CN114748420A - Preparation method of amphiphilic polymer micelle with charge self-reversal activation mitochondrial targeting effect - Google Patents
Preparation method of amphiphilic polymer micelle with charge self-reversal activation mitochondrial targeting effect Download PDFInfo
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- 239000000693 micelle Substances 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 230000008685 targeting Effects 0.000 title claims abstract description 15
- 230000002438 mitochondrial effect Effects 0.000 title claims description 15
- 230000004913 activation Effects 0.000 title claims description 7
- 230000000694 effects Effects 0.000 title description 8
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 27
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 claims abstract description 16
- 239000002105 nanoparticle Substances 0.000 claims abstract description 15
- 238000000502 dialysis Methods 0.000 claims abstract description 11
- JRBPAEWTRLWTQC-UHFFFAOYSA-N dodecylamine Chemical compound CCCCCCCCCCCCN JRBPAEWTRLWTQC-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000006116 polymerization reaction Methods 0.000 claims abstract description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 51
- 239000000243 solution Substances 0.000 claims description 48
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 43
- 238000003756 stirring Methods 0.000 claims description 31
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- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 6
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- KZYWBYBQPIOWIY-UHFFFAOYSA-N [3-(hydroxymethyl)-2-nitrophenyl]methanol Chemical compound OCC1=CC=CC(CO)=C1[N+]([O-])=O KZYWBYBQPIOWIY-UHFFFAOYSA-N 0.000 claims description 6
- 230000001376 precipitating effect Effects 0.000 claims description 6
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 5
- 239000012359 Methanesulfonyl chloride Substances 0.000 claims description 5
- UWTDFICHZKXYAC-UHFFFAOYSA-N boron;oxolane Chemical compound [B].C1CCOC1 UWTDFICHZKXYAC-UHFFFAOYSA-N 0.000 claims description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- QARBMVPHQWIHKH-UHFFFAOYSA-N methanesulfonyl chloride Chemical compound CS(Cl)(=O)=O QARBMVPHQWIHKH-UHFFFAOYSA-N 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- CAHWDGJDQYAFHM-UHFFFAOYSA-N 2-nitroisophthalic acid Chemical compound OC(=O)C1=CC=CC(C(O)=O)=C1[N+]([O-])=O CAHWDGJDQYAFHM-UHFFFAOYSA-N 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 4
- HDFQKJQEWGVKCQ-UHFFFAOYSA-N 1,3-dimethyl-2-nitrobenzene Chemical compound CC1=CC=CC(C)=C1[N+]([O-])=O HDFQKJQEWGVKCQ-UHFFFAOYSA-N 0.000 claims description 3
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- HFBMWMNUJJDEQZ-UHFFFAOYSA-N acryloyl chloride Chemical compound ClC(=O)C=C HFBMWMNUJJDEQZ-UHFFFAOYSA-N 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
- 239000013078 crystal Substances 0.000 claims description 3
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 239000000155 melt Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
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- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
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- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 18
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 6
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- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
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- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
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- LQNUZADURLCDLV-IDEBNGHGSA-N nitrobenzene Chemical group [O-][N+](=O)[13C]1=[13CH][13CH]=[13CH][13CH]=[13CH]1 LQNUZADURLCDLV-IDEBNGHGSA-N 0.000 description 1
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- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
- C08G73/02—Polyamines
- C08G73/0206—Polyalkylene(poly)amines
- C08G73/0213—Preparatory process
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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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
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- 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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- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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Abstract
The invention provides a preparation method of a charge self-reversal activated mitochondrion targeted amphiphilic polymer micelle. By preparing PBMDA and TPP-PEG-NH2And carrying out melt polymerization reaction with dodecylamine to obtain an amphiphilic polymer, and then adding the amphiphilic polymer into a DMSO solution containing upconversion nanoparticles (UCNPS), and then carrying out dialysis to obtain TPP/NIR @ Ce 6/UCNPS. The preparation method has simple operation, mild reaction conditions and preparationThe micelle of the polymer micelle has good size, and can expose a mitochondrion targeting group by charge self-reversal in a lysosome, thereby delivering the drug to the mitochondrion in a targeted way.
