CN107412159B - Preparation method and application of triblock polymer micelle - Google Patents
Preparation method and application of triblock polymer micelle Download PDFInfo
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- CN107412159B CN107412159B CN201710209266.0A CN201710209266A CN107412159B CN 107412159 B CN107412159 B CN 107412159B CN 201710209266 A CN201710209266 A CN 201710209266A CN 107412159 B CN107412159 B CN 107412159B
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61K47/00—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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
- A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
- A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
Abstract
The invention provides a preparation method of a triblock polymer micelle, which comprises the following steps: (1) reacting chitosan oligosaccharide with dithiodipropionic acid under the action of an activator to obtain a first intermediate; (2) reacting the first intermediate with polyethyleneimine under the action of an activating agent to obtain a second intermediate; (3) reacting the second intermediate with imidazole acrylic acid under the action of an activating agent to obtain the triblock polymer; (4) and (4) dissolving the triblock polymer obtained in the step (3) in water to obtain the triblock polymer micelle. The triblock polymer micelle has excellent tissue permeability and enhanced permeation and retention effects, so that the triblock polymer micelle has a natural passive targeting effect and provides a new candidate system for the development of nano-carriers.
Description
Technical Field
The invention relates to the technical field of polymers, in particular to a preparation method and application of a triblock polymer micelle.
Background
At present, the treatment of tumors becomes a global problem to be solved urgently, chemotherapy means is generally adopted for treatment in clinical treatment, adverse effects of different degrees are caused on the physical conditions of patients, adverse reactions such as vomiting and hair dropping are easy to occur, drug resistance is easy to generate, albumin nanoparticles and the like on the market at present are expensive in manufacturing cost, the tumor targeting is not ideal, the loaded drugs or genes cannot be quickly and effectively released, and the chemotherapy means is easy to generate adverse phenomena such as multi-drug resistance, the tumors cannot be completely resected, and the tumors are quickly invaded and transferred. The polymer micelle is used as a novel drug carrier, shows huge application potential in a plurality of fields such as medicine and the like, has unique advantages, has higher stability and better biocompatibility, can increase the solubility of insoluble drugs, can be used as an effective carrier of genes, reduces toxic and side effects, has passive and active targeting effects, and improves the curative effect of the drugs. According to the characteristics of tumor microenvironment (such as pH value inside and outside tumor cells, glutathione concentration, active oxygen concentration, temperature change and the like), some intelligent polymer micelles can generate stimulation response in the tumor cells, so that the structure is damaged, and the loaded chemotherapeutic drugs or genes are released, such as Genex-PM, which is successfully marketed, and in addition, a plurality of polymer micelle chemotherapeutic preparations are in clinical research.
Most chemotherapy drugs have poor water solubility, lack selectivity on tumor parts and have strong toxic and side effects on normal tissues or organs; at present, the phenomena of multidrug resistance, accelerated apoptosis, easy transfer and the like of tumors are reversed by interfering the expression of genes in cells by using siRNA, but the siRNA has the defects of large molecular weight, strong negative charge, easy degradation by nuclease and the like, so that the selection of a proper gene/drug carrier is very important. In the non-viral vector, the cationic polymer has good stability, mature preparation, adjustable and easily controlled structure, convenient modification and the like, and is a main vector of gene medicines or antitumor medicines. Many types of cationic polymers are reported, such as Polyethyleneimine (PEI), polyamide, polylysine, chitosan, and the like. The surfaces of polyethyleneimine and polyamide are rich in amino groups, positive charge nanoparticles can be prepared, the positive charge nanoparticles interact with negatively charged cell membranes, and the uptake of tumor cells to carriers is increased, but the polymers generally have high cytotoxicity, so that the application of the polymers is limited to a certain extent.
Disclosure of Invention
In order to solve the above technical problems, the present invention aims to provide a triblock polymer micelle with excellent tissue permeability and retention effect, and an application of the micelle in a tumor treatment drug.
The preparation method of the triblock polymer micelle comprises the following steps:
(1) reacting chitosan oligosaccharide (CSO) with dithiodipropionic acid (-ss-) under the action of an activating agent to obtain a first intermediate chitosan oligosaccharide-dithiodipropionic acid (CSO-ss-).
(2) And (2) reacting the first intermediate obtained in the step (1) with Polyethyleneimine (PEI) under the action of an activating agent to obtain a second intermediate chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine (CSO-ss-PEI).
