WO2011150671A1

WO2011150671A1 - Magnetic composite, preparation method and use thereof

Info

Publication number: WO2011150671A1
Application number: PCT/CN2011/000892
Authority: WO
Inventors: 刘克良; 张权; 王晨宏; 贾启燕; 冯思良; 孟庆斌; 李思成
Original assignee: 中国人民解放军军事医学科学院毒物药物研究所; 成都一平医药科技发展有限公司
Priority date: 2010-06-03
Filing date: 2011-05-24
Publication date: 2011-12-08
Also published as: CN102266566B; CN102266566A

Abstract

A magnetic composite, preparation method and use thereof are provided. The magnetic composite is multilayer core-shell architecture with Fe₃O₄ nanoparticle cores and an A-b-poly(ethylene glycol)-b-poly(glycerol mono(meth)acrylate) triblock copolymer shell, in which "A" is poly(basic amino acid) segment and its derivative. The magnetic composite has low toxicity, can penetrate the cell membrane and can be used for the drug delivery carriers.

Description

Magnetic composite and preparation method and use thereof

Technical field

The invention belongs to the field of medicine and pharmacy, and relates to a magnetic composite. Specifically, the magnetic composite is a core-shell structure, that is, the outer layer is a block copolymer of a chemically bonded polybasic amino acid or a derivative thereof. The inner core is a metal oxide particle. The invention further relates to a process and use of the magnetic composite. Background technique

The cell membrane is a barrier between the internal and external environment of the cell. The components in the cell need to be excreted through secretion, exocytosis, etc., and the extracellular components need to be transported into the cell through receptors, ion channels, and the like. It is precisely because of the selective permeation of the cell membrane to the substance that the macromolecular drug is difficult to pass through the biofilm to reach the focal area. For example, cerebrovascular diseases often require protective agents such as growth factors and superoxide dismutase, which are difficult to penetrate. The blood-brain barrier does not reach an effective therapeutic concentration. Therefore, the application of many easily degradable drugs, peptides, proteins and various types of RM, DNA and fragments thereof in the pharmaceutical field is greatly limited.

Peptide drugs, RNA, DNA and fragments thereof are difficult to enter cells and are easily degraded by the body or cells, and thus are difficult to express, so that it is necessary to carry them into cells and make them less susceptible to degradation and expression. Currently, nucleic acid/drug carriers mainly include viral vectors and non-viral vectors. Viral vectors have high transfection efficiency, but there are safety hazards such as poor orientation, low carrying capacity, immunogenicity and potential tumorigenicity. For example, in 2000, patients at the University of Pennsylvania died of viral vectors used in gene therapy. In 2002, a clinical trial of innate immune insufficiency gene therapy in France occurred in the clinical trial of leukemia. Both of these failed cases are attributed to the insecurity of the viral vector.

Non-viral carrier materials mainly include liposome carrier materials and polymer carrier materials. The class has the characteristics of low price, simple operation and high safety, so it has been widely studied and applied. The liposome or lipid complex utilizes a phospholipid having a hydrophobic head hydrophobic tail, which can form a water molecule layer sphere in an aqueous solution, and encapsulates the DNA with a liposome to protect against nuclease degradation. There are some excellent liposome and liposome transfection kits sold at home and abroad. The carrier has the characteristics of simple operation and low immunogenicity, but its disadvantage is that it has certain toxicity and low transfection rate. For example, Xiamen Sun Horse Bioengineering Co., Ltd. has studied DNA transfection reagents. Its main component is cationic liposome polymer. However, due to its high toxicity and low transfection efficiency, it cannot be accepted by the market. Various series of gene vectors from Invi trogen, USA. Most of them use cationic liposome and neutral phospholipid as the basic materials, which are highly toxic. Polymer carrier materials include naturally occurring and synthetic cationic high molecular polymers such as polylysine (PLL), polyethyleneimine (PEI), polyamide-amine dendrimer, and deacetylation. Chi tosan, cationic polyester (PAGA), cationic poly-brartinate (PPE) and polyvinylpyridine salt. PLL is a cationic polymer used earlier for gene transfer, and genetically targets hepatocytes after binding to asialoglycoprotein. It can be degraded by trypsin in the body to produce amino acids in the human body, so there is no danger of retention. Unlike cationic liposomes, the efficiency of not using receptor-mediated PLL transfer into genes is quite low if reagents that lyse endosomes or reagents that lysosomal (such as chloroquine) are not added (see MD Brown, Et al. Bioconjugate Chemi s try, 2000, 11, 880-891. ). In terms of transfer efficiency, PEI is the most powerful and most studied cationic polymer, but PEI exhibits greater cytotoxicity when used in vivo or in vitro, thus limiting its clinical application. PEI 25 kDa has high transfection efficiency, but the cytotoxicity is very large; low molecular weight PEI cells have little toxicity, but almost no transfection effect (refer to A. Kichler, The Journa l of Gene Medicine, 2004, 6, S3) -S10. ).

