Preparation method and application of PLGA-curcumin nanoparticles

Abstract

The invention discloses a preparation method and application of PLGA (poly (lactic-co-glycolic acid))-curcumin nanoparticles. The invention relates to the technical field of biomedicine, and the preparation method comprises the following steps: taking a polyvinyl alcohol (PVA) aqueous solution as a water phase and a dichloromethane solution of polylactic acid-glycolic acid copolymer (PLGA) and curcumin (Cur) as an oil phase, preparing curcumin-entrapped PLGA nanoparticles (PLGA at Cur NPs) by adopting a water-in-oil-in-water double emulsion method, and the prepared PLGA nanoparticles are spherical, uniform in particle size and uniform in particle size. The carrier PLGA has good biocompatibility and biodegradability and is non-toxic and non-irritant, and a surfactant does not need to be added in the preparation process of the nanoparticles, so that the toxicity is further reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine, in particular to a preparation method and application of PLGA-curcumin nanoparticles. BACKGROUND

[0002] Curcumin (molecular formula C 21 H 20 O6) is a hydrophobic polyphenolic compound extracted from the rhizomes of Curcuma longa, accounting for 3-6% of the dry weight of turmeric. Its molecular structure is connected by two adjacent methoxy phenol rings through an α, β-unsaturated β-diketone bridge, giving it a unique electronic conjugated system and diverse biological activities.

[0003] As the core component of traditional Chinese medicine, curcumin has attracted much attention due to its broad-spectrum pharmacological effects: anti-tumor effect: by inhibiting the NF-κB signaling pathway, down-regulating the expression of pro-oncogenes (such as COX-2, MMP-9), and inducing tumor cell apoptosis; antioxidant activity: directly scavenging free radicals (DPPH clearance rate IC50 is 11.3 μM), activating the Nrf2 / ARE pathway, and enhancing the activity of cellular antioxidant enzymes (SOD, GSH-Px); anti-inflammatory mechanism: blocking the secretion of inflammatory factors such as TNF-α and IL-6, inhibiting the activation of inflammasome NLRP3 (IL-1β in RAW264.7 macrophages decreased by 67%); immune regulation: regulating T cell differentiation, promoting the polarization of anti-inflammatory Th2 cells, and inhibiting excessive immune response. However, the clinical application of curcumin is limited by its physicochemical property defects. Low water solubility (solubility in water <1 μg / mL) leads to low cellular uptake efficiency (intestinal absorption rate <1%); chemical instability: easily degrades into inactive products such as trans-dihydrocurcumin under physiological pH (>7.4) and light conditions; significant first-pass effect: oral bioavailability is only about 0.1%, and half-life is short (about 1.5 hours).

[0004] To overcome the above problems, researchers have developed various delivery strategies based on nanocarriers: liposome encapsulation: curcumin is wrapped by a phospholipid bilayer to improve its water dispersibility (solubility increased by 400 times), but there are problems of low encapsulation efficiency (<60%) and poor storage stability; polymeric micelles: using amphiphilic block copolymers (such as Pluronic F127) to self-assemble into micelles, with a drug loading capacity of up to 15%, but they are prone to rapid dissociation in blood; inorganic nanoparticles: mesoporous silica (MSN) and metal-organic frameworks (MOF) achieve high drug loading through pore adsorption, but their biodegradability is limited. Among them, polylactic acid-glycolic acid copolymer (PLGA) as an FDA-approved biodegradable material, shows unique advantages: excellent biocompatibility: the hydrolysis products lactic acid and glycolic acid can be metabolized through the tricarboxylic acid cycle without systemic toxicity; precise controlled release ability:

[0005] Studies have shown that PLGA nanoparticles can increase the bioavailability of curcumin by 20-40 times, and significantly prolong its circulation time in vivo (AUC increases by 15 times). For example, PLGA-encapsulated curcumin showed a 52% reduction in inflammatory factors IL-6 and TNF-α levels in a colitis model, and no liver and kidney function damage. SUMMARY

[0006] The purpose of the present application is to provide a preparation method and application of curcumin-loaded PLGA nanoparticles. The prepared PLGA nanoparticles are spherical, with uniform particle size. The carrier PLGA has good biocompatibility and biodegradability, is non-toxic and non-irritating, and does not require the addition of a surfactant in the preparation process of the nanoparticles, further reducing toxicity.