(3) And (3) reacting the second intermediate obtained in the step (2) with imidazole acrylic acid (UA) under the action of an activating agent to obtain the triblock polymer chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine-imidazole acrylic acid (CSO-ss-PEI-UA) shown in the formula (I).
Wherein n is 18-32, m is 232-;
(4) and (4) dissolving the triblock polymer obtained in the step (3) in water to obtain the triblock polymer micelle.
Wherein, in the step (1):
the reaction temperature is 30-45 deg.C, preferably 40 deg.C.
The reaction is carried out in a solvent, wherein the solvent is one or more of water, DMF, methanol, chloroform and acetone, and water and DMF are preferred.
The reaction time is 10-16h, preferably 12 h.
The mass ratio of CSO to dithiodipropionic acid is 1: 2-3.
In the step (2):
the reaction temperature is 20-30 deg.C, preferably 20 deg.C.
The reaction is carried out in a solvent, which is one or both of water and a phosphate buffer, preferably water, preferably a phosphate buffer of 7.4.
The reaction time is 10-16h, preferably 12 h.
The mass ratio of the chitosan oligosaccharide-dithiodipropionic acid to the PEI is 1:1.5-3, preferably 1: 2.
In the step (3):
the reaction is carried out in a solvent at a temperature of 40-55 deg.C, preferably 50 deg.C.
The solvent is one or more of water, DMF, methanol, chloroform and acetone, preferably water and DMF.
The reaction time is 10-16h, preferably 12 h.
The mass ratio of the chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine to the imidazole acrylic acid is 1.2-1.5:1, preferably 1.3-1.4: 1.
In the step (4):
the reaction temperature is 20-30 ℃.
The concentration of the triblock polymer is 0.8 to 1.2g/ml, preferably 1 mg/ml.
The preparation method of the triblock polymer micelle comprises the following steps:
(1) dissolving chitosan oligosaccharide (CSO) in water to obtain chitosan oligosaccharide aqueous solution, adjusting pH to 7-8, slowly dropping into mixed solution of activating agent and dithiodipropionic acid (-ss-) DMF solution under stirring, and stirring at 30-45 deg.C (preferably 40 deg.C) for 10-16h (preferably 12h) to obtain first intermediate chitosan oligosaccharide-dithiodipropionic acid (CSO-ss-).
After the reaction is completed, the reaction mixture is dialyzed against distilled water for 1 to 2 days, preferably for 24 hours (MWCO 1000), the dialysate is filtered, and the filtrate is freeze-dried, whereby the yield is about 95%.
The molecular weight of the CSO is 3-5 kDa.
The concentration of the chitosan oligosaccharide in the chitosan oligosaccharide water solution is 20-40mg/ml, preferably 25-35mg/ml, and further preferably 30 mg/ml.
The concentration of dithiodipropionic acid in the DMF solution of dithiodipropionic acid is 65-80mg/ml, preferably 70-80mg/ml, and more preferably 70 mg/ml.
The water is preferably distilled water.
The pH value can be adjusted by adding a trace amount of NaOH.
(2) Dissolving the first intermediate obtained in the step (1) in water to obtain a first intermediate aqueous solution, mixing the first intermediate aqueous solution with an activating agent and a Polyethyleneimine (PEI) aqueous solution, and stirring and reacting at 20-30 ℃ for 10-16h, preferably 12h to obtain a second intermediate chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine (CSO-ss-PEI).
Wherein, after the reaction is finished, dialyzing with dialysis bag (MWCO ═ 1000) for 1-2 days, preferably for 2 days, and lyophilizing to obtain a yield of about 90%.
The aqueous PEI solution was added to the aqueous first intermediate solution at a pH of about 6 and the remaining aqueous PEI solution was added after about 30 minutes.
The molecular weight of PEI is 600 g/mol.
The concentration of the first intermediate aqueous solution (chitosan oligosaccharide-dithiodipropionic acid aqueous solution) is 5-15mg/ml, preferably 8-12mg/ml, and more preferably 10 mg/ml.
The concentration of the polyethyleneimine in the polyethyleneimine aqueous solution is 350-450mg/ml, preferably 400-450mg/ml, and further preferably 400 mg/ml.
The water is preferably distilled water.
(3) And (3) dissolving the second intermediate obtained in the step (2) in water to obtain a second intermediate (chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine) water solution, slowly dropwise adding the second intermediate into a mixed solution of an activating agent and an imidazole acrylic acid (UA) DMF solution, and stirring and reacting for 10-16-h, preferably 12h at the temperature of 40-55 ℃ (preferably 50 ℃) to obtain a triblock polymer, namely chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine-imidazole acrylic acid (CSO-ss-PEI-UA) of the formula (I).
