CN114081953B - Prodrug dendrimer nano-carrier and preparation method and application thereof - Google Patents

Prodrug dendrimer nano-carrier and preparation method and application thereof Download PDF

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CN114081953B
CN114081953B CN202111217352.9A CN202111217352A CN114081953B CN 114081953 B CN114081953 B CN 114081953B CN 202111217352 A CN202111217352 A CN 202111217352A CN 114081953 B CN114081953 B CN 114081953B
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lys
prodrug
peg
dendrimer
fmoc
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CN114081953A (en
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颜廷胜
刘忠华
许淼
胡岚馨
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Northeast Agricultural University
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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
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • 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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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers

Abstract

A prodrug dendrimer nano-carrier, a preparation method and application thereof belong to the field of nano-drug carrier materials, wherein the nano-carrier is prepared by a step-by-step peptide method, hydrophobic self-assembly and crosslinking reaction. The system has multifunctional targeting molecule ligands, and the targeting ligands can be used as targeting elements, so that the active targeting capability of the carrier is improved, and the targeting molecules can be used as size conversion elements, so that the penetration capability of drug tumors is improved. The drug delivery system can change the local microenvironment of the tumor through the primary drug release, thereby triggering the secondary drug release and realizing the cascade treatment of the tumor. Aiming at multiple biological barriers and complex dynamic microenvironments in the drug delivery process, the method adopts multiple targeting, size conversion and drug graded release, and is expected to realize accurate and efficient tumor cascade amplification treatment guided by images.

Description

Prodrug dendrimer nano-carrier and preparation method and application thereof
Technical Field
The invention belongs to the field of nano drug carrier materials, and particularly relates to a prodrug dendrimer nano carrier, a preparation method and application thereof.
Background
Limited tumor penetration and ineffective cell internalization are important factors limiting tumor drug therapy. Small size nanoparticles have a greater opportunity to overcome interstitial transport barriers and diffuse deep into tumor tissue. However, due to tumor vascular extravasation and poor pharmacokinetic profile, it is difficult to achieve satisfactory drug delivery. Particles with too small a size are likely to be cleared by renal excretion, and large size nanoparticles, while able to bypass renal clearance, are generally less permeable to their tumor. Size-switchable Drug Delivery Systems (DDS) can be used to solve the above problems, and there are research groups reporting a size/charge double-switched "trojan horse" nanoparticle that can maintain a larger size in the blood circulation to improve stability; after reaching the tumor part, the nano-particles can be rapidly decomposed into ultra-small nano-particles, so that the tumor penetration capability of the nano-preparation is effectively improved. Several studies have shown that DDS with naked boric acid groups can bind sialic acid overexpressed on the surface of brain tumor cells, improving the binding and efficient internalization of brain tumor cells. However, the cascade activation mechanism of these vectors still needs to be perfect to fully exploit their potential in tumor therapy.
Cascade amplification treatment may cooperate with the therapeutic action between different agents to achieve complementary therapeutic effects. Possible joint mechanisms for these methods include: (1) upregulation of ROS stress; (2) inhibiting a repair mechanism of radiation-induced DNA damage; (3) relieving tumor anoxia, etc. There are studies on encapsulation of anticancer drug 7-ethyl-10-hydroxycamptothecin (SN-38) using pegylated hollow tantalum oxide (H-TaOx) nanoshells for chemical/radiation cascade treatment of tumors. SN-38 can induce apoptosis of tumor cells not only by inhibiting topoisomerase I, but also by inducing cell cycle arrest and forcing tumor cells into the radiation-sensitive phase. The nano platform can obviously improve the efficacy of subsequent radiotherapy by combining the energy deposition capability of Ta atoms and the radiosensitization of SN-38. The sequential interactions between PDT and chemotherapy, in addition to direct destruction of tumor cells, can also destroy key proteins such as P-glycoprotein (P-gp) associated with the development of drug resistance.
