CN110124054B - Preparation method and application of targeted nano particles self-assembled layer by layer - Google Patents
Preparation method and application of targeted nano particles self-assembled layer by layer Download PDFInfo
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- CN110124054B CN110124054B CN201910540490.7A CN201910540490A CN110124054B CN 110124054 B CN110124054 B CN 110124054B CN 201910540490 A CN201910540490 A CN 201910540490A CN 110124054 B CN110124054 B CN 110124054B
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
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- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
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- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
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- 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
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- 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
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- A61P35/00—Antineoplastic agents
Abstract
The invention relates to a preparation method of targeted nanoparticles by layer-by-layer self-assembly, which is characterized in that slightly soluble hydrophobic drugs are connected on hyaluronic acid through chemical bonds, and the same or different hydrophobic drugs are encapsulated by self-assembly to obtain self-assembled nanoparticles; connecting polylysine and targeting ligand molecule via chemical bond; the targeted nano particles are obtained by respectively covering hyaluronic acid, polylysine or polylysine-targeted ligand on the outermost layer of the nano particles by layer self-assembly according to needs. The targeted nano particle prepared by the method has high drug loading, can realize passive and active targeting of tumors through an EPR effect and a targeting ligand, has functionality by the selected carrier material, and has tumor targeting effect by combining with a CD44 receptor highly expressed at the tumor part; the targeting nanoparticles prepared by the method can be coated with two drugs with synergistic effect, so that the stability and targeting property of the drugs are improved, and the dosage and toxicity are reduced.
Description
Technical Field
The invention belongs to the technical field of polymer drug carriers, and particularly relates to a preparation method and application of targeted nanoparticles self-assembled layer by layer.
Background
The anti-tumor drugs clinically applied at present are mainly cytotoxic chemotherapeutic drugs, but the drugs have low selectivity and large toxic and side effects, are easy to damage normal cells and tissues, and are easy to generate drug resistance in the treatment process. Therefore, the research and development of antitumor drugs with definite targets and small side effects is urgent.
Layer-by-layer self-assembly (LbL) technology provides a multifunctional method that can functionalize the surface of a charged drug carrier without being affected by its internal design. This design may facilitate targeting or enhance cellular uptake, stimulate responsive release, and also facilitate maintenance of biomolecule activity, reducing early degradation during vector transport. LbL technology is the deposition of oppositely charged polyelectrolytes or other charged entities to form multilayer structures with nanometer precision. The driving forces involved in multilayer assembly are primarily electrostatic, but may also involve hydrogen bonding, hydrophobic interactions, van der waals forces, covalent bonding or click chemistry modifications LbL technology can design carriers for drug delivery that can significantly improve drug steric stability and prolong circulation time in vivo.
Through searching, the following patent publications related to the patent application of the invention are found:
1. a preparation method (CN105748441A) of a layer-by-layer self-assembly nano targeting carrier containing camptothecin uses Hydroxycamptothecin (HCPT) as a model drug, uses natural nontoxic degradable biological material chitosan as a base material, and utilizes a glycyrrhetinic acid receptor-mediated tumor active targeting technology to prepare the layer-by-layer self-assembly nano particles capable of responding to special redox and pH environments of tumor tissues and tumor cells. The targeted nanocapsule prepared by the method has high monodispersity, controllable capsule wall thickness and cavity volume, and reductive responsiveness, can release the antitumor drug in the inner cavity in a controlled manner aiming at the microenvironment of tumor cells, can be used as a targeted transportation tool of the antitumor drug, and has a great application prospect in the field of targeted treatment of tumors.