Wherein, after the reaction is finished, the liquid is placed in a dialysis bag (MW 1000), the solution is dialyzed for 2-3d, preferably 2d, the dialyzate is filtered, and the supernatant is freeze-dried, so that the yield is about 90%.
The molecular weight of UA is 138.12 g/mol.
The concentration of the chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine in the chitosan oligosaccharide-dithiodipropionic acid-polyethyleneimine aqueous solution is 4-6mg/ml, preferably 4-5mg/ml, and further preferably 4.8 mg/ml.
The concentration of the imidazole acrylic acid in the imidazole acrylic acid DMF solution is 15-20mg/ml, preferably 17-18mg/ml, and further preferably 17.2 mg/ml.
The water is preferably distilled water.
The reaction formulae of steps (1) to (3) are as follows:
wherein n is 18-32, m is 232-.
(4) Dissolving the triblock polymer obtained in the step (3) in water at 20-30 ℃, and filtering by a filter membrane to obtain the triblock polymer micelle.
Further, in the step (1), the dithiodipropionic acid is activated by a carboxyl group through an activating agent and then reacts with the chitosan oligosaccharide.
In the step (2), the first intermediate reacts with polyethyleneimine after activating carboxyl by an activating agent.
In the step (3), the imidazole acrylic acid reacts with the second intermediate after activating carboxyl by an activating agent.
Further, the activating agent is one or more of hydrochloride of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) and Dicyclohexylcarbodiimide (DCC).
EDC and NHS are preferred, in a molar ratio of 1:1.
further, in step (1), the molar ratio of activator to-ss-is from 0.8 to 1.2:1, preferably 1:1, the molar excess of carboxyl in dithiodipropionic acid can ensure that only one carboxyl is reacted.
In step (1), the dithiodipropionic acid solution is activated for carboxyl in an activator for about 30min at 30-45 ℃, preferably 40 ℃.
Further, in the step (2), the molar ratio of the activator to the first intermediate CSO-ss-is 0.8 to 1.2:1, preferably 1:1.
in the step (2), the first intermediate CSO-ss-aqueous solution and the activating agent aqueous solution are mixed and stirred for reaction, and carboxyl activation is carried out.
Further, in the step (3), the molar ratio of the activating agent to the imidazole acrylic acid is 0.8-1.2:1, preferably 1:1.
in the step (3), the imidazole acrylic acid solution is activated for carboxyl in an activating agent for about 30min at 40-55 ℃.
Further, in the step (4), the triblock polymer is added into water, and forms triblock polymer micelles under the ultrasonic condition.
Sonication in an ice bath is preferred.
The probe is preferably sonicated 50 times (400w, 2s on duty 2s off 2 s).
The triblock polymer micelle prepared by the preparation method is applied as a carrier of tumor drugs and/or genes, and the triblock polymer micelle carrying the tumor drugs and/or the genes is a triblock polymer drug-carrying micelle.
Furthermore, the gene is one or more of siRNA, Toll-like 4 receptor gene, multidrug resistance gene, Bcl-2 anti-apoptosis gene, programmed death ligand gene and Mcl-1 anti-apoptosis gene.
Furthermore, the tumor drug is one or more of adriamycin, osthole, paclitaxel, peganum harmala, camptothecin, oxaliplatin, etoposide and cetuximab.
The preparation method of the triblock polymer drug-loaded micelle comprises the following steps: and (3) uniformly mixing the drug solution and/or the gene solution with the triblock polymer micelle solution prepared by the method to obtain the triblock polymer drug-loaded micelle.
The preparation method of the triblock polymer drug-loaded micelle comprises the following steps: slowly dripping a drug solution (such as a DMSO solution of adriamycin, if adriamycin hydrochloride is adopted, hydrochloric acid of the adriamycin hydrochloride needs to be neutralized by triethylamine) into a triblock polymer micelle solution, carrying out ultrasonic treatment for 20-40min, preferably 30min, carrying out magnetic stirring for 1-3h, preferably 2h, then putting the triblock polymer micelle solution into a dialysis bag for dialysis for 4h, changing distilled water once every 1h, filtering dialysate by using a 0.45 mu m microporous filter membrane, and carrying out freeze drying to obtain a triblock polymer drug-loaded micelle; or dissolving gene lyophilized powder water solution (such as siRNA lyophilized powder 0.5OD and 62.5 μ L DEPC water to form 20 μ M stock solution, storing at-20 deg.C), mixing with triblock polymer micelle solution/triblock polymer drug-loaded micelle solution uniformly, and incubating at 20-30 deg.C for 30min to obtain triblock polymer drug-loaded micelle with gene; wherein the gene is one or more of siRNA, programmed death ligand gene and Mcl-1 anti-apoptosis gene; the medicine is one or more of adriamycin, osthole, paclitaxel, peganum harmala, camptothecin, oxaliplatin, etoposide and cetuximab.
