CN115300483A - Preparation method of mussel-like ultra-small lipid nanoparticles with high cell phagocytosis rate - Google Patents
Preparation method of mussel-like ultra-small lipid nanoparticles with high cell phagocytosis rate Download PDFInfo
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- 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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- 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
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Abstract
The invention provides a preparation method of an ultra-small mytilus edulis lipid nanoparticle with high cell phagocytosis rate, a prepared product and application of a medicament. The polydopamine modified lipid nanoparticle prepared by the invention has better biocompatibility, cell affinity and cell adhesion, improves the water dispersibility of the nanoparticle, optimizes the particle size of the nanoparticle, enhances the in vivo cell delivery capability and improves the bioavailability.
Description
Technical Field
The invention relates to the technical field of preparation of biological materials, in particular to a preparation method of ultra-small lipid nanoparticles with high phagocytosis rate.
Background
Intracellular drug delivery systems of Lipid Nanoparticles (LNPs) are currently a clinically effective and versatile non-viral delivery technology. LNPs can encapsulate and deliver a variety of bioactive agents, including small molecule drugs, proteins, peptides, and nucleic acids. Lipid-based delivery systems have the advantage of simple formulation procedures and compositions, and combine good biocompatibility with high bioavailability. To date, lipid-based drug formulations are common nano-drugs approved by the FDA. Since lipid-based nanocarriers can interact with cells in a variety of ways, including endocytosis, fusion with cell membranes, etc.
However, in practical clinical application, LNP has defects in size, structure and charge, and the phagocytic efficiency of cells is often greatly reduced due to biological barriers. LNPs are mostly spherical structures composed of lipids and emulsifiers with diameters in the range of about 40-1000nm, and the type of lipid and emulsifier selected for their preparation affects the size of these structures. In conventional oral administration, in order to achieve absorption of the drug, a mucus barrier, consisting of mucus glycoproteins forming a three-dimensional network that prevent macromolecules from penetrating it, up to 100 μm thick, covering the epithelium of the gastrointestinal tract, must be overcome. Although the mesh size is sufficient to allow drug permeation in the 100-200nm range, ionic interactions, hydrogen bonding or hydrophobic interactions can also limit drug diffusion in mucus. In gene therapy, lipid nanoparticle @ mRNA formulations also need to overcome multiple intracellular and extracellular barriers in order to function in vivo. Firstly, the mRNA needs to be protected from nuclease degradation in physiological fluids, secondly the lipid nanoparticle @ mRNA system needs to reach the target tissue and then be phagocytosed by the target cell, and finally the mRNA molecule must escape the endosome to reach the cytoplasm, so that translation takes place. Efficient cellular phagocytosis and endosomal escape are critical for mRNA delivery. Although the mechanism is not fully understood, positively charged lipids may contribute to electrostatic interactions and fusion with negatively charged endosomal membranes, resulting in leakage of mRNA molecules into the cytoplasm. Therefore, the preparation method of the particles in the LNP delivery system is improved, the particles in the LNP delivery system are adjusted in size, structure, charge and the like, and the realization of efficient intracellular delivery becomes the key point in practical application.
Polydopamine (PDA), formed by oxidative polymerization of monomeric Dopamine (DA) under alkaline conditions, is an emerging coating for biomedical applications because of its biocompatibility, surface adhesion properties that promote cell adhesion, and ease of immobilization of biomolecules. In nanomedicine, PDA-modified nanoparticles have found important applications in gene delivery, molecular diagnostics, etc. PDA has been reported in a large number as a modification of nanoparticles such as metal, semiconductor, inorganic, etc., but its binding to lipid nanoparticles has been studied only rarely. And the combination of polydopamine with nano-materials has the problem that the particle size is larger after combination, and some insoluble black precipitates tend to deposit at the bottom of a reaction vessel during solution oxidation, so that the surface modification of PDA lacks uniformity and the required thickness is difficult to maintain. The purification methods of the polydopamine nanoparticles reported at present are high-speed centrifugation methods, but the high-speed centrifugation methods have poor efficiency and low yield, and can cause irreversible aggregation of the polydopamine nanoparticles, so that the storage stability of the nanoparticles is poor. As a novel photothermal therapy (PTT) reagent, polydopamine nanoparticles can convert light energy into heat energy, but the conversion efficiency is low, so that the limitation is great, and the practical application is realized. In addition, the dopamine is used as a neurotransmitter, the interaction between PDA and human body cells, and the effectiveness and safety of in vivo drug delivery are also the problems which are widely concerned at present as drug carriers in the biomedical field,
disclosure of Invention
The invention provides a preparation method of a mussel-like nanoparticle with an ultra-small particle size, high cell phagocytosis efficiency and good biocompatibility.