[0007] The preparation method of PLGA-curcumin nanoparticles comprises the following steps:

[0008] Step one, dissolve polylactic acid-glycolic acid copolymer PLGA and a certain mass of curcumin Cur in dichloromethane DCM to form a mixed solution, then slowly add to a 5% (mass fraction) polyvinyl alcohol PVA aqueous solution, and use a 100W ultrasonic disrupter to ultrasonicate to obtain an initial emulsion;

[0009] Step two, slowly add the initial emulsion in step one to a 1% (mass fraction) polyvinyl alcohol PVA aqueous solution through a membrane, and use a 100W ultrasonic disrupter to ultrasonicate to obtain a re-emulsion;

[0010] Step three, remove the residual dichloromethane DCM in the re-emulsion by stirring at room temperature for 12h, then use a centrifuge to centrifuge at a speed of 1200r / min for 10min to separate and obtain PLGA@Cur NPs. Wash the PLGA@Cur NPs with ultrapure water 3 times for purification.

[0011] Preferably, the concentration of polylactic acid-glycolic acid copolymer PLGA in step one is 2.5-50mg / mL.

[0012] Preferably, the mass ratio of dichloromethane DCM and polyvinyl alcohol PVA in step one is 1:3.

[0013] Preferably, the ultrasonic disrupter used in step one is a 100W ultrasonic disrupter, and the ultrasonic time is 1min.

[0014] Preferably, the ultrasonic disrupter used in step two is a 100W ultrasonic disrupter, and the ultrasonic time is 3min.

[0015] Preferably, a microporous filter membrane is used in step two, and the pore size of the microporous filter membrane is 0.22-0.45μm.

[0016] 0.22 μm-0.45 μm.

[0017] Application of PLGA-curcumin nanoparticles as drug nanocarriers.

[0018] Advantages of the present application:

[0019] The PLGA nanoparticles prepared in the present application are spherical, the particle size is controllable, the particle size is uniform, the carrier PLGA has good biocompatibility and biodegradability, PLGA hydrolysis produces glycolic acid and lactic acid, which have biological inertness to growing cells and are cleared from the body through common metabolic pathways, have good stability, are conducive to stable release in the body, are non-toxic and non-irritating, and do not need to add surfactants in the preparation process of the nanoparticles, further reducing the toxicity; the present application overcomes the problem of poor solubility of curcumin, improves the bioavailability of curcumin, increases the curative effect, has good sustained-release effect, and compared with naked drug curcumin, the nanoparticles are more easily taken up by cells and have stronger inhibitory effect on tumor cell growth; the preparation method of the present application is simple and convenient to operate. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a PLGA-curcumin nanoparticle observed by SEM;

[0021] Figure 2 is a table of Zeta potential, particle size and PDI of the drug-loaded nanoparticles measured by dynamic light scattering particle size instrument (DLS);

[0022] Figure 3 is a table of Cur loading rate of the prepared series of drug-loaded nanoparticles;

[0023] Figure 4 is a stability diagram of the prepared PLGA-curcumin nanoparticles;

[0024] Figure 5 is an antioxidant diagram of the prepared PLGA-curcumin nanoparticles;

[0025] Figure 6 is a biocompatibility diagram of the prepared PLGA-curcumin nanoparticles;

[0026] Figure 7 is an antioxidant performance diagram of the PLGA-curcumin nanoparticles at the cellular level. DETAILED DESCRIPTION

[0027] The present application is further described in detail below in conjunction with the examples, and it is particularly pointed out that: in the following examples, the specific conditions are not specified, and the conventional conditions or the conditions recommended by the manufacturer are used, and in the following examples, the raw materials used can be obtained from ordinary commercial sources unless otherwise specified.

