CN102266566B - Magnetic compound, and preparation method and purpose thereof - Google Patents
Magnetic compound, and preparation method and purpose thereof Download PDFInfo
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Abstract
The invention belongs to the field of medicament and pharmaceutics, and relates to a magnetic compound. Specifically, the magnetic compound has a nuclear-casing structure, which means that an external layer is a segmented copolymer of chemical bond gathered basic amino acids or derivatives thereof, and a nuclear is metal oxide particles. A first block of the segmented copolymer is polyethylene glycol, and a second block is polyacrylic acid monoglyceride or polymethacrylic acid monoglyceride, wherein the second block is combined with surfaces of Fe3O4 particles. The invention also relates to a preparation method and a purpose of the magnetic compound. The magnetic compound of the invention not only has low toxicity, but also can permeate a cell membrane or even a karyotheca to enter a nucleus, or even pass through a blood cerebral barrier (BBB), so the magnetic compound can be used as a conveying carrier of various nucleic acids / medicaments.
Description
Technical field
The invention belongs to medicine and pharmaceutics field, relate to a kind of magnetic composite, particularly, described magnetic composite is nucleocapsid structure, i.e. the outer block copolymer that gathers the basic amino acid or derivatives thereof for chemical bond-linking, and kernel is metal oxide particle.The invention still further relates to preparation method and the purposes of described magnetic composite.
Background technology
Cell membrane is the barrier of cell internal and external environment, and intracellular component need to just can be excreted by effects such as secretion, exocytosis, and the extracellular composition need pass through the effects such as receptor, ion channel, just can be transported in cell.Exactly because the selective permeation effect of cell membrane to material; make macromolecular drug be difficult to pass biomembrane and arrive focal zone; usually need the protective agents such as somatomedin, superoxide dismutase as cerebrovascular disease, because it is difficult to see through blood brain barrier, can not reach effective treatment concentration.Therefore, the medicine of many easy degradeds, polypeptide, protein and all kinds of RNA, DNA with and fragment greatly limited in the application of pharmaceutical field.
Polypeptide drug, RNA, DNA and fragment thereof are difficult to enter cell and easily by body or cell degradation, thereby beyond expression of words, enter cell so need to carry it, and make it be difficult for being degraded, and express the carrier that improves.At present, the carrier of nucleic acid/medicine mainly contains viral vector and non-virus carrier two classes.Virus type carrier transfection efficiency is higher, but have that guidance quality is poor, carrying capacity is low, the safety hidden danger such as immunogenicity and potential oncogenicity.For example, 2000, the University of Pennsylvania had patient to use in viral vector dead in gene therapy.In France, innate immunity insufficiency disorder Gene Therapy Clinical Trials occuring again in 2002, leukemic side effect occurs.The case of these two failures is all by the insecurity owing to viral vector.
The non-virus carrier material mainly contains liposome vectors material and polymer carrier materials two classes, has the characteristics such as price is low, simple to operate, safety is high, therefore obtains broad research and application.Liposome or lipid complex utilize phospholipid to have hydrophilic hydrophobic tail, can form the characteristics of water molecule layer spheroid in aqueous solution, and DNA is got up with liposome, can avoid the degraded of nuclease.Both at home and abroad more existing good liposomees and lipofectamine box are sold, and this carrier has simple to operate, the characteristics that immunogenicity is low, but its shortcoming be have certain toxicity and transfection efficiency lower.For example, there was research in Xiamen sun horse bio-engineering corporation to the DNA transfection reagent, and its main component is the polymer of cationic-liposome, but because its product toxicity is high, and transfection efficiency is low and can not be accepted by market.The genophore of the various series of American I nvitrogen company.What major part adopted is that cationic-liposome and neutral phospholipid are stock, and toxicity is very high.Polymer carrier materials comprises natural formation and cationic high molecular polymer synthetic, as poly-D-lysine (PLL), polymine (PEI), the tree-like macromolecule of polyamide-amide (dendrimer), chitosan (chitosan), cationic polyester (PAGA), cation poly phosphate (PPE) and polyvinylpyridine salt etc.PLL early is used for the cationic polymer of gene transfer, is combined with Asialoglycoprotein rear hepatocyte to be carried out gene target.It can generate the aminoacid that exists in human body by the interior trypsin degradation of body, therefore there is no the hazardness of delay.Different from cationic-liposome, if do not add the reagent of tniema endosome or the reagent of lysosome class (as chloroquine etc.), not using that the efficient of receptor-mediated PLL transgene is quite low (can be with reference to M.D.Brown, et al.Bioconjugate Chemistry, 2000,11,880-891.).With regard to transfer efficiency, PEI is that function is also a kind of cationic polymer of most study the strongest the time, but PEI in vivo or show larger cytotoxicity during external application, therefore limited its clinical practice.PEI 25kDa has higher transfection efficiency, but cytotoxicity is very large; Low-molecular-weight PEI cytotoxicity is little, but almost do not have the transfection effect (can be with reference to A.Kichler, The Journal of Gene Medicine, 2004,6, S3-S10.).