2. A magnetic polyelectrolyte microcapsule and its preparation method (CN103462806A), use porous or hollow layer by layer self-assembly polyelectrolyte microcapsule as carriers, modify magnetic nanoparticle AFe2O4(A is divalent metal ion) to make its surface carry electric charge, prepare magnetic polyelectrolyte microcapsule through the electrostatic interaction among different electric charges that polyelectrolyte of microcapsule outermost layer and modified magnetic nanoparticle carry; the AFe2O4 nano particles account for 15-50% of the mass of the polyelectrolyte microcapsule. The particle size of the microcapsule and the AFe2O4 nano particle is adjustable, the particle size of the microcapsule is 0.1-10 mu m, the particle size of the AFe2O4 nano particle is 1-100 nm, and the magnetic carrying capacity is high, so that the magnetic responsiveness is good. The invention utilizes polyelectrolyte microcapsule electrostatic adsorption to modify AFe2O4 magnetic nanoparticles to prepare the targeted magnetic microcapsule, wherein the particle size and the magnetic carrying capacity of the AFe2O4 nanoparticles are controllable, the operation is simple and convenient, and the targeted magnetic microcapsule has good application prospect in the fields of magnetic materials, cell biology, molecular biology, medicine and the like.
3. The polysaccharide/inorganic nanoparticle hybrid micro-nano drug-loaded capsule (CN101703490A) has the following structural characteristics: (1) an oil-in-water microemulsion capsule core formed by a fat-soluble medicine A or a fat-soluble medicine B dissolved in an oily solvent, a negatively charged OSA modified polysaccharide derivative dispersant and water; (2) the polysaccharide and the inorganic nano particles with negative electricity form a capsule wall through layer-by-layer self-assembly based on electrostatic interaction; (3) the particle size of the micro-nano medicine carrying capsule is 50-1000 nm. The micro-nano medicine-carrying capsule provided by the invention has high medicine-carrying rate of in-situ embedding of medicines, and the raw materials are all made of biodegradable materials with good biocompatibility and biodegradability. In addition, the drug delivery system has dual 'intelligent' functions of targeting and controlled release.
By contrast, the present patent application is substantially different from the above patent publications.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method and application of a layer-by-layer self-assembled targeted nanoparticle, the targeted nanoparticle prepared by the method has high drug loading, passive and active targeting of tumors can be realized through an EPR effect and a targeting ligand, and the selected carrier material also has the functionality: polylysine facilitates interaction with and penetration of cell membranes, while hyaluronic acid also has tumor targeting effects by binding to highly expressed CD44 receptors at tumor sites.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for preparing targeted nanoparticles self-assembled layer by layer is characterized in that a slightly-soluble hydrophobic drug is connected to hyaluronic acid through a chemical bond, and the same or different hydrophobic drugs are encapsulated through self-assembly to obtain self-assembled nanoparticles; connecting polylysine and targeting ligand molecule via chemical bond; the targeted nano particles are obtained by respectively covering hyaluronic acid, polylysine or polylysine-targeted ligand on the outermost layer of the nano particles by layer self-assembly according to needs.
Moreover, the hydrophobic drug is a paclitaxel drug, a camptothecin drug or a podophyllotoxin drug.
Moreover, the slightly soluble hydrophobic drug is one or more of paclitaxel drugs, camptothecin drugs and podophyllotoxin drugs.
Furthermore, the targeting ligand molecule is folic acid, RGD, iRGD, NGR, cell penetrating peptide, CIELLQAR, IELLQAR, CRAQLLEI, RAQLLEIC, (D) -CIELLQAR, (D) -IELLQAR, (D) -CRAQLLEI or (D) -RAQLLEICD, and one or more targeting ligand molecules can be attached to polylysine.
Furthermore, the molecular weight of the hyaluronic acid and polylysine is 3000-2000000 Da.