Polymer/magnetic nanoparticle composites combine polymers and magnetic inorganic particles The advantages, both magnetic responsiveness and polymer functionality, have become the focus of research on nucleic acid/drug delivery vehicles. Alkaline co-precipitation of Fe ² 7Fe ³⁺ (molar ratio of 1: 2) in the presence of a diblock copolymer of glyceryl monoacrylate or methacrylic acid monoester and acrylic acid or methacrylic acid to obtain dispersion in water The diblock copolymer encapsulates the composite nanoparticles of Fe ₃ 0 ₄ nanoparticles, wherein the polyacrylic acid monoglyceride or polymethacrylic acid monoglyceride block is attached to the surface of the Fe ₃ 0 ₄ nanoparticle, and the polyacrylic acid or polyfluorene The acrylic block is on the surface of the composite nanoparticle (refer to Chinese patent ZL200410058599. 0). The composite nanoparticle does not carry a functional group of a nucleic acid/drug, and the surface of the composite nanoparticle is negatively charged under neutral or acidic conditions and is cytotoxic. Therefore, it is not suitable as a carrier for nucleic acids/drugs. Summary of the invention

One aspect of the invention relates to a magnetic composite of the order of nanometers (average particle size of from one nanometer to several hundred nanometers, such as greater than or equal to c-

Fe ₃ 0 ₄ particles having an HH of less than 1000 nm, such as 1 nm to 100 nm, are cores, and the outer layer is a compound of formula 1 below O CI, HH

2

Formula 1

In the above formula 1,

z is one or more of the same or different chemical functional groups or functional fragments selected from the group consisting of polybasic amino acids, amino acid fragments having a basic amino acid number greater than 50%, polybasic amino acids linked to a fluorescent label, Anticancer drugs, biotin, transferrin, methyl; b is a linkage bond between a biological functional group selected from the following: an ester bond (a C00-), an amide bond (a C0NR-, H, CH ₃ , or -CH ₂ - ), a disulfide bond (a S—S—), ether bond (a 0—), ,

CH ₃ , or CH ₃ CH _{2 ,} etc.), 1, 3-triazole ring (

The formula 1 comprises a block copolymer structure, the first block is polyethylene glycol, and the average degree of polymerization X is from 1 to 300;

The second block is polyacrylic acid monoglyceride or polyglyceryl monoglyceride, y =

1 - 500, r = H or CH ₃ ,

Wherein the second block is bonded to the surface of the core of the Fe ₃ 0 ₄ particle, and Z forms the outermost layer of the magnetic composite, and the average particle size of the magnetic composite is in the range of one nanometer to several tens of micrometers.

The content of the basic amino acid is greater than 50% of the amino acid fragment. Specifically, the content of the basic amino acid may be greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or greater than 99%. Amino acid fragment.

In one embodiment of the invention, the Fe ₃ 0 ₄ particles have an average particle diameter of from several nanometers to several tens of nanometers, specifically, more than ten nanometers.

In one embodiment of the present invention, the magnetic composite has an average particle diameter of 3 to 300 nm, and the basic amino acid is one or more selected from the group consisting of histidine, arginine, and lysine. The amino acid fragment having a polybasic amino acid or a basic amino acid number greater than 50% is 3 to 39 amino acids in length.

In one embodiment of the invention, the amino acid fragment having a polybasic amino acid or a basic amino acid number greater than 50% is selected from the group consisting of RRRRRRRRR (SEQ ID NO: 1), KKKKKKKKKK (SEQ ID NO: 2), RRRRRAAGG ( SEQ ID NO: 3), RRRRRAAGGKKK (SEQ ID NO: 4), RRRRRAAGGKRRR (SEQ ID NO: 5), RRRRRAAGKCC (SEQ ID NO: 6), GRKKRRQRRRGCG (SEQ ID NO: 7), GRRRQRR KRGCG (SEQ ID NO: 8), GCGGGYGRKKRRQRRR (SEQ ID NO: 9), and RRRQIKIWFQNRRMKWKK (SEQ ID NO: 10), specifically, selected from RRRRRRRRR (SEQ ID NO: 1), GRKKRRQRRRGCG (SEQ ID) NO: 7), and GCGGGYGRKKRRQRRR (SEQ ID NO: 9).

In one embodiment of the present invention, the fluorescent label is a fluorescein series, a Cy series fluorescent dye, an Alexa Fluor series fluorescent dye, specifically, the fluorescein series is selected from FITC, TRITC, FAM, or the like. The Cy series fluorescent dye is selected from the group consisting of Cy5, Cy5.5, Cy7, or an analogue thereof, and more specifically, the fluorescent label is selected from the group consisting of FITC, TRITC, FAM, or the like.

In one embodiment of the invention, the Z is two or more than two different chemical functional groups or functional fragments selected from the group consisting of: polybasic amino acids, amino acids having a basic amino acid number greater than 50% a fragment, a polybasic amino acid linked to a fluorescent label, an anticancer drug, a biotin, a transferrin, a methyl group; wherein the polybasic amino acid or a basic amino acid having a content of more than 50% of the amino acid fragment and the antibiotic The cancer drug or the molar ratio to biotin or to methyl is 50:1 to 1:50.

The chemically bonded polybasic amino acid block copolymer encapsulates the second block polyacrylic acid monoglyceride or polymethacrylic acid monoester of the block copolymer in the FeA particle to bond with the surface of the Fe ₃ 0 ₄ particle; magnetic composite The secondary outer layer of the material is polyethylene glycol, which makes the magnetic composite have good biocompatibility; the polybasic amino acid is free at the outermost layer of the magnetic composite, capable of carrying the magnetic complex through the cell membrane, even the nuclear membrane. Entering the nucleus, even through the blood-brain barrier (BBB), can be used as a delivery vehicle for various nucleic acids/drugs.

A macromolecular conjugate of a polybasic amino acid capable of binding to a drug (such as methotrexate, cyclophosphamide) or a bioactive substance (such as biotin, transferrin) of polyethylene glycol-poly(A) a bis-dienoic acid monoester block copolymer combination of Fe ₃ 0 ₄ particles (the molar ratio of the two can be from 50: 1 to 1: 50), therefore, not only can be profitable Targeting specific tissues and organs can be achieved with biologically active substances that are freed at the outermost layer of the magnetic composite, and can also carry drugs into the cells. The polybasic amino acid is a cationic polymer, which can form a complex with a negatively charged DNA molecule, and the macromolecular conjugate of a polybasic amino acid can be combined with polyethylene glycol monomethyl ether-poly(meth)acrylic acid. The monoglyceride block copolymer is coated with Fe ₃ 0 ₄ particles (the molar ratio of the two can be from 50: 1 to 1: 50), so the polyethylene glycol monomethyl ether-poly(meth)acrylic acid can be adjusted. The molecular weight of polyethylene glycol monomethyl ether in the monoglyceride block copolymer serves to protect and shield the polybasic amino acids and nucleic acids from enzymatic hydrolysis.