Description
Technical Field
The invention relates to the technical field of mitochondrion targeting, in particular to a preparation method of an amphiphilic polymer micelle with a mitochondrion targeting effect activated by charge self-reversal.
Background
Photodynamic therapy (PDT) is an emerging modality for the treatment of tumors. It is a novel non-invasive tumor therapy by delivering photosensitizers to tumor tissue and using light excitation of appropriate wavelength band to generate energy, which causes oxygen around the tumor tissue to generate highly reactive singlet oxygen, thereby killing tumor cells. PDT has the characteristics of low toxicity and high safety, and can be simultaneously carried out with other treatment means, such as operation, radiotherapy and chemotherapy. PDT also has the advantage of high selectivity, generally for the treatment of tumors at specific sites, without affecting other normal tissues. However, most of photosensitizers in PDT are hydrophobic organic molecules, have low solubility in physiological environments, and can be rapidly eliminated in blood, so that the application of PDT is greatly limited. In recent years, researchers have been working on developing various nano-drug carriers to improve PDT efficacy. The nano-drug carrier has the following advantages in drug delivery: (1) the bioavailability of the medicine is improved; (2) improving the solubility of hydrophobic drugs; (3) the targeted drug delivery can be realized after the modification of the targeted group, and the side effect is reduced; (4) controlled release of the drug is achieved by internal or external stimulation; (5) improve the solubility and stability of the medicine and enhance the anti-tumor effect.
The presence of various site-specific biostimulations (i.e., pH, redox, active oxygen, enzymes, or a combination of both) can be used to trigger charge reversal of the nanoparticle. Among these stimuli, the pH gradient between the physiology of the tumor and the extracellular environment is one of the most frequently used trigger signals. The pH of the physiological environment is about 7.4, whereas the pH of the extracellular environment of solid tumors is typically between 6.4 and 6.8. Furthermore, the pH in the endo/lysosomal interval is typically between 4.5 and 5.5. The existence of such a pH gradient between the extracellular and intracellular environment of the tumor provides an opportunity for the precise design of an intelligent drug delivery system to the tumor site. Therefore, using this specific site pH, it is feasible to develop a pH-responsive drug delivery system that can effectively bypass the complex biological barriers of the tumor extracellular or intracellular microenvironment, and effectively release the loaded drug to the target site within the cancer cell for maximal therapeutic effect. In the pH-induced negative charge-flipping strategy, nanocarriers consisting of polymers or decorated with pH-dependent charge-flipping molecules are fabricated. These molecules exhibit a negative charge at physiological pH values and become positively charged at acidic pH values.
Subcellular localization of photosensitizers is a key factor affecting the efficacy of PDT because it determines the initial site of photodynamic damage. As an energy factory of cells, mitochondria provide most direct energy Adenosine Triphosphate (ATP) for the metabolism of cells at any moment, play a crucial role in the growth, proliferation and death of the cells, control the metabolism of the cells and are closely related to the carcinogenic mechanism of the cells. Mitochondria are important targets for photodynamic therapy and photosensitizers localized to mitochondria can kill tumor cells more efficiently than photosensitizers localized to other organelles. The initial reaction after illumination is to form singlet oxygen on mitochondria, and then the active oxygen on the mitochondria induces the permeability change of the mitochondria so as to lead to depolarization and deformation of the mitochondria, release of cytochrome c and finally lead to apoptosis. Therefore, if a functional nano drug-carrying system can be designed and prepared, the photodynamic therapeutic agent can be selectively accumulated in mitochondria, and the PDT curative effect can be enhanced.
Disclosure of Invention
The invention aims to provide a preparation method of a charge self-reversal activated mitochondrion targeted amphiphilic polymer micelle for delivering a photosensitizer to a related subcellular organelle (mitochondrion). The method is simple to operate, the reaction condition is mild, the prepared polymer micelle is good in size, and the charge can be automatically reversed in lysosomes to expose mitochondria targeting groups, so that the drug is delivered to mitochondria in a targeted manner. The specific technical scheme is as follows.
