CN110755633A - Preparation method of double-pH-sensitive nano delivery system loaded with chemotherapeutic drug daunorubicin - Google Patents

Preparation method of double-pH-sensitive nano delivery system loaded with chemotherapeutic drug daunorubicin Download PDF

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CN110755633A
CN110755633A CN201911155318.6A CN201911155318A CN110755633A CN 110755633 A CN110755633 A CN 110755633A CN 201911155318 A CN201911155318 A CN 201911155318A CN 110755633 A CN110755633 A CN 110755633A
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daunorubicin
methacrylate
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molar ratio
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CN110755633B (en
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郑化
宋亚静
李单
廖健洪
刘灿
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Wuhan University of Technology WUT
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    • 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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    • C08F293/005—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule using free radical "living" or "controlled" polymerisation, e.g. using a complexing agent
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    • C08F2438/00—Living radical polymerisation
    • C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]

Abstract

The invention relates to a preparation method of a double-pH-sensitive nano delivery system loaded with a chemotherapeutic drug daunorubicin. The method comprises the following specific steps: daunorubicin reacts with p-alkynylbenzaldehyde to generate imine bond daunorubicin; then preparing a polymer carrier of oligoethylene glycol methyl ether methyl methacrylate-poly (p-azido methyl acrylate benzyl ester-co-aminoethyl methacrylate), and introducing an amphiphilic block, azido and amino; carrying out click chemical reaction on alkynyl of imine bond daunorubicin and azido of the polymer carrier to obtain a daunorubicin polymer prodrug intermediate; and (3) reacting the amino group of the high-molecular prodrug intermediate with 2, 3-dimethylmaleic anhydride to obtain the daunorubicin high-molecular prodrug, and then carrying out self-assembly to obtain the polymer micelle. The polymer micelle obtained by the method can realize charge reversal according to different pH conditions in normal systemic circulation and cancer cell environments, improve targeting property, realize accurate transfer of medicaments, reduce toxic and side effects of the medicaments and enhance anti-tumor curative effect.

Description

Preparation method of double-pH-sensitive nano delivery system loaded with chemotherapeutic drug daunorubicin
Technical Field
The invention belongs to the field of nano-drug preparations and the field of polymer chemistry, and particularly relates to a preparation method of a double-pH-sensitive nano delivery system loaded with a chemotherapeutic drug daunorubicin.
Background
With the rapid increase of the incidence and mortality of tumors, the search for efficient and safe treatment methods is not easy. The traditional chemotherapy drugs used at present are mainly micromolecular drugs, and have many defects: low selectivity, easy metabolism, easy generation of multidrug resistance and the like. Moreover, many drugs are hydrophobic, and thus, the traditional administration mode causes serious toxic and side effects. Modern drug delivery nano-systems have rapidly developed to address these challenges. Compared with the traditional chemotherapy drugs, the nano-drug has the following advantages: (1) the medicine is absorbed by endocytosis, so that targeted medication is realized, and the bioavailability of the medicine is improved; (2) improving the solubility of hydrophobic drugs; (3) after the targeting group is modified, the targeting drug delivery can be realized, the drug dosage is reduced, and the side effect is reduced; (4) the half-life period of the medicine is prolonged, and the effective blood concentration and the medicine effect are improved; (5) controlling the constant release of the drug and improving the pharmacokinetic parameters of the drug; (6) eliminating the limitation of body barriers (such as blood brain barrier, blood eye barrier and cell biomembrane barrier) on the action of the medicine; nanocarriers generally have unique physical and chemical properties and good biocompatibility with cells. The polymer micelle is used as a carrier type of a drug delivery system, the amphiphilic segmented copolymer forms a core in an aqueous medium through a hydrophobic segment, a shell is formed through a hydrophilic segment, and the stable core-shell polymer micelle is formed through self-assembly. The hydrophilic shell enhances water solubility by encapsulating the drug in the core by hydrophilic-hydrophobic interactions. And the polymer micelle with the size less than 200nm has the effects of enhancing permeation and retention at a tumor part, so that the polymer micelle is broken in response at the tumor part to release the medicament, thereby reducing the toxic and side effects of the medicament.
Daunorubicin, as a first-line anticancer drug in clinical use for tumor treatment. The mechanism of action is to inhibit the nucleic acid synthesis process of the cell. However, the carbonyl reductase I existing in human liver can convert daunorubicin into a new daunorubicin alcohol, and the new alcohol metabolite not only reduces the activity of anticancer drugs, but also has cardiotoxicity to cause damage to cardiac cells, thus greatly limiting the application due to the defect.