Target-specific release and activation are key to the success of cascade amplification therapies, and tumor metabolic abnormalities create specific microenvironments that provide the possibility for drug release and activation. However, the microenvironment is constantly changing during the development of tumors, and the novel DDS is not only adapted to the tumor microenvironment, but also needs to enhance the controllability of drug release and cascade activation by regulating the physiological state of the tumor.
Disclosure of Invention
Aiming at the defects existing in the background technology, the invention provides a prodrug dendrimer nano-carrier, a preparation method and application thereof, wherein the nano-carrier has the characteristics of high drug loading, multiple targeting, variable size, good biocompatibility and the like; the device also has the performances of size conversion and medicine grading release, and is expected to realize image-guided accurate and efficient brain tumor cascade treatment.
The invention constructs a prodrug dendrimer diagnosis and treatment platform aiming at multiple physiological barriers and complex dynamic microenvironments in the tumor drug delivery process, and can be applied to the visualized cascade amplification treatment of various tumors.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a prodrug dendrimer nanocarrier having the structural formula:
the preparation method of the prodrug dendrimer nanocarrier comprises the following steps: the nano-carrier is prepared from an antitumor prodrug dendrimer modified by a bifunctional ligand by a self-assembly method; the antitumor prodrug dendrimer is formed by covalently bonding a basic dendrimer with a borated modified diagnosis and treatment reagent through a phosphate bond.
Specifically, the method comprises the steps of:
step one: synthesis of polyethylene glycol-lysine base block polymer Lys by using N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole system m -PEG-Lys n Wherein m=1 to 20, n=1 to 20, and the molecular weight of peg is 1 to 20KDa;
step two: lys is to be Lys m -PEG-Lys n One end of the mixture is connected with 3, 4-dihydroxybenzoic acid Catechol to obtain Catechol 2m -Lys m -PEG-Lys n -Fmoc dendrimer, wherein m = 1 to 20;
step three: anticancer drug or prodrug molecule containing boric acid group and Catechol 2m -Lys m -PEG-Lys n -Fmoc reaction in dichloromethane solution, wherein the molar ratio of anticancer drug or prodrug molecule to block polymer is 2 m-4 m:1 obtaining an amphiphilic prodrug dendrimer PD based on borate linkages 2m -Catechol 2m -Lys m -PEG-Lys n -Fmoc；
Step four: modification of bifunctional ligand, specifically, modification of boric acid group-containing Compound with PD through an amide bond 2m -Catechol 2m -Lys m -PEG-Lys n Fmoc Lys end coupling to obtain bifunctional ligand-modified anti-tumor prodrug dendrimer PD 2m -PEG-Lys n -BA 2n Wherein m=1 to 20, n=1 to 20; by reacting compounds containing ortho-dihydroxyl groups with PD via an amide bond 2m -Catechol 2m -Lys m -PEG-Lys n Fmoc Lys end coupling to obtain bifunctional ligand-modified anti-tumor prodrug dendrimer PD 2m -PEG-Lys n -DH 2n Wherein m=1 to 20, n=1 to 20; dialyzing the obtained two prodrug dendrimers, and freeze-drying;
step five: PD 2m -PEG-Lys n -BA 2n And PD 2m -PEG-Lys n -DH 2n Mixing and dissolving in polar solvent, and volatilizing or reprecipitating the solvent to prepare the borate crosslinking integrated nano carrier.
An application of the prodrug dendrimer nanocarrier in tumor cascade treatment.
Compared with the prior art, the invention has the beneficial effects that:
(1) The drug delivery system has a multifunctional targeting molecule ligand. The targeting ligands can be used as targeting elements, so that the active targeting capability of a carrier system is improved; on the other hand, they can be used as elements for size conversion of the carrier system. Under normal tissue physiological conditions, a stable cross-linked state is maintained, and the stability in blood circulation is improved; when they reach the tumor tissue, the sensitive bonds are broken under the stimulation of the local microenvironment, and the carrier releases the ultra-small nano particles to improve the penetration capacity of the tumor.