As a first aspect, the present invention provides a method for preparing an amphiphilic polymer micelle having a charge self-reversal activation mitochondrial targeting efficacy, the method comprising the steps of:
s1, preparing PBMDA;
s101, 9.0g/0.060mol of 1, 3-dimethyl-2-nitrobenzene is added to a 0.2M/500mL NaOH solution stirred at 95 ℃; then 40g/0.25mol KMnO was slowly added4Refluxing the mixture for several hours, and collecting the reflux filtrate; acidifying the filtrate to pH 1 with hydrochloric acid, and filtering to obtain 2-nitro-1, 3-benzenedicarboxylic acid;
s102, dissolving 8g/38mmol of 2-nitro-1, 3-phthalic acid in 50mL of anhydrous tetrahydrofuran, and cooling to 4 ℃; slowly adding 1.0M tetrahydrofuran borane complex under nitrogen, and stirring for several hours; then 40mL of methanol was added dropwise, followed by filtration and drying under vacuum to obtain a residue; the residue was redissolved in ethyl acetate, extracted with 4X 100mL of saturated sodium chloride solution, the organic layer dried over anhydrous magnesium sulfate for several hours, and then further purified by silica gel chromatography to give 2-nitro-1, 3-benzenedimethanol;
s103, dropwise adding 0.1mol of triethylamine into a 50mL anhydrous dichloromethane solution of 7.3g, 40mmol of 2-nitro-1, 3-benzenedimethanol under nitrogen gas at a constant pressure within a plurality of hours; slowly adding acryloyl chloride to the reaction mixture at constant pressure, stirring the mixture for several hours and filtering; drying the filtrate under vacuum to obtain a residue, and re-dissolving the residue in ethyl acetate; washing with 3 × 100mL of saturated sodium chloride solution; further purification by silica gel chromatography gave white crystals of PBMDA.
S2，TPP-PEG-NH2Preparing;
s201, dissolving 16g of PEG2000 in 20mL of anhydrous dichloromethane, adding 4mL of methanesulfonyl chloride and 4mL of triethylamine at low temperature, reacting for several hours in nitrogen, and stirring for several hours; adding ether for precipitation to obtain a light yellow solid, adding 200mL of 25% ammonia water, stirring for a plurality of hours, and then standing for a plurality of hours;
s202, adjusting the pH value of the solution obtained in the step S201 to 13 by using sodium hydroxide, extracting the solution for a plurality of times by using dichloromethane, concentrating the extract, precipitating the extract by using ether and filtering the precipitate under reduced pressure to obtain NH2-PEG-NH2(ii) a Dissolving 2.62g of triphenylphosphine in 10mL of acetonitrile, dissolving 1.95g of 6-bromoacetic acid in 20mL of acetonitrile, dropping the 6-bromoacetic acid into the acetonitrile solution of triphenylphosphine under nitrogen, and heating and refluxing for several hours; will be 0265g TPP-COOH, 0.576g DMAP, 1.008g DCC were dissolved in 40mL anhydrous dichloromethane and activated with stirring for several hours; 30mL of a solution containing 12g of NH2-PEG-NH was added2Stirring the anhydrous dichloromethane solution for a plurality of hours, and precipitating and drying the solution by diethyl ether to obtain TPP-PEG-NH2。
S3, preparation of amphiphilic polymer: NPBMDA, TPP-PEG-NH2Mixing with dodecylamine at a molar feed ratio of 10:1:9, and carrying out melt polymerization reaction under nitrogen for several hours; then dialyzing the reacted mixture with distilled water for several hours, and then freeze-drying to obtain an amphiphilic polymer;
s4, preparation of polymer micelle: 5mg of Ce6 was dispersed in 5mL of DMSO, after which it was added to 1mL of DMSO solution containing 0.5mg of upconverting nanoparticles, stirred for several hours, then 50mg of amphiphilic polymer was added; dripping the mixed solution into 20ml of distilled water, stirring for a plurality of hours, and dialyzing the solution for a plurality of hours; after dialysis, the dialysate was freeze dried to give TPP/NIR @ Ce 6/UCNPS.
Further, the method is carried out.