Further, the N/P ratio of the gene to the triblock polymer micelle is 4-20: 1, N is amino in triblock polymer micelle, and P is phosphate radical in gene.
Wherein the mass ratio of the tumor medicament to the triblock polymer micelle is 0.1-0.5: 1-3.
By the scheme, the invention at least has the following advantages:
the CSO adopted by the invention has good biocompatibility and water solubility, can be used as a hydrophilic shell, but is extremely unstable when used alone as a carrier; PEI is widely applied to gene vectors, has a proton sponge effect, but has higher toxicity when being used alone, so in order to solve the problem, two carboxyl groups in dithiodipropionic acid are activated and used as connecting arms to respectively link CSO and PEI, the grafting ratio of CSO is 3.33% and the grafting ratio of UA is 44.4% compared with PEI, imidazole acrylic acid (UA) is connected to one end of PEI as a hydrophobic inner core for encapsulating indissolvable drugs, a layer of CSO is coated outside a disulfide bond (-ss-) in dithiodipropionic acid at the other end for maintaining the whole polymer to be positive, the toxicity of PEI is reduced at the same time, the polymer can be self-assembled in water to form nano particles with pH sensitivity and reduction sensitivity, so the toxicity of the polymer is reduced, and the reduction sensitivity (-ss-) and the pH sensitivity (proton sponge effect) of the polymer in a tumor microenvironment are reflected, PEI is adopted as a main chain for adsorbing siRNA (carboxyl is activated by EDC and NHS among units, and amino and carboxyl react to form amido bond), the carrier formed by the PEI can realize drug and gene combination treatment of tumors, and a novel carrier is provided for a drug delivery system of tumors.
The particle size of the nano particles formed by the polymer micelle is about 140nm, and the nano particles have excellent tissue permeability and enhanced permeation and retention effects, so that the nano particles have a natural passive targeting effect and provide a new candidate system for the development of nano carriers; the polymer has unique polymer particles with three-layer structures, and genes can be compressed into a PEI layer after the particles are formed, so that the size of micelle particles and the large-scale production of the micelle particles are favorably controlled, and a chemical structure reference is provided for constructing a nano carrier and developing a novel drug-carrying system based on an acid-sensitive polymer.
The polymer drug-loaded micelle nano particle prepared by the invention can be rapidly broken under the action of high-concentration Glutathione (GSH) in a tumor cell through hydrophilic and hydrophobic property change caused by PEI protonation, so that the polymer micelle nano particle has certain acid sensitivity and reduction sensitivity, and the conformation is changed under the acidic environment of the tumor cell to release loaded genes and drugs; can be used for transfecting genes into target cells and regulating the expression of target proteins in the presence of serum, and can simultaneously carry anti-tumor drugs and genes, thereby jointly playing an anti-tumor role.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings.
Drawings
FIG. 1 shows the results of CSO (a), CSO-ss- (b), PEI (c), CSO-ss-PEI (d), CSO-ss-PEI-UA (e) in example 1 of the present invention1An H-NMR spectrum;
FIG. 2 is a graph showing fluorescence intensity of triblock polymers at different concentrations in example 1 of the present invention;
FIG. 3 shows a graphical representation of the triblock polymer at λ in example 1 of the invention1At 372nm and λ3Quotient of fluorescence intensity at 383nm (I)1/I3) The ratio to the logarithm of the corresponding concentration;
FIG. 4 shows graphically the change in particle size of the triblock polymer as detected by dynamic light scattering particle size analyzer DLS in example 1 of the present invention;
FIG. 5 is a transmission electron microscope image of a triblock polymer micelle prepared in example 1 of the present invention;
FIG. 6 is a graph of the in vitro release profile of Dox-micell in example 1 of the present invention;
FIG. 7 is an electrophoretogram for determining the binding ability of siRNA to triblock polymer micelle in example 1 of the present invention;
FIG. 8 is an electrophoretogram for measuring the enzyme protection effect of triblock polymer micelle on siRNA in example 1 of the present invention;
FIG. 9 is an electrophoretogram of serum stability after binding of siRNA to triblock polymer micelle as determined in example 1 of the present invention;
fig. 10 is a graph of the in vitro release of siRNA-micell in example 1 of the present invention, wherein a is pH5.3, B is pH5.3 + GSH, C is pH7.4, and D is pH7.4 + GSH.
