CN113440498A - Gambogic acid nanoparticles and preparation method and application thereof - Google Patents
Gambogic acid nanoparticles and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5192—Processes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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Abstract
The invention discloses a gambogic acid polyethylene glycol polylactic acid segmented copolymer nanoparticle preparation, and a preparation method and application thereof, belonging to the technical field of pharmaceutical preparations. The gambogic acid nanoparticles prepared by the method are round nanoparticles with the average particle size of 338.9nm, are uniform in size and uniform in dispersion, can improve the water solubility of gambogic acid, and show anti-inflammatory activity obviously superior to that of raw material medicines in a mouse body.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a preparation method of gambogic acid polyethylene glycol polylactic acid segmented copolymer nanoparticles and application of the nanoparticles in treating inflammation.
Background
Gambogic acid is a caged xanthone compound extracted from gamboge, is one of main active ingredients of gamboge, and has been proved to have various biological activities such as tumor cell proliferation inhibition, anti-inflammation, antivirus and neuroprotection at present. As a chemical component of a natural medicine source, the gambogic acid also has the advantages of high efficiency, low toxicity, low drug resistance, difficult residue and the like. All of these have made gambogic acid have great potential as a new drug. However, the extremely low water solubility of gambogic acid makes it difficult to apply to clinical research and development as an in vivo anti-inflammatory drug. Although the solubility of gambogic acid can be improved under strong alkaline conditions, when the pH value is too large, degradation of gambogic acid is caused. The current method of adding solubilizer also causes a series of adverse reactions, which all bring a plurality of obstacles and limitations to the production and clinical application development of gambogic acid. Therefore, researches and developments of gambogic acid nanoparticles capable of improving the bioavailability of gambogic acid are carried out to solve the problem of water insolubility of the gambogic acid, and the targeting characteristic of the nanoparticles is combined, so that the gambogic acid nanoparticles can exert the drug effect to the maximum extent and have very important significance.
Disclosure of Invention
In view of the above, the present invention provides a gambogic acid nanoparticle for improving the water solubility of gambogic acid, and the second object of the present invention is to provide a method for preparing a gambogic acid nanoparticle preparation; the invention also aims to provide the application of the gambogic acid nanoparticle preparation in treating inflammation.
In order to achieve the purpose, the invention provides the following technical scheme:
a preparation method of gambogic acid nanoparticles mainly comprises the following steps:
(1) dissolving Gambogic Acid (GA) and polyethylene glycol polylactic acid block copolymer (mPEG-PLA) in a dichloromethane-acetone mixed solvent to obtain an organic phase;
(2) taking 1-3% (m/v) of polyvinyl alcohol (PVA) aqueous solution as a water phase, and uniformly mixing the water phase with the organic phase obtained in the step (1);
(3) ultrasonically treating in ice-water bath to obtain colostrum;
(4) taking a polyvinyl alcohol (PVA) aqueous solution as a continuous phase, and slowly dropwise adding the primary emulsion obtained in the step (3) into the stirred PVA aqueous solution;
(5) removing the organic solvent and part of water by using a rotary evaporator;
(6) and (3) after ultracentrifugation, taking the precipitate, washing the precipitate for 1-6 times by using distilled water, and dispersing the precipitate in water to obtain gambogic acid nanoparticles (GA-mPEG-PLA-NPs).
Further, the volume ratio of dichloromethane to acetone in the dichloromethane-acetone mixed solvent in the step (1) is 1: 10-10: 1.
Further, the concentration of the polyethylene glycol polylactic acid block copolymer in the step (1) is 1-50 mg/mL, and the weight ratio of the gambogic acid to the polyethylene glycol polylactic acid block copolymer is 1: 35-1: 5.
Further, the volume ratio of the organic phase to the aqueous phase in the step (2) is 1: 5-5: 1.
Further, the ultrasonic condition in the step (3) is that the amplitude is 100%, each time of ultrasonic is 1-10 s, the interval is 1-10 s, and the total ultrasonic time is 30-100 s.
Further, the concentration of the polyvinyl alcohol in the step (4) is 0.1-1% (m/v), and the stirring time is 10-200 min.
Further, the centrifugation in the step (6) is carried out under the conditions that the centrifugation speed is 10000-40000 rpm and the centrifugation time is 0.5-2 h;
on the one hand, the invention provides gambogic acid nanoparticles prepared by the preparation method.
On the other hand, the invention provides the application of the gambogic acid nanoparticles in preparing medicaments for treating inflammation.
Compared with the prior art, the invention has the following beneficial effects:
1. the gambogic acid nanoparticles prepared by the invention are round nanoparticles with the average particle size of 338.9nm, have smooth and flat surfaces, uniform size and uniform dispersion, have negative charges on the surfaces, and have extremely high encapsulation efficiency and sustained-release effect.