Refluxing the mixture as described in S101 for several hours, which is 24 hours;
the cooling in S102 is to 4 ℃ by ice bath;
slowly adding 1.0M tetrahydrofuran borane complex under nitrogen in S102, and stirring for several hours, wherein the several hours are 48 hours;
drying the organic layer of the organic layer in the S102 by using anhydrous magnesium sulfate for several hours, wherein the several hours are 12 hours;
0.1mol of triethylamine is added dropwise to 7.3g under nitrogen with a constant pressure over several hours, which is 1 hour for several hours, as described in S103;
the mixture in S103 is stirred for 18 hours and filtered, several hours of which are 18 hours;
in S201, 4mL of methanesulfonyl chloride and 4mL of triethylamine are added at low temperature, then the mixture reacts for a plurality of hours in nitrogen, and then is stirred for a plurality of hours, wherein the reaction time is 2 hours, and the stirring time is 24 hours;
the mixture is stirred for a plurality of hours and then placed for a plurality of hours in S201, wherein the stirring time is 96 hours, and the placing time is 72 hours;
heating and refluxing for several hours in S202, wherein the several hours are 24 hours;
stirring and activating for several hours in S202, wherein the several hours are 1 hour;
adding 30mL of anhydrous dichloromethane solution containing 12g of NH2-PEG-NH2 into the S202, and stirring for several hours, wherein the several hours are 12 hours;
the melt polymerization under nitrogen in S3 is carried out for several hours, and the several hours are 12 hours;
dialyzing the reacted mixture against distilled water for several hours, which is 48 hours, as described in S3;
s4, then adding it to 1mL of DMSO solution containing 0.5mg of upconverting nanoparticles and stirring for several hours, which is 0.5 hour;
in S4, the mixed solution is dropped into 20ml of distilled water and stirred for several hours, and then the solution is dialyzed for several hours, wherein the stirring for several hours is 8 hours, and the dialysis for several hours is 48 hours.
Further, the conditions of silica gel chromatography were: n-hexane, ethyl acetate 1:1, v/v as eluent.
Preferably, the dialysis of the solution as described in S4 is performed by transferring the solution to a 3500KDa dialysis bag.
The beneficial effects of the invention are: the method is simple to operate, the reaction condition is mild, the size of the prepared polymer micelle is good, the polymer micelle shows electronegativity under physiological conditions, long-time circulation and stability in blood are facilitated, after the polymer micelle is endocytosed by tumor tissues, charges are automatically reversed in lysosomes/endosomes due to the reduction of the pH value, and mitochondrial targeting groups are exposed, so that the drug is delivered to mitochondria in a targeted manner.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.
FIG. 1 is the synthesis path of NPBMDA;
FIG. 2 is a synthetic route of TPP-PEG-NH 2;
FIG. 3 is a synthetic route for amphiphilic polymers;
FIG. 4 is a nuclear magnetic spectrum of dinitro-1, 3-phthalic acid;
FIG. 5 is a nuclear magnetic spectrum of dinitro-1, 3-benzenedimethanol;
FIG. 6 is a nuclear magnetic spectrum of NPBMDA;
FIG. 7 shows TPP-PEG-NH2Nuclear magnetic spectrum of (a);
FIG. 8 is a nuclear magnetic spectrum of an amphiphilic polymer;
FIG. 9 is a TEM image of TPP/NIR @ Ce 6/UCNPS;
FIG. 10 shows the charge of amphiphilic polymer micelles in PBS at different pH values;
FIG. 11 is a schematic diagram showing the high and low system energy levels at the distance from the TPP group to the main chain in a theoretical simulated polymer;
figure 12 is a schematic representation of the distribution of Ce6 in 4T1 cells after treatment with different nanoparticles assessed with CLSM.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are some, but not all embodiments of the present application. The components of the embodiments of the present application, generally described and illustrated in the figures herein, can be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the claimed invention, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1
As shown in FIG. 1, the preparation method of PBMDA is as follows:
1, 3-dimethyl-2-nitrobenzene (9.0g, 0.060mol) was added to a stirred solution of NaOH (0.2M, 500mL) at 95 ℃. Slow addition of KMnO4(40g, 0.25mol) and the mixture was refluxed for 24 hours. Acidifying the filtrate to pH 1 with hydrochloric acid, and filtering to obtain the product 2-nitro-1, 3-benzenedioic acid.
