CN115300483B - Preparation method of mussel-like ultra-small lipid nanoparticle with high cell phagocytosis rate - Google Patents
Preparation method of mussel-like ultra-small lipid nanoparticle with high cell phagocytosis rate 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
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
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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
- A61K9/5192—Processes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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Abstract
The invention provides a preparation method of ultra-small mussel-like lipid nanoparticles with high cell phagocytosis rate, a prepared product and application of medicine application. The polydopamine modified lipid nanoparticle prepared by the method has good biocompatibility, cell affinity and cell adhesiveness, improves the water dispersibility of the nanoparticle, optimizes the particle size of the nanoparticle, enhances the in-vivo cell delivery capacity, and improves the bioavailability.
Description
Technical Field
The invention relates to the technical field of biological material preparation, in particular to a preparation method of ultra-small lipid nano particles with high cell phagocytosis rate.
Background
Intracellular drug delivery systems of Lipid Nanoparticles (LNP) are currently clinically effective and versatile non-viral delivery technologies. LNP 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 procedure and composition, and combine good biocompatibility with high bioavailability. To date, lipid-based pharmaceutical 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, and the like.
However, in practical clinical applications, LNP tends to be greatly reduced in phagocytic efficiency due to biological barriers due to defects in its size, structure, and charge. LNP 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 chosen for their preparation affects the size of these structures. In the case of traditional oral administration, however, in order to achieve drug absorption, it is necessary to overcome a mucous barrier up to 100 μm thick covering the epithelium of the gastrointestinal tract, which consists of mucous glycoproteins, forming a three-dimensional network, impeding the penetration of macromolecules. Although the mesh size is in the range of 100-200nm sufficient to allow drug permeation, ionic interactions, hydrogen bonding or hydrophobic interactions, etc. also limit drug diffusion in mucus. In gene therapy, the lipid nanoparticle @ mRNA formulation also needs to overcome multiple intracellular and extracellular barriers in order to function in vivo. Firstly, it is necessary to protect the mRNA from nuclease degradation in physiological fluids, secondly the lipid nanoparticle @ mRNA system needs to reach the target tissue, then be phagocytized by the target cells, and finally the mRNA molecules must escape from the endosome to reach the cytoplasm, where translation takes place. Efficient cell 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, improving the preparation method of the particles in the LNP delivery system, adjusting the particles in the LNP delivery system in terms of size, structure, charge and the like, and realizing efficient intracellular delivery becomes a key in practical application.
Polydopamine (PDA), formed by oxidative polymerization of monomeric Dopamine (DA) under alkaline conditions, is an emerging biomedical application coating, because of its biocompatibility, surface adhesion that promotes cell adhesion and ease of immobilization of biomolecules. PDA modified nanoparticles have found important applications in nanomedicine, in gene delivery, molecular diagnostics, and the like. PDA has been widely reported as modification of nanoparticles such as metal, semiconductor, and inorganic, but its binding to lipid nanoparticles has been studied. And the problem that the particle size tends to be larger after the polydopamine is combined with the nano material often exists, and some insoluble black precipitates tend to be deposited at the bottom of a reaction vessel during the solution oxidation process, so that the PDA surface modification is lack of uniformity and the required thickness is difficult to maintain. The currently reported purification methods of polydopamine nanoparticles are high-speed centrifugation methods, but the high-speed centrifugation methods have poor efficiency and low yield, and can cause irreversible aggregation of polydopamine nanoparticles, so that the storage stability of the nanoparticles is poor. As a novel photo-thermal treatment (PTT) agent, polydopamine nanoparticles can convert light energy into heat energy, but the conversion efficiency is low, which greatly limits its practical application. Furthermore, dopamine itself acts as a neurotransmitter, PDA interactions with human cells, and the effectiveness and safety of in vivo drug delivery are also currently a concern for its use in biomedical fields as a drug carrier,
disclosure of Invention
The invention provides a preparation method of simulated mussel nano-particles with ultra-small particle size, high cell phagocytic efficiency and good biocompatibility.