The magnetic composite of the Fe ₃ 0 ₄ particles encapsulated by the block copolymer of the polybasic amino acid synthesized by the invention has low cytotoxicity, and under the experimental conditions, the magnetic complex and the human cervical cancer HeLa cells are incubated together 24 . At hours, cell viability is above 90% (see Figure 1). And magnetic composites with surface-bound polybasic amino acids have a high ability to cross cell membranes, even through the nuclear membrane into the nucleus (see Figure 2, A~B). Fluorescence confocal microscopy showed that the fluorescently labeled magnetic complex with no polybasic amino acid modification on the surface could not pass through the cell membrane (see Figure 3, AD), while the fluorescently labeled magnetic complex with polybasic amino acid modification on the surface could Pass through the cell membrane (see Figure 4, AD). Another aspect of the invention relates to a method of preparing the above magnetic composite, comprising the steps of:

1) Preparation of Fe ₃ 0 ₄ magnetic fluid stabilized with inorganic or organic acids (for reference, for example, R. Massart, IEEE Transactions on Magnetics, 1981, MAG-17, 1247-1248.)

2) An aqueous solution of the compound of the formula 1 is added to the magnetic fluid prepared in 1), and the reaction is carried out at a temperature of from O'C to 90'C, and the reaction time is from 1 minute to 50 hours.

Preferably, the number of moles of the compound represented by the formula 1: is a large number of moles of Fe ₃ 0 ₄ At 1.

Or include the following steps:

1) preparing a Fe ₃ 0 ₄ magnetic fluid stabilized with an inorganic or organic acid,

2) encapsulating the magnetic fluid Fe ₃ 0 ₄ particles prepared in 1) with the block copolymer represented by the block copolymer structure in Formula 1,

3) bonding a polybasic amino acid to the surface of the block copolymer encapsulating the Fe ₃ i i via a reactive reactive group on the above block copolymer at a reaction temperature of from O'C to 90 ^e C, reaction time It is from 1 minute to 50 hours.

Preferably, the number of moles of the block copolymer: the number of moles of Fe ₃ 0 ₄ is greater than 1. In one embodiment of the invention, the inorganic acid is perchloric acid, hydrochloric acid, nitric acid, or sulfuric acid, and the organic acid is formic acid, acetic acid, or trifluoroacetic acid.

The synthesis of the block copolymer used in the present invention can be carried out by any conventional method for synthesizing a block copolymer, such as living radical polymerization (including atom transfer radical polymerization (ATRP), reversible addition-cleavage chain transfer radical polymerization (RAFT). Etc.), anionic polymerization, etc. A further aspect of the invention relates to the use of said magnetic complex as a pharmaceutical carrier, RNA vector, or DNA vector. Specifically, the drug is an anticancer drug, an antiviral drug, a drug for treating diabetes, or a drug for treating cardiovascular and cerebrovascular diseases. Specifically, the anticancer drug is methotrexate and/or cyclophosphamide.

Still another aspect of the invention relates to the use of the magnetic composite for the preparation of an anticancer drug, an antiviral drug, a medicament for treating diabetes, or a medicament for treating cardiovascular and cerebrovascular diseases.

A further aspect of the invention relates to a pharmaceutical composition comprising the magnetic composite of any of the invention, and a pharmaceutically acceptable excipient.

A further aspect of the invention relates to a method of treating and/or preventing cancer comprising the step of administering an effective amount of the magnetic composite of the invention, wherein said magnetic complex The compound serves as a carrier for the anticancer drug; specifically, the anticancer drug is methotrexate and/or cyclophosphamide. The administration method may be injection or oral administration, and the administration amount may be determined according to the administration amount of the anticancer drug (for example, methotrexate and/or cyclophosphamide), or may be determined by the doctor according to the specific condition of the patient (for example, the patient's condition, Age, gender, etc.) OK. DRAWINGS

F i g. 1 : Relative survival rate of human cervical cancer HeLa cells cultured for 24 hours in the presence of different concentrations of magnetic complexes.

F i g. 2: Microscopic photo of polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ magnetic complex into human cervical cancer HeLa cells (Prussian blue stained iron, saffron T stained nucleus). Figure 2A: 400 X; Figure 2B: Photograph of a high power microscope (Ι ΟΟΟ χ ).

F i g. 3: Fluorescence confocal microscopy photographs demonstrate that FITC-PEG-PGA-Fe ₃ 0 ₄ cannot pass through the Caco-2 cell membrane. Figure 3A: FITC fluorescence imaging; Figure 3B: Rhodamine-Phalloidin stained cell membrane; Figure 3C: Hoeches t 33258 stained nuclei; Figure 3D: Merged map of the first three images.