Polymer/magnetic nano-particle composite combines the advantage of polymer and magnetic inorganic particle, has the functional of magnetic responsiveness and polymer concurrently, has become the focus of nucleic acid/drug conveying carrier research.Under the existence of the diblock copolymer of acrylic acid monoglyceride or methacrylic acid monoglyceride and acrylic or methacrylic acid, alkaline co-precipitation Fe
2+/ Fe
3+(mol ratio is 1: 2) obtains being scattered in the diblock copolymer parcel Fe in water
3O
4The composite nanoparticle of nanoparticle, wherein polyacrylic acid glycerol monoesters or polymethylacrylic acid monoglyceride block are attached to Fe
3O
4On nanoparticle surface, and polyacrylic acid or polymethylacrylic acid block are in the surface (with reference to Chinese patent ZL200410058599.0) of composite nanoparticle.This composite nanoparticle does not carry the function base of nucleic acid/medicine, and the surface band negative charge of composite nanoparticle, have cytotoxicity under neutrality or acid condition.Therefore, be not suitable as the carrier of nucleic acid/medicine.
Summary of the invention
One aspect of the present invention relates to a kind of magnetic composite, and it is take the Fe of nanoscale (mean diameter is as a nanometer to the hundreds of nanometer)
3O
4Particle is kernel, and skin is the compound shown in following formula 1,
Formula 1
In top formula 1,
Z is selected from following one or more identical or different chemical functional group or function fragment: the content of poly-basic amino acid, basic amino acid number greater than 50% amino acid fragment, be connected with poly-basic amino acid, cancer therapy drug, biotin, transferrins, the methyl of fluorescent marker;
B is selected from following biological function group and the connecting key of block copolymer: ester bond (COO-), amido link (CONR
1-, R
1=H, CH
3, or-CH
2-), disulfide bond (S-S-), ehter bond (O-), carbonnitrogen bond (C-N (R
2)-, R
2=H, CH
3, or CH
3CH
2Deng), 1,3-triazole ring
And
The structure that comprises block copolymer in formula 1, the first block are Polyethylene Glycol, and its average degree of polymerization x is 1-300;
The second block is polyacrylic acid glycerol monoesters or polymethylacrylic acid monoglyceride, y=1-500, r=H or CH
3,
Wherein the second block and Fe
3O
4The combination of particle core surface, the outermost layer of Z-shaped one-tenth magnetic composite, the mean diameter of described magnetic composite is in the scope of nanometer to tens micron.
The content of described basic amino acid number is greater than 50% amino acid fragment, particularly, can be for the content of basic amino acid number 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, described Fe
3O
4The mean diameter of particle is a few nanometer to tens nanometers, particularly, is tens nanometers.
In one embodiment of the invention, the mean diameter of described magnetic composite is 3～300nm, described basic amino acid is selected from one or more in histidine, arginine and lysine, and the content of described poly-basic amino acid or basic amino acid number is 3-39 aminoacid greater than the length of 50% amino acid fragment.
in one embodiment of the invention, the content of described poly-basic amino acid or basic amino acid number is selected from RRRRRRRRR (SEQ ID NO:1) greater than 50% amino acid fragment, 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), GRRRQRRKKRGCG (SEQ ID NO:8), GCGGGYGRKKRRQRRR (SEQ ID NO:9), and RRRQIKIWFQNRRMKWKK (SEQ ID NO:10), particularly, be selected from RRRRRRRRR (SEQ ID NO:1), GRKKRRQRRRGCG (SEQ ID NO:7), and GCGGGYGRKKRRQRRR (SEQ ID NO:9).