Therefore, it is important to design a drug capable of responsively releasing daunorubicin at a tumor site and to select a non-toxic and biodegradable carrier material.
Disclosure of Invention
The invention aims to provide a preparation method of a double-pH sensitive nano delivery system for loading chemotherapeutic drug daunorubicin, which can realize charge reversal, improve targeting property, realize accurate delivery of drugs, reduce toxic and side effects of drugs and enhance anti-tumor curative effect in normal systemic circulation and tumor environment.
In order to solve the technical problems, the invention adopts the following technical scheme:
the preparation method of the double-pH-sensitive nano delivery system loaded with the chemotherapeutic drug daunorubicin is provided, and specifically comprises the following steps:
(1) under the conditions of light and oxygen free, the Schiff base reaction is carried out on the aldehyde group of p-alkynylbenzaldehyde and the amino group of Daunorubicin (DNR), and the imine bond daunorubicin (imine-DNR) is obtained through post-treatment, wherein methanol is used as a reaction solvent, triethylamine neutralizes free hydrochloric acid in daunorubicin, and the reaction formula is as follows:
Figure BDA0002284642230000021
(2) under the anaerobic condition, polyethylene glycol methyl ether methyl methacrylate (OEGMA) with molecular weight of 300 and 4-cyano-4- (phenylthioformylthio) pentanoic acid (CTP) take a bulk polymerization reaction by taking 1, 4-dioxane as a solvent and Azobisisobutyronitrile (AIBN) as an initiator to generate oligoethylene glycol methyl ether methyl methacrylate (POEGMA); under the oxygen-free condition, using N, N-Dimethylformamide (DMF) as a solvent and AIBN as an initiator to perform reversible addition fragmentation chain transfer radical polymerization (RAFT) reaction on POEGMA, 4-Azido Benzyl Methacrylate (ABMA) and Aminoethyl Methacrylate (AMA), and obtaining a high molecular carrier, namely oligoethylene glycol methyl ether methyl methacrylate-poly (p-azido benzyl methacrylate-co-aminoethyl methacrylate) (POPAA) after dialysis, freezing and drying, wherein the reaction formula is as follows:
(3) under the conditions of light and oxygen free, taking DMF as a solvent, in the presence of sodium ascorbate (NaAsc) and copper sulfate, carrying out click chemical reaction on azido in the high molecular carrier POPAA obtained in the step (2) and alkynyl of imine bond daunorubicin imine-DNR obtained in the step (1), dialyzing, freezing and drying to generate a daunorubicin high molecular prodrug intermediate POPPA @ imine-DNR, wherein the reaction formula is as follows:
Figure BDA0002284642230000031
(4) dissolving the POPPA @ imine-DNR obtained in the step (3) and 2, 3-dimethyl maleic anhydride (DA) in dimethyl sulfoxide (DMSO) for reaction at room temperature, dialyzing, and freeze-drying to obtain a daunorubicin macromolecule prodrug DA-POPAA @ imine-DNR, wherein the reaction formula is as follows:
Figure BDA0002284642230000032
(5) and (4) preparing the DA-POPAA @ imine-DNR obtained in the step (4) by adopting an ultrasonic self-assembly method to obtain a polymer micelle, namely the double-pH-sensitive nano delivery system loaded with chemotherapeutic drugs daunorubicin.
According to the scheme, the step (1) is specifically operated as follows: adding triethylamine into a methanol solution of daunorubicin to react under an oxygen-free environment, wherein the reaction temperature is 25-35 ℃, and the reaction time is 2-5 h; and then adding a methanol solution of p-alkynylbenzaldehyde, continuously reacting for 24-36h in the dark, carrying out rotary evaporation on the solvent, adding methanol for redissolving, adding acetonitrile for precipitation, centrifuging, and carrying out vacuum drying to obtain the product imine-DNR.
According to the scheme, in the step (1), the molar ratio of daunorubicin to p-alkynylbenzaldehyde is 1 (1-1.2).