Detailed Description
The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
Preparation of a triblock polymer comprising the steps of:
(1) synthesis of CSO-ss-
Dissolving 1.578g dithiodipropionic acid, 1.437g EDC and 0.861g NHS in 20ml DMF, activating at 40 deg.C for 30 min; weighing 0.6g of CSO, dispersing in 20mL of distilled water, adding a trace amount of NaOH to adjust the pH value to 7-8, slowly dropping the mixture into dithiodipropionic acid solution under the condition of stirring, stirring at 40 ℃ for reaction for 12h, dialyzing for 1d (MWCO is 1000) by using distilled water, filtering the dialyzate, and freeze-drying the filtrate to obtain CSO-ss-, wherein the yield is 95%.
(2) Synthesis of CSO-ss-PEI
Dissolving 1g of CSO-ss-prepared in the step (1) in 100mL of distilled water, and stirring to form an aqueous solution; dissolving 2.935g EDC and 1.435g NHS in 5ml distilled water to form an activator aqueous solution, adding the activator aqueous solution into the CSO-ss-aqueous solution for activation for 30min, dissolving 2g PEI in 5ml distilled water, adding the PEI into the activated CSO-ss-aqueous solution, stopping adding when the pH value reaches about 6, adding the rest PEI aqueous solution after 30min, stirring and reacting for 12h at 20 ℃, dialyzing for 2 days by a 1000 dialysis bag (MWCO ═ 1000), filtering the dialyzate, and freeze-drying the filtrate to obtain the CSO-ss-PEI, wherein the yield is 90%.
(3) Synthesis of CSO-ss-PEI-UA
0.43g of UA, 1.19g of EDC and 0.71g of NHS were added to a flask containing 25ml of DMF and stirred for 30min until it dissolved; dissolving 0.6g of CSO-ss-PEI-UA prepared in the step (2) in 125mL of distilled water, slowly dripping the solution into the flask, reacting at 50 ℃ for 12h, placing the liquid in the flask into a dialysis bag (MW 1000) after the reaction is finished, dialyzing for 2d with distilled water, filtering the dialyzate, taking the supernatant, and freeze-drying to obtain CSO-ss-PEI-UA with the yield of 90%.
(4) Synthesis of triblock polymer micelle
Dissolving a polymer CSO-ss-PEI-UA in 5mL of deionized water, carrying out ultrasonic treatment for 50 times (400w, 2s stop for 2s) by using an ice bath probe to prepare 1mg/mL-1Blank triblock polymer micelle solution (blank-micell).
(5) Synthesis of triblock polymer drug-loaded micelle
And (3) slowly adding 0.5mg of a DMSO solution of doxorubicin hydrochloride (Dox) (adding triethylamine to neutralize hydrochloric acid in the Dox, wherein the molar ratio of the Dox to the triethylamine is 1:2) dropwise into the micelle solution obtained in the step (4), carrying out ultrasonic treatment for 30min, carrying out magnetic stirring for 2h, then placing the micelle solution in a dialysis bag for dialysis for 4h, changing distilled water every 1h, filtering the dialysate by using a 0.45-micrometer microporous filter membrane, and carrying out freeze drying to obtain the triblock polymer drug-loaded micelle (in the embodiment, the doxorubicin-loaded micelle Dox-micelle).
(6) Synthesis of triblock polymer drug-loaded micelle with gene
Adding 62.5 μ L of Diethylpyrocarbonate (DEPC) (DEPC is diethyl coke, DEPC water is water treated by diethyl coke and sterilized at high temperature and high pressure, or DEPC water can be changed into sterile water) into 0.5OD of siRNA freeze-dried powder, dissolving with water sterilized at high temperature and high pressure to form 20 μ M stock solution, and storing at-20 deg.C; mixing Dox-micell and blank-micell with siRNA respectively according to the N/P (N is amino in triblock polymer micelle, P is phosphate radical in gene, and N/P can be understood as the molar ratio of carrier to gene) ratio of 1-8, incubating for 30min at room temperature to obtain Dox-siRNA-micell and siRNA-micell, wherein the gun tips and EP tubes used in the reaction are both treated by DEPC.