2. The gambogic acid nanoparticles prepared by the invention improve the water solubility of gambogic acid, and show anti-inflammatory activity obviously superior to that of raw material medicines in a mouse body.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described below.
FIG. 1 is a TEM image of GA-mPEG-PLA-NPs.
FIG. 2 is a graph showing the distribution of the particle size of GA-mPEG-PLA-NPs.
FIG. 3 is a surface potential map of GA-mPEG-PLA-NPs.
FIG. 4 is a graph showing in vitro release of GA-mPEG-PLA-NPs.
FIG. 5 is a graph showing the effect of GA-mPEG-PLA-NPs on the content of inflammatory mediators and inflammatory factors in an in vitro model of inflammation.
FIG. 6 is a graph showing the effect of GA-mPEG-PLA-NPs on the levels of inflammatory mediators and inflammatory factors in an in vivo model of inflammation.
Detailed Description
The present invention is described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto, and it is obvious that the examples in the following description are only some examples of the present invention, and it is obvious for those skilled in the art to obtain other similar examples without inventive exercise and falling into the scope of the present invention.
Example 1
A preparation method of gambogic acid nanoparticles comprises the following steps:
(1) dissolving 35mg mPEG-PLA and 1mg Gambogic Acid (GA) in 3ml dichloromethane and acetone solution (the volume ratio of dichloromethane to acetone is 1: 1);
(2) adding the organic solution into 6mL of 2.5% (m/v) polyvinyl alcohol solution, and uniformly mixing;
(3) immediately putting the solution obtained in the step (2) into an ice bath, and carrying out ultrasonic treatment with the amplitude of 100%, 3s every time and 3s intervals, wherein the total ultrasonic time is 40 s;
(4) after the ultrasonic treatment is finished, dropwise adding the obtained emulsion into 20mL of 0.5% (m/v) polyvinyl alcohol solution under high-speed magnetic stirring;
(5) continuously stirring for 92min after dripping;
(6) under the condition of room temperature, removing the organic solvent and part of water by rotary evaporation;
(7) centrifuging to collect gambogic acid nanoparticles, washing for 3 times by using deionized water, wherein the centrifugation speed is 40000rpm, and the centrifugation time is 1 h;
(8) and (4) taking the precipitate obtained in the step (7), washing with water for three times, and then re-dispersing with deionized water to obtain the catalyst.
Observing the morphology of the gambogic acid nanoparticles by using a Transmission Electron Microscope (TEM), and measuring the particle size and zeta potential of the gambogic acid nanoparticles by using a laser particle size analyzer and a potentiometer.
The TEM image of the gambogic acid nanoparticles is shown in fig. 1, and it can be seen from the figure that the prepared gambogic acid nanoparticles are spherical, the surface is smooth and flat, and the dispersion is uniform. The particle size and zeta potential of gambogic acid nanoparticles are shown in fig. 2 and fig. 3, and it can be seen that the average particle size of the gambogic acid nanoparticles is 338.9nm, and the zeta potential is-10.2 mv.
Through determination, the entrapment rate of the gambogic acid nanoparticles reaches 85%.
Example 2
In vitro release of gambogic acid nanoparticles
The in vitro release profile of GA-mPEG-PLA-NPs was determined by dialysis. Soaking the dialysis bag in boiling water for 5min, and washing with deionized water. And (3) placing the nanoparticle suspension into a dialysis bag, placing the dialysis bag into PBS (pH 7.4) buffer solution which is stirred at the constant temperature of 37 ℃, rotating at the speed of 100r/min, taking 600 mu L of buffer solution at a specified time, detecting, supplementing the buffer solution with the same volume, and calculating the ratio of the released medicine to the medicine in the nanoparticle suspension to obtain the release degree.
The result is shown in fig. 4, it can be seen from the figure that the release rate of the gambogic acid nanoparticles in the first 16h is high, the release rate is stable in 24h, and the release rate in 48h is 52.38%, so that the slow release effect is achieved.
Example 3
Evaluation of in vitro anti-inflammatory activity of gambogic acid nanoparticles
Taking RAW264.7 cells in logarithmic growth phase, adding 1 × 10 cells into each well of six-well plate61.2mL of cell suspension per mL was incubated at 37 ℃ for 1 hour. The test was divided into seven groups. Addition of blank control and LPS model group0.4mL of physiological saline, three drug groups with low, medium and high (0.5 mu M, 1 mu M and 2 mu M) GA-mPEG-PLA-NPs with different concentrations, a positive control group with 2 mu M dexamethasone sodium phosphate (DEX), and a bulk drug control group with 0.3 mu M gambogic acid for 1 h. Adding 400 μ L of LPS with concentration of 5 μ g/mL to the other groups except the blank control group, and adding complete culture medium to the blank control group; standing at 37 deg.C for 5% CO2Continuously culturing in cell culture box, centrifuging culture solution at 2500rpm and 4 deg.C for 10min for 18 hr, collecting supernatant, and detecting inflammation mediators (NO, PGE)2) And the content of inflammatory factors (IL-1 beta/IL-6/IL-10/TNF-alpha) are used as the basis for evaluating the anti-inflammatory activity of the gambogic acid nanoparticles.