The method comprises the following specific steps:
the preparation method comprises the steps of taking 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride A as an activating agent, and connecting hyaluronic acid B with adipic acid dihydrazide C to obtain modified hyaluronic acid; wherein, substance A: b: the proportion mol of C: mol: mol is 10-50: 1: 2-10;
connecting succinic anhydride E and a drug F under the alkaline condition of N, N-diisopropylethylamine D to enable the succinic anhydride E and the drug F to carry carboxyl groups, so as to obtain a drug intermediate; wherein, substance D: e: the proportion mol of F: mol: mol is 0.5-2: 1-2: 1;
connecting the modified medicine H and the modified hyaluronic acid I by using 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride A and hydroxysuccinimide G as activating agents to obtain an amphiphilic hyaluronic acid-medicine polymer; wherein, substance A: g: h: ratio mol of I: mol: mol: mol is 10-50: 10-50: 2-10: 1;
fourthly, reacting polylysine J and 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfo succinimide ester sodium salt K in a PBS buffer solution with the pH value of 7.0-7.8 to modify an active group with a double bond, modifying a targeting ligand L with a sulfydryl group, reacting in PBS with the pH value of 6.0-6.8, dialyzing and purifying to obtain a polylysine-targeting ligand molecule; wherein substance J: k: the proportion mol of L: mol: mol is 1: 2-20: 2-20;
fifthly, dissolving the amphiphilic hyaluronic acid-drug polymer, stirring, adding the drug with the concentration of 0.5-50 mg/mL, stirring, dialyzing for 12 hours, carrying out ultrasonic treatment by using a cell disruption instrument, and passing through a membrane to obtain the self-assembled nanoparticles;
sixthly, taking the self-assembled nanoparticles carrying the medicine, adding 0.5-50 mg/mL polylysine or polylysine-targeting ligand aqueous solution, and stirring at room temperature to enable the self-assembled nanoparticles to be deposited on the surfaces of the nanoparticles; removing unadsorbed polylysine by ultrafiltration and centrifugation to obtain a layer of deposited nanoparticles with a diameter of 80-400 nm; then continuously adding 0.5-50 mg/mL hyaluronic acid aqueous solution, stirring to enable the hyaluronic acid aqueous solution to be deposited on the surfaces of the nanoparticles, performing ultrasonic treatment under a cell disruption instrument, and passing through a membrane to obtain two layers of deposited nanoparticles with the diameter of 80-400nm, namely obtaining layer-by-layer self-assembled targeted nanoparticles; according to the requirement, the steps are repeated, and the target nano particles self-assembled layer by layer can be obtained by multiple alternate deposition, wherein the diameter of the target nano particles is 80-800 nm.
The nanoparticles prepared by the preparation method of the targeted nanoparticles self-assembled layer by layer are applied to preparation of antitumor drugs and targeted antitumor drugs.
The invention has the advantages and positive effects that:
1. the targeted nano particles prepared by the method have high drug loading, can realize passive and active targeting of tumors through an EPR effect and a targeting ligand, and the selected carrier material also has the following functions: the polylysine is favorable for interacting with cell membranes and penetrating the cell membranes, and the hyaluronic acid also has a tumor targeting effect by combining with a CD44 receptor highly expressed at a tumor part; the targeting nanoparticles prepared by the method can be coated with two drugs with synergistic effect, so that the stability and targeting property of the drugs are improved, and the dosage and toxicity are reduced.
2. The method of the invention connects hyaluronic acid with hydrophobic drugs by a chemical synthesis method, so that the hyaluronic acid has amphipathy, can self-assemble and wrap one or more drugs, obtains nanoparticles with higher drug loading rate and encapsulation rate, and improves the water solubility and stability of the drugs.
3. According to the method, a plurality of targeting ligand molecules are connected to the polylysine by a chemical synthesis method, so that the targeting effect of the polylysine is enhanced.
4. According to the method, the number of deposition layers can be controlled according to needs by a layer-by-layer self-assembly method, hyaluronic acid, polylysine or polylysine-targeting ligand can be deposited on the outermost layer of the nano particles, the targeting capability or the membrane permeation capability of the nano particles is improved, the stability of the system can be improved by multi-layer deposition, and the biocompatibility is good.