(2) The functionalization of the carrier prodrug structural molecule. The polymer constituting the main component of the carrier contains the active ingredient of the prodrug, and is used as a diagnostic and therapeutic agent to play the roles of imaging and inhibiting tumor growth after the prodrug is activated; can be used as a hydrophobic core in the self-assembly process to maintain the stable state of the carrier.
(3) The drug delivery system can change the local microenvironment of the tumor through the primary drug release, thereby triggering the secondary drug release and realizing the cascade treatment of the tumor.
Drawings
FIG. 1 is a schematic diagram of a process for preparing a prodrug dendrimer nanocarrier;
FIG. 2 is a graph of DOX prodrug dendrimer nanocarrier particle size distribution;
FIG. 3 is a transmission electron microscope image of DOX prodrug dendrimer nanocarriers.
Detailed Description
The technical scheme of the present invention is further described below with reference to the examples and the accompanying drawings, but is not limited thereto, and modifications and equivalents of the technical scheme of the present invention should be included in the protection scope of the present invention without departing from the spirit and scope of the technical scheme of the present invention.
The prodrug dendrimer nano-carrier is prepared by a progressive peptide method, hydrophobic self-assembly and crosslinking reaction. The system has multifunctional targeting molecule ligands, and the targeting ligands can be used as targeting elements, so that the active targeting capability of the carrier is improved, and the targeting molecules can be used as size conversion elements, so that the penetration capability of drug tumors is improved. The drug delivery system can change the local microenvironment of the tumor through the primary drug release, thereby triggering the secondary drug release and realizing the cascade treatment of the tumor. Aiming at multiple biological barriers and complex dynamic microenvironments in the drug delivery process, the method adopts multiple targeting, size conversion and drug graded release, and is expected to realize accurate and efficient tumor cascade amplification treatment guided by images.
The first embodiment is as follows: the present embodiment describes a prodrug dendrimer nanocarrier having the following structural formula:
the second embodiment is as follows: a method for preparing a prodrug dendrimer nanocarrier according to one embodiment, the method specifically comprises: the nano-carrier is prepared from an antitumor prodrug dendrimer modified by a bifunctional ligand by a self-assembly method; the antitumor prodrug dendrimer is formed by covalently bonding a basic dendrimer with a borated modified diagnosis and treatment reagent through a phosphate bond.
And a third specific embodiment: the preparation method of the prodrug dendrimer nanocarrier in the second embodiment comprises the following steps:
step one: synthesis of polyethylene glycol-lysine base Block Polymer Lys Using N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole (DIC/HOBt) System m -PEG-Lys n Wherein m=1 to 20, n=1 to 20, and the molecular weight of peg is 1 to 20KDa;
step two: lys is to be Lys m -PEG-Lys n One end of the mixture is connected with 3, 4-dihydroxybenzoic acid Catechol to obtain Catechol 2m -Lys m -PEG-Lys n -Fmoc dendrimer, wherein m = 1 to 20;
step three: will contain boric acidGroup anti-cancer drug or prodrug molecules and Catechol 2m -Lys m -PEG-Lys n -Fmoc reaction in dichloromethane solution, wherein the molar ratio of anticancer drug or prodrug molecule to block polymer is 2 m-4 m:1 obtaining an amphiphilic prodrug dendrimer PD based on borate linkages 2m -Catechol 2m -Lys m -PEG-Lys n Fmoc (abbreviated as PD 2m -PEG-Lys n -Fmoc)；
Step four: modification of bifunctional ligand, specifically, modification of boric acid group-containing Compound with PD through an amide bond 2m -Catechol 2m -Lys m -PEG-Lys n Fmoc Lys end coupling to obtain bifunctional ligand-modified anti-tumor prodrug dendrimer PD 2m -PEG-Lys n -BA 2n (abbreviated as PD 2m -PEG-BA 2n ) Wherein m=1 to 20, n=1 to 20; by reacting compounds containing ortho-dihydroxyl groups with PD via an amide bond 2m -Catechol 2m -Lys m -PEG-Lys n Fmoc Lys end coupling to obtain bifunctional ligand-modified anti-tumor prodrug dendrimer PD 2m -PEG-Lys n -DH 2n (abbreviated as PD 2m -PEG-DH 2n ) Wherein m=1 to 20, n=1 to 20; dialyzing the obtained two prodrug dendrimers, and freeze-drying;
step five: PD 2m -PEG-Lys n -BA 2n And PD 2m -PEG-Lys n -DH 2n Mixing and dissolving in polar solvent, and volatilizing or reprecipitating the solvent to prepare the borate crosslinking integrated nano carrier.