2-Nitro-1, 3-benzenedicarboxylic acid (8g, 38mmol) was dissolved in anhydrous tetrahydrofuran (50mL) and cooled to 4 ℃ in an ice bath. In N2Then, 1.0M tetrahydrofuran borane complex was slowly added by syringe and stirred at room temperature for 48 hours. Methanol (40mL) was then added dropwise to the mixture, followed by filtration and drying under vacuum. The residue was redissolved in ethyl acetate and extracted with saturated sodium chloride solution (4X 100 mL). The organic layer was dried over anhydrous magnesium sulfate for 12 hours, and then further purified by silica gel chromatography (n-hexane: ethyl acetate 1:1, v/v as an eluent) to obtain 2-nitro-1, 3-benzenedimethanol.
Triethylamine (0.1mol) was added dropwise to a solution of 2-nitro-1, 3-benzenedimethanol (7.3g, 40mmol) in dry Dichloromethane (DCM) (50mL) over 1 hour under nitrogen using a constant pressure funnel. Acryloyl chloride was added slowly to the reaction mixture using a constant pressure funnel. The mixture was stirred at room temperature for 18 hours and filtered. The filtrate was dried under vacuum and the residue was redissolved in ethyl acetate. Wash with saturated sodium chloride solution (3 × 100 mL). Further purification by silica gel chromatography (n-hexane: ethyl acetate 1:1, v/v as eluent) gave white crystals of PBMDA.
As shown in FIG. 2, its TPP-PEG-NH2The preparation method comprises the following steps: 16g PEG2000 was dissolved in 20mL of anhydrous dichloromethane, and 4mL methanesulfonyl chloride and 4mL triethylamine were added in an ice bath, and reacted under nitrogen for 2h, followed by stirring at room temperature for 24 h. Precipitation with a large amount of diethyl ether to give a pale yellow solid, 200mL of 25% aqueous ammonia was added, and the mixture was stirred at room temperature for 4 days and then left open at room temperatureStanding for 3 days. Adjusting pH to 13 with sodium hydroxide, extracting with dichloromethane for three times, concentrating the extractive solution, precipitating with diethyl ether, and filtering under reduced pressure to obtain NH2-PEG-NH2. 2.62g of triphenylphosphine and 1.95g of 6-bromoacetic acid were dissolved in 10mL and 20mL of acetonitrile, respectively, and the 6-bromoacetic acid was added dropwise to the triphenylphosphine solution in acetonitrile under nitrogen, followed by heating and refluxing for 24 hours. 265g TPP-COOH, 0.576g DMAP, 1.008g DCC were dissolved in 40mL anhydrous dichloromethane and activated by stirring for 1 hour. 30mL of a solution containing 12g of NH were added2-PEG-NH2Stirring the obtained solution at room temperature for 12 hours, and precipitating and drying the obtained product by using diethyl ether to obtain TPP-PEG-NH2。
As shown in FIG. 3, the amphiphilic polymer is prepared as follows: NPBMDA, TPP-PEG-NH2And dodecylamine were mixed in a 10:1:9 molar feed ratio in a two-necked flask. Under the protection of nitrogen, the melt polymerization reaction was carried out for 12 hours. Then, the reacted mixture was dialyzed against distilled water for 48 hours, followed by freeze-drying to obtain an amphiphilic polymer.
The preparation method of the polymer micelle comprises the following steps: ce6(5mg) was dispersed in DMSO (5mL), and then added to a DMSO (1mL) solution containing up-converting nanoparticles (UCNPS) (0.5mg), stirred at room temperature for 0.5 hours, then added with an amphiphilic polymer (50mg), the above mixed solution was dropped into 20mL of distilled water, stirred at room temperature for 8 hours, and then the solution was transferred to a dialysis bag (3500KDa) and dialyzed for 48 hours. After dialysis, the dialysate was freeze dried to give TPP/NIR @ Ce 6/UCNPS.
TPP/NIR @ Ce6/UCNPS undergoes charge reversal from negative to positive at pH 5.1. While the pH of the endosome is 5.0-6.0 and the pH of the lysosome is 4.0-5.0, so that the polymer micelle can perform charge reversal after escaping from the capture of the lysosome and the endosome. Theoretical calculation shows that the polymer in the lysosome/endosome environment has lowered pH value, protonation effect and TPP group far away from the main chain, so that the energy of the whole polymer micelle system tends to be lowered. When no protonation occurs, the closer the TPP group is to the main chain, the lower the system energy, and the more stable it is. Therefore, experiments prove that the polymer micelle can perform self-reversal of charges after passing through lysosomes and endosomes, expose TPP groups and target mitochondria.