The invention provides a preparation method of mussel-like ultra-small lipid nanoparticles with high cell phagocytic rate, which is characterized by comprising the following steps:
step 1, preparing a polyvinyl alcohol solution: preparing polyvinyl alcohol into solution by deionized water under the condition of heating to 90 ℃, cooling to room temperature, and then placing in an ice bath at 4 ℃;
step 2, preparing a pre-polymerized polyphenol substance solution: preparing polyphenol hydrochloride into an aqueous solution to obtain a polyphenol solution, adding a medicament required by treatment, adjusting the PH to be alkaline, and reacting for 30min to obtain a pre-polymerized polyphenol 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 obtain a water phase;
step 4, preparing lipid solution: adding lipid into the mixed solution of acetone and ethanol, stirring at 50deg.C for dissolving 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 rapidly stirring for 10min;
step 6, adjusting the PH to be less than 7 by using 0.1N hydrochloric acid;
and 7, centrifuging to neutrality to obtain the PDA modified lipid nanoparticle.
Further, the preparation method of the ultra-small mussel-like lipid nanoparticle with high cell phagocytosis rate comprises the step 1, wherein the concentration of the polyvinyl alcohol solution is 0.2wt%.
Furthermore, the preparation method of the ultra-small mussel-like lipid nanoparticle with high cell phagocytosis rate is characterized in that the concentration of the polyphenol substance solution in the step 2 is 5-10wt%.
Further, the preparation method of the ultra-small mussel-like lipid nanoparticle with high cell phagocytosis rate comprises the following steps: one of dopamine, gallic acid, tannic acid, EGCG and tea polyphenols.
Further, the preparation method of the ultra-small mussel-like lipid nanoparticle with high cell phagocytosis rate comprises the following steps that the pH range in the step 2 is 7-12, and the alkaline solution is selected from the following steps: sodium hydroxide, tris solution.
Further, the preparation method of the ultra-small mussel-like lipid nanoparticle with high cell phagocytosis rate is characterized in that the stirring time in the step 3 is 10-15 min; the temperature was 4 ℃.
Furthermore, the lipid in the step 4 is a single lipid, and preferably the single lipid is selected from one or a mixture of several of glyceryl monostearate, phospholipids and sterols.
Furthermore, in the preparation method of the ultra-small mussel-like lipid nanoparticle with high cell phagocytosis rate, the ratio of acetone to absolute ethyl alcohol in the step 4 is 1:1.
Furthermore, the preparation method of the ultra-small mussel-like lipid nanoparticle with high cell phagocytosis rate is characterized in that the ratio of the oil phase to the water phase in the step 5 is 1:10.
Further, the preparation method of the ultra-small mussel-like lipid nanoparticle with high cytophagy rate comprises the following steps: small molecule drugs, proteins, therapeutic nucleic acids; preferably, the therapeutic nucleic acid is an oligonucleotide, a messenger RNA.
The second aspect of the invention is the ultra-small mussel-like lipid nanoparticle with high cell phagocytic rate prepared and obtained based on the method provided by the first aspect.
The third aspect of the invention is the application of the ultra-small mussel-like lipid nanoparticle with high cell phagocytic rate prepared based on the method provided by the first aspect as a drug delivery carrier.
The beneficial effects of the invention are as follows:
(1) The invention adopts the polydopamine modified lipid nanoparticle with high reactivity, enhances the affinity of the nanoparticle to cells, adjusts the dispersibility of the lipid nanoparticle in water, and endows the lipid nanoparticle 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 the PDA and a cell lipid membrane, and the problem of low bioavailability caused by biological barrier is solved;
drawings
FIG. 1 shows XPS patterns of lipid nanoparticles (SLNs) and polydopamine modified lipid nanoparticles (PSLNs) in examples 1 and 2 according to the present invention;
FIG. 2 is an SEM of nanoparticles of example 1 of the invention;
FIG. 3 is an SEM of nanoparticles of example 2 of the invention;
FIG. 4 shows the phagocytosis of pure lipid nanoparticles according to example 5 of the present invention;
FIG. 5 shows the cellular phagocytosis of gelatin-lipid nanoparticles according to example 5 of the present invention;
FIG. 6 shows the phagocytosis of polydopamine modified lipid nanoparticles (PSLN) according to example 5 of the present invention;
FIG. 7 is a graph comparing the average fluorescence intensity of three nanoparticles.
Detailed Description
The invention will be further described with reference to the drawings and specific examples.