The solvent volatilization method specifically comprises the following steps: the mixed solution formed in the polar solvent was transferred to a round bottom flask. The solvent was evaporated under vacuum to form a thin film. Thereafter, 1 to 5mL of Phosphate Buffered Saline (PBS) buffer was added to rehydrate the film, followed by 3 minutes of sonication. Upon self-assembly in PBS, a borate ester linkage is formed between BA and DH of adjacent terminal dendrimers, resulting in the formation of crosslinked nanoparticles.
The reprecipitation method specifically comprises the following steps: a certain amount of mixed solution is sucked by a micro-syringe, slowly dripped into PBS buffer solution and continuously stirred for 2-8 h. The obtained nanoparticles were dialyzed and lyophilized.
The specific embodiment IV is as follows: the preparation method of the prodrug dendrimer nanocarrier in the third embodiment comprises the following steps: coupling of (Fmoc) Lys (Boc) -OH to NH using N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole (DIC/HOBt) as coupling agents 2 -PEG-NH 2 Adding cold diethyl ether to precipitate the pegylated molecules, and then washing twice with cold diethyl ether; removing Fmoc group by treatment with 5-50% (v/v) of Dimethylformamide (DMF) solution containing 4-methylpiperidine, and washing with cold ether for 3 times again; the white powder was dried in vacuo and subjected to 1 to 3 (Fmoc) Lys (Boc) -OH couplings and 3 to 10 (Fmoc) Lys (Fmoc) -OH couplings, respectively, to give Fmoc-Lys dendrimer terminated with Fmoc groups m -PEG-Lys n -Fmoc, wherein m=1 to 20, n=1 to 20, and peg has a molecular weight of 1 to 20KDa.
Fifth embodiment: the preparation method of the prodrug dendrimer nanocarrier of the third embodiment, wherein in the third step, the anticancer drug R containing boric acid groups comprises, but is not limited to, bortezomib, boric acid modified doxorubicin or boric acid modified pheophorbide A.
Specific embodiment six: the method for preparing a prodrug dendrimer nanocarrier of the third embodiment, wherein in the fourth step, the bifunctional ligand has tumor targeting and crosslinking self-assembly properties. The compound containing the boric acid group is one or more of 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid or 3-carboxyl-5-nitrobenzeneboronic acid; the compound containing the ortho-dihydroxyl group is one or more of 3, 4-dihydroxyl benzoic acid, maltobionic acid or sialic acid.
Seventh embodiment: the method for preparing a prodrug dendrimer nanocarrier of the third embodiment, wherein in the fifth step, the polar solvent is one or more of dimethylformamide, methanol, ethanol, acetone, isopropanol, pyridine, n-butanol or tetrahydrofuran; the PD 2m -PEG-Lys n -BA 2n And PD 2m -PEG-Lys n -DH 2n The mass ratio of (2) is 1:0.1 to 10.
Eighth embodiment: use of a prodrug dendrimer nanocarrier of any one of embodiments one to seven in cascaded tumor therapy.
Detailed description nine: the application of the prodrug dendrimer nanocarrier in tumor cascade treatment is described in the eighth embodiment, wherein the prodrug dendrimer nanocarrier is prepared by respectively encapsulating a hydrophilic agent and a hydrophobic agent in a hydrophobic core of an ultra-small nanoparticle by a layered encapsulation method or a one-pot method.