[0028] The preparation method of the PLGA-curcumin nanoparticles comprises the following steps:

[0029] Step one, dissolve polylactic acid-glycolic acid copolymer PLGA and a certain mass of curcumin Cur in dichloromethane DCM to form a mixed solution, then slowly add to 5% (mass fraction) polyvinyl alcohol PVA aqueous solution, and use a 100W ultrasonic cell disruptor to ultrasonicate to obtain an initial emulsion;

[0030] Step two, slowly add the initial emulsion in step one to 1% (mass fraction) polyvinyl alcohol PVA aqueous solution through membrane filtration with pure water, and use a 100W ultrasonic cell disruptor to ultrasonicate to obtain a re-emulsion;

[0031] Step three, remove the residual dichloromethane DCM in the re-emulsion by stirring at room temperature for 12h, then centrifuge at a speed of 1200r / min for 10min to separate to obtain PLGA@Cur NPs, and wash the PLGA@Cur NPs with ultrapure water for 3 times for purification.

[0032] The concentration of the polylactic acid-glycolic acid copolymer PLGA in step one is 2.5-50mg / mL.

[0033] The mass ratio of dichloromethane DCM and polyvinyl alcohol PVA in step one is 1:3.

[0034] The 100W ultrasonic cell disruptor is used for ultrasonication for 1min in step one.

[0035] The 100W ultrasonic cell disruptor is used for ultrasonication for 3min in step two.

[0036] A microporous filter membrane is used in step two, and the pore size of the microporous filter membrane is 0.22-0.45μm.

[0037] The application of the PLGA-curcumin nanoparticles as a drug nanocarrier.

[0038] Preparation Example 1:

[0039] The preparation of the PLGA-curcumin nanoparticles comprises the following steps:

[0040] Step one, dissolve 50mg PLGA and Cur in 1mL dichloromethane to form a mixed solution, and the mass ratio of Cur and PLGA is 1:25, then slowly add to 3mL 5% (mass fraction) PVA aqueous solution, and use a 100W ultrasonic cell disruptor to ultrasonicate for 1min to obtain an initial emulsion;

[0041] Step two, the initial emulsion in step one is slowly added to 50 mL of 1% (mass fraction) PVA aqueous solution, and is ultrasonically broken for 3 min by using a 100W ultrasonic cell disruptor to obtain a re-emulsion;

[0042] Step three, the residual dichloromethane in the re-emulsion is removed by stirring at room temperature for 12 h. Then, the PLGA@Cur NPs are separated by centrifugation at a speed of 1200 r / min for 10 min. The PLGA@Cur NPs are purified by washing with 15 mL of ultrapure water for 3 times to obtain PLGA@Cur 1;

[0043] Preparation Example 2:

[0044] The preparation of PLGA-curcumin nanoparticles includes the following steps:

[0045] Step one, 50 mg of PLGA and Cur are dissolved in 1 mL of dichloromethane to form a mixed solution, and the mass ratio of Cur to PLGA is 1:10. Then, the mixed solution is slowly added to 3 mL of 5% (mass fraction) PVA aqueous solution, and is ultrasonically broken for 1 min by using a 100W ultrasonic cell disruptor to obtain an initial emulsion;

[0046] Step two, the initial emulsion in step one is slowly added to 50 mL of 1% (mass fraction) PVA aqueous solution, and is ultrasonically broken for 3 min by using a 100W ultrasonic cell disruptor to obtain a re-emulsion;

[0047] Step three, the residual dichloromethane in the re-emulsion is removed by stirring at room temperature for 12 h. Then, the PLGA@Cur NPs are separated by centrifugation at a speed of 1200 r / min for 10 min. The PLGA@Cur NPs are purified by washing with 15 mL of ultrapure water for 3 times to obtain PLGA@Cur 2;

[0048] Preparation Example 3:

[0049] The preparation of PLGA-curcumin nanoparticles includes the following steps:

[0050] Step one, 50 mg of PLGA and Cur are dissolved in 1 mL of dichloromethane to form a mixed solution, and the mass ratio of Cur to PLGA is 1:5. Then, the mixed solution is slowly added to 3 mL of 5% (mass fraction) PVA aqueous solution, and is ultrasonically broken for 1 min by using a 100W ultrasonic cell disruptor to obtain an initial emulsion;