The technical effects of the present invention will be explained based on experimental evidence.
As shown in FIG. 4, in1H NMR (DMSO-d6): d 14.2(-COOH),8.17(m,2H, ArH),7.79(m,1H, ArH) under the conditions of nuclear magnetic spectrum. Nuclear magnetic spectrum of dinitro-1, 3-phthalic acid. 1H NMR (DMSO-d6): chemical shift 14.2 is the characteristic peak of-COOH, 8.17 is the characteristic peak of H at meta position on benzene ring, and 7.79 is the characteristic peak of H at para position on benzene ring. By means of nuclear magnetic spectrum we can prove the successful preparation of dinitro-1, 3-phthalic acid.
As shown in fig. 5, in1H NMR(DMSO-d6):d 7.68(m,3H,ArH),5.56(t,2H,-OH),4.70(d,-4H,-CH2OH) condition, a nuclear magnetic spectrum of dinitro-1, 3-benzenedimethanol. Nuclear magnetic spectrum of dinitro-1, 3-benzene dimethanol. 1H NMR (DMSO-d6) chemical shift 7.68 is characteristic peak of H on benzene ring, 5.56 is characteristic peak of-OH, 4.70 is characteristic peak of methylene on-CH 2 OH. By means of nuclear magnetic spectrum we can prove the successful preparation of dinitro-1, 3-benzenedimethanol.
As shown in fig. 6, in1H NMR(DMSO-d6)：δ7.62(m，3H，ArH)，6.45(d，2H，-CH＝CH2)，6.11(dd，2H，-CH＝CH2)，5.35(s，4H，ArCH2Nuclear magnetic spectrum of O-) NPBMDA. We can demonstrate the successful preparation of NPBMDA by nuclear magnetic spectroscopy.
As shown in FIG. 7, in1H NMR (DMSO-d6): delta 3.61 (CH-in PEG repeat units)2)，7.6-7.85(ArH)TPP-PEG-NH2Nuclear magnetic spectrum of (1). Shows the nuclear magnetic spectrum of TPP-PEG-NH2, 1H NMR (DMSO-d6): chemical shift 3.61 is the characteristic peak of-CH 2 in PEG repeating unit, and 7.6-7.85 is the characteristic peak on the benzene ring of triphenylphosphine. The successful preparation of TPP-PEG-NH2 can be proved by nuclear magnetic spectrum.
As shown in FIG. 8, in1H NMR (DMSO-d6): delta 1.3 (. delta. -CH in dodecylamine repeat unit)2) 3.55 (CH in PEG repeat Unit)2)，5.35(s，4H，ArCH2O-)7.6Nuclear magnetic spectrum of 2(m, 3H, ArH), 7.69 (ArH on triphenylphosphine) amphiphilic polymer. 1H NMR (DMSO-d6): chemical shifts 1.3 is the characteristic peak of-CH 2 in the dodecylamine repeating unit, 3.55 is the characteristic peak of-CH 2 in the PEG repeating unit, 5.35 is the characteristic peak of CH 2O-on the benzene ring, 7.62 is the characteristic peak on the nitrobenzene ring, and 7.69 is the characteristic peak on the benzene ring of triphenylphosphine. By means of nuclear magnetic spectrum, we can prove the successful preparation of amphiphilic polymer.
NIR @ Ce6/UCNPS can be prepared by the above method according to the above conditions, and the TEM image of NIR @ Ce6/UCNPS is shown in FIG. 9. As can be seen from the TEM image, the polymer micelle has a regular spherical morphology and a uniform size, and the size is about 140-160 nm.
From FIG. 10, it can be seen that the charge was-8.2 mV at pH 7.4, -4mV at pH 6.5, -2mV at pH 5.5, and-1 mV at pH 5.2, 1.5mV at pH 5, and 3.5mV at pH 4.5, and it was found from the experimental results that the charge changed from negative to positive as the pH decreased. At pH 5.1 the charge is reversed.