Detailed description ten: the application of the prodrug dendrimer nanocarrier of the ninth embodiment in tumor cascade treatment, wherein the hydrophilic agent is one of indocyanine green (ICG), cabazitaxel (CBA) or doxorubicin hydrochloride (DOX HCl), cy7.5, di or gadofacinic acid; the hydrophobic agent is one of Methotrexate (MTX), camptothecine (CPT), beta-lapachone, vincristine (VCR) or Paclitaxel (PTX).
The specific method for encapsulating the hydrophilic and hydrophobic agent comprises the following steps: under the condition of room temperature, dissolving the hydrophilic reagent A in ultrapure water, wherein the concentration range is 0.1-10 mg/mL; then PD 2m -PEG-BA 2n And PD 2m -PEG-DH 2n According to the following steps of 1:0.1 to 10 (wherein the mass of the hydrophilic agent A does not exceed PD 2m -PEG-BA 2n And PD 2m -PEG-DH 2n 50% of the total mass of the two components is mixed and dissolved in the solution; ultrasonic treatment is carried out for 1 to 10 minutes, the mixture is transferred into a round-bottomed flask, and water is evaporated in vacuum to form a film alpha; further dissolving the hydrophobic reagent B in anhydrous chloroform, wherein the concentration range is 0.1-10 mg/mL; uniformly dispersing the film alpha into anhydrous chloroform dissolved with a hydrophobic reagent B (wherein the mass of the hydrophobic reagent B is not more than 40% of the mass of the film alpha), and evaporating in vacuum to form a film beta; then, adding 1-5 mL Phosphate Buffered Saline (PBS) buffer solution to rehydrate the film, and performing ultrasonic treatment for 3-8 minutes; transferring the nanoparticle solution loaded with the drug into a centrifugal filter tube (MWCO: 3-5 kDa), centrifuging at 6000-10000 rpm, filteringRemoving free reagent that is not loaded.
Example 1:
preparation of Bortezomib (BTZ) prodrug dendrimer nanocarriers (figure 1)
(1) The concentration of the preparation is 0.25mM NH 2 -PEG 2000 -NH 2 4 times the amount (molar ratio) of N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole (DIC/HOBt) coupling reagent and 4 times the amount (molar ratio) of (Fmoc) Lys (Boc) -OH were added for a coupling reaction of 120 minutes. After the reaction, cold diethyl ether was added to precipitate the pegylated molecules, which were then washed twice with cold diethyl ether. Fmoc groups were removed by treatment with a solution of Dimethylformamide (DMF) containing 10% (v/v) 4-methylpiperidine and washed 3 times again with cold diethyl ether. The white powder was dried in vacuo and subjected to 3 (Fmoc) Lys (Boc) -OH couplings and 1 (Fmoc) Lys (Fmoc) -OH couplings, respectively, to give Fmoc group-terminated dendrimer (Fmoc-Lys) 8 -PEG 2000 -Lys 8 -Fmoc)。
(2) Fmoc-Lys 8 -PEG 2000 -Lys 8 One end of Fmoc is connected with 3, 4-dihydroxybenzoic acid (Catechol) to obtain Catechol 16 -Lys 8 -PEG 2000 -Lys 8 -Fmoc dendrimer.
(3) Bortezomib and Catechol 16 -Lys 8 -PEG 2000 -Lys 8 Fmoc reaction in dichloromethane solution (bortezomib with Catechol 16 -Lys 8 -PEG 2000 -Lys 8 -Fmoc molar ratio 32:1) to obtain amphiphilic prodrug dendrimer based on borate linkages (BTZ) 16 -PEG 2000 -Lys 8 -Fmoc)。
(4) 4-carboxyphenylboronic acid is bonded to BTZ via an amide bond 16 -PEG 2000 -Lys 8 Lys end coupling of Fmoc to obtain BTZ 16 -PEG 2000 -CPA 16 (4-carboxyphenylboronic acid and BTZ) 16 -PEG 2000 -Lys 8 -Fmoc molar ratio 20: 1) The unreacted reagent was removed by dialysis and freeze-dried.