[0051] Step two, the initial emulsion in step one is slowly added to 50 mL of 1% (mass fraction) PVA aqueous solution, and is ultrasonically broken for 3 min by using a 100W ultrasonic cell disruptor to obtain a re-emulsion;

[0052] Step three, remove the residual dichloromethane in the re-emulsion by stirring at room temperature for 12 h. Then centrifuge at 1200 r / min for 10 min to separate the PLGA@Cur NPs. Wash the PLGA@Cur NPs with 15 mL ultrapure water for 3 times to purify, and obtain PLGA@Cur 3;

[0053] Preparation Example 4:

[0054] Preparation of PLGA-curcumin nanoparticles, including the following steps:

[0055] Step one, dissolve 50 mg of PLGA and Cur in 1 mL of dichloromethane to form a mixed solution, and the mass ratio of Cur and PLGA is 3:10, then slowly add to 3 mL of 5% (mass fraction) PVA aqueous solution, and use a 100W ultrasonic crusher to ultrasonic for 1 min to obtain an initial emulsion;

[0056] Step two, slowly add the initial emulsion in step one to 50 mL of 1% (mass fraction) PVA aqueous solution through a membrane, and use a 100W ultrasonic crusher to ultrasonic for 3 min to obtain a re-emulsion;

[0057] Step three, remove the residual dichloromethane in the re-emulsion by stirring at room temperature for 12 h. Then centrifuge at 1200 r / min for 10 min to separate the PLGA@Cur NPs. Wash the PLGA@Cur NPs with 15 mL ultrapure water for 3 times to purify, and obtain PLGA@Cur 4;

[0058] Preparation Example 5:

[0059] Preparation of PLGA-curcumin nanoparticles, including the following steps:

[0060] Step one, dissolve 50 mg of PLGA and Cur in 1 mL of dichloromethane to form a mixed solution, and the mass ratio of Cur and PLGA is 3:10, then slowly add to 3 mL of 5% (mass fraction) PVA aqueous solution, and use a 100W ultrasonic crusher to ultrasonic for 1 min to obtain an initial emulsion;

[0061] Step two, slowly add the initial emulsion in step one to 50 mL of 1% (mass fraction) PVA aqueous solution through a membrane, and use a 100W ultrasonic crusher to ultrasonic for 3 min to obtain a re-emulsion;

[0062] Step three, remove the residual dichloromethane in the double emulsion by stirring at room temperature for 12h. Then separate the PLGA@Cur NPs by centrifugation at 1200r / min for 10min. Purify the PLGA@Cur NPs by washing with 15mL ultrapure water for 3 times to obtain PLGA@Cur 5;

[0063] As can be seen from the SEM images of the PLGA@Cur NPs in FIG. 1, the PLGA@Cur NPs are spherical in shape, smooth in surface, and uniform in size. Figure 1 As can be seen from the SEM images of the PLGA@Cur NPs in FIG. 1, the PLGA@Cur NPs are spherical in shape, smooth in surface, and uniform in size.

[0064] Figure 2 As can be seen from the SEM images of the PLGA@Cur NPs in FIG. 1, the PLGA@Cur NPs are spherical in shape, smooth in surface, and uniform in size.

[0065] The hydrodynamic size of the PLGA@Cur NPs is about 300nm, which is consistent with the SEM result. The PDI of the PLGA@Cur NPs is less than 0.3, indicating that the PLGA@Cur NPs have good monodispersity.

[0066] As can be seen from the SEM images of the PLGA@Cur NPs in FIG. 1, the PLGA@Cur NPs are spherical in shape, smooth in surface, and uniform in size. Figure 3 As can be seen from the SEM images of the PLGA@Cur NPs in FIG. 1, the PLGA@Cur NPs are spherical in shape, smooth in surface, and uniform in size.

[0067] Figure 4 As can be seen from the SEM images of the PLGA@Cur NPs in FIG. 1, the PLGA@Cur NPs are spherical in shape, smooth in surface, and uniform in size.