According to the scheme, the operation of the step (2) is as follows: dissolving polyethylene glycol methyl ether methacrylate (OEGMA), 4-cyano-4- (phenylthiocarbonylthio) pentanoic acid and azobisisobutyronitrile in 1, 4-dioxane, reacting for 20-32h at 70-80 ℃ after three cycles of freezing-air extraction-thawing, and dialyzing, and freeze-drying to obtain POEGMA; dissolving POEGMA, 4-Azido Benzyl Methacrylate (ABMA), Aminoethyl Methacrylate (AMA) and Azobisisobutyronitrile (AIBN) in DMF, performing freeze-suction-thawing cycle for 3 times, reacting at 70-80 ℃ for 40-60h, dialyzing, and freeze-drying to obtain POPAA.
According to the scheme, the molar ratio of the polyethylene glycol methyl ether methacrylate to the 1, 4-cyano-4- (phenylthiocarbonylthio) pentanoic acid is (30-50): 1, the molar ratio of the polyethylene glycol methyl ether methacrylate to the azodiisobutyronitrile is (150-) -250): 1; the molar ratio of 4-azido benzyl methacrylate, aminoethyl methacrylate to oligoethylene glycol monomethyl ether methyl methacrylate is (30-60): (30-60): 1, the molar ratio of the oligomeric ethylene glycol monomethyl ether methyl methacrylate to the azobisisobutyronitrile is (4-5): 1.
according to the scheme, the number average molecular weight of the POPAA is 20-28kDa, wherein the number average molecular weight of the oligomeric ethylene glycol monomethyl ether methyl methacrylate is 9-12kDa, the number average molecular weight of the aminoethyl methacrylate is 4-6kDa, and the number average molecular weight of the benzyl p-azidomethacrylate is 7-10 kDa. Polyethylene glycol monomethyl ether methyl methacrylate and aminoethyl methacrylate are hydrophilic blocks, and benzyl p-azidomethacrylate is a hydrophobic block.
According to the scheme, the operation of the step (3) is as follows: and (3) dissolving the POPAA obtained in the step (2) and the imine-NDR obtained in the step (1) in DMF, adding sodium ascorbate and copper sulfate pentahydrate, reacting at 25-35 ℃ in an oxygen-free atmosphere in a dark place for 36-48h, and dialyzing, freezing and drying to obtain a daunorubicin macromolecule prodrug intermediate POPPA @ imine-DNR.
According to the scheme, in the step (3), the molar ratio of azide to imine-NDR in POPAA is 1 (0.2-1), the molar ratio of sodium ascorbate to copper sulfate pentahydrate is 1 (1-1.5), and the molar ratio of imine-NDR to sodium ascorbate is as follows: (5-10): 1.
according to the scheme, the reaction time of the step (4) is 2-6 h.
According to the scheme, in the step (4), the molar ratio of amino to 2, 3-dimethylmaleic anhydride in the POPPA @ imine-DNR is 1: (1-3).
According to the scheme, the step (5) is specifically operated as follows: and (3) dissolving the DA-POPAA @ imine-DNR obtained in the step (4) in a phosphate buffer solution with the pH value of 7.4 to prepare a daunorubicin prodrug solution of 0.5-1mg/mL, carrying out ultrasonic treatment for 5-10min, and filtering through a 0.45 mu m pinhole type filter membrane to obtain the polymer micelle.
According to the scheme, the size of the polymer micelle obtained in the step (5) is 50-200 nm.
In the process of preparing the polymer micelle, 2, 3-Dimethylmaleic Anhydride (DA) is selected to modify amino and is modified into a β -carboxyl amide structure, so that charge conversion under different pH conditions can be realized, and the polymer micelle has double pH sensitivity-The micelle is negatively charged, so that nonspecific combination with serum protein can be reduced, and thrombosis can be prevented, so that hemolysis is inhibited, and the blood circulation time of the nano-drug is prolonged; while in the weakly acidic environment (pH 6.5-5.0) of the tumor, DA is detached, and the ionic groups around the micelles are separated from COO-To NH3 +The reversal of charge is realized, and the micelle with positive charge can be adsorbed on the cell membrane with negative charge through electrostatic adsorption, so that the uptake of cancer cells to the micelle is enhanced, the drug absorption is promoted, and the anti-tumor curative effect is enhanced.
The invention has the beneficial effects that:
1. the polymer micelle provided by the invention has double pH sensitivity, can realize charge reversal according to different pH conditions in normal systemic circulation and cancer cell environments, improves targeting property, realizes accurate transfer of medicaments, reduces toxic and side effects of the medicaments, and enhances the anti-tumor curative effect.