5. The method of the invention selects hyaluronic acid and polylysine as negative and positive polyelectrolytes, and connects targeting ligand molecules through chemical bonds to achieve the aim of multiple targeting. The method can entrap one or more hydrophobic drugs, can obtain higher drug-loading rate and encapsulation efficiency, provides possibility for simultaneously delivering two drugs with synergistic effect, and the hyaluronic acid has the capacity of targeting CD44 molecules on the surface of tumor cells, while the polylysine can be connected with one or more targeting ligand molecules by a chemical synthesis method, so that the targeting capacity can be further improved.
Drawings
FIG. 1 is a MALDI-TOF spectrum of a polylysine-targeting ligand prepared in example 4 of the present invention;
FIG. 2 is a DLS particle size distribution diagram of the nanomicelle prepared in example 5 of the present invention;
FIG. 3 is a graph of zeta potential of nanomicelles prepared in example 5 of the present invention;
FIG. 4 is a DLS particle size distribution diagram of the nanomicelle prepared in example 6 of the present invention;
FIG. 5 is a graph of zeta potential of nanomicelles prepared in example 6 of the present invention;
FIG. 6 is a DLS particle size distribution diagram of the nanomicelle prepared in example 7 of the present invention;
FIG. 7 is a graph of zeta potential of nanomicelles prepared in example 7 of the present invention;
FIG. 8 is a DLS particle size distribution diagram of nanomicelle prepared in example 8 of the present invention;
FIG. 9 is a graph of zeta potential of nanomicelles prepared in example 8 of the present invention;
FIG. 10 is a DLS particle size distribution diagram of nanomicelle prepared in example 9 of the present invention;
FIG. 11 is a graph of zeta potential of nanomicelles prepared in example 9 of the present invention;
FIG. 12 is a DLS particle size distribution diagram of nanomicelle prepared in example 10 of the present invention;
fig. 13 is a zeta potential diagram of the nanomicelle prepared in example 10 of the present invention.
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A method for preparing targeted nanoparticles self-assembled layer by layer is characterized in that a slightly-soluble hydrophobic drug is connected to hyaluronic acid through a chemical bond, and the same or different hydrophobic drugs are encapsulated through self-assembly to obtain self-assembled nanoparticles; connecting polylysine and targeting ligand molecule via chemical bond; the targeted nano particles are obtained by respectively covering hyaluronic acid, polylysine or polylysine-targeted ligand on the outermost layer of the nano particles by layer self-assembly according to needs.
Preferably, the hydrophobic drug is a paclitaxel drug, a camptothecin drug or a podophyllotoxin drug.
Preferably, the slightly soluble hydrophobic drug is one or more of paclitaxel drugs, camptothecin drugs and podophyllotoxin drugs.
Preferably, the targeting ligand molecule is folate, RGD, iRGD, NGR, a cell penetrating peptide, CIELLQAR, IELLQAR, CRAQLLEI, RAQLLEIC, (D) -CIELLQAR, (D) -IELLQAR, (D) -CRAQLLEI or (D) -RAQLLEICD, and one or more targeting ligand molecules can be attached to polylysine.
Preferably, the molecular weight of the hyaluronic acid and the polylysine is 3000-.