[0068] As can be seen from the SEM images of the PLGA@Cur NPs in FIG. 1, the PLGA@Cur NPs are spherical in shape, smooth in surface, and uniform in size. Figure 5 As can be seen from the SEM images of the PLGA@Cur NPs in FIG. 1, the PLGA@Cur NPs are spherical in shape, smooth in surface, and uniform in size.

[0069] Figure 6 As can be seen from the SEM images of the PLGA@Cur NPs in FIG. 1, the PLGA@Cur NPs are spherical in shape, smooth in surface, and uniform in size.

[0070] Application example:

[0071] ​​​RAW 264.7 cells were seeded into 96-well plates at a density of 8000 cells / well and incubated at 37°C in a 5% CO2 incubator for 12 h. The following experimental groups were established: (1) Blank control group (D): No treatment was given, and normal culture was performed; (2) Model group (A): 10 μL of 1 mmol / L TTBHP was added to the cells and cultured for 24 h to induce oxidative stress; (3) Free curcumin group (B): 10 μL of curcumin solution was added to the cells under TBHP induction conditions and cultured for 24 h; (4) PLGA@CurNPs group (C): 10 μL of 1.2 mg / mL PLGA@CurNPs was added to the cells under TBHP induction conditions and cultured for 24 h. After the treatment of each group, H2DCFDA fluorescent probe was added to stain the cells, PBS was used to wash away excess dye, and then fluorescence microscopy was used for imaging.

[0072] Depend on Figure 5 The results showed that the model group (A) exhibited the strongest green fluorescence, indicating a significant increase in reactive oxygen species (ROS) levels. The free curcumin group (B) showed a decrease in fluorescence, suggesting that curcumin has a certain ROS scavenging effect. The PLGA@Cur NPs group (C) showed a significant decrease in fluorescence signal, indicating that the nano-formulation can more effectively reduce intracellular ROS levels. The blank control group (D) showed almost no fluorescence signal. These results demonstrate that PLGA@Cur NPs exhibits superior antioxidant performance compared to free curcumin at the cellular level.

[0073] The above description is merely an exemplary embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A process for the preparation of PLGA-curcumin nanoparticles, characterized in that: The method comprises the following steps: Step one, dissolving polylactic acid-glycolic acid copolymer PLGA and a certain mass of curcumin Cur in dichloromethane DCM to form a mixed solution, then slowly adding to 5% (mass fraction) polyvinyl alcohol PVA aqueous solution, and using a 100W ultrasonic cell disruptor to ultrasonically obtain an initial emulsion; Step two, slowly adding the initial emulsion in step one to 1% (mass fraction) polyvinyl alcohol PVA aqueous solution, and using a 100W ultrasonic cell disruptor to ultrasonically obtain a re-emulsion; Step three, removing residual dichloromethane DCM in the re-emulsion by stirring at room temperature for 12h, then using a centrifuge to centrifuge at a speed of 1200r / min for 10min to separate to obtain PLGA@Cur NPs, and washing the PLGA@Cur NPs with ultrapure water for 3 times for purification.

2. The method of claim 1, wherein the PLGA-curcumin nanoparticles are prepared by: The concentration of polylactic acid-glycolic acid copolymer PLGA in step one is 2.5-50mg / mL.

3. The method of claim 1, wherein the PLGA-curcumin nanoparticles are prepared by: The mass ratio of dichloromethane DCM and polyvinyl alcohol PVA in step one is 1:

3.

4. The method of claim 1, wherein the PLGA-curcumin nanoparticles are prepared by: The 100W ultrasonic cell disruptor is used for ultrasonic treatment for 1min in step one.

5. The method of claim 1, wherein the PLGA-curcumin nanoparticles are prepared by: The 100W ultrasonic cell disruptor is used for ultrasonic treatment for 3min in step two.

6. The method of claim 1, wherein the PLGA-curcumin nanoparticles are prepared by: A microporous filter membrane is used in step two, and the pore size of the microporous filter membrane is 0.22-0.45μm.

7. A PLGA-curcumin nanoparticle prepared by the preparation method of the PLGA-curcumin nanoparticle in claims 1-6, which is used as a drug nanocarrier.

Images