2. The high-molecular-weight POPPA obtained through RAFT polymerization reaction has narrow molecular weight distribution and functional group designability, on one hand, 2, 3-Dimethylmaleic Anhydride (DA) can be selected to modify amino in the POPPA and modify the amino into β -carboxyamide structure, so that charge reversal of an intracellular environment of a polymer micelle is realized, on the other hand, azido is introduced into the POPPA and undergoes click chemical reaction with alkynyl of imine-DNR to prepare a daunorubicin high-molecular prodrug intermediate POPPA @ imine-DNR, and the method is simple in operation, easy in condition realization and high in drug loading.
3. The high-molecular carrier POPPA used in the invention has good biocompatibility and biodegradability, and the obtained daunorubicin high-molecular prodrug DA-POPAA @ imine-DNR can be self-assembled to form a polymer micelle with small particle size, good dispersity and stable structure.
4. The polymer micelle has an amphiphilic multi-block copolymer, polyethylene glycol monomethyl ether methyl methacrylate and aminoethyl methacrylate are hydrophilic blocks, and p-azido benzyl methacrylate is a hydrophobic block, so that the water solubility of the daunorubicin is remarkably improved, the long circulation characteristic and the space stability of the polymer micelle are ensured, and the circulation time of the polymer micelle in blood is prolonged.
5. The polymer micelle has a proper size of 50-200nm, and can be effectively enriched to a tumor part through an EPR effect.
Drawings
FIG. 1 is a particle size distribution diagram (A) of the polymer micelle prepared in example 1, a transmission electron micrograph (B) of the polymer micelle, and a surface potential distribution diagram (C) of the polymer micelle at pH7.4 and 6.5, respectively.
FIG. 2 is a nuclear magnetic diagram (1H-NMR) of the intermediate POEGMA of the polymer carrier, POPAA of the polymer carrier and POPAA @ imine-DNR of the polymer prodrug of daunorubicin prepared in example 1.
FIG. 3 is a nuclear magnetic diagram (1H-NMR) of daunorubicin DNR and imine-linked daunorubicin-DNR prepared in example 1.
FIG. 4 is a graph showing in vitro release data of the polymer micelle prepared in example 1.
FIG. 5 is a distribution diagram of the particle size of the polymer micelle prepared in example 2.
FIG. 6 is a distribution diagram of the particle size of the polymer micelle prepared in example 3.
Detailed Description
The technical solutions of the present invention are further illustrated below by specific embodiments, and it should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as specifically limiting the present invention.
Example 1
A preparation method of a double-pH sensitive nano delivery system loaded with chemotherapeutic drugs daunorubicin comprises the following specific steps:
1) synthesis of imine bond daunorubicin:
dissolving daunorubicin (195mg, 0.035mmol) in 20mL methanol, adding triethylamine (285 μ L, 2.075mmol), sealing, vacuumizing, introducing nitrogen, placing in a heat-collecting constant-temperature heating magnetic stirrer, and stirring at 30 deg.C for 2h to obtain solution A; p-alkynylbenzaldehyde (54mg, 0.04mmol) was dissolved in methanol to obtain a solution B; and adding the solution B into the solution A through an injector, continuously reacting for 24 hours in the dark, carrying out rotary evaporation on the solvent, adding about 1mL of methanol for redissolving, adding about 3mL of acetonitrile for precipitation, centrifuging, removing the supernatant, and carrying out vacuum drying to obtain the product of the imine bond daunorubicin imine-DNR.
2) Synthesis of the polymer carrier:
polyethylene glycol methyl ether methacrylate (OEGMA) (1200mg, 4mmol), 4-cyano-4- (phenylthioformylthio) pentanoic acid (CTP) (27.9mg, 0.1mmol) and Azobisisobutyronitrile (AIBN) (3.28mg, 0.02mmol) are weighed and dissolved in 3mL of 1, 4-dioxane, and the POEGMA product is obtained after three times of freezing-air extraction-melting cycle, placed in a heat collection type constant temperature heating magnetic stirrer, reacted for 24h at 70 ℃, dialyzed and freeze-dried. Dissolving the freeze-dried product POEGMA (300mg, 0.025mmol), 4-Azido Benzyl Methacrylate (ABMA) (272.5mg, 1.25mmol), Aminoethyl Methacrylate (AMA) (206.2mg, 1.25mmol) and Azobisisobutyronitrile (AIBN) (0.82mg, 0.005mmol) in 5mL of DMF, performing freeze-suction-thawing cycle for three times, placing in a heat collection type constant temperature heating magnetic stirrer at 70 ℃ for reaction for 48h, dialyzing, and freeze-drying to obtain the product, namely the high molecular carrier POPAA.