The invention provides a preparation method of a mussel-imitated ultra-small lipid nanoparticle with high cell phagocytosis rate, which is characterized by comprising the following steps:
step 1 preparation of polyvinyl alcohol solution: preparing polyvinyl alcohol into a solution by using deionized water under the condition of heating to 90 ℃, cooling to room temperature, and placing in an ice bath at 4 ℃;
step 2, preparing a prepolymerized polyphenol substance solution: preparing polyphenol substance hydrochloride into an aqueous solution to obtain a polyphenol substance solution, adding a medicament required by treatment, adjusting the pH value to be alkaline, and reacting for 30min to obtain a pre-polymerized polyphenol substance solution;
step 3, uniformly mixing the pre-polymerized polyphenol substance solution obtained in the step 2 with the polyvinyl alcohol solution obtained in the step 1 to jointly serve as a water phase;
step 4 preparation of lipid solution: adding lipid into mixed solution of acetone and ethanol, and stirring at 50 deg.C to dissolve to obtain oil phase;
step 5, slowly adding the lipid solution obtained in the step 4 into the uniform mixed solution obtained in the step 3, and quickly stirring for 10min;
step 6, adjusting the pH value to be less than 7 by using 0.1N hydrochloric acid;
and 7, centrifuging to be neutral to obtain the PDA modified lipid nanoparticles.
Further, in the preparation method of the ultra-small mussel lipid nanoparticles with high phagocytosis rate, the concentration of the polyvinyl alcohol solution in the step 1 is 0.2wt%.
Further, in the preparation method of the ultra-small mussel lipid nanoparticles with high cell phagocytosis rate, the concentration of the polyphenol substance solution in the step 2 is 5-10 wt%.
Further, the preparation method of the ultra-small mytilus edulis lipid nanoparticle with high phagocytosis rate comprises the following steps of 2, wherein the polyphenols are selected from: one of dopamine, gallic acid, tannic acid, EGCG and tea polyphenol.
Further, in the preparation method of the ultra-small mussel lipid nanoparticle with high phagocytosis rate, the pH range in step 2 is 7-12, and the alkaline solution is selected from: sodium hydroxide, tris solution.
Further, in the preparation method of the ultra-small mytilus edulis Linnaeus lipid nanoparticle with high cell phagocytosis rate, the stirring time in the step 3 is 10-15 min; the temperature was 4 ℃.
Further, in the preparation method of the ultra-small mytilus edulis Linnaeus lipid nanoparticle with high cell phagocytosis rate, the lipid in the step 4 is a monoester, and preferably the monoester is one or a mixture of more of glyceryl monostearate, phospholipid and sterol.
Further, in the preparation method of the ultra-small mytilus edulis Linnaeus lipid nanoparticles with high cell phagocytosis rate, the ratio of acetone to absolute ethyl alcohol in the step 4 is 1:1.
Further, the method for preparing the ultra-small biomimetic mussel lipid nanoparticles with high cell phagocytosis rate comprises the step 5 that the ratio of the oil phase to the water phase is 1.
Further, the preparation method of the ultra-small mytilus edulis lipid nanoparticle with high cell phagocytosis rate comprises the step 2, wherein the drug required for treatment is selected from the group consisting of: small molecule drugs, proteins, therapeutic nucleic acids; preferably, the therapeutic nucleic acid is an oligonucleotide, messenger RNA.
The second aspect of the invention is the ultra-small mytilus edulis lipid nanoparticles with high phagocytosis rate prepared based on the method provided by the first aspect.
The third aspect of the invention is an application of the ultra-small biomimetic mussel lipid nanoparticles with high phagocytosis rate prepared based on the method provided by the first aspect as a drug delivery carrier.
The invention has the beneficial effects that:
(1) The invention adopts polydopamine with high reaction activity to modify the lipid nanoparticles, enhances the affinity of the nanoparticles to cells, adjusts the dispersibility of the lipid nanoparticles in water, and endows the lipid nanoparticles with smaller particle size.
(2) The technical means for preparing the nano particles adopted by the invention is simple and mild, and can effectively encapsulate and deliver various therapeutic drugs, such as molecular drugs, proteins, peptides, nucleic acids and the like.