F i g. 4: Fluorescence confocal microscopy photographs demonstrate that FITC-polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ can pass through the Caco-2 cell membrane. Figure 4A: FITC fluorescence imaging; Figure 4B: Rhodamine-Phalloidin stained cell membrane; Figure 4C: Hoeches t 33258 stained nuclei; Figure 4D: Merged map of the first three images. detailed description

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings, however, If no specific conditions are specified in the examples, they are carried out according to the general conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained commercially. The abbreviations used in the following examples are: PEG, polyethylene glycol; mPEG, polyethylene glycol monomethyl ether; CH ₃ S0 ₃ -PEG-0H, single-ended methanesulfonyl polyethylene glycol; N ₃ -PEG- 0H, single-ended azido polyethylene glycol; NH ₂ -PEG- 0H, single-ended amino polyethylene glycol; FMAL-PEG-Br, one-furan protects the maleimide end group, and the other end is α - polyethylene glycol bromoisobutyrate; MAL, maleimide end group; SA, 1,1-dimethyl-1, 3-dioxolan-4-methacrylate, SMA, A 2,2-dimethyl-1,3-dioxopentan-4-sterol acrylate; PGA, polyacrylic acid monoglyceride; PGMA, polyglyceryl monoglyceride; PMDETA, 1, 1, 4 , 7, 7 -pentamethyldiethylenetriamine; Fmoc, fluorenylmethoxycarbonyl; Boc, tert-butoxycarbonyl; Pbf, 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5- Sulfonyl; Tr t, trityl; FITC, fluorescein isothiocyanate; MTX, methotrexate. Further, in the following examples, for the convenience of expression, an amino acid fragment having a content of a basic amino acid of more than 50% is sometimes referred to as a polybasic amino acid. In the following examples, the polybasic amino acid linked to FITC is "GCGGGYGRKKRRQRRR (SEQ ID NO: 9)", and the polybasic amino acid not linked to FITC is "GRKKRRQRRRGCG (SEQ ID NO: 7), Example 1: Preparation of CH ₃ S0 ₃ -PEG- 0H

3. 8 mL of methylsulfonyl chloride was dissolved in 50 mL of tetrahydrofuran, and then slowly added dropwise to 200 mL of tetrahydrofuran dissolved in 10 g of PEG (average molecular weight of 200) and 7 mL of triethylamine, and the addition was completed after 3 hours. Stir at room temperature overnight. The reaction solution was filtered, and the solvent was evaporated to remove the solvent, which was evaporated to silica gel column to afford CH ₃ S0 ₃ - PEG-0H. Example 2: Preparation of Ns-PEG-OH

2. 8 g of CH3SO3-PEG-OH (average molecular weight of 200) and 1 g of sodium azide were dissolved in 25 mL of water, and the reaction solution was reacted at 80 ° C for 24 hours. After the reaction was completed, it was cooled to room temperature, and the solvent water was distilled off under reduced pressure, and 100 mL of dichloromethane was added, and organic phase was used. Dry over anhydrous sodium sulfate, filter, and then remove the solvent by rotary distillation.

N plant PEG-OHo Example 3: Preparation of NH ₂ -PEG-0H

1 g of N3-PEG-OH (average molecular weight of 200) was placed in a reaction flask, 0.11 g of water was added, and 1.3 g of triphenylphosphine was dissolved in 35 mL of tetrahydrofuran and added to the reaction flask. , stir at room temperature for 10 hours. After completion of the reaction, filtered and the solvent was removed by rotary evaporation, isolated by silica gel column to give NH ₂ -PEG-0H. Example 4: Preparation of other molecular weight NH ₂ -PEG- 0H

The PEG having an average molecular weight of 600, 2000 and 5000 was used instead of the PEG having an average molecular weight of 200 in Example 1, Example 1 and Example 3, and the same operation was carried out to obtain NH ₂ -PEG-0H of the corresponding molecular weight, respectively. . Example 5: Preparation of FMAL-PEG-0H

2.84 g of 4,10 dioxatricyclo[5. 2. 1. 0 (2,6) ]癸-8-ene-3, 5-dione was dissolved in 70 mL of methanol, and the solution was cooled. To 0 ° C, then 3. 31 g of NH ₂ -PEG-0H (average molecular weight of 200) prepared in Example 3 was dissolved in 30 mL of methanol and added to the above reaction solution at 0 ° C. After stirring for 10 minutes, stirring at room temperature for 45 minutes, and finally refluxing for 4 hours, the mixture was cooled to room temperature, and the solvent was evaporated to dryness. After overnight, filter, remove the solvent by rotary distillation, and separate through silica gel column.

Example 6: Preparation of FMAL-PEG-Br

2. 4 g of FMAL-PEG-0H prepared in Example 5 (the average molecular weight of PEG is 200) Dissolved in 70 mL of dry tetrahydrofuran, then 1.2 mL of triethylamine. The reaction solution was cooled to 0 ° C, and then 1.0 mL of α-bromoisobutyryl bromide was dissolved in 20 mL of dry tetrahydrofuran, added dropwise to the reaction solution, and reacted at 0 ° C for 1 hour, stirred at room temperature for 24 hours, and filtered to remove white precipitate. The solvent was removed by rotary evaporation, and the residue was purified by silica gel column to afford FMAL-PEG-Br. Example 7: Preparation of other molecular weight FMAL-PEG-Br

The FMAL-PEG-OH having a PEG average molecular weight of 200 in Example 6 was replaced by FMAL-PEG-OH having a PEG average molecular weight of 600, 2000 and 5000, respectively. The other operation was the same as in Example 6, respectively, to obtain a corresponding molecular weight of FMAL-PEG. - Br. Example 8: Preparation of MAL-PEG-PGA

0.2 g of FMAL-PEG-Br prepared in Example 6, 58 mg of CuBr, 3.04 g of SA was dissolved in 1.5 mL of anisole, 87 μΐ of PMDETA was added under argon, the solution became light green, and the immersion was preheated. The reaction was stirred for 3 hours in an oil bath at 65 ° C to give a green viscous material. The obtained green polymer was dissolved in tetrahydrofuran, and the solution was passed through a neutral A1 ₂ ₃ column. The filtrate was collected, concentrated by rotary evaporation, and precipitated with ten times of petroleum ether (60-90 ° C) to obtain a slightly yellow polymer. Then, the polymer was dissolved in 20 mL of toluene, heated under reflux for 7 hours, and the solvent was removed by rotary evaporation. Finally, the obtained polymer was dissolved in 1.0 mol/L hydrochloric acid/dioxane (1:3, v/v), stirred at 0 ° C for 1 hour, stirred at room temperature for 24 hours, and transferred to a dialysis bag. (The upper limit of molecular weight is 3,500) Repeated dialysis to obtain a colorless transparent solution. Freeze drying to give a white solid MAL-PEG-PGA. Example 9: Preparation of MAL-PEG-PGMA