3) Synthesis of daunorubicin macromolecule prodrug intermediate:
and (3) putting the freeze-dried POPAA (100mg, 0.0038mmol), imine-DNR (50mg, 0.07mmol), sodium ascorbate (2mg, 0.01mmol) and copper sulfate pentahydrate (2.5mg, 0.01mmol) in 10mL of mixed solution of DMF and water (DMF: water ═ 1: 1) in a heat collection type constant-temperature heating magnetic stirrer at 70 ℃ for reaction for 24h, dialyzing, freezing and drying to obtain a daunorubicin polymer prodrug intermediate POPPA @ imine-DNR, wherein the molar ratio of azide in the POPAA to imine-DNR is 1: 0.5.
4) Synthesis of daunorubicin macromolecule prodrug:
taking 60mg of POPPA @ imine-DNR and 5.04mg of 2, 3-dimethyl maleic anhydride obtained in the step 3), dissolving in DMSO, continuing to react for 2h, dialyzing, and freeze-drying to obtain the daunorubicin macromolecule prodrug DA-POPAA @ imine-DNR, wherein the molar ratio of amino in the POPPA @ imine-DNR to the 2, 3-dimethyl maleic anhydride is 1: 2.
5) Preparation of prodrug nanoparticles with dual pH sensitivity:
dissolving 5mg of DA-POPAA @ imine-DNR obtained in the step 4) in 5mL of phosphate buffer solution with the pH value of 7.4, performing ultrasonic treatment for 5min, passing through a 0.45-micrometer pinhole type filter membrane to obtain a polymer micelle, namely a nano delivery system of the double-pH sensitive load chemotherapeutic drug daunorubicin, and determining the particle size and the dispersity in a Malvern particle sizer.
FIG. 1 is a graph (A) showing the particle size distribution of the polymer micelle prepared in this example, a graph (B) obtained by transmission electron microscopy, and a graph (C) showing the surface potential distribution of the polymer micelle at pH7.4 and 6.5, respectively. As can be seen from the figure: fig. 1(a) shows that the prepared polymer micelle particle size and dispersity are respectively size ═ 137nm, PDI ═ 0.168; PDI represents the stability of the micelle, and the value is between 0 and 1, and the closer to 0, the more stable and uniform the micelle is, and the result shows that the micelle has good stability. FIG. 1(B) is a diagram of the morphology of micelles, and it can be observed that the micelles are uniform in size and distribution; FIG. 1(C) shows the surface potentials of the micelles at pH7.4 and pH6.5, respectively, of-9.05 and 5.76mV, respectively, indicating that the micelles can achieve negative to positive charge reversal in different pH environments.
FIG. 2 is a nuclear magnetic map (1H-NMR) of the intermediate POEGMA of the polymer carrier, POPAA of the polymer carrier and POPAA @ imine-DNR of the polymer prodrug of daunorubicin prepared in example 1, wherein the map shows the assignment of H atoms. The figure shows that: in POEGMA nuclear magnetic spectrum, the chemical shifts of delta 3.37and delta 4.07ppm are respectively-OCH in POEGMA3And (O-CH)2-CH2-O) signal peaks, which can prove the successful synthesis of POEGMA; in a POPAA nuclear magnetic spectrum, delta 7.39,7.09 and 4.92ppm are respectively signal peaks of an aromatic ring and a benzylidene group of ABMA, and can indicate the successful synthesis of POPAA; in a nuclear magnetic spectrum of POPAA @ imine-DNR, delta 7.94 and delta 7.1-7.2 respectively correspond to benzene ring hydrogen and imidazole ring hydrogen of the medicine daunorubicin, and the successful bonding of the imine bond daunorubicin to the polymer carrier can be proved.
FIG. 3 is a nuclear magnetic map (1H-NMR) of DNR and the imine-bonded daunorubicin imine-DNR prepared in example 1, the map showing the assignment of H atoms. The figure shows that: DNR shows hydrogen of benzene ring at two chemical shifts of 7.90(s, 2H), 7.65(s, 1H); in an imine-DNR nuclear magnetic spectrum, 5 chemical shifts appear in 7.85(s, 2H), 7.58(s, 1H), 7.74(s, 1H), 7.51(s, 2H) and 8.35(s, 2H), and the successful synthesis of imine-DNR can be proved by chemical shift at 8.35.