(3) The PDA modified lipid nanoparticle prepared by the invention can complete more cell phagocytosis in a short time through various interactions of PDA and a cell lipid membrane, and the problem of low bioavailability caused by biological barriers is solved;
drawings
FIG. 1 is an XPS Spectrum of Lipid Nanoparticles (SLNs) and polydopamine modified lipid nanoparticles (PSLNs) in examples 1 and 2 of the present invention;
FIG. 2 is an SEM of nanoparticles of example 1 of the present invention;
FIG. 3 is an SEM of nanoparticles of example 2 of the present invention;
FIG. 4 is the phagocytosis of pure lipid nanoparticles according to example 5 of the present invention;
FIG. 5 is the phagocytosis of gelatin-lipid nanoparticles in example 5 of the present invention;
FIG. 6 is the phagocytosis of polydopamine modified lipid nanoparticle (PSLN) in example 5 of the present invention;
FIG. 7 is a graph comparing the average fluorescence intensity of three nanoparticles.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
Example 1
A preparation method of ultra-small mytilus edulis lipid nanoparticles with high cell phagocytosis rate comprises the following steps:
preparation of lipid nanoparticles: 0.24g of polyvinyl alcohol was dissolved in 120mL of deionized water at a temperature of 90 ℃ to obtain a 0.2% PVA solution. After cooling to room temperature, it was placed in an ice bath at 4 ℃. At 50 ℃, 200mg of glyceryl monostearate is added into 12mL of mixed solution of acetone and absolute ethyl alcohol for dissolution, wherein the ratio of the acetone to the absolute ethyl alcohol is 1:1. The lipid solution was then slowly added to the PVA solution and stirred rapidly for 10min. The nanoparticle precipitate was then collected by centrifugation at 10000r/min for 15min with 0.1N hydrochloric acid to adjust pH =2 and washed to neutrality with deionized water. Freeze drying to obtain nanometer particle.
Fig. 1 is an XPS Spectrum of Lipid Nanoparticles (SLNs) and polydopamine-modified lipid nanoparticles (PSLNs) in examples 1 and 2 of the present invention. Qualitative analysis is carried out on the surface elements of the nano particles through an X-ray Photoelectron Spectroscopy (XPS), and from the result of a high-resolution spectrogram, compared with the simple lipid nano particles, the lipid nano particles modified by adding the PDA have a more obvious N peak, so that the modification of the PDA in the lipid nano particles is confirmed.
FIG. 2 is an SEM of nanoparticles of example 1 of the present invention. It can be seen from the figure that the unmodified nanoparticles are in an irregular spherical structure, the particle size of the nanoparticles is mostly distributed around 100nm, and the nanoparticles are agglomerated.
Example 2
A preparation method of an ultra-small biomimetic mussel lipid nanoparticle with high cell phagocytosis rate comprises the following steps:
preparing the polydopamine modified lipid nanoparticle: 0.24g of polyvinyl alcohol was dissolved in 120mL of deionized water at a temperature of 90 ℃ to give a 0.2% PVA solution. After cooling to room temperature, it was placed in an ice bath at 4 ℃.0.1g dopamine was dissolved in 10mL deionized water and 300. Mu.L sodium hydroxide solution (50 wt%) was added for prepolymerization at room temperature for 30min. Then adding the PVA solution into the mixture, and stirring the mixture evenly. At 50 ℃, 200mg of glyceryl monostearate is added into 12mL of mixed solution of acetone and absolute ethyl alcohol for dissolution, wherein the ratio of the acetone to the absolute ethyl alcohol is 1:1. The lipid solution was then slowly added to the PVA solution and stirred rapidly for 10min. The nanoparticle precipitate was then collected by centrifugation at 10000r/min for 15min with 0.1N hydrochloric acid to adjust pH =2 and washed to neutrality with deionized water. Freeze drying to obtain nanometer particle.
FIG. 3 is an SEM of nanoparticles of example 2 of the present invention. As can be seen from the figure, compared with unmodified nanoparticles, the morphology of the nanoparticles modified by PDA is not greatly changed, but the particle size of the nanoparticles is greatly reduced to be within 20-50nm, meanwhile, the agglomeration phenomenon of the nanoparticles is also obviously improved, and the dispersion degree of the nanoparticles is better.
Example 3
A preparation method of ultra-small mytilus edulis lipid nanoparticles with high cell phagocytosis rate comprises the following steps:
preparation of MTX @ dopamine modified lipid nanoparticles: 0.24g of polyvinyl alcohol was dissolved in 120mL of deionized water at a temperature of 90 ℃ to obtain a 0.2% PVA solution. After cooling to room temperature, it was placed in an ice bath at 4 ℃.0.1g of dopamine was dissolved in 10mL of deionized water, and 300. Mu.L of sodium hydroxide solution (50 wt%) was added, followed by addition of 5mg of Methotrexate (MTX), followed by prepolymerization at room temperature for 30min. Then adding the PVA solution into the mixture, and stirring the mixture evenly. At 50 ℃, 200mg of glyceryl monostearate is added into 12mL of mixed solution of acetone and absolute ethyl alcohol for dissolution, wherein the ratio of the acetone to the absolute ethyl alcohol is 1:1. The lipid solution was then slowly added to the PVA solution and stirred rapidly for 10min. The nanoparticle precipitate was then collected by centrifugation at 10000r/min for 15min with 0.1N hydrochloric acid to adjust pH =2 and washed to neutrality with deionized water. Freeze drying to obtain nanometer particle.