The polymerized monomer SA in Example 8 was replaced with SMA, and the other operations were the same as in Example 8. A white powdery solid MAL-PEG-PGMA was obtained. Example 10: Preparation of MAL-PEG-PGA and MAL-PEG-PGMA of other molecular weights

The same operation was carried out by using FMAL-PEG-Br with PEG average molecular weights of 600, 2000 and 5000 instead of FMAL-PEG-Br of Example 8 and Example 9, respectively, to obtain MAL-PEG-PGA of the corresponding molecular weight, respectively. MAL- PEG- PGMA. Example 11: Preparation of polybasic amino acids

The MBHA resin was selected and the Fmoc synthesis strategy was used to extend the C-terminal to the N-terminus according to the sequence of the polybasic amino acid GRKKRRQRRRGCG (SEQ ID NO: 7), and the solution was removed with 25% piperidine/N,N-dimethylformamide. For the protection, the condensation method is benzotriazole-^^,1^'-tetramethylurea hexafluorophosphate 0^1 ¹ 11)/ 1-hydroxybenzotriazole (HOBt) method. The detection method uses a ninhydrin indicator. The α-amino group of the amino acid used is protected by Fmoc, and the amino acids having a side chain protecting group are: Fmoc-Lys (Boc)-0H, Fmoc-Arg(Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc- Cys (Trt) - 0H. Trifluoroacetic acid/water/m-cresol/ 1,2-ethanedithiol=90/5/3/2 (volume ratio) The polybasic amino acid was cleaved from the resin and then subjected to reversed phase liquid chromatography. Separation and purification to obtain a pure polybasic amino acid. Example 12: Preparation of FITC-Polybasic Amino Acid Derivatives

Solid phase synthesis of polybasic amino acids, terminated with FITC, the amino acid sequence of the derivative is FITC-(ω-GHBA) -GCGGGYGRKKRRQRRR (SEQ ID NO: 9), ω-GHBA: ω-aminocaproic acid, other operations and implementation In the same manner as in Example 11, a FITC-polybasic amino acid was obtained. Example 13: Preparation of polybasic amino acid-PEG-PGA conjugate 0. 1 g of MAL-PEG-PGA prepared in Example 8 was dissolved in 4 mL of deionized water, 40 mg of polybasic amino acid (GRKKRRQRRRGCG, SEQ ID NO: 7) was added, stirred at room temperature overnight, and the reaction solution was transferred to a dialysis bag. (The upper limit of molecular weight is 3500) The dialysis was repeated and freeze-dried to obtain a polybasic amino acid-PEG-PGA conjugate. Example 14: Preparation of FITC-Polybasic Amino Acid-PEG-PGA Conjugate The FITC-polybasic amino acid derivative was used in place of the polybasic amino acid of Example 13, and the other procedures were the same as in Example 13, to prepare FITC. - Polybasic amino acid-PEG-PGA conjugate. Example 15: Blocking of other basic molecular weight polybasic amino acid-PEG-PGA conjugates

The MAL-PEG-PGA of Example 13 was replaced with MAL-PEG-PGA having a PEG average molecular weight of 600, 2000 and 5000, respectively, and the other operations were the same as those in Example 13, respectively, to obtain a polybasic amino acid-PEG-PGA conjugate of the corresponding molecular weight. Compound. Example 16: Preparation of other molecular weight FITC-polybasic amino acid-PEG-PGA conjugate

The MAL-PEG-PGA of Example 14 was replaced by MAL-PEG-PGA having an average molecular weight of 600, 2000 and 5000, respectively, and the other operations were the same as in Example 14, respectively, to obtain a FITC-polybasic amino acid-PEG- PGA conjugate. Example 17: Preparation of Polybasic Amino Acids of Other Molecular Weights - PEG PGMA Conjugates

MAL-PEG-PGMA of Example 15 was replaced with MAL-PEG-PGMA having a PEG average molecular weight of 600, 2000 and 5000, respectively, and the other operations were the same as in Example 15, respectively. A polybasic amino acid-PEG-PGMA conjugate of the corresponding molecular weight. Example 18: Preparation of FITC-Polybasic Amino Acid-PEG-PGMA Conjugates of Other Molecular Weights

The MAL-PEG-PGA of Example 16 was replaced by 1^^-?£0-?01^ with PEG average molecular weights of 600, 2000 and 5000, respectively, and the other operations were the same as in Example 16, respectively, to obtain FITC-poly of the corresponding molecular weight. Basic amino acid-PEG-PGMA conjugate. Example 19: Preparation of Perchloric Acid Stabilized Magnetic Fluid Aqueous Solution

Dissolve 2 g of FeC l ₂ · 4H ₂ 0 in 25 mL of 1 mol / L hydrochloric acid solution and 5. 4 g of FeCls · 6H ₂ 0 in 25 raL of deionized water in a three-necked bottle. Argon. 160 mL of 1.5 5 ml / L ammonia water was added dropwise to the three-necked flask, and a black precipitate was formed in the solution, and the reaction was further stirred at room temperature for 24 hours. The supernatant was poured by a magnet and the supernatant was decanted and washed 3 times with water. Under a argon atmosphere, 2. 0 mol / L of HC10 ₄ 50 mL was added dropwise to a three-necked flask, and the mixture was stirred at room temperature for 15 minutes. After standing for 10 minutes, the supernatant was decanted according to the above method, and the apparatus was reconstituted, and then argon gas was passed for 30 minutes. 2.0 mL of HCIO4 50 mL was added dropwise from a constant pressure funnel, and the mixture was stirred at room temperature for 15 minutes. After the stirring was stopped, the reaction product was centrifuged for 30 minutes, the solution was decanted, and the precipitate was quickly transferred to a dialysis bag, and dialyzed repeatedly with deionized water to obtain a perchloric acid-stabilized aqueous solution of magnetic fluid, and the concentration of Fe ₃ 0 ₄ was measured as follows: 32 mg/mL. Example 20: Preparation of polybasic amino acid by direct method - PEG-PGA-Fe ₃ 0 ₄