(5) 3, 4-dihydroxybenzoic acid is bonded with BTZ through an amide bond 16 -PEG 2000 -Lys 8 Lys end coupling of Fmoc to obtain BTZ 16 -PEG 2000 -DDA 16 (3, 4-dihydroxybenzoic acid and BTZ) 16 -PEG 2000 -Lys 8 -Fmoc molar ratio 20: 1) The unreacted reagent was removed by dialysis and freeze-dried.
(6) BTZ is processed 16 -PEG 2000 -CPA 16 And BTZ 16 -PEG 2000 -DDA 16 The mass ratio is 1:1 were mixed and dissolved in DCM and transferred to a round bottom flask. The solvent was evaporated under vacuum to form a thin film. Thereafter, 5mL of Phosphate Buffered Saline (PBS) buffer was added to rehydrate the film, followed by 3 minutes of sonication. After self-assembly in PBS, crosslinked nanoparticles were formed. The cross-linked nanoparticles prepared in this example had a narrower particle size distribution with an average particle size of 150.+ -.20 nm. The nanoparticles were dispersed in DMEM medium containing 10% Fetal Bovine Serum (FBS) at 37 ℃ and incubated for 72h without significant change in particle size. The nano particles synthesized by the method have good biocompatibility and do not show obvious toxic effect on HUVEC cells in the concentration range of less than 1mg/mL in vitro. IC of nanoparticle pair K-562 cells 50 The value was 2.5. Mu.g/mL (BTZ equivalent).
Example 2:
preparation of boric acid modified Doxorubicin (DOX) prodrug dendrimer nanocarriers (FIGS. 2 and 3)
This example differs from example 1 in that bortezomib in step (3) of example 1 above was replaced with boric acid-modified doxorubicin. Boric acid modified doxorubicin and Catechol 16 -Lys 8 -PEG-Lys 8 -Fmoc reaction molar ratio 28:1.
The average particle diameter of the boric acid modified doxorubicin prodrug dendrimer nano-particles prepared in the embodiment is 100-130 nm, and the boric acid modified doxorubicin prodrug dendrimer nano-particles are in a spherical or ellipsoidal aggregation state. The nanoparticle was dispersed in PBS and incubated at 37 ℃ for 28 days without significant change in particle size. The nanoparticle has good biocompatibility and does not show obvious toxic effect on HUVEC cells in a concentration range of less than 1mg/mL. The IC50 value of the nanoparticle to HepG2 cells was 2.8. Mu.g/mL (DOX equivalent). The Mean Residence Time (MRT) value of DOX in vivo in the boric acid modified DOX prodrug dendrimer nanocarrier is prolonged by nearly 10 times compared to free DOX. The elimination rate constant (Kel) is reduced to 1/10 of the original value, and the carrier is proved to be capable of effectively prolonging the residence time of the medicine in blood circulation.
Example 3:
CPT encapsulation by bortezomib prodrug dendrimer nanocarriers
BTZ under room temperature conditions 16 -PEG 2000 -CPA 16 And BTZ 16 -PEG 2000 -DDA 16 The mass ratio is 1:1 were mixed and dissolved in DCM and transferred to a round bottom flask. Evaporating the solvent under vacuum to form a film α; CPT was dissolved in anhydrous chloroform at a concentration of 5mg/mL. The film α was uniformly dispersed in anhydrous chloroform in which the hydrophobic reagent B was dissolved, and evaporated in vacuo to form film β. Thereafter, 5mL of Phosphate Buffered Saline (PBS) buffer was added to rehydrate the film and sonicated for 8 minutes. The drug loaded nanoparticle solution was transferred to a centrifugal filter tube (molecular weight cut off (MWCO): 3 kDa), and centrifuged at 8000rpm to remove the free reagent that was not loaded. The drug loading rate of the nano particles prepared by the embodiment to CPT can reach more than 30%. At H 2 O 2 In PBS at a concentration of 0.01mM, the crosslinked nanoparticles maintained extremely low release of CPT for 48 h; at H 2 O 2 The release of crosslinked nanoparticles to ICG48h was 75.3% in PBS at a concentration of 1.00 mM.