In one embodiment of the invention, described fluorescent marker is fluorescein series, the serial fluorescent dye of Cy, Alexa Fluor series fluorescent dye, particularly, described fluorescein series is selected from FITC, TRITC, FAM or its analog, described Cy series fluorescent dye is selected from Cy5, Cy5.5, Cy7 or its analog, more specifically, described fluorescent marker is selected from FITC, TRITC, FAM or its analog.
In one embodiment of the invention, described Z is selected from following two or more than different chemical functional group or the function fragment of two: the content of poly-basic amino acid, basic amino acid number greater than 50% amino acid fragment, be connected with poly-basic amino acid, cancer therapy drug, biotin, transferrins, the methyl of fluorescent marker; Wherein, the content of described poly-basic amino acid or basic amino acid number greater than 50% amino acid fragment and cancer therapy drug or with biotin or with the mol ratio of methyl be 50: 1 to 1: 50.
Another aspect of the present invention relates to the preparation method of above-mentioned magnetic composite, comprises the steps:
1) prepare with mineral acid or the stable Fe of organic acid
3O
4Magnetic fluid (can reference, for example: R.Massart, IEEE Transactions on Magnetics, 1981, MAG-17,1247-1248.),
2) to 1) in add the aqueous solution of the compound shown in formula 1 in the magnetic fluid of preparation, react, reaction temperature is 0 ℃ to 90 ℃, the response time is 1 minute to 50 hours.
Preferably, the molal quantity of the compound shown in described formula 1: Fe
3O
4Molal quantity greater than 1.
Perhaps comprise the steps:
1) prepare with mineral acid or the stable Fe of organic acid
3O
4Magnetic fluid,
2) Fe of the magnetic fluid of preparation the block copolymer parcel 1 shown in the block copolymer structure in use formula 1)
3O
4Particle,
3) will gather basic amino acid and link parcel Fe by the active reactive group key on above-mentioned block copolymer
3O
4On the surface of the block copolymer of particle, reaction temperature is 0 ℃ to 90 ℃, and the response time is 1 minute to 50 hours.
Preferably, the molal quantity of described block copolymer: Fe
3O
4Molal quantity greater than 1.
In one embodiment of the invention, described mineral acid is perchloric acid, hydrochloric acid, nitric acid or sulphuric acid, and described organic acid is formic acid, acetic acid or trifluoracetic acid.
The method of synthetic available existing any synthetic segmented copolymer of the present invention's block copolymer used is as active free radical polymerization (comprising atom transfer radical polymerization (ATRP), reversible addition-cracking chain transferring free-radical polymerization (RAFT) etc.), anionic polymerisation etc.
Also aspect of the present invention relates to described magnetic composite as the purposes of pharmaceutical carrier, RNA carrier or DNA vector.Particularly, described medicine is the medicine of cancer therapy drug, antiviral drugs, treatment diabetes or the medicine for the treatment of cardiovascular and cerebrovascular disease, and particularly, described cancer therapy drug is methotrexate and/or cyclophosphamide.
The poly-basic amino acid block copolymer parcel of chemical bond-linking Fe
3O
4In particle, the second block polypropylene acid glycerol monoesters of block copolymer or the effect of polymethylacrylic acid monoglyceride are and Fe
3O
4The particle surface combination; Time skin of magnetic composite is Polyethylene Glycol, makes magnetic composite have good biocompatibility; Poly-basic amino acid is free in the outermost layer of magnetic composite, can carry magnetic composite and pass cell membrane, or even nuclear membrane enters nucleus, even by blood brain barrier (BBB), can be used as the delivery vehicles of all kinds of nucleic acid/medicines.