0.12 g of the polybasic amino acid-PEG-PGA conjugate prepared in Example 13 was dissolved in 3 mL of deionized water, and the pH was adjusted to be acidic with hydrochloric acid. The argon gas was evacuated, and the oxygen in the reaction flask was removed three times, and 0.4 g of perchloric acid-stabilized magnetic fluid was added. After 12 hours of reaction, magnetic separation, a magnetic composite of a polybasic amino acid-PEG-PGA conjugate coated with Fe ₃ 0 ₄ particles (polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ ) was obtained. Aqueous solution. Example 21: Preparation of polybasic amino acid-PEG-PGA-Fe ₃ 0 _{4 by} indirect method

The MAL-PEG-PGA prepared in Example 8 was dissolved in 3 mL of deionized water, vacuum-filled with argon, and the oxygen in the reaction flask was removed three times, and 0.4 mL of perchloric acid stabilized magnetic was added. fluid. After reacting for 12 hours, magnetic separation was carried out to obtain a magnetic complex (MAL-PEG-PGA-Fe ₃ 0 ₄ ) aqueous solution of MAL-PEG-PGA-coated Fe ₃ 0 ₄ particles. The polybasic amino acid (GRKKRRQRRRGCG, SEQ ID NO: 7) was dissolved in 0.2 mL of deionized water, the pH of the solution was adjusted to be acidic with hydrochloric acid, and then 2 mL of MAL-PEG-PGA-Fe ₃ 0 ₄ aqueous solution was added to the solution. The reaction solution was stirred at room temperature overnight, and magnetically separated to obtain a magnetic composite (polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ ) aqueous solution of the polybasic amino acid-PEG-PGA conjugate-coated Fe ₃ 0 ₄ particles. . Example 22: Direct preparation of FITC-polybasic amino acid - PEG-PGA-Fe ₃ 0 ₄ Replace the polybasic amino acid of Example 20 with PEG-PGA with FIT-polybasic amino acid-PEG-PGA conjugate The conjugate was the same as in Example 20. An aqueous solution of a magnetic composite (FITC-polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ ) encapsulating Fe ₃ 0 ₄ particles of FITC-polybasic amino acid-PEG-PGA conjugate was prepared by direct method. Example 23: Direct preparation of polybasic amino acids - PEG-PGMA-Fe ₃ 0 ₄ and FITC-polybasic amino acids - PEG-PGMA-Fe ₃ 0 ₄

The polybasic amino acid-PEG-PGA conjugate and the FITC-polybasic amino acid-PEG-PGMA conjugate were replaced by the polybasic amino acid-PEG-PGMA conjugate and the polybasic amino acid-PEG-PGA conjugate of Example 20, respectively. 20. Directly preparing the magnetic composite (polybasic amino acid-PEG-PGMA-Fe ₃ 0 ₄ ) aqueous solution and FITC-polybasic amino acid of the polybasic amino acid-PEG-PGMA conjugate coated Fe ₃ 0 ₄ particles PEG-PGMA conjugation The magnetic composite (FITC-polybasic amino acid-PEG-PGMA-Fe ₃ 04 ) aqueous solution of Fe ₃ 0 ₄ particles was wrapped. Example 24: Preparation of magnetic composites in which Fe ₃ 0 ₄ particles were encapsulated by mPEG-PGA and polybasic amino acid-PEG-PGA conjugates

Preparation of magnetic composites of mPEG-PGA and polybasic amino acid-PEG-PGA conjugates in combination with Fe ₃ 0 ₄ particles: The synthesis of mPEG-PGA (average molecular weight of mPEG 2000) can be referred to: S. Wan, et al . J, Ma t er. Chem., 2005, 15, 3424-3430. The mPEG-PGA and the polybasic amino acid-PEG-PGA conjugate were separately coated with Fe ₃ 0 ₄ particles in various ratios from 50:1 to 1:50, and the same operation as in Example 20 was carried out. A magnetic composite in which the mPEG-PGA and the polybasic amino acid-PEG-PGA conjugate were combined to form the Fe ₃ 0 ₄ particles was separately prepared. Example 25: Preparation of magnetic composites of other molecular weight mPEG-PGA and polybasic amino acid-PEG-PGA conjugates in combination with Fe ₃ 0 ₄ particles

The mPEG-PGA of Example 24 was replaced with mPEG-PGA having an average molecular weight of 200 and 5000, respectively, and the same operation as in Example 24 was carried out. They were prepared to give the corresponding molecular weight mPEG- PGA basic amino acid and poly - magnetic composition PEG- PGA package conjugate composition Fe ₃ 0 ₄ particles. Example 26: Preparation of magnetic composites in which Fe ₃ 0 ₄ particles were encapsulated by mPEG-PGMA and polybasic amino acid-PEG-PGMA conjugate

The mPEG-PGA and the polybasic amino acid-PEG-PGA conjugate in Example 24 were replaced with mPEG-PGMA (the average molecular weight of mPEG was 2000) and polybasic amino acid-PEG-PGMA conjugate, respectively. Example 24. Preparation of mPEG-PGMA and polybasic amino acid-PEG-PGMA conjugates respectively to form Fe ₃ 0 ₄ particles Magnetic composite. Example 27: Preparation of magnetic composites of other molecular weights of mPEG-PGMA and polybasic amino acid-PEG-PGMA conjugates in combination with Fe ₃ 0 ₄ particles