Preferably, the specific steps are as follows:
the preparation method comprises the steps of taking 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride A as an activating agent, and connecting hyaluronic acid B with adipic acid dihydrazide C to obtain modified hyaluronic acid; wherein, substance A: b: the proportion mol of C: mol: mol is 10-50: 1: 2-10;
connecting succinic anhydride E and a drug F under the alkaline condition of N, N-diisopropylethylamine D to enable the succinic anhydride E and the drug F to carry carboxyl groups, so as to obtain a drug intermediate; wherein, substance D: e: the proportion mol of F: mol: mol is 0.5-2: 1-2: 1;
connecting the modified medicine H and the modified hyaluronic acid I by using 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride A and hydroxysuccinimide G as activating agents to obtain an amphiphilic hyaluronic acid-medicine polymer; wherein, substance A: g: h: ratio mol of I: mol: mol: mol is 10-50: 10-50: 2-10: 1;
fourthly, reacting polylysine J and 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfo succinimide ester sodium salt K in a PBS buffer solution with the pH value of 7.0-7.8 to modify an active group with a double bond, modifying a targeting ligand L with a sulfydryl group, reacting in PBS with the pH value of 6.0-6.8, dialyzing and purifying to obtain a polylysine-targeting ligand molecule; wherein substance J: k: the proportion mol of L: mol: mol is 1: 2-20: 2-20;
fifthly, dissolving the amphiphilic hyaluronic acid-drug polymer, stirring, adding the drug with the concentration of 0.5-50 mg/mL, stirring, dialyzing for 12 hours, carrying out ultrasonic treatment by using a cell disruption instrument, and passing through a membrane to obtain the self-assembled nanoparticles;
sixthly, taking the self-assembled nanoparticles carrying the medicine, adding 0.5-50 mg/mL polylysine or polylysine-targeting ligand aqueous solution, and stirring at room temperature to enable the self-assembled nanoparticles to be deposited on the surfaces of the nanoparticles; removing unadsorbed polylysine by ultrafiltration and centrifugation to obtain a layer of deposited nanoparticles with a diameter of 80-400 nm; then continuously adding 0.5-50 mg/mL hyaluronic acid aqueous solution, stirring to enable the hyaluronic acid aqueous solution to be deposited on the surfaces of the nanoparticles, performing ultrasonic treatment under a cell disruption instrument, and passing through a membrane to obtain two layers of deposited nanoparticles with the diameter of 80-400nm, namely obtaining layer-by-layer self-assembled targeted nanoparticles; according to the requirement, the steps are repeated, and the target nano particles self-assembled layer by layer can be obtained by multiple alternate deposition, wherein the diameter of the target nano particles is 80-800 nm.
The nanoparticles prepared by the preparation method of the targeted nanoparticles self-assembled layer by layer can be applied to preparation of antitumor drugs and targeted antitumor drugs.
According to the invention, the hyaluronic acid is connected with the hydrophobic drug through a chemical bond, and the indissoluble drug is further encapsulated by self-assembly, so that the water solubility and the stability of the drug are improved, and one or more drugs with synergistic effects can be encapsulated for combined administration; the polylysine and the targeting ligand molecule are connected through a chemical bond so as to increase the active targeting capability; hyaluronic acid, polylysine or polylysine-targeting ligands can be respectively deposited on the surface of the nano particles by layer-by-layer self-assembly, so that the stability of the nano particles can be improved by multilayer deposition, the multi-targeting capability can be improved, and the chance of reaching tumor parts by the medicament can be increased.
The related embodiments of the invention are as follows:
example 1: synthesis of modified hyaluronic acid
100mg of hyaluronic acid was sufficiently dissolved in water, 5 times equivalent of adipic acid dihydrazide was added, the pH of the solution was adjusted to 4.75 with 0.1M hydrochloric acid, and 5 times equivalent of EDC was added to the system to maintain the pH of the system at 4.75 within one hour. After one hour, the reaction was quenched by adjusting the pH to 7 with 0.1M sodium hydroxide. The resulting solution was dialyzed with 3500 dialysis bags in 0.1M sodium chloride solution for 12 hours, 25% ethanol solution for 12 hours and water for 24 hours, respectively. The resulting mixture was dried in a freeze-dryer for 12 hours to obtain 97.1mg of modified hyaluronic acid.
Example 2: synthesis of paclitaxel intermediates
50mg of paclitaxel was dissolved in dichloromethane, 1.5 equivalents of succinic anhydride and 1 equivalent of DIEA were added, and the mixture was reacted at room temperature with dichloromethane: anhydrous methanol 17: detecting the reaction, when the product is completely generated, adding dichloromethane to dilute the system, then extracting with saturated ammonium chloride solution for three times, then extracting with saturated sodium chloride solution for two times, finally drying with anhydrous sodium sulfate, spin-drying the liquid, and purifying the product by column chromatography to obtain a paclitaxel intermediate 48.8mg with the yield of 87.36%.