The macromole conjugate that key connects poly-basic amino acid can connect with key polyethylene glycol-(methyl) the acrylic acid monoglyceride block copolymer combination parcel Fe of medicine (as methotrexate, cyclophosphamide) or bioactive substance (as biotin, transferrins)
3O
4Particle (both make up molar ratio can from 50: 1 to 1: 50), therefore, not only can utilize to be free in the outermost bioactive substance of magnetic composite, realize the targeting to particular organization and organ, and can carry medicine and enter in cell.Poly-basic amino acid is cationic polymer, can form complex with electronegative DNA molecular, and the macromole conjugate that key connects poly-basic amino acid can make up parcel Fe with poly glycol monomethyl ether-poly-(methyl) acrylic acid monoglyceride block copolymer again
3O
4Particle (both make up molar ratio can from 50: 1 to 1: 50); so, can play by the molecular weight of regulating poly glycol monomethyl ether in poly glycol monomethyl ether-poly-(methyl) acrylic acid monoglyceride block copolymer the effect that enzymolysis is avoided in protection and the poly-basic amino acid of shielding and nucleic acid.
The key that the present invention synthesizes connects the block copolymer parcel Fe of poly-basic amino acid
3O
4The magnetic composite of particle has lower cytotoxicity, and under the condition of experiment, magnetic composite and HeLa Cells were hatched 24 hours jointly, and cell survival rate is higher than 90% (seeing accompanying drawing 1).And the magnetic composite that surperficial key connects poly-basic amino acid has the high ability of passing cell membrane, or even can pass nuclear membrane and enter in nucleus and (to see accompanying drawing 2, A～B).Test by confocal microscope and proved that fluorescent-labeled magnetic complex that the poly-basic amino acid of surperficial nothing is modified can pass not cell membrane and (see accompanying drawing 3, A～D), and the fluorescent-labeled magnetic complex that the surface has poly-basic amino acid to modify can pass cell membrane and (sees accompanying drawing 4, A～D).
Description of drawings
Fig. 1: the relative survival rate of HeLa Cells after cultivating 24 hours under variable concentrations magnetic composite existence condition.
Fig. 2: poly-basic amino acid-PEG-PGA-Fe
3O
4Magnetic composite enters the microphotograph (prussian blue staining ferrum, safranine T transfect cell core) of HeLa Cells.Fig. 2 A:400 *; Fig. 2 B: be high power microscope photo (1000 *).
Fig. 3: confocal microscope photo proof FITC-PEG-PGA-Fe
3O
4Can not pass the Caco-2 cell membrane.Fig. 3 A:FITC fluorescence imaging; Fig. 3 B: the cell membrane of rhodamine-phalloidin dyeing; The nucleus of Fig. 3 C:Hoechest 33258 dyeing; Fig. 3 D: first three plants the combined diagram of imaging.
Fig. 4: the poly-basic amino acid-PEG-PGA-Fe of confocal microscope photo proof FITC-
3O
4Can pass the Caco-2 cell membrane.Fig. 4 A:FITC fluorescence imaging; Fig. 4 B: the cell membrane of rhodamine-phalloidin dyeing; The nucleus of Fig. 4 C:Hoechest 33258 dyeing; Fig. 4 D: first three plants the combined diagram of imaging.
The specific embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment carries out according to the condition of normal condition or manufacturer's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Used being abbreviated as: PEG in the following examples, Polyethylene Glycol; MPEG, poly glycol monomethyl ether; CH
3SO
3-PEG-OH, single-ended mesyl Polyethylene Glycol; N
3-PEG-OH, single-ended azido Polyethylene Glycol; NH
2-PEG-OH, single-ended amino Polyethylene Glycol; FMAL-PEG-Br, an end furan protection maleimide amine end groups, the other end is the Polyethylene Glycol of α-isobutyl bromide ester; MAL, the maleimide amine end groups; SA, acrylic acid 2,2-dimethyl-1,3-dioxolane-4-methanol ester; SMA, methacrylic acid 2,2-dimethyl-1,3-dioxolane-4-methanol ester; PGA, the polyacrylic acid glycerol monoesters; PGMA, the polymethylacrylic acid monoglyceride; PMDETA, 1, Isosorbide-5-Nitrae, 7,7-pentamethyl-diethylenetriamine; Fmoc, fluorenylmethyloxycarbonyl; Boc, tertbutyloxycarbonyl; Pbf, 2,2,4,6,7-pentamethyl Dihydrobenzofuranes-5-sulfonyl; Trt, trityl; FITC, Fluorescein isothiocyanate; MTX, methotrexate.In addition, in the following embodiments, for the convenience of explaining, sometimes also the content of basic amino acid number used is called poly-basic amino acid greater than 50% amino acid fragment.In the following embodiments, the poly-basic amino acid that is connected with FITC does not connect the poly-basic amino acid of FITC take " GRKKRRQRRRGCG (SEQ ID NO:7) " as example take " GCGGGYGRKKRRQRRR (SEQ ID NO:9) " as example.