The mPEG-PGMA of Example 26 was replaced with mPEG-PGMA having an average molecular weight of 200 and 5000 mPEG, respectively, and the other operation was the same as in Example 26. The magnetic composites of the respective molecular weights of mPEG-PGMA and polybasic amino acid-PEG-PGMA conjugates were combined to form Fe ₃ 0 ₄ particles. Example 28: Preparation of Magnetic Complex of MTX-PEG-PGA Conjugate and Polybasic Amino Acid-PEG-PGA Conjugate in Combination with Fe ₃ 0 ₄ Particles

Preparation of magnetic composites of MTX-PEG-PGA conjugate and polybasic amino acid-PEG-PGA conjugate in combination with Fe ₃ 0 ₄ particles: MTX-PEG-PGA conjugate (average molecular weight of PEG of 200) For synthesis, please refer to: Q. Zhang, et a l. J. Mater. Chem., 2009, 19, 8393-8402. The MTX-PEG-PGA conjugate and the polybasic amino acid-PEG-PGA conjugate were separately coated with Fe ₃ 0 ₄ particles in various ratios from 50:1 to 1:50, and the same operation example 20. A magnetic composite in which the MTX-PEG-PGA conjugate and the polybasic amino acid-PEG-PGA conjugate were combined to form the Fe ₃ 0 ₄ particles was separately prepared. Example 29: Preparation of magnetic composites of other molecular weight MTX-PEG-PGA conjugates and polybasic amino acid-PEG-PGA conjugates in combination with Fe ₃ 0 ₄ particles

The MTX-PEG-PGA conjugate of Example 28 was replaced with the MTX-PEG-PGA conjugate having a PEG average molecular weight of 2000 and 5000, respectively, and the other operation was the same as in Example 28. A magnetic composite in which the corresponding molecular weight of the MTX-PEG-PGA conjugate and the polybasic amino acid-PEG-PGA conjugate were combined to encapsulate the Fe ₃ 0 ₄ particles was separately prepared. Example 30: Preparation of Magnetic Complex of MTX-PEG-PGMA Conjugate and Polybasic Amino Acid-PEG-PGMA Conjugate in Combination with Fe ₃ 0 ₄ Particles

The MTX-PEG-PGA conjugate and the polybasic amino acid in Example 28 were replaced with MTX-PEG-PGMA conjugate (average molecular weight of PEG of 200) and polybasic amino acid-PEG-PGMA conjugate, respectively. The PEG-PGA conjugate was the same as Example 28. A magnetic composite in which the MTX-PEG-PGMA conjugate and the polybasic amino acid-PEG-PGMA conjugate were combined to encapsulate the Fe ₃ 0 ₄ particles was separately prepared. Example 31: Preparation of magnetic composites of other molecular weight MTX-PEG-PGMA conjugates and polybasic amino acid-PEG-PGMA conjugates in combination with Fe ₃ 0 ₄ particles were prepared using PEG average molecular weights of 2000 and 5,000, respectively. The MTX-PEG-PGMA conjugate was substituted for the MTX-PEG-PGMA conjugate in Example 30, and the other operation was the same as in Example 30. Magnetic composites of the corresponding molecular weight MTX-PEG-PGMA conjugate and polybasic amino acid-PEG-PGMA conjugate combined with Baoquan Fe ₃ 0 ₄ particles were separately prepared. Example 32: Experiment of cytotoxicity of magnetic complexes

The polybasic amino acid obtained in Example 20 -? 80-?0 person 6 ₃ 0 ₄ and the MAL-PEG-PGA-Fe ₃ 0 ₄ mentioned in Example 21 were separately added to the culture solution for cell culture experiments, and then the relative survival rate of the cells was determined by the MTS method. The experimental method can be referred to: S. Wan, et al. J. B i omed. Ma ter. Res. A, 2007, 80, 946-954. Figure 1 is a comparison of cytotoxicity of MAL-PEG-PGA-Fe ₃ 0 ₄ and polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ . The magnetic complex was incubated with human cervical cancer HeLa cells for 24 hours, and the cell survival rate was higher than 90%.

Similarly, the magnetic composite of the present invention prepared in other examples was verified, and the results showed that the cell survival rate was high, indicating that the magnetic complex was very toxic. Little or no toxicity. Example 33: Staining method to demonstrate the passage of magnetic composites through cell membranes

The polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ obtained in Example 20 was added to the culture solution for cell culture experiments, and then the presence of iron in the cells was determined by Prussian blue staining, and the nucleus was counterstained by saffron T. Figure 2 (A~B) is a staining photograph of the polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ entering the nucleus. It can be seen that the surface-modified polybasic amino acid magnetic complex can carry Fe ₃ 0 ₄ particles into the nucleus. .

Similarly, the magnetic composites of the present invention prepared in the other examples were verified, and as a result, they all passed through the cell membrane. Example 34: Fluorescent labeling demonstrates that FITC-PEG-PGA-Fe ₃ 0 ₄ cannot pass through Caco-2 cell membrane

The FITC-PEG-PGA-Fe ₃ 0 _{4 was} synthesized by a method similar to that of Example 20 or 21 except that the polybasic amino acid was replaced with FITC, and then it was added to the culture solution for cell culture experiments, and then by fluorescence confocal. Spectral imaging determines the presence of intracellular fluorescent substances. Figure 3 (A~D) demonstrates that FITC-PEG-PGA-Fe ₃ 0 ₄ cannot pass through the Caco-2 cell membrane. Example 35: Fluorescent labeling method to demonstrate FITC-polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ crossing cell membrane

The FITC-polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ obtained in Example 22 was added to the culture solution for cell culture experiments, and then the presence of intracellular fluorescent substances was determined by fluorescence confocal spectrum imaging. Figure 4 (A~D) is a fluorescent confocal photograph of FITC-polybasic amino acid-PEG-PGA-Fe ₃ 0 ₄ entering Caco-2 cells. It can be seen that the surface-modified FITC-labeled polybasic amino acid magnetic composite Can carry Fe ₃ 0 ₄ The child enters the cell.

Similarly, the magnetic composites of the present invention prepared in the other examples were verified, and as a result, they all passed through the cell membrane. Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and substitutions may be made to those details in light of the teachings of the invention, which are within the scope of the invention. The full scope of the invention is indicated by the appended claims and any equivalents thereof.

Claims

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A magnetic composite having a core of Fe ₃ 0 ₄ particles as a core and an outer layer of a compound represented by Formula 1 below.

In the above formula 1,

Z is one or more of the same or no chemical groups or functional fragments selected from the group consisting of: a polybasic amino acid, an amino acid fragment having a basic amino acid number greater than 50%, and a polylinker linked to a fluorescent label Basic amino acids, anticancer drugs, biotin, transferrin, methyl;

b is a linkage of a biological functional group selected from the group consisting of an ester bond (a C00-), an amide bond (a CON-, R, = H, CH ₃ , or -CH ₂ - ), disulfide Key (―S—S—), ether bond (—0—), ,

CH ₃ , or CH ₃ CH ₂ ) , 1, 3-triazole ring (

The second block is polyacrylic acid monoglyceride or polymethyl methacrylate monoester, y =

1 - 500 , rH or CH ₃ ,

Wherein the second block is bonded to the surface of the core of the Fe ₃ 0 ₄ particle, and Z forms the outermost layer of the magnetic composite, and the average particle size of the magnetic composite is from one nanometer to several tens of micrometers. In the range.

The magnetic composite according to claim 1, wherein the magnetic composite has an average particle diameter of 3 to 300 nm, and the basic amino acid is selected from the group consisting of histidine, arginine, and lysine. One or more of the acids, the polybasic amino acid or the amino acid fragment having a number of basic amino acids greater than 50% is 3 to 39 amino acids in length.

3. The magnetic composite according to claim 2, wherein the amino acid fragment having a polybasic amino acid or a basic amino acid number greater than 50% is selected from the group consisting of RRRRRRRRR, KKKKKKKKKK, RRRRRAAGG, RRRRRAAGGKKK, RRRRRAAGGKRRR, RRRRRAAG CC, GRKKR QRRRGCG, GRRRQRRKKRGCG, GCGGGYGRKKRRQRRR, and RRRQIKIWFQNRRMKWKK, specifically, selected from RRRRRRRRR, GRKKRRQRRRGCG, and GCGGGYGRKKRRQRRR.

The magnetic composite according to claim 1 , wherein the fluorescent label is a fluorescein series, a Cy series fluorescent dye, or an Alexa F luor series fluorescent dye, specifically, the fluorescein series is selected from FITC, TRITC, FAM, or the like, the Cy series fluorescent dye is selected from Cy5, Cy5.5, Cy7, or an analogue thereof, more specifically, the fluorescent label is selected from FITC, TRITC, FAM, or the like. Things.

The magnetic composite according to claim 1, wherein the Z is two or more than two different chemical functional groups or functional fragments selected from the group consisting of polybasic amino acids and basic amino acid numbers. An amino acid fragment having a content greater than 50%, a polybasic amino acid linked to a fluorescent label, an anticancer drug, biotin, transferrin, methyl; wherein the content of the polybasic amino acid or basic amino acid is greater than 50 The molar ratio of % amino acid fragment to anticancer drug or to biotin or thiol is 50: 1 to 1: 50.

The magnetic composite according to claim 1, wherein the anticancer drug is methotrexate and/or cyclophosphamide.

7. The method of preparing a magnetic composite according to claim 1, comprising the steps of: 1) preparing a Fe ₃ 0 ₄ magnetic fluid stabilized with an inorganic or organic acid,

2) An aqueous solution of the compound of the formula 1 is added to the magnetic fluid prepared in 1), and the reaction is carried out at a temperature of from O'C to 90 ° C for a reaction time of from 1 minute to 50 hours.

8. The method of preparing a magnetic composite according to claim 1, comprising the steps of:

1) preparing a Fe ₃ 0 fluid stabilized with an inorganic or organic acid,

3) bonding the polybasic amino acid to the surface of the block copolymer encapsulating the Fe ₃ 0 ₄ particles through the active reactive group on the above block copolymer, the reaction temperature is O 'C to 90 ° C, reaction time It is from 1 minute to 50 hours.

The method according to claim 7 or 8, wherein the inorganic acid is perchloric acid, hydrochloric acid, nitric acid, or sulfuric acid; and the organic acid is formic acid, acetic acid, or trifluoroacetic acid.

The use of the magnetic composite according to any one of claims 1 to 6 as a drug carrier, an RNA carrier, or a DNA carrier, specifically, the drug is an anticancer drug, an antiviral drug, a drug for treating diabetes Or a medicament for treating cardiovascular and cerebrovascular diseases. Specifically, the anticancer drug is guanidin and/or cyclophosphamide.

The use of the magnetic composite according to any one of claims 1 to 6 for the preparation of an anticancer drug, an antiviral drug, a medicament for treating diabetes, or a medicament for treating cardiovascular and cerebrovascular diseases.

A pharmaceutical composition comprising the magnetic composite according to any one of claims 1 to 6, and a pharmaceutically acceptable adjuvant.

13. A method of treating and/or preventing cancer comprising the step of administering an effective amount of the magnetic composite of claim 6.