Example 1
The preparation method of the ultra-small mussel-like lipid nanoparticle 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 give a 0.2% PVA solution. After cooling to room temperature, it was placed in an ice bath at 4 ℃.200mg of glyceryl monostearate was added to 12mL of a mixed solution of acetone and absolute ethanol at 50℃to dissolve, wherein the ratio of acetone to absolute ethanol was 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. And freeze-drying to obtain the nano particles.
FIG. 1 shows XPS Spectra of Lipid Nanoparticles (SLNs) and polydopamine modified lipid nanoparticles (PSLNs) in examples 1 and 2 according to the present invention. Qualitative analysis is carried out on the surface elements of the nano particles by an X-ray photoelectron spectrometer (X-ray Photoelectron Spectroscopy, XPS), and compared with the simple lipid nano particles, the lipid nano particles modified by the PDA have a more obvious N peak from the high-resolution spectrogram result, and the modification of the PDA in the lipid nano particles is confirmed.
Fig. 2 is an SEM of nanoparticles in example 1 of the present invention. From the figure, the unmodified nano particles are in an irregular spherical structure, the particle size of the nano particles is mostly distributed around 100nm, and the agglomeration phenomenon exists among the nano particles.
Example 2
The preparation method of the ultra-small mussel-like lipid nanoparticle with high cell phagocytosis rate comprises the following steps:
preparation of polydopamine modified lipid nanoparticles: 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 of dopamine was dissolved in 10mL of deionized water, and 300. Mu.L of sodium hydroxide solution (50 wt%) was added for prepolymerization at room temperature for 30min. Then adding the mixture into PVA solution, and stirring uniformly. 200mg of glyceryl monostearate was added to 12mL of a mixed solution of acetone and absolute ethanol at 50℃to dissolve, wherein the ratio of acetone to absolute ethanol was 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. And freeze-drying to obtain the nano particles.
Fig. 3 is an SEM of nanoparticles in example 2 of the present invention. The graph shows that compared with unmodified nanoparticles, the PDA modified nanoparticles have unchanged morphology, but the particle size of the nanoparticles is greatly reduced and is mostly within 20-50nm, meanwhile, the agglomeration phenomenon of the nanoparticles is obviously improved, and the dispersity of the nanoparticles is better.
Example 3
The preparation method of the ultra-small mussel-like lipid nanoparticle 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 give 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, 300. Mu.L of sodium hydroxide solution (50 wt%) was added, followed by 5mg of Methotrexate (MTX) and prepolymerization at room temperature for 30min. Then adding the mixture into PVA solution, and stirring uniformly. 200mg of glyceryl monostearate was added to 12mL of a mixed solution of acetone and absolute ethanol at 50℃to dissolve, wherein the ratio of acetone to absolute ethanol was 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. And freeze-drying to obtain the nano particles.
Example 4: gelatin (Gel) modified lipid nanoparticles
A control was prepared using gelatin flow-type 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 give 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 deionized water at 40 ℃.200mg of glyceryl monostearate was added to 12mL of a mixed solution of acetone and absolute ethanol at 50℃to dissolve, wherein the ratio of acetone to absolute ethanol was 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 PVA solution, stirring rapidly for 10min, centrifuging at 10000r/min for 15min, collecting nanoparticle precipitate, and washing twice with deionized water. And freeze-drying to obtain the nano particles.
Example 5: verification of phagocytic function of ultra-small mussel-like 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 1-4 above were blended with 5mg 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 inoculated in 48-well plates and attached for 12h. The three rhodamine-labeled lipid nanoparticles (red) described above were then added separately, incubated for 6h, the medium was aspirated, and the medium was carefully rinsed twice with PBS to remove non-phagocytized nanoparticles. And observing the phagocytosis of the nano particles by using a laser confocal microscope.
Experimental results
FIG. 4 is a graph showing phagocytic effect of pure lipid nanoparticles in example 5 of the present invention. By staining the cytoskeleton (green) with FITC-podobicyclic peptides and staining the nuclei with DAPI (blue), it can be clearly observed from the figure that only a very small fraction of the lipid nanoparticles (red) can be phagocytized by cells.
FIG. 5 is a graph showing phagocytosis effects of gelatin-lipid nanoparticles according to example 5 of the present invention. The cell can be clearly observed under the bright field condition, the cell nucleus (blue) is stained by DAPI, and no obvious red fluorescence exists in the cell under a microscope, which indicates that the cell phagocytosis efficiency of the Gel-lipid nanoparticle is low.