Example 4: gelatin (Gel) modified lipid nanoparticles
The control example was prepared using gelatin flow-through lipid nanoparticles as follows:
preparation of gelatin (Gel) modified lipid nanoparticles: 0.24g of polyvinyl alcohol was dissolved in 120mL of deionized water at a temperature of 90 ℃ to obtain a 0.2% PVA solution. After cooling to room temperature, it was placed in an ice bath at 4 ℃.200mg of gelatin was dissolved in 10mL of deionized water at 40 ℃. At 50 ℃, 200mg of glyceryl monostearate is added into 12mL of mixed solution of acetone and absolute ethyl alcohol for dissolution, wherein the ratio of the acetone to the absolute ethyl alcohol is 1:1. The lipid solution was then slowly added to the gelatin solution and stirred for 30min to form a primary emulsion. Then adding the primary emulsion into a PVA solution, quickly stirring for 10min, centrifuging at 10000r/min for 15min, collecting the nano-particle precipitate, and washing twice with deionized water. Freeze drying to obtain nanometer particle.
Example 5: phagocytic function verification of ultra-small mussel-simulated lipid nanoparticles
The experimental method comprises the following steps:
the pure lipid nanoparticles, PDA-lipid nanoparticles and gelatin-lipid nanoparticles (30 mL) prepared in examples 1 to 4 above were blended with 5mg of rhodamine, respectively, and stirred at room temperature for 24 hours under dark conditions. Deionized water was dialyzed (3500 Da) against light for 3 days to remove unlabeled rhodamine.
RAW264.7 cells at 5X 10 per well 4 The density was seeded in 48-well plates and attached for 12h. Then, the three rhodamine-labeled lipid nanoparticles (red) were added separately, incubated for 6h, the medium was aspirated and carefully washed twice with PBS to remove non-phagocytized nanoparticles. And observing the phagocytosis condition of the nanoparticles by adopting a laser confocal microscope.
Results of the experiment
FIG. 4 is a graph showing the phagocytic effect of the pure lipid nanoparticle of example 5 of the present invention. By staining the cytoskeleton (green) and the nuclei (blue) with FITC-Coprine and DAPI, it can be clearly seen from the figure that only a very small fraction of the lipid nanoparticles (red) can be phagocytosed by the cells.
FIG. 5 is a graph showing the phagocytic effect of the gelatin-lipid nanoparticle of example 5 of the present invention. Under the condition of a bright field, cells can be clearly observed, DAPI stains cell nuclei (blue), and no obvious red fluorescence is seen in the cells under a microscope, which indicates that the cell phagocytosis efficiency of the Gel-lipid nanoparticles is low.
FIG. 6 is a graph showing the phagocytic effect of PDA-lipid nanoparticles in example 5 of the present invention. The cellular skeleton (green) and the cell nucleus (blue) are stained by FITC-ghost-cyclopeptide, red fluorescence in the cells can be clearly observed from the figure, and the result shows that the PDA-lipid nanoparticles successfully enter the cells, and the phagocytosis rate of the cells is greatly improved.
Fig. 7 is a comparison graph of the mean fluorescence intensities of the three nanoparticles, and the data shows that the mean fluorescence intensity of the lipid nanoparticles modified with dopamine is increased by 4-5 times as compared with that of the unmodified nanoparticles and gelatin-modified nanoparticles, indicating that the efficiency of the nanoparticles entering cells is greatly increased by the modification of PDA.