Example 4:
packaging of indocyanine green (ICG) by bortezomib prodrug dendrimer nanocarriers
ICG was dissolved in ultrapure water at a concentration of 1mg/mL at room temperature. Thereafter BTZ is added 16 -PEG 2000 -CPA 16 And BTZ 16 -PEG 2000 -DDA 16 The mass ratio is 2:1 were mixed and dissolved in the above solution and transferred to a round bottom flask. The solvent was evaporated under vacuum to form a thin film. 5mL of Phosphate Buffered Saline (PBS) buffer was added to rehydrate the film and sonicated for 8 minutes. Transferring the nanoparticle solution loaded with the drug to a separation deviceThe free reagent that was not loaded was removed by centrifugation at 8000rpm in a heart filter tube (molecular weight cut-off (MWCO): 2 kDa). The drug loading rate of the nano particles prepared in the embodiment to ICG can reach more than 35%. At H 2 O 2 In PBS at a concentration of 0.01mM, the crosslinked nanoparticles maintained extremely low release of ICG for 48 h; at H 2 O 2 The release of crosslinked nanoparticles to ICG48h was 89.3% in PBS at a concentration of 1.00 mM.
Example 5:
co-encapsulation of DOX HCl and PTX by bortezomib prodrug dendrimer nanocarriers
DOX HCl was dissolved in ultrapure water at room temperature to a concentration of 2mg/mL. Thereafter BTZ is added 16 -PEG 2000 -CPA 16 And BTZ 16 -PEG 2000 -DDA 16 The mass ratio is 9:1 are mixed and dissolved in the solution. Ultrasonic treatment is carried out for 5 minutes, transferred into a round bottom flask, and water is evaporated in vacuum to form a film alpha; PTX was further dissolved in anhydrous chloroform at a concentration ranging from 1mg/mL. The film α was uniformly dispersed in anhydrous chloroform in which PTX was dissolved, and evaporated in vacuo to form film β. Thereafter, 5mL of Phosphate Buffered Saline (PBS) buffer was added to rehydrate the film and sonicated for 5 minutes. The drug loaded nanoparticle solution was transferred to a centrifugal filter tube (molecular weight cut off (MWCO): 5 kDa), and centrifuged at 10000rpm to remove the free reagent that was not loaded. The drug loading of the nano particles prepared in the embodiment to DOX, HCl and PTX respectively reaches more than 20% and 25%. At H 2 O 2 In a Simulated Body Fluid (SBF) at a concentration of 0.01mM, only 12.7% of DOX and 13.6% of PTX were released after 48h incubation. At 10mMH 2 O 2 Under the stimulation, the bortezomib prodrug dendrimer nano-carrier can rapidly release the loaded drug, the release amount of DOX is 89.5% and the release amount of PTX is 84.3% after 48h of incubation.