Embodiment 1:CH
3SO
3The preparation of-PEG-OH
The 3.8mL methylsufonyl chloride is dissolved in the 50mL oxolane, then slowly is added drop-wise in the 200mL oxolane that is dissolved with 10g PEG (mean molecule quantity is 200) and 7mL triethylamine, dropwise stirred overnight at room temperature after 3 hours.Filtering reacting liquid, the desolventizing of filtrate rotary evaporation separates obtaining CH through silicagel column
3SO
3-PEG-OH.
The preparation of embodiment 2:N3-PEG-OH
With 2.8g CH
3SO
3-PEG-OH (mean molecule quantity is 200) and 1g sodium azide are dissolved in 25mL water, and reactant liquor reacted 24 hours under 80 ℃.Reaction is cooled to room temperature after finishing, and decompression steams aqueous solvent, adds the 100mL dichloromethane, and organic facies is spent the night with anhydrous sodium sulfate drying, filters, and revolves the steaming desolventizing, separates obtaining N through silicagel column
3-PEG-OH.
Embodiment 3:NH
2The preparation of-PEG-OH
With 1g N
3-PEG-OH (mean molecule quantity is 200) is placed in reaction bulb, adds 0.13g water, then the 1.3g triphenylphosphine is dissolved in the 35mL oxolane, joins in reaction bulb stirring at room 10 hours.After reaction finishes, filter, revolve the steaming desolventizing, separate obtaining NH through silicagel column
2-PEG-OH.
Embodiment 4: the NH of other molecular weight
2The preparation of-PEG-OH
Respectively with mean molecule quantity be 600,2000 and 5000 PEG to replace mean molecule quantity in embodiment 1, embodiment 2 and embodiment 3 be 200 PEG, carry out identical operation, make respectively the NH of corresponding molecular weight
2-PEG-OH.
The preparation of embodiment 5:FMAL-PEG-OH
With 4,10 dioxas three rings [5.2.1.0 (2,6)] last of the ten Heavenly stems of 2.84g-8-alkene-3, the 5-diketone is dissolved in 70mL methanol, and solution is cooled to 0 ℃, then, then with the NH of 3.31g embodiment 3 preparations
2-PEG-OH (mean molecule quantity is 200) is dissolved in 30mL methanol and is added dropwise in top reactant liquor, reactant liquor stirred 10 minutes under 0 ℃, and stirring at room 45 minutes refluxed 4 hours at last, be cooled to room temperature, revolve the steaming desolventizing, residue is dissolved in the 200mL dichloromethane, with saturated common salt water washing three times, organic facies is spent the night with anhydrous magnesium sulfate drying, filter, revolve the steaming desolventizing, separate obtaining FMAL-PEG-OH through silicagel column.
The preparation of embodiment 6:FMAL-PEG-Br
The FMAL-PEG-OH (the PEG mean molecule quantity is 200) of 2.4g embodiment 5 preparations is dissolved in the oxolane of 70mL drying, then adds the 1.2mL triethylamine.Reactant liquor is cooled to 0 ℃, then 1.0mL α-bromine isobutyl acylbromide is dissolved in the oxolane of 20mL drying, is added dropwise in reactant liquor, 0 ℃ was reacted 1 hour, and stirring at room 24 hours removes by filter white precipitate, revolve the steaming desolventizing, separate obtaining FMAL-PEG-Br through silicagel column.
Embodiment 7: the preparation of the FMAL-PEG-Br of other molecular weight
Respectively with the PEG mean molecule quantity be 600,2000 and 5000 FMAL-PEG-OH to replace PEG mean molecule quantity in embodiment 6 be 200 FMAL-PEG-OH, other operates with embodiment 6, makes respectively the FMAL-PEG-Br of corresponding molecular weight.