The compounds in this example were characterized and the properties of the products were determined as follows:
1. nuclear magnetic characterization
Dissolving CSO, CSO-ss-, PEI, CSO-ss-PEI and CSO-ss-PEI-UA in D respectively2In O, tested on a 400MHz NMR spectrometer,1the H-NMR spectrum is shown in FIG. 1: in CSO, delta is 4.67(H1)3.20-4.00ppm (sugar ring), 2.01 (-CH)2) (ii) a The signal at δ ═ 2.74, 2.93ppm is assigned to a particular proton peak of the pentaheterocyclic group in dithiodipropionic acid, tableThe Mindithiodipropionic acid was successfully grafted to CSO; the introduction of PEI is confirmed by a proton peak at delta 2.3-2.7ppm in a CSO-ss-PEI conjugate spectrum; signals of UA are located at δ ═ 8.23(Ha), δ ═ 7.61(Hb), δ ═ 7.5(Hc) and δ ═ 6.51(H1), indicating successful introduction of CSO-ss-PEI by UA; given the spectrum of CSO-ss-PEI-UA, the peaks assigned to all the above results imply a successful synthesis of CSO-ss-PEI-UA copolymer and a successful preparation of CSO-ss-PEI-UA polymer.
2. Determination of the Critical Micelle Concentration (CMC) of polymers
The CMC value of the polymer at pH7.4 was measured using pyrene as a hydrophobic fluorescent probe, the logarithm of the concentration (lgC) of each sample solution was measured as the abscissa, and the fluorescence intensity of the polymer at different concentrations was measured as λ of each sample solution as shown in FIG. 21At 372nm and λ3Fluorescent intensity ratio at 383nm (I)1/I3) As a ordinate, a scattergram is plotted, and as shown in fig. 3, a horizontal tangent of the data point and a tangent of the mutation curve are drawn according to each point, and the polymer concentration corresponding to the intersection of the two tangents is the Critical Micelle Concentration (CMC).
As can be seen from FIG. 3, when the polymer concentration is low, I1/I3The value remained unchanged, indicating that the polymer did not form micelles; when the concentration reaches a certain value, I1/I3The value drops sharply, indicating that the polymer begins to form micelles at this concentration. Compared with the small molecular surfactant, the CMC of the polymer can reach 7.94 multiplied by 10-3mg.mL-1This indicates that the polymer forms micelles that are relatively stable during dilution, and that they have the potential to act as drug carriers.
3. Determination of pH-and reduction-sensitivity of polymers
To investigate the pH and redox response behavior of polymer nanoparticles prepared by probe sonication (2mg/mL), we detected the size change of the particle size with a dynamic light scattering particle size analyzer DLS, as shown in fig. 4. The polymeric nanoparticles tested were dissolved in different solutions under the following conditions: (i) at pH7.4, (ii) at pH5.3, (iii) with GSH (100mM) at pH7.4 and (iv) GSH (100mM) at pH 5.3.
After 1 hour incubation of the polymeric nanoparticles with Phosphate Buffered Saline (PBS) (pH 7.4) + GSH and PBS (pH 5.3), respectively, the swelling from 124.6nm to 365.3 and 622.6nm resulted in a bimodal peak for PBS (pH 5.3) + GSH, suggesting that micelle stability was disrupted. The change in particle size is due to protonation of the imidazole groups in acidic solution and the cleavage of disulfide bonds in the presence of GSH, which results in rapid breakdown of the hydrophilic/hydrophobic balance and promotes rapid intracellular release of drugs and genes.
4. Transmission electron microscopy images of triblock polymer micelles
As shown in fig. 5, the micelle morphology is rounded, consistent with the results of particle size analysis, and can be accumulated in tumor sites by using the EPR effect to realize passive targeted therapy.