Claims (4)

1. A preparation method of a prodrug dendrimer nano-carrier is characterized by comprising the following steps: the method specifically comprises the following steps: the nano-carrier is prepared from an antitumor prodrug dendrimer modified by a bifunctional ligand by a self-assembly method; the antitumor prodrug dendrimer is formed by covalently bonding a basic dendrimer with a boration modified diagnosis and treatment reagent through a boric acid ester bond; the structural formula of the bifunctional ligand modified prodrug dendrimer is as follows:
；
the method comprises the following steps:
step one: synthesis of polyethylene glycol-lysine base block polymer Lys by using N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole system m -PEG-Lys n Wherein m=1 to 20, n=1 to 20; the first step is specifically as follows: coupling of (Fmoc) Lys (Boc) -OH to NH Using N, N' -diisopropylcarbodiimide and 1-hydroxybenzotriazole as coupling agent 2 -PEG-NH 2 Adding cold diethyl ether to precipitate the pegylated molecules, and then washing twice with cold diethyl ether; treating with a dimethylformamide solution containing 5-50% (v/v) of 4-methylpiperidine to remove Fmoc groups, and washing with cold ether for 3 times again; the white powder was dried in vacuo and subjected to 1-3 (Fmoc) Lys (Boc) -OH couplings and 3-10 (Fmoc) Lys (Fmoc) -OH couplings, respectively, to give Fmoc-Lys dendrimer terminated with Fmoc groups m -PEG-Lys n -Fmoc, wherein m = 1-20, n = 1-20;
step two: lys is to be Lys m -PEG-Lys n One end of the mixture is connected with 3, 4-dihydroxybenzoic acid Catechol to obtain Catechol 2m -Lys m -PEG-Lys n -Fmoc dendrimer, wherein m = 1-20;
step three: anticancer drug or prodrug molecule containing boric acid group and Catechol 2m -Lys m -PEG-Lys n -Fmoc reaction in dichloromethane solution, wherein the molar ratio of anticancer drug or prodrug molecule to block polymer is 2 m-4 m:1 obtaining an amphiphilic prodrug dendrimer PD based on borate linkages 2m -Catechol 2m -Lys m -PEG-Lys n -Fmoc; the anticancer drug containing boric acid group comprises bortezomib, boric acid modified doxorubicin orOne of boric acid modified pheophorbide A;
step four: modification of bifunctional ligand, specifically, modification of boric acid group-containing Compound with PD through an amide bond 2m -Catechol 2m -Lys m -PEG-Lys n Fmoc Lys end coupling to obtain bifunctional ligand-modified anti-tumor prodrug dendrimer PD 2m -PEG-Lys n -BA 2n Wherein m=1 to 20, n=1 to 20; by reacting compounds containing ortho-dihydroxyl groups with PD via an amide bond 2m -Catechol 2m -Lys m -PEG-Lys n Fmoc Lys end coupling to obtain bifunctional ligand-modified anti-tumor prodrug dendrimer PD 2m -PEG-Lys n -DH 2n Wherein m=1 to 20, n=1 to 20; dialyzing the obtained two antitumor prodrug dendrimers, and freeze-drying; the compound containing the boric acid group is one or more of 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, 4-carboxyphenylboronic acid or 3-carboxyl-5-nitrobenzeneboronic acid; the compound containing the ortho-dihydroxyl group is one or more of 3, 4-dihydroxyl benzoic acid, maltobionic acid or sialic acid;
step five: PD 2m -PEG-Lys n -BA 2n And PD 2m -PEG-Lys n -DH 2n Mixing and dissolving in polar solvent, and volatilizing or reprecipitating the solvent to prepare prodrug dendrimer nano carrier; the polar solvent is one or more of dimethylformamide, methanol, ethanol, acetone, isopropanol, pyridine, n-butanol or tetrahydrofuran; the PD 2m -PEG-Lys n -BA 2n And PD 2m -PEG-Lys n -DH 2n The mass ratio of (2) is 1:0.1 to 10.
2. An application of the prodrug dendrimer nanocarrier prepared by the preparation method of claim 1 in preparing a tumor cascade therapeutic drug.
3. The use of a prodrug dendrimer nanocarrier according to claim 2 for preparing a drug for cascade treatment of tumors, wherein: the prodrug dendrimer nano-carrier is prepared by respectively encapsulating hydrophilic and hydrophobic reagents in the hydrophobic core and the crosslinking cavity of the ultra-small nano-particles by using a layered encapsulation method or a one-pot method.
4. The use of a prodrug dendrimer nanocarrier according to claim 3 for preparing a drug for cascade treatment of tumors, wherein: the hydrophilic reagent is one of indocyanine green, cabazitaxel or doxorubicin hydrochloride, cy7.5, diD or gadolinium-spray acid; the hydrophobic reagent is one of methotrexate, camptothecine, beta-lapachone, vincristine or taxol.
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