As can be seen from fig. 11, the energy is low when the TPP group is far from the polymer chain at the time of protonation. When not protonated, the TPP group is low in energy when adsorbed to a polymer chain. Thus, at pH 7.4 the TPP groups are embedded in the backbone, while at lysosomes the pH is lowered and a protonation effect occurs and the TPP groups are exposed.
As shown in fig. 12, the nucleus was blue, the mitochondria was green, and the red color was Ce6 under 630nm excitation. After the group of nanoparticles NIR @ Ce6/UCNPS without TPP groups enters 4T1 cells, Ce6 in the mitochondrial site in the cells shows a certain red fluorescence, but the red fluorescence is very weak, which indicates that Ce6 is enriched in mitochondria but the nanoparticles do not have a mitochondrial targeting group, so that the enrichment in the cells is very small, particularly in the mitochondrial site. As can be seen from the b in fig. 12, the confocal results of the TPP @ Ce6/UCNPS group showed significantly more enrichment at the mitochondrial site than the NIR @ Ce6/UCNPS group, which is probably due to the fact that the positively charged nanoparticles are more easily endocytosed by the cells at the cellular level and that the mitochondrial targeting group acts upon entry into the cells, making the nanoparticles more enriched in mitochondria. It can be seen from the observation of c in fig. 12 that the TPP/NIR @ Ce6/UCNPS group is greatly enriched at the mitochondrial site because the charge of the nanoparticle is reversed after endocytosis by the cell and escape from lysosome and endosome, and the mitochondrial targeting group encapsulated in the nanoparticle is exposed, so that the nanoparticle can be actively targeted to the mitochondria.
It should be understood that the above examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. It should also be understood that various changes and modifications can be made by one skilled in the art after reading the disclosure of the present invention, and equivalents fall within the scope of the invention as defined by the appended claims.
Claims (4)
1. A preparation method of amphiphilic polymer micelle with charge self-reversal activation and mitochondrial targeting efficacy is characterized by comprising the following steps:
s1, preparing PBMDA;
s101, adding 9.0g/0.060mol of 1, 3-dimethyl-2-nitrobenzene into 0.2M/500mL of NaOH solution which is stirred at the temperature of 95 ℃; then 40g/0.25mol of KMnO are slowly added4Refluxing the mixture for several hours, and collecting the reflux filtrate; acidifying the filtrate to pH 1 with hydrochloric acid, and filtering to obtain 2-nitro-1, 3-benzenedicarboxylic acid;
s102, dissolving 8g/38mmol of 2-nitro-1, 3-phthalic acid in 50mL of anhydrous tetrahydrofuran, and cooling to 4 ℃; slowly adding 1.0M tetrahydrofuran borane complex under nitrogen, and stirring for several hours; then 40mL of methanol was added dropwise, followed by filtration and drying under vacuum to obtain a residue; the residue was redissolved in ethyl acetate, extracted with 4X 100mL of saturated sodium chloride solution, the organic layer dried over anhydrous magnesium sulfate for several hours, and then further purified by silica gel chromatography to give 2-nitro-1, 3-benzenedimethanol;
s103, dropwise adding 0.1mol of triethylamine into a solution of 7.3g of 40mmol of 2-nitro-1, 3-benzenedimethanol in 50mL of anhydrous dichloromethane under the condition of nitrogen and at a constant pressure within a plurality of hours; slowly adding acryloyl chloride to the reaction mixture at constant pressure, stirring the mixture for several hours and filtering; drying the filtrate under vacuum to obtain a residue, and re-dissolving the residue in ethyl acetate; washing with 3 × 100mL saturated sodium chloride solution; further purification by silica gel chromatography gave white crystals of PBMDA.