Determination of drug loading of high molecular weight polymer prodrug:
weighing about 10mg of freeze-dried polymeric prodrug DA-POPAA @ imine-DNR, adding a proper amount of deionized water, then adding a certain amount of 0.1mol/L hydrochloric acid, stirring until the mixture is dissolved, and stirring overnight at 40-50 ℃ in the dark. The above solution was then subjected to UV-vis absorbance measurement. Then the drug loading was calculated to be 12.6% according to the daunorubicin standard curve and the following formula.
Figure BDA0002284642230000081
Wherein:
CDNR-concentration of DNR (μ g/mL);
Vsolution-volume of polymeric prodrug solution (mL);
mprodrug-mass of polymeric prodrug (mg);
in vitro release of polymeric micelles: weighing a proper amount of freeze-dried polymer micelle powder, respectively dissolving the polymer micelle powder in buffers with different pH values (pH7.4, 6.5 and 5.0), transferring the micelle solution into a corresponding dialysis bag (DW ═ 3500) after the micelle is dissolved, and putting the dialysis bag into a centrifuge tube containing a proper amount of corresponding medium. Placing the mixture in a constant-temperature shaking incubator, respectively sucking 3mL of dialysate at fixed time, supplementing an equal amount of fresh buffer solution with corresponding pH value, and avoiding light in the whole process. The concentration of daunorubicin was determined according to UV-vis and standard curves, each group of samples was determined 3 times, and the mean value was taken.
FIG. 4 is the in vitro release data curve of the polymer micelle of the nano-delivery system of the dual pH-sensitive loaded chemotherapeutic drug daunorubicin prepared in example 1, showing: the micelle releases less than 13% in 24h under the condition of pH7.4, which indicates that the micelle can keep stability in normal tissues; the release amount is about 22% in 24h under the condition of pH6.5, and the release amount reaches 70% in 48h under the condition of pH5.0, which indicates that the micelle has acid sensitivity, is beneficial to promoting the drug absorption in the tumor environment, and can control the release amount in normal tissues.
Example 2
1) Synthesis of imine bond daunorubicin: same as example 1
2) Synthesis of the polymer carrier: same as example 1
3) Synthesis of daunorubicin macromolecule prodrug intermediate:
and (3) taking the POPAA (100mg, 0.0038mmol) and the imine-DNR (20mg, 0.03mmol) after freeze-drying, taking sodium ascorbate (1mg, 0.005mmol) and copper sulfate pentahydrate (1.25mg, 0.005mmol), dissolving in a mixed solution of DMF and water (DMF: water ═ 1: 1), placing in a heat collection type constant-temperature heating magnetic stirrer at 70 ℃ for reaction for 24 hours, dialyzing, freezing and drying to obtain a daunorubicin macromolecule prodrug intermediate POPPA @ imine-DNR, wherein the molar ratio of azide in the POPAA to the imine-DNR is 1: 0.2.
4) Synthesis of daunorubicin macromolecule prodrug:
taking 60mg of POPPA @ imine-DNR and 5.04mg of 2, 3-dimethyl maleic anhydride obtained in the step 3), dissolving in DMSO, continuing to react for 2h, dialyzing, and freeze-drying to obtain the daunorubicin macromolecule prodrug DA-POPAA @ imine-DNR, wherein the molar ratio of amino in the POPPA @ imine-DNR to the 2, 3-dimethyl maleic anhydride is 1: 2.
5) Preparation of prodrug nanoparticles with dual pH sensitivity:
dissolving 5mg of DA-POPAA @ imine-DNR obtained in the step 4) in 5mL of phosphate buffer solution with the pH value of 7.4, performing ultrasonic treatment for 5min, and passing through a 0.45-micrometer pinhole type filter membrane to obtain a polymer micelle, namely the double-pH-sensitive nano delivery system for loading the chemotherapeutic drug daunorubicin.
As a result of measuring the particle size and the degree of dispersion in a malvern particle sizer, the particle size and the degree of dispersion were 78.95nm and 0.056, respectively, and the micelle particle size was small and the stability was good, as shown in fig. 5.
Determination of drug loading of high molecular weight polymer prodrug:
weighing about 10mg of the freeze-dried macromolecule prodrug, adding a proper amount of deionized water, then adding a certain amount of 0.1mol/L hydrochloric acid, stirring until the mixture is dissolved, and stirring overnight at 40-50 ℃ in a dark place. The above solution was then subjected to UV-vis absorbance measurement. Then, the drug loading was calculated to be 7.2% according to the daunorubicin standard curve and the drug loading calculation formula in example 1.