5. In vitro release profiles of Dox-micell in different buffers (PBS)
As shown in FIG. 6, Dox-release from Dox-micell was suppressed at pH7.4PBS, with a cumulative release of 26.71% + -1.96% over 48 hours. However, rapid drug release was achieved in PBS or Glutathione (GSH) (10mM) at pH 5.3. The cumulative release rate of Dox in Dox-micell in pH5.3 PBS with GSH (10mM) was as high as 72.92% ± 3.22%. This release rate was almost twice that of PBS at pH 7.4. Protonation of the imidazole groups in an acidic environment facilitates rapid release, resulting in increased hydrophilicity of the hydrophobic core of the micelle. The cleavage of disulfide bonds under reducing conditions is also a factor leading to the rapid breakdown of the hydrophilic/hydrophobic balance. The results show that the polymer micelle has the characteristics of extracellular stability and rapid drug release in cells, realizes the effective controlled release of the drug and is beneficial to the treatment of tumors.
6. Agarose gel electrophoresis is utilized to determine the binding capacity of siRNA and the triblock polymer micelle
0.25g of agarose is weighed and dissolved in 25mL of 1 Xelectrophoresis buffer (TAE) buffer solution, after the agarose is heated and dissolved in a microwave oven, 2.5 mu L of nucleic acid gel dye (GelRed) is added and mixed evenly, then the solution is poured into a gel making groove for making gel, and after the agarose is cooled to room temperature, the gel is placed into an electrophoresis groove for preparing electrophoresis.
0.025nmolsiRNA was taken into 6 EP tubes each containing no ribonuclease (RNase), then 17. mu.L of DEPC-treated water, 1. mu.L of triblock polymer micelle solution, 2. mu.L of triblock polymer micelle solution, 4. mu.L of triblock polymer micelle solution, 8. mu.L of triblock polymer micelle solution, 16. mu.L of triblock polymer micelle solution were added, and each group was made up to 18. mu.L with DEPC-treated water. After the solutions in the EP tubes are fully mixed, the mixture is kept at room temperature for 30min, 15 μ L of the mixture is respectively added into 3 μ L of 6 × loading buffer, the mixture is loaded, 100V is carried out, the experiment result is observed by a gel imager after electrophoresis for 15min, as shown in figure 7, the N/P ratio of siRNA and polymer in each lane in the figure is respectively 1, 2, 4, 5 and 6, as can be seen from the figure, more siRNA is electrostatically adsorbed by the carrier along with the increase of the N/P ratio, the fluorescence intensity of a free band is weakened, the encapsulation rate is about 90% when the N/P ratio is 5, and the encapsulation rate is about 98% when the N/P ratio is 6.
7. The enzyme protection effect of the triblock polymer micelle on siRNA is determined by agarose gel electrophoresis
The ability of micelles to protect siRNA against nuclease degradation was examined by agarose gel electrophoresis, with free siRNA as a control. 0.12nmolsiRNA was added to 6 EP tubes without RNase, and 17. mu.L of DEPC-treated water was added to tubes No. 1-2, respectively; respectively adding 4 mu L of triblock polymer micelle solution and 13 mu L of DEPC water into the 3-6 tubes, fully mixing the solutions in the EP tubes, and standing at room temperature for 30 min; adding 1U of RNase I/μ g siRNA into 2-6 tubes, and incubating at room temperature for 30min, 60min, 90min and 120 min; and adding heparin sodium into the 3-6 tubes respectively, incubating for 15min at room temperature, and dissociating the siRNA from the triblock polymer micelle. Respectively taking 15 mu L of No. 1-6 tube samples and 3 mu L of 6 × loading buffer, mixing uniformly, loading, carrying out electrophoresis for 15min, observing by a gel imaging system, and analyzing the result, wherein the result is shown in figure 8.
The results show that naked siRNA and 1U of RNase I/mu g siRNA are degraded after being incubated for 30min (band 2), while siRNA-ACPU micolle and RNase I are hardly degraded after being incubated for 30min (band 3), and the integrity of siRNA can be well maintained after being incubated for 120min (band 6). The result shows that the prepared micelle can slow down the degradation of the siRNA by nuclease to a certain extent.
8. Determination of serum stability after combination of siRNA and triblock polymer micelle by agarose gel electrophoresis
And (3) comparing the degradation condition of the siRNA-micell after incubation in serum for different time by adopting agarose gel electrophoresis, and taking free siRNA as a control. 0.012nmolsiRNA was added to 6 EP tubes without RNase, and 17. mu.L of DEPC-treated water was added to tubes No. 1-2, respectively; respectively adding 4 mu L of triblock polymer micelle solution and 13 mu L of DEPC water into the 3-6 tubes, fully mixing the solutions in the EP tubes, and standing at room temperature for 30 min; adding 20% fetal calf serum into 2-6 tubes, respectively, and incubating at room temperature for 30min, 60min, 90min, and 120 min; and adding heparin sodium into the 3-6 tubes respectively, incubating for 20min at room temperature, and dissociating the siRNA from the triblock polymer micelle. Respectively taking 15 mu L of No. 1-6 tube samples and 3 mu L of 6 × loading buffer, mixing uniformly, loading, carrying out electrophoresis for 15min, observing by a gel imaging system, and analyzing the result, wherein the result is shown in figure 9.
As can be seen from the figure, after the siRNA combined with the triblock polymer micelle is subjected to heparin decomplexation, the siRNA moves from the negative electrode to the positive electrode in an electric field, and is stained by GelRed on a gel plate, so that a white bright band appears. Naked siRNA degraded after 30min incubation with 20% serum (lane 2), whereas siRNA-micell maintained siRNA integrity well after 120min incubation with 20% serum (lane 6). The naked siRNA is proved to be easily degraded by enzyme in serum after being incubated with the serum, and the serum instability is shown; after the siRNA-ACPU micolle and the serum are incubated together, the degradation of the siRNA is reduced, the degradation time is prolonged, and the result shows that the micelle can reduce the degradation of the siRNA by the enzyme in the serum. The result shows that the prepared micelle has a certain protection effect on siRNA.
9. Release of SiRNA in micelles
Selecting PBS (pH 5.3), PBS (pH 5.3) + GSH, PBS (pH 7.4) and PBS (pH 7.4) + GSH as release media respectively to examine the release characteristics of the siRNA-micell under different pH environments.
Diluting siRNA-micell with different release media, respectively placing the diluted siRNA-micell into centrifuge tubes, wherein each group of the three samples are parallel, and the temperature is 37 ℃ and the rpm is 100 min-1Performing in vitro release experiment under the condition of 0.5h, 1h, 2h,Sampling for 6h, 12h, 24h and 48h at 4 deg.C and 50000rpm min-1Centrifuging for 45min, collecting supernatant, measuring the emission spectrum of the solution to be measured at excitation wavelength of 480nm within the range of 500nm-600nm, wherein the bandwidth of excitation and emission slits is 10nm, and the result is shown in FIG. 10.
Under the medium of pH7.4 and pH7.4 + GSH, the siRNA is adsorbed to the PEI layer, and the release of the siRNA from the siRNA-micell is effectively prevented under the condition; however, micelles became unstable at pH5.3 and pH5.3 + GSH to release siRNA. These results indicate that the siRNA-micell can protect siRNA from enzymatic degradation in physiological environment and enhance intracellular siRNA release, and siRNA released in targeting cytoplasm can silence transcription of target gene and down-regulate expression of target protein.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A preparation method of a triblock polymer micelle is characterized by comprising the following steps:
(1) reacting chitosan oligosaccharide with dithiodipropionic acid under the action of an activator to obtain a first intermediate;
(2) reacting the first intermediate obtained in the step (1) with polyethyleneimine under the action of an activating agent to obtain a second intermediate;
(3) reacting the second intermediate obtained in the step (2) with imidazole acrylic acid under the action of an activating agent to obtain a triblock polymer of a formula (I);
wherein n =18-32, m = 232-;
(4) dissolving the triblock polymer obtained in the step (3) in water to obtain the triblock polymer micelle;
in the step (1), the dithiodipropionic acid reacts with the chitosan oligosaccharide after activating carboxyl by an activating agent;
in the step (2), the first intermediate is activated by carboxyl through an activating agent and then reacts with polyethyleneimine;
in the step (3), the imidazole acrylic acid reacts with the second intermediate after being activated by carboxyl through an activating agent;
the activating agent is one or more of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and dicyclohexylcarbodiimide;
in the step (4), the triblock polymer is added into water to form a triblock polymer micelle under an ultrasonic condition.
2. The method of claim 1, wherein: in the step (1), the mol ratio of the activating agent to the dithiodipropionic acid is 0.8-1.2: 1.
3. the method of claim 1, wherein: in the step (2), the mol ratio of the activating agent to the first intermediate chitosan oligosaccharide-dithiodipropionic acid is 0.8-1.2: 1.
4. use of the triblock polymer micelle prepared by the preparation method according to any one of claims 1 to 3 as a carrier of adriamycin or siRNA.
5. The preparation method of the triblock polymer drug-loaded micelle is characterized by comprising the following steps: mixing adriamycin or siRNA with the triblock polymer micelle prepared according to any one of claims 1 to 3 to obtain the triblock polymer drug-loaded micelle.
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