Example 3: synthesis of amphiphilic hyaluronic acid-paclitaxel polymer
50mg of modified hyaluronic acid is dissolved in formamide, four times equivalent of paclitaxel intermediate is dissolved in DMF, 20 times equivalent of EDC and NHS is added, carboxyl is activated for 2 hours at room temperature, then the modified hyaluronic acid is dripped in and reacts for 48 hours at room temperature under the protection of inert gas. After the reaction, the mixture was dialyzed in a dialysis bag of 3500 against a 25% ethanol solution for 24 hours, against water for 48 hours, and then dried in a freeze-dryer for 12 hours, to obtain 94.8mg of an amphiphilic hyaluronic acid-paclitaxel polymer.
Example 4: preparation of polylysine-targeting ligand molecules
60mg of polylysine was dissolved in 5ml PBS (pH 7.4) buffer solution and stirred at room temperature. Dissolving 2.5 times of equivalent of sulfo-SMCC in a small amount of water, carrying out ultrasonic treatment in an ultrasonic cleaner, carrying out warm bath at 50 ℃ for a while, diluting the solution to 3mL by using PBS (pH 7.4) buffer solution, slowly dropping the solution into the solution of polylysine under the condition of salt solution, reacting for 5 hours under the protection of inert gas, removing most of water by using a rotary evaporator in the water bath at 55 ℃, dropping the rest small amount of liquid into ethanol, precipitating powdery solid, centrifuging at 3000rpm for 10 minutes, pouring out supernatant, washing twice by using ethanol, and finally drying the solid at room temperature by using inert gas.
50mg of the above compound was dissolved in 9ml PBS (pH 6.5) buffer solution and stirred at room temperature. 5-fold equivalent of the targeting polypeptide cielllqar was dissolved well with 6mL of acetonitrile and 9mL of a buffer solution of pbs (pH 6.5), and then slowly added dropwise to the above solution. And reacting at room temperature for 24 hours under the protection of inert gas. Dialyzing out the salt of the system by using a 3500 dialysis bag, recrystallizing the product by using ethanol, separating out powdery solid, centrifuging at 3000rpm for 10 minutes, pouring out the supernatant, washing twice by using ethanol, and finally drying the solid by using inert gas at room temperature. 50mg of polylysine-targeting ligand molecule was obtained. And finally connecting 1-3 targeting ligand molecules by using MALDI-TOF analysis. The MALDI-TOF spectrum of the prepared polylysine-targeting ligand is shown in figure 1, and as can be seen from figure 1, polylysine is successfully connected with 1-3 targeting ligand molecules.
Example 5: preparation of paclitaxel-loaded hyaluronic acid-paclitaxel nanoparticles
Dissolving 18mg of amphiphilic hyaluronic acid-paclitaxel polymer in 3ml of ultrapure water, and stirring at room temperature for 30 min; dissolving completely, dropwise adding paclitaxel dissolved in 330 μ L ethanol into the solution under stirring, gradually changing the solution from transparent solution to turbid solution with the addition of paclitaxel, stirring at room temperature for one hour to stabilize micelle, rotating at 55 deg.C for 5 min with a rotary evaporator to remove ethanol as added organic solvent, performing ultrasonic treatment with probe under ice bath for 30min with power of 15%, opening for 2s, closing for 2s, filtering with 0.45 μ L filter membrane, and storing at 4 deg.C. The nanometer particle diameter of the micelle measured by a Malvern particle sizer is 148.4nm, and the zeta potential is-24.3 mV. HPLC found drug encapsulation > 95%. The DLS particle size distribution diagram and zeta potential diagram of the prepared nano-micelle are shown in FIGS. 2 and 3, and it can be seen from the diagrams that the nano-particle size of the micelle is 148.4nm and the zeta potential is-24.3 mV.