Example 3
1) Synthesis of imine bond daunorubicin: same embodiment 1
2) Synthesis of the polymer carrier: same embodiment 1
3) Synthesis of daunorubicin macromolecule prodrug intermediate:
the POPAA (100mg, 0.0038mmol) and the imine-DNR (100mg, 0.15mmol) after freeze-drying are taken, sodium ascorbate (3mg, 0.015mmol) and copper sulfate pentahydrate (3.75mg, 0.015mmol) are taken, dissolved in a mixed solution of DMF and water (DMF: water ═ 1: 1), and the mixture is placed in a heat collection type constant temperature heating magnetic stirrer at 70 ℃ for reaction for 24 hours. Dialyzing, freezing and drying to obtain the daunorubicin macromolecule prodrug intermediate POPPA @ imine-DNR, wherein the molar ratio of the azide in the POPAA to the imine-DNR is 1: 1.
4) Synthesis of daunorubicin macromolecule prodrug:
taking 60mg of POPPA @ imine-DNR and 5.04mg of 2, 3-dimethyl maleic anhydride obtained in the step 3), dissolving in DMSO, continuing to react for 2h, dialyzing, and freeze-drying to obtain the daunorubicin macromolecule prodrug DA-POPAA @ imine-DNR, wherein the molar ratio of amino in the POPPA @ imine-DNR to the 2, 3-dimethyl maleic anhydride is 1: 2.
5) Preparation of prodrug nanoparticles with dual pH sensitivity:
dissolving 5mg of DA-POPAA @ imine-DNR obtained in the step 4) in 5mL of phosphate buffer solution with the pH value of 7.4, performing ultrasonic treatment for 5min, and passing through a 0.45-micrometer pinhole type filter membrane to obtain a polymer micelle, namely the double-pH-sensitive nano delivery system for loading the chemotherapeutic drug daunorubicin.
As a result of measuring the particle size and the degree of dispersion in a malvern particle sizer, the particle size and the degree of dispersion were 149.3nm and PDI was 0.116, respectively, and the micelle particle size was small and the stability was good, as shown in fig. 5.
Determination of drug loading of high molecular weight polymer prodrug:
about 10mg of the lyophilized polymeric prodrug is weighed, added with an appropriate amount of deionized water, then added with an amount of 0.1mol/L hydrochloric acid, stirred to dissolve, and stirred overnight at 40-50 ℃ in the dark. The above solution was then subjected to UV-vis absorbance measurement. Then the drug loading was calculated to be 18% according to the daunorubicin standard curve and the drug loading calculation formula in example 1.

Claims (10)

1. A preparation method of a double-pH sensitive nano delivery system loaded with chemotherapeutic drugs daunorubicin is characterized by comprising the following steps:
(1) under the conditions of light and oxygen free, the Schiff base reaction is carried out on the aldehyde group of p-alkynylbenzaldehyde and the amino group of daunorubicin, and imine bond daunorubicin is obtained through post-treatment, wherein methanol is used as a reaction solvent, triethylamine neutralizes free hydrochloric acid in daunorubicin, and the reaction formula is as follows:
Figure FDA0002284642220000011
(2) under the anaerobic condition, the polyethylene glycol methyl ether methyl methacrylate with the molecular weight of 300 and 4-cyano-4- (phenylthiocarbonylthio) valeric acid take self-bulk polymerization reaction by taking 1, 4-dioxane as a solvent and azodiisobutyronitrile as an initiator to generate oligoethylene glycol methyl ether methyl methacrylate; the method comprises the following steps of carrying out reversible addition fragmentation chain transfer free radical polymerization on methyl oligoethylene glycol methyl methacrylate, 4-azido benzyl methacrylate and aminoethyl methacrylate under an anaerobic condition by using DMF as a solvent and azodiisobutyronitrile as an initiator, dialyzing, freezing and drying to obtain a high-molecular carrier, namely, methyl oligoethylene glycol methyl methacrylate-poly (benzyl p-azidomethacrylate-co-aminoethyl methacrylate), wherein the reaction formula is as follows:
Figure FDA0002284642220000012
(3) under the conditions of light and oxygen free, taking DMF as a solvent, carrying out click chemical reaction on azido in the polymer carrier obtained in the step (2) and alkynyl of imine bond daunorubicin obtained in the step (1) in the presence of sodium ascorbate and copper sulfate, dialyzing, freezing and drying to generate a daunorubicin polymer prodrug intermediate, wherein the reaction formula is as follows:
Figure FDA0002284642220000013
(4) dissolving the daunorubicin macromolecule prodrug intermediate obtained in the step (3) and 2, 3-dimethylmaleic anhydride in dimethyl sulfoxide for reaction at room temperature, dialyzing, and freeze-drying to obtain the daunorubicin macromolecule prodrug, wherein the reaction formula is as follows:
Figure FDA0002284642220000021
(5) and (4) preparing the daunorubicin macromolecule prodrug obtained in the step (4) by adopting an ultrasonic self-assembly method to obtain a polymer micelle, namely the double-pH sensitive nano delivery system loaded with the chemotherapeutic drug daunorubicin.