FIG. 6 is a graph showing the phagocytic effect of PDA-lipid nanoparticles in example 5 of the present invention. The cytoskeleton (green) is dyed by FITC-podobicyclic peptide, the nucleus (blue) is dyed by DAPI, and red fluorescence in cells can be clearly observed from the graph, which indicates that PDA-lipid nano particles successfully enter cells, and the phagocytosis rate of the cells is greatly improved.
Fig. 7 is a graph comparing the average fluorescence intensity of three nanoparticles, and the data shows that the average fluorescence intensity of the lipid nanoparticles modified by dopamine is 4-5 times higher than that of the lipid nanoparticles modified by unmodified nanoparticles and the lipid nanoparticles modified by gelatin, which indicates that the efficiency of the nanoparticles entering cells is greatly improved after modification by PDA.
Table 1: different modified nanoparticle phagocytic effects
The experiment conclusion shows that the invention firstly utilizes dopamine to modify lipid nano particles, improves the dispersibility of the lipid nano particles in water and reduces the particle size of the lipid nano particles; and enhances nanoparticle biocompatibility and cell affinity. Secondly, due to the characteristic of the 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 nano particles by cells is promoted. The invention utilizes the dopamine modified lipid nanoparticle, can carry out in vivo cell drug delivery by loading various drugs, improves the loss caused by biological barrier in physiological environment, and improves the bioavailability of the drugs.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The preparation method of the mussel-like ultra-small lipid nanoparticle with high cell phagocytosis rate is characterized by comprising the following steps of:
step 1, preparing a polyvinyl alcohol solution: preparing polyvinyl alcohol into solution by deionized water under the condition of heating to 90 ℃, cooling to room temperature, and then placing in an ice bath at 4 ℃;
step 2, preparing a pre-polymerized polyphenol substance solution: preparing polyphenol hydrochloride into an aqueous solution to obtain a polyphenol solution, adding a medicament required by treatment, adjusting the PH to be alkaline, and reacting for 30min to obtain a pre-polymerized polyphenol solution, wherein the polyphenol is dopamine;
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 obtain a water phase;
step 4, preparing lipid solution: adding lipid into a mixed solution of acetone and ethanol, stirring and dissolving at 50 ℃ to obtain an oil phase, wherein the lipid is glycerol monostearate;
step 5, slowly adding the lipid solution obtained in the step 4 into the uniform mixed solution obtained in the step 3, and rapidly stirring for 10min;
step 6, adjusting the pH to be less than 7 by using 0.1N hydrochloric acid;
and 7, centrifuging and washing with deionized water to neutrality to obtain the PDA modified lipid nanoparticle.
2. The method for preparing ultra-small mussel-like lipid nanoparticles with high cytophagocytic efficiency according to claim 1, wherein the concentration of the polyvinyl alcohol solution in step 1 is 0.2. 0.2wt%.
3. The method for preparing the ultra-small mussel-like lipid nanoparticle with high cytophagy rate according to claim 2, wherein the concentration of the polyphenol substance solution in the step 2 is 5wt% -10wt%.
4. The method for preparing the ultra-small mussel-like lipid nanoparticles with high cytophagy rate as claimed in claim 3, wherein the pH range in the step 2 is 7-12.
5. The method for preparing the ultra-small mussel-like lipid nanoparticle with high cytophagy rate as claimed in claim 4, wherein the stirring time in the step 3 is 10-15 min; the temperature was 4 ℃.
6. The method for preparing ultra-small mussel-like lipid nanoparticles with high cytophagy according to claim 5, wherein the ratio of acetone to absolute ethanol in step 4 is 1:1.
7. The method for preparing the ultra-small mussel-like lipid nanoparticle with high cytophagy according to claim 6, wherein the ratio of the oil phase to the water phase in the step 5 is 1:10.
8. The method for preparing ultra-small mussel-like lipid nanoparticles with high cytophagy according to any one of claims 1 to 7, wherein the drug required for the treatment in step 2 is a small molecule drug.
9. The ultra-small mussel-like lipid nanoparticle with high cytophagocytic efficiency obtained by the method according to any one of claims 1 to 8.
10. Use of the ultra-small mussel-like lipid nanoparticle with high cytophagy according to claim 9 for the preparation of a drug delivery vehicle.
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| 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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