should be noted that, unless otherwise clearly specified and defined, the terms "connected" and "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0026] Example 1

[0027] As Figure 1 shown, the PLGA-coated curcumin nanoparticles are spherical in shape, with a particle size of 200 - 235 nm, an encapsulation efficiency of 40% - 80%, and the particle size is stable within 7 days in phosphate buffer solution (PBS), aqueous solution, and 10% fetal bovine serum (FBS). Specifically, in this example, the PLGA-coated curcumin nanoparticles were sampled every day for 7 consecutive days to measure the particle size of the nanoparticles, and their stability was evaluated and analyzed. As Figure 2 shown, Size (nm) represents the particle size of the nanoparticles, Time (day) represents the number of days of placement, Water represents the aqueous solution, PBS represents the phosphate buffer solution, and FBS represents the fetal bovine serum; the inset in the upper right corner is a physical picture of the nanoparticles. The particle sizes of the nanoparticles placed in the three different solutions are uniform and there is no obvious difference, indicating that the prepared PLGA-coated curcumin nanoparticles have good stability.

[0028] The preparation method of the PLGA-coated curcumin nanoparticles is as follows:

[0029] S1. Dissolve PLGA and curcumin in DMSO solution respectively to form basic solutions with a mass concentration of 20 mg / mL;

[0030] S2. Based on S1, under the state of water bath ultrasound, slowly add the basic solution containing PLGA to the basic solution containing curcumin to obtain a mixed solution A, where the mass ratio of curcumin to PLGA is 1:2.5 - 1:10;

[0031] S3. Based on S2, slowly add the mixed solution A in step S2 into an aqueous solution of PVA with a concentration of 2% under the condition of water bath ultrasonic treatment to obtain a mixed solution B, where the volume ratio of the mixed solution A to the mixed solution B is 1:5 to 1:5.5;

[0032] S4. Based on S3, dialyze the mixed solution B in an aqueous solution overnight to obtain a PLGA-encapsulated curcumin nanoformulation.

[0033] In this example, for the preparation method of the PLGA-encapsulated curcumin nanoformulation, in step S1, 20 mg of PLGA and 20 mg of curcumin are respectively weighed and dissolved in 1 mL of DMSO solution (the concentrations are respectively 20 mg / mL);

[0034] In step S2, curcumin and PLGA are mixed at mass ratios of 1:2.5, 1:5, and 1:10 respectively, and slowly added to the PLGA solution under the condition of water bath ultrasonic treatment to obtain three different mixed solutions A with different mass ratios;

[0035] The particle sizes of the PLGA-encapsulated curcumin nanoformulations prepared by the above method are measured by a laser particle size analyzer (equipment model: Malvern, Zetasizer Nano-ZS90). The particle sizes of the nanoformulations prepared with 3 different mass ratios are approximately 225.5, 205.6, and 212.2 nm respectively, and their encapsulation efficiencies are 40%, 60%, and 80% respectively. The results are shown in Table 1. Considering factors such as the particle size and encapsulation efficiency of the nanoformulation comprehensively, the nanoformulation synthesized with a mass ratio of 1:5 is selected for subsequent experiments in the later stage. The morphology of the nanoformulation is observed by a transmission electron microscope. The electron microscope results show spherical particles with uniform morphology and size, and the diameter is approximately 200 nm, as Figure 1 shown.

[0036] Table 1 Particle size measurement results of PLGA-encapsulated curcumin nanoformulations

[0037]

[0038] When the concentration of the PLGA-encapsulated curcumin nanoformulation is 60 - 100 μM, it can inhibit the PEDV virus titer of 3.84 - 4.33 LgTCID50 / mL.

[0039] Specifically, porcine epidemic diarrhea virus (PEDV) with a multiplicity of infection of 0.1 (MOI = 0.1) was added to Vero cells for infection for 1 hour, discarded and washed three times with PBS, and PLGA-encapsulated curcumin nanoformulations were diluted to final concentrations of 60 μM, 80 μM, and 100 μM, respectively, and added to a culture medium containing 2% FBS. The culture medium was added to the cell culture wells, and the virus supernatant was collected after culturing for 24 hours, and the TCID50 of the virus was determined. Figure 4 The results show that, Virustiter represents the virus titer, TCID50 represents the infection dose of half the tissue cells, and Hours post infection (hpi) represents the infection time. The PLGA-encapsulated curcumin nanoformulation prepared in this example has a very significant inhibitory effect on PEDV. The PLGA-encapsulated curcumin nanoformulations with concentrations of 60μM, 80μM, and 100μM can inhibit 3.84Lg TCID 50 、4.17LgTCID 50 、4.33Lg TCID 50 virus.