Example 6: the surface of the hyaluronic acid-paclitaxel nano particle loaded with paclitaxel is coated with a layer of polylysine
10mg of polylysine was dissolved in ultrapure water, slowly added to 1mL of the micelle prepared in example 5, and stirred for 0.5 h. Centrifuging at 1900rpm for 3min in 50000Da ultrafiltering centrifuge tube, and repeating centrifuging for about ten times. Passing through a filter membrane with the pore diameter of 0.45 mu L, and storing at 4 ℃. The particle size of the nanoparticles was 177.2nm as measured by a Malvern particle sizer, and the zeta potential was +26.6 mV. HPLC found drug encapsulation > 95%. The DLS particle size distribution diagram and zeta potential diagram of the prepared nano-micelle are shown in FIGS. 4 and 5, and it can be seen from the diagrams that the nano-particle size of the micelle is 177.2nm and the zeta potential is +26.6 mV.
Example 7: the surface of the hyaluronic acid-paclitaxel nano particle loaded with paclitaxel is coated with a layer of polylysine-targeting ligand molecules
20mg of polylysine-targeting ligand molecule was dissolved in ultrapure water, slowly added to 1mL of the micelle prepared in example 5, and stirred for 0.5 h. Performing ultrasonic treatment with probe under ice bath for 2min with power of 15% for 2s, turning on for 2s, turning off for 2s, centrifuging with 50000Da ultrafiltration centrifuge tube at 1900rpm for 3min, and repeating centrifuging for about ten times. Passing through a filter membrane with the pore diameter of 0.45 mu L, and storing at 4 ℃. The particle size of the nanoparticles was 149.5nm as measured by a Malvern particle sizer, and the zeta potential was +18.3 mV. HPLC found drug encapsulation > 95%. The DLS particle size distribution diagram and zeta potential diagram of the prepared nano-micelle are shown in FIGS. 6 and 7, and it can be seen from the diagrams that the nano-particle size of the micelle is 149.5nm and the zeta potential is +18.3 mV.
Example 8: the surface of the hyaluronic acid-paclitaxel nano particle loaded with paclitaxel is coated with a layer of polylysine and a layer of hyaluronic acid
20mg of hyaluronic acid was dissolved in ultrapure water, slowly added to 1mL of the micelle prepared in example 6, and stirred for 0.5 h. Performing ultrasonic treatment with probe under ice bath for 10min with power of 15%, 2s on, and 2s off. Passing through a filter membrane with the pore diameter of 0.45 mu L, and storing at 4 ℃. The particle size of the nano-particles measured by a Malvern particle sizer is 128.5nm, and the zeta potential is-20.2 mV. HPLC found drug encapsulation > 95%. The DLS particle size distribution diagram and zeta potential diagram of the prepared nano-micelle are shown in FIGS. 8 and 9, and it can be seen from the diagrams that the nano-particle size of the micelle is 128.5nm and the zeta potential is-20.2 mV.
Example 9: the surface of the hyaluronic acid-paclitaxel nano particle loaded with paclitaxel is coated with a layer of polylysine, and a layer of hyaluronic acid is further coated with a layer of polylysine
20mg of polylysine was dissolved in ultrapure water, slowly added to 1mL of the micelle prepared in example 8, and stirred for 0.5 h. Centrifuging at 1900rpm for 3min in 50000Da ultrafiltering centrifuge tube, and repeating centrifuging for about ten times. Passing through a filter membrane with the pore diameter of 0.45 mu L, and storing at 4 ℃. The particle size of the nanoparticles was 185.6nm as measured by a Malvern particle sizer, and the zeta potential was +24.4 mV. HPLC found drug encapsulation > 95%. The DLS particle size distribution diagram and zeta potential diagram of the prepared nano-micelle are shown in FIGS. 10 and 11, and it can be seen from the graphs that the nano-particle size of the micelle is 185.6nm and the zeta potential is +24.4 mV.