Table 1: phagocytic effect of differently modified nanoparticles
Experimental conclusion, as can be seen from the description of the above examples, the present invention firstly modifies lipid nanoparticles with dopamine to improve the dispersibility in water and reduce the particle size; and enhances the biocompatibility and cell affinity of the nanoparticles. Secondly, due to the characteristics of dopamine zwitterion, the dopamine zwitterion can generate various interactions with the cell surface, so that the dopamine zwitterion has good cell adhesion performance, and the phagocytosis of the nanoparticles by cells is promoted. The dopamine-modified lipid nanoparticle can be used for carrying various medicines to carry out medicine delivery of in-vivo cells, so that the loss caused by biological barriers in the delivery of the lipid nanoparticle in a physiological environment is improved, and the bioavailability of the medicine is improved.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (12)
1. A preparation method of a mussel-like ultra-small lipid nanoparticle with high cell phagocytosis rate is characterized by comprising the following steps:
step 1 preparation of polyvinyl alcohol solution: preparing polyvinyl alcohol into a solution by using deionized water under the condition of heating to 90 ℃, cooling to room temperature, and placing in an ice bath at 4 ℃;
step 2, preparing a pre-polymerized polyphenol substance solution: preparing polyphenol substance hydrochloride into an aqueous solution to obtain a polyphenol substance solution, adding a medicament required by treatment, adjusting the pH value to be alkaline, and reacting for 30min to obtain a pre-polymerized polyphenol substance solution;
step 3, uniformly mixing the pre-polymerized polyphenol substance solution obtained in the step 2 with the polyvinyl alcohol solution obtained in the step 1 to jointly serve as a water phase;
step 4 preparation of lipid solution: adding lipid into mixed solution of acetone and ethanol, and stirring at 50 deg.C to dissolve to obtain oil phase;
step 5, slowly adding the lipid solution obtained in the step 4 into the uniform mixed solution obtained in the step 3, and quickly stirring for 10min;
step 6, adjusting the pH value to be less than 7 by using 0.1N hydrochloric acid;
and 7, centrifuging to be neutral to obtain the PDA modified lipid nanoparticles.
2. The method for preparing the ultra-small mytilus edulis Linnaeus lipid nanoparticle with high phagocytosis rate as claimed in claim 1, wherein the concentration of the polyvinyl alcohol solution in step 1 is 0.2wt%.
3. The method for preparing the ultra-small biomimetic mussel lipid nanoparticles with high phagocytosis rate according to claim 2, wherein the concentration of the polyphenol substance solution in step 2 is 5wt% to 10wt%.
4. The method for preparing the ultra-small mytilus edulis Linnaeus lipid nanoparticle with high phagocytosis rate as claimed in claim 3, wherein the polyphenols in step 2 are selected from: one of dopamine, gallic acid, tannic acid, EGCG and tea polyphenol.
5. The method for preparing the ultra-small mytilus edulis Linnaeus lipid nanoparticle with high phagocytosis rate as claimed in claim 4, wherein the pH in step 2 is in the range of 7-12, and the alkaline solution is selected from the group consisting of: sodium hydroxide, tris solution.
6. The method for preparing the ultra-small mytilus edulis Linnaeus lipid nanoparticle with high phagocytosis rate as claimed in claim 5, wherein the stirring time in step 3 is 10-15 min; the temperature was 4 ℃.
7. The method for preparing the ultra-small mytilus edulis Linnaeus lipid nanoparticle with high phagocytosis rate as claimed in claim 6, wherein the lipid in step 4 is a monoester, preferably the monoester is selected from one or more of glyceryl monostearate, phospholipid and sterol.
8. The method for preparing the ultra-small mytilus edulis Linnaeus lipid nanoparticle with high phagocytosis rate as claimed in claim 7, wherein the ratio of acetone to absolute ethanol in step 4 is 1:1.
9. The method for preparing the ultra-small mytilus edulis Linnaeus lipid nanoparticle with high phagocytosis rate as claimed in claim 8, wherein the ratio of the oil phase to the water phase in step 5 is 1.
10. The method for preparing the ultra-small mytilus edulis Linnaeus lipid nanoparticle with high phagocytosis rate as claimed in any of claims 1-9, wherein the drug required for treatment in step 2 is selected from the group consisting of: small molecule drugs, proteins, therapeutic nucleic acids; preferably, the therapeutic nucleic acid is an oligonucleotide, messenger RNA.
11. The ultra-small mytilus edulis lipid nanoparticles with high phagocytic rate prepared according to any of the methods of claims 1-10.
12. Use of the ultra-small mytilus edulis lipid nanoparticles with high phagocytic rate of claim 11 as a drug delivery vehicle.
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| US20080206341A1 (en) * | 2004-06-09 | 2008-08-28 | Maria Rosa Gasco | Lipid Nanoparticles as Vehicles for Nucleic Acids, Process for Their Preparation and Use |
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| US20080206341A1 (en) * | 2004-06-09 | 2008-08-28 | Maria Rosa Gasco | Lipid Nanoparticles as Vehicles for Nucleic Acids, Process for Their Preparation and Use |
| CN111053757A (en) * | 2018-10-15 | 2020-04-24 | 吉优诺（上海）基因科技有限公司 | Lipid nanoparticles targeting hepatic stellate cells, preparation method and application thereof |
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