2. The method of claim 1, wherein step (1) is specifically operated as: adding triethylamine into a methanol solution of daunorubicin to react under an oxygen-free environment, wherein the reaction temperature is 25-35 ℃, and the reaction time is 2-5 h; and then adding a methanol solution of p-alkynylbenzaldehyde, continuously reacting for 24-36h in the dark, rotationally evaporating the solvent, adding methanol for redissolving, adding acetonitrile for precipitation, centrifuging, and drying in vacuum to obtain the product of the imine bond daunorubicin.
3. The process according to claim 1, wherein in the step (1), the molar ratio of daunorubicin to p-alkynylbenzaldehyde is 1 (1-1.2).
4. The method of claim 1, wherein the step (2) is specifically operated as: dissolving polyethylene glycol methyl ether methacrylate, 4-cyano-4- (phenylthiocarbonylthio) pentanoic acid and azobisisobutyronitrile into 1, 4-dioxane, reacting for 20-32h at 70-80 ℃ after three cycles of freezing-air extraction-melting, and then dialyzing, freezing and drying to obtain oligoethylene glycol methyl ether methacrylate; dissolving oligo-ethylene glycol methyl ether methyl methacrylate, 4-azido benzyl methacrylate, aminoethyl methacrylate and azobisisobutyronitrile in DMF, performing freezing-air extraction-thawing cycle for 3 times, reacting at 70-80 ℃ for 40-60h, dialyzing, and freeze-drying to obtain the polymer carrier.
5. The method according to claim 1, wherein in the step (2), the molar ratio of polyethylene glycol methyl ether methacrylate to 1, 4-cyano-4- (phenylthiocarbonylthio) pentanoic acid is (30-50): 1, the molar ratio of the polyethylene glycol methyl ether methacrylate to the azodiisobutyronitrile is (150-) -250): 1; the molar ratio of 4-azido benzyl methacrylate, aminoethyl methacrylate to oligoethylene glycol monomethyl ether methyl methacrylate is (30-60): (30-60): 1, the molar ratio of the oligomeric ethylene glycol monomethyl ether methyl methacrylate to the azobisisobutyronitrile is (4-5): 1.
6. the method of claim 1, wherein the step (3) is specifically operated as: and (3) dissolving the high molecular carrier obtained in the step (2) and the imine bond daunorubicin obtained in the step (1) in DMF, adding sodium ascorbate and copper sulfate pentahydrate, reacting at 25-35 ℃ in an oxygen-free atmosphere in the dark for 36-48h, and dialyzing, freezing and drying to obtain a daunorubicin high molecular prodrug intermediate.
7. The preparation method according to claim 1, wherein in the step (3), the molar ratio of azide to imine bond daunorubicin in the polymeric carrier is 1 (0.2-1), the molar ratio of sodium ascorbate to copper sulfate pentahydrate is 1 (1-1.5), and the molar ratio of imine bond daunorubicin to sodium ascorbate is: (5-10): 1.
8. the method according to claim 1, wherein the reaction time of step (4) is 2-6 h.
9. The preparation method according to claim 1, wherein in the step (4), the molar ratio of the amino group to the 2, 3-dimethylmaleic anhydride in the daunorubicin polymeric prodrug intermediate is 1: (1-3).
10. The method of claim 1, wherein the step (5) is specifically operated as: and (3) dissolving the daunorubicin macromolecule prodrug obtained in the step (4) in a phosphate buffer solution with the pH value of 7.4 to prepare a daunorubicin prodrug solution of 0.5-1mg/mL, carrying out ultrasonic treatment for 5-10min, and filtering through a 0.45-micrometer pinhole type filter membrane to obtain the polymer micelle.
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