Example 10: the surface of the hyaluronic acid-paclitaxel nano particle loaded with paclitaxel is coated with a layer of polylysine, and a layer of hyaluronic acid is further coated with a layer of polylysine-targeting ligand molecules
40mg of polylysine-targeting ligand molecule was dissolved in ultrapure water, slowly added to 1mL of the micelle prepared in example 8, and stirred for 0.5 h. Performing ultrasonic treatment with probe under ice bath for 2min with power of 15% for 2s, turning on for 2s, turning off for 2s, centrifuging with 50000Da ultrafiltration centrifuge tube at 1900rpm for 3min, and repeating centrifuging for about ten times. Passing through a filter membrane with the pore diameter of 0.45 mu L, and storing at 4 ℃. The particle size of the nanoparticles was 159.9nm as measured by a Malvern particle sizer and the zeta potential was +18.4 mV. HPLC found drug encapsulation > 95%. The DLS particle size distribution diagram and zeta potential diagram of the prepared nano-micelle are shown in FIGS. 12 and 13, and it can be seen from the graphs that the nano-particle size of the micelle is 159.9nm and the zeta potential is +18.4 mV.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Claims (2)
1. A preparation method of targeted nanoparticles by layer-by-layer self-assembly is characterized by comprising the following steps: the method comprises the following specific steps:
the method comprises the steps of taking 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride A as an activating agent, and connecting hyaluronic acid B with adipic acid dihydrazide C to obtain modified hyaluronic acid I; wherein, substance A: b: the proportion mol of C: mol: mol is 10-50: 1: 2-10;
under the alkaline condition of N, N-diisopropylethylamine D,will be provided withThe succinic anhydride E is connected with the drug F to enable the succinic anhydride E to have carboxyl groups, so that a drug intermediate H is obtained; wherein, substance D: e: the proportion mol of F: mol: mol is 0.5-2: 1-2: 1;
connecting the drug intermediate H and the modified hyaluronic acid I by using 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride A and hydroxysuccinimide G as activating agents to obtain an amphiphilic hyaluronic acid-drug polymer; wherein, substance A: g: h: ratio mol of I: mol: mol: mol is 10-50: 10-50: 2-10: 1;
fourthly, reacting polylysine J and 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfo succinimide ester sodium salt K in a PBS buffer solution with the pH value of 7.0-7.8 to modify an active group with a double bond, modifying a targeting ligand L with a sulfydryl group, reacting in PBS with the pH value of 6.0-6.8, dialyzing and purifying to obtain a polylysine-targeting ligand molecule; wherein substance J: k: the proportion mol of L: mol: mol is 1: 2-20: 2-20;
fifthly, dissolving the amphiphilic hyaluronic acid-drug polymer, stirring, adding the drug with the concentration of 0.5-50 mg/mL, stirring, dialyzing for 12 hours, carrying out ultrasonic treatment by using a cell disruption instrument, and passing through a membrane to obtain the self-assembled nanoparticles;
sixthly, taking the self-assembled nanoparticles carrying the medicine, adding polylysine with the concentration of 0.5-50 mg/mL or the polylysine-targeting ligand aqueous solution, and stirring at room temperature to enable the polylysine-targeting ligand aqueous solution to be deposited on the surfaces of the nanoparticles; removing unadsorbed polylysine or polylysine-targeting ligand by ultrafiltration and centrifugation to obtain a layer of deposited nanoparticles with a diameter of 80-400 nm; then continuously adding 0.5-50 mg/mL hyaluronic acid aqueous solution, stirring to enable the hyaluronic acid aqueous solution to be deposited on the surfaces of the nanoparticles, performing ultrasonic treatment under a cell disruption instrument, and passing through a membrane to obtain two layers of deposited nanoparticles with the diameter of 80-400nm, namely obtaining layer-by-layer self-assembled targeted nanoparticles; according to the requirement, the steps are repeated, and the target nano particles self-assembled layer by layer can be obtained by multiple alternate deposition, wherein the diameter of the target nano particles is 80-800 nm.
2. The application of the nanoparticles prepared by the layer-by-layer self-assembly targeting nanoparticle preparation method of claim 1 in preparing antitumor drugs.
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