The results are shown in FIG. 5, which shows that compared to the control experiment, the inflammatory mediators (NO, PGE) of the gambogic acid nanoparticle experimental group2) And the inhibitor and proinflammatory factors (IL-1 beta/IL-6/TNF-alpha) are effectively inhibited, and the content of the inflammation inhibitor IL-10 is obviously improved.
Example 4
Evaluation of in vivo anti-inflammatory Activity of Gambogic acid nanoparticles
LPS was used to establish a mouse acute lung injury model (ALI). The intraperitoneal injection was started 3 days after the mice were acclimatized. The test components were divided into seven groups: 5. 3, 1mg/kg.b.w concentration of drug group, blank control group, positive control group (dexamethasone sodium phosphate solution 2mg/kg.b.w), LPS control group and raw material drug control group (gambogic acid 3 mg/kg.b.w). After 5 days of continuous injection, seven groups of mice were instilled with 10 μ LLPS (2mg/kg. b.w) by bronchial instillation 30min after the last administration. After 6h, all mice were sacrificed by cervical dislocation and the content of lung inflammatory factors and inflammatory mediators were examined.
The results are shown in FIG. 6, and the experimental results show that the GA-mPEG-PLA-NPs low (1mg/kg. b.w), medium (3mg/kg. b.w) and high (5mg/kg. b.w) dose groups can greatly reduce the content of inflammatory mediator NO in LPS-induced ALI mice. The PGE can be obviously reduced in the medium and high dose groups2The content of (a). The low, medium and high dose groups can remarkably reduce the content of TNF-alpha and IL-1 beta in lung lavage fluid of mice. The middle-high dose group can greatly reduce the content of IL-6 and greatly increase the content of IL-10. And the in vivo anti-inflammatory activity of GA-mPEG-PLA-NPs is better than that of rattan(ii) a xanthic acid.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A preparation method of gambogic acid nanoparticles is characterized by mainly comprising the following steps:
(1) dissolving gambogic acid and polyethylene glycol polylactic acid block copolymer in a dichloromethane-acetone mixed solvent to obtain an organic phase;
(2) taking 1-3% (m/v) of polyvinyl alcohol aqueous solution as a water phase, and uniformly mixing the water phase with the organic phase obtained in the step (1);
(3) ultrasonically treating the mixed solution prepared in the step (2) under the ice-water bath condition to obtain primary emulsion;
(4) slowly dripping the primary emulsion obtained in the step (3) into a polyvinyl alcohol aqueous solution under stirring;
(5) removing the organic solvent and part of water in the solution obtained in the step (4) by using a rotary evaporator;
(6) and (4) centrifuging, taking the precipitate, washing the precipitate for 1-6 times by using distilled water, and dispersing the precipitate in water to obtain the water-dispersible solid.
2. The preparation method according to claim 1, wherein the volume ratio of dichloromethane to acetone in the dichloromethane-acetone mixed solvent in step (1) is 1:10 to 10: 1.
3. The preparation method according to claim 1, wherein the concentration of the polyethylene glycol polylactic acid block copolymer in the step (1) is 1 to 50mg/mL, and the weight ratio of the gambogic acid to the polyethylene glycol polylactic acid block copolymer is 1:35 to 1: 5.
4. The method according to claim 1, wherein the volume ratio of the organic phase to the aqueous phase in step (2) is 1:5 to 5: 1.
5. The preparation method according to claim 1, wherein the ultrasonic treatment in the step (3) is performed under the condition that the amplitude is 100%, each ultrasonic treatment is performed for 1-10 s at an interval of 1-10 s, and the total ultrasonic treatment time is 30-100 s.
6. The method according to claim 1, wherein the concentration of the polyvinyl alcohol in the step (4) is 0.1 to 1% (m/v), and the stirring time is 10 to 200 min.
7. The method according to any one of claims 1 to 6, wherein the centrifugation in the step (6) is carried out at a centrifugation rate of 10000 to 40000rpm for 0.5 to 2 hours.
8. A gambogic acid nanoparticle obtained by the production method according to any one of claims 1 to 7.
9. Use of gambogic acid nanoparticles according to claim 8 in the preparation of a medicament for the treatment of inflammation.
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