The preparation of embodiment 8:MAL-PEG-PGA
With the FMAL-PEG-Br of 0.2g embodiment 6 preparations, 58mg CuBr, 3.04g SA are dissolved in the 1.5mL methyl phenyl ethers anisole; add 87 μ L PMDETA under argon shield, solution becomes light green color, immerses in the oil bath that has been preheating to 65 ℃; stirring reaction 3 hours gets green dope.With oxolane dissolving gained green polymer, solution is crossed neutral Al
2O
3Post is collected filtrate, revolves to steam fully to concentrate, and precipitates it with ten times of amount petroleum ether (60-90 ℃), gets little yellow polymer.Then, this polymer is dissolved in 20mL toluene, reflux 7 hours is revolved the steaming desolventizing.At last the polymer that obtains is dissolved in 1.0mol/L hydrochloric acid/dioxane (1: 3, v/v) in, at 0 ℃ of lower stirring reaction after 1 hour, stirring at room 24 hours, transfer to (be limited to 3500 on molecular weight) in bag filter repeatedly the dialysis, get colourless transparent solution.Lyophilization gets white solid MAL-PEG-PGA.
The preparation of embodiment 9:MAL-PEG-PGMA
With the polymerization single polymerization monomer SA in SMA replacement embodiment 8, other operates with embodiment 8.Make white powder solid MAL-PEG-PGMA.
Embodiment 10: the MAL-PEG-PGA of other molecular weight and the preparation of MAL-PEG-PGMA
Be that 600,2000 and 5000 FMAL-PEG-Br replaces the FMAL-PEG-Br in embodiment 8 and embodiment 9 with the PEG mean molecule quantity respectively, carry out identical operation, make respectively MAL-PEG-PGA and the MAL-PEG-PGMA of corresponding molecular weight.
Embodiment 11: the preparation of poly-basic amino acid
Select mbha resin; adopt the Fmoc synthesis strategy; sequence GRKKRRQRRRGCG (SEQ ID NO:7) by poly-basic amino acid; extend to the N end from the C end, with 25% piperidines/DMF solution deprotection; method of condensing adopts BTA-N; N, N ', N '-tetramethylurea hexafluorophosphoric acid ester (HBTU)/1-hydroxy benzo triazole (HOBt) method.Detection method adopts the 1,2,3-indantrione monohydrate indicator.Amino acid whose alpha-amido used all with the Fmoc protection, has the aminoacid of Side chain protective group to be: Fmoc-Lys (Boc)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Cys (Trt)-OH.Trifluoroacetic acid/water/metacresol/1，2-ethandithiol=90/5/3/2 (volume ratio) will be gathered basic amino acid cracking from the resin and be got off, and then carry out separation and purification with reversed phase liquid chromatography, obtain the poly-basic amino acid of sterling.
The preparation of the poly-basic amino acid derivant of embodiment 12:FITC-
Adopt the poly-basic amino acid of solid phase synthesis, use the FITC end-blocking, the derivant aminoacid sequence is FITC-(ω-GHBA)-GCGGGYGRKKRRQRRR (SEQ ID NO:9), ω-GHBA: omega-amino-caproic acid, other operation is identical with embodiment 11, makes the poly-basic amino acid of FITC-.
Embodiment 13: the preparation of poly-basic amino acid-PEG-PGA conjugate
0.1g the MAL-PEG-PGA of embodiment 8 preparations is dissolved in the 4mL deionized water, add 40mg to gather basic amino acid (GRKKRRQRRRGCG, SEQ ID NO:7), stirred overnight at room temperature, reactant liquor is transferred to the dialysis repeatedly that (is limited to 3500 on molecular weight) in bag filter, and lyophilization obtains poly-basic amino acid-PEG-PGA conjugate.
Embodiment 14:FITC-gathers the preparation of basic amino acid-PEG-PGA conjugate
With the poly-basic amino acid in the poly-basic amino acid derivant replacement of FITC-embodiment 13, other operation is identical with embodiment 13, makes the poly-basic amino acid of FITC--PEG-PGA conjugate.
Embodiment 15: the preparation of the poly-basic amino acid of other molecular weight-PEG-PGA conjugate
Be that 600,2000 and 5000 MAL-PEG-PGA replaces MAL-PEG-PGA in embodiment 13 with the PEG mean molecule quantity respectively, other operates with embodiment 13, makes respectively the poly-basic amino acid of corresponding molecular weight-PEG-PGA conjugate.