S2，TPP-PEG-NH2Preparing;
s201, dissolving 16g of PEG2000 in 20mL of anhydrous dichloromethane, adding 4mL of methanesulfonyl chloride and 4mL of triethylamine at low temperature, reacting for several hours in nitrogen, and stirring for several hours; adding ether for precipitation to obtain a light yellow solid, adding 200mL of 25% ammonia water, stirring for a plurality of hours, and then standing for a plurality of hours;
s202, adjusting the pH value of the solution obtained in the step S201 to 13 by using sodium hydroxide, extracting the solution for a plurality of times by using dichloromethane, concentrating the extract, precipitating the concentrated extract by using ether and filtering the precipitate under reduced pressure to obtain NH2-PEG-NH2(ii) a Dissolving 2.62g of triphenylphosphine in 10mL of acetonitrile, dissolving 1.95g of 6-bromoacetic acid in 20mL of acetonitrile, dropping the 6-bromoacetic acid into the acetonitrile solution of triphenylphosphine under nitrogen, and heating and refluxing for several hours; dissolving 0.265g of TPP-COOH, 0.576g of DMAP and 1.008g of DCC in 40mL of anhydrous dichloromethane, and stirring and activating for a plurality of hours; 30mL of a solution containing 12gNH was added2-PEG-NH2Stirring the anhydrous dichloromethane solution for a plurality of hours, and precipitating and drying the solution by diethyl ether to obtain TPP-PEG-NH2。
S3, preparation of amphiphilic polymer: mixing NPBMDA, TPP-PEG-NH2 and dodecylamine at a molar feed ratio of 10:1:9, and carrying out melt polymerization under nitrogen for several hours; then dialyzing the reacted mixture with distilled water for several hours, and then freeze-drying to obtain an amphiphilic polymer;
s4, preparation of polymer micelle: 5mg of Ce6 was dispersed in 5mL of DMSO, after which it was added to 1mL of DMSO solution containing 0.5mg of upconverting nanoparticles, stirred for several hours, then 50mg of amphiphilic polymer was added; dripping the mixed solution into 20ml of distilled water, stirring for a plurality of hours, and dialyzing the solution for a plurality of hours; after dialysis, the dialysate was freeze dried to give TPP/NIR @ Ce 6/UCNPS.
2. The method for preparing the amphiphilic polymer micelle with the charge self-reversal activation and mitochondrial targeting efficacy according to claim 1,
refluxing the mixture as described in S101 for several hours, which is 24 hours;
the cooling described in S102 to 4 ℃ is to 4 ℃ by ice bath;
slowly adding 1.0M tetrahydrofuran borane complex under nitrogen in S102, and stirring for several hours, wherein the several hours are 48 hours;
drying the organic layer of the organic layer in the S102 by using anhydrous magnesium sulfate for several hours, wherein the several hours are 12 hours;
0.1mol of triethylamine is added dropwise to 7.3g under nitrogen with a constant pressure over several hours, which are 1 hour for several hours, as described in S103;
the mixture in S103 is stirred for 18 hours and filtered, several hours of which are 18 hours;
in S201, 4mL of methanesulfonyl chloride and 4mL of triethylamine are added at low temperature, then the mixture reacts for a plurality of hours in nitrogen, and then is stirred for a plurality of hours, wherein the reaction time is 2 hours, and the stirring time is 24 hours;
the mixture is stirred for a plurality of hours and then placed for a plurality of hours in S201, wherein the stirring time is 96 hours, and the placing time is 72 hours;
heating and refluxing for several hours in S202, wherein the several hours are 24 hours;
stirring and activating for several hours in S202, wherein the several hours are 1 hour;
30mL of a solution containing 12gNH was added as described in S2022-PEG-NH2Stirring the anhydrous dichloromethane solution for a plurality of hours, wherein the plurality of hours are 12 hours;
the melt polymerization under nitrogen in S3 is carried out for several hours, and the several hours are 12 hours;
dialyzing the reacted mixture against distilled water for several hours, which is 48 hours, as described in S3;
s4, then adding it to 1mL of DMSO solution containing 0.5mg of upconverting nanoparticles and stirring for several hours, which is 0.5 hour;
in S4, the mixed solution is dropped into 20ml of distilled water and stirred for several hours, and then the solution is dialyzed for several hours, wherein the stirring for several hours is 8 hours, and the dialysis for several hours is 48 hours.
3. The method for preparing the amphiphilic polymer micelle with the charge self-reversal activation and mitochondrial targeting efficacy according to claim 1 or 2, wherein the conditions of silica gel chromatography are as follows: n-hexane, ethyl acetate 1:1, v/v as eluent.
4. The method for preparing amphiphilic polymer micelle with charge self-reversal activation and mitochondrial targeting efficacy according to claim 3, wherein the dialysis of the solution in S4 is performed by transferring the solution into a 3500KDa dialysis bag.
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