[0040] The biocompatibility of PLGA-encapsulated curcumin nanoformulations was evaluated by hemolysis experiments in vitro. Fresh blood from BLAB / c mice was centrifuged at 4°C to obtain red blood cells, which were then washed three times with PBS. 25 μL of red blood cells were added with ddH2O (positive control), PBS (negative control) and different concentrations of PLGA-encapsulated curcumin (20, 40, 80 μg / mL, the converted concentration units correspond to 50, 100, 200 μM) and mixed with 25 μL of red blood cells, and incubated at 37°C for 4 hours. After centrifugation of the liquid, the supernatant was taken and the absorbance at 540 nm was detected by an enzyme-linked microplate reader. The hemolysis rate was calculated as follows: Hemolysis rate (%) = (OD 样品 -OD ddH2O ) / (OD 样品 -OD PBS )×100.

[0041] like Figure 3 As shown, the vertical axis (Hemolysis) represents the hemolysis rate, and the illustration in the upper right corner is an actual image of the hemolysis experiment. The hemolysis rate of the positive control group treated with ddH2O was 100%, and none of the PBS groups showed hemolysis. The hemolysis rates of the three groups treated with PLGA-encapsulated curcumin at different concentrations were all less than 2%. Microscopically, the erythrocyte morphology was normal. These results demonstrate that the PLGA-encapsulated curcumin nanoformulation has good biocompatibility and safety.

[0042] Application of PLGA-coated curcumin nanoparticles, application of PLGA-coated curcumin nanoparticles in the preparation of drugs against porcine epidemic diarrhea virus, and animal experiments were carried out using the nanoparticles prepared in Example 1.

[0043] Ten 5-day-old healthy piglets negative for PEDV antibody and pathogen detection were selected and divided into an experimental group and a control group (5 piglets / group). Before virus challenge, each piglet in the experimental group was orally administered PLGA-coated curcumin nanoparticles (used at a dose of 1 mg / kg body weight) 12 h in advance. Each piglet in the experimental group and the control group was orally administered 4 mL of 1×10 6.0 TCID 50 / mL PEDV virus solution. Two hours after oral administration of the virus solution, each piglet in the experimental group was orally administered PLGA-coated curcumin nanoparticles twice a day for 4 consecutive days. Each group was raised in isolation and continuously observed for 7 days until the end of the experiment, and the diarrhea conditions of each group were recorded.

[0044] The protective effect of PLGA-coated curcumin nanoparticles on PEDV-infected piglets is shown in Table 2. As the results show, the diarrhea rate of piglets in the control group was 100% (5 / 5 piglets). The experimental group orally administered PLGA-coated curcumin nanoparticles could effectively protect piglets from PEDV infection, significantly reduce the diarrhea rate of piglets, and its protection rate reached 80% (4 / 5 piglets), indicating that PLGA-coated curcumin nanoparticles have a good protective effect on piglets against PEDV infection.

[0045] Table 2 Protective effect of PLGA-coated curcumin nanoparticles on PEDV-infected piglets

[0046]

[0047] The present invention effectively overcomes the problems of poor water solubility and low oral bioavailability of curcumin, has a good protective effect on piglets against PEDV infection, effectively reduces the diarrhea rate of piglets, and can be widely used for the prevention and control of PEDV in livestock and poultry farms.

[0048] The above embodiments are only the preferred embodiments of the present invention and cannot be used to limit the scope of the rights of the present invention. Therefore, modifications, equivalent changes, improvements, etc. made according to the scope of the patent application of the present invention still fall within the scope covered by the present invention.

Claims

1. A PLGA-coated curcumin nanoformulation and its preparation method, characterized in that: The PLGA-coated curcumin nanoformulation is spherical in shape, with a particle size of 200 - 235 nm, an encapsulation efficiency of 40% - 80%, and the particle size remains stable within 7 days in phosphate buffer solution, aqueous solution, and 10% fetal bovine serum; The preparation method of the PLGA-coated curcumin nanoformulation is as follows: S1. Dissolve PLGA and curcumin in DMSO solution respectively to form a basic solution with a mass concentration of 20 mg / mL; S2. Based on S1, under the state of water bath sonication, slowly add the basic solution containing PLGA to the basic solution containing curcumin to obtain a mixed solution A, where the mass ratio of curcumin to PLGA is 1:2.5 - 1:10; S3. Based on S2, slowly add the mixed solution A in step S2 to a 2% PVA aqueous solution under the state of water bath sonication to obtain a mixed solution B, where the volume ratio of the mixed solution A to the mixed solution B is 1:5 - 1:5.5; S4. Based on S3, dialyze the mixed solution B in an aqueous solution overnight to obtain the PLGA-coated curcumin nanoformulation.

2. Use of the PLGA-coated curcumin nanoformulation according to claim 1, characterized in that: The application of the PLGA-coated curcumin nanoformulation in the preparation of drugs against porcine epidemic diarrhea virus.

3. Use of the PLGA-coated curcumin nanoformulation according to claim 2, characterized in that: When the concentration of the curcumin-loaded PLGA nanoparticles is 60 - 100 μM, it can inhibit the PEDV virus titer of 3.84 - 4.33 Lg TCID 50 / mL.

4. Use of the PLGA-coated curcumin nanoformulation according to claim 2 or 3, characterized in that: The PLGA-coated curcumin nanoformulation is administered via the oral route, with a dose of 1 mg / kg body weight, used twice a day, and orally administered continuously for 4 days.

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