Embodiment 16: the FITC-of other molecular weight gathers the preparation of basic amino acid-PEG-PGA conjugate
Be that 600,2000 and 5000 MAL-PEG-PGA replaces MAL-PEG-PGA in embodiment 14 with the PEG mean molecule quantity respectively, other operate with embodiment 14, and the FITC-that makes respectively corresponding molecular weight gathers basic amino acid-PEG-PGA conjugate.
Embodiment 17: the preparation of the poly-basic amino acid of other molecular weight-PEG-PGMA conjugate
Be that 600,2000 and 5000 MAL-PEG-PGMA replaces MAL-PEG-PGA in embodiment 15 with the PEG mean molecule quantity respectively, other operates with embodiment 15, makes respectively the poly-basic amino acid of corresponding molecular weight-PEG-PGMA conjugate.
Embodiment 18: the FITC-of other molecular weight gathers the preparation of basic amino acid-PEG-PGMA conjugate
Be that 600,2000 and 5000 MAL-PEG-PGMA replaces MAL-PEG-PGA in embodiment 16 with the PEG mean molecule quantity respectively, other operate with embodiment 16, and the FITC-that makes respectively corresponding molecular weight gathers basic amino acid-PEG-PGMA conjugate.
Embodiment 19: the preparation of the magnetic fluid aqueous solution that perchloric acid is stable
With 2g FeCl
24H
2O is dissolved in solution and the 5.4gFeCl in 25mL 1mol/L hydrochloric acid
36H
2The solution that O is dissolved in the 25mL deionized water is placed in mixing in there-necked flask, logical argon.Drip 160mL 1.5mol/L ammonia in there-necked flask, produce black precipitate in solution, continued stirring reaction 24 hours under room temperature.Hold the incline supernatant and washing with water 3 times of precipitation by Magnet.Drip 2.0mol/L HClO in there-necked flask under argon shield
450mL, stirring at room 15 minutes.Standing 10 minutes, by the supernatant of inclining of upper method, restoring means, more logical argon 30 minutes dripped 2.0mol/L HClO from constant pressure funnel
450mL, stirring at room 15 minutes.After stopping stirring with centrifugal 30 minutes of reactant, the solution that inclines, precipitation is transferred to rapidly in bag filter, repeatedly dialyses with deionized water, makes the stable magnetic fluid aqueous solution of perchloric acid, records Fe
3O
4Concentration be: 32mg/mL.
Embodiment 20: the poly-basic amino acid-PEG-PGA-Fe of direct method preparation
3O
4
The poly-basic amino acid of 0.12g embodiment 13 preparation-PEG-PGA conjugate is dissolved in the 3mL deionized water, regulates pH value to acid with hydrochloric acid.Evacuation applying argon gas, triplicate remove the oxygen in reaction bulb, add the stable magnetic fluid of 0.4mL perchloric acid.Reacted 12 hours, Magnetic Isolation obtains poly-basic amino acid-PEG-PGA conjugate parcel Fe
3O
4The magnetic composite of particle (poly-basic amino acid-PEG-PGA-Fe
3O
4) aqueous solution.
Embodiment 21: the poly-basic amino acid-PEG-PGA-Fe of indirect method preparation
3O
4
The MAL-PEG-PGA of 0.12g embodiment 8 preparations is dissolved in the 3mL deionized water, and evacuation applying argon gas, triplicate remove the oxygen in reaction bulb, add the stable magnetic fluid of 0.4mL perchloric acid.Reacted 12 hours, Magnetic Isolation obtains MAL-PEG-PGA parcel Fe
3O
4Magnetic composite (the MAL-PEG-PGA-Fe of particle
3O
4) aqueous solution.Poly-basic amino acid (GRKKRRQRRRGCG, SEQ ID NO:7) is dissolved in the 0.2mL deionized water, to acid, then adds 2mL MAL-PEG-PGA-Fe with the hydrochloric acid conditioning solution pH value in solution
3O
4Aqueous solution, reactant liquor at room temperature stir and spend the night, and Magnetic Isolation obtains poly-basic amino acid-PEG-PGA conjugate parcel Fe
3O
4The magnetic composite of particle (poly-basic amino acid-PEG-PGA-Fe
3O
4) aqueous solution.
Embodiment 22: direct method prepares the poly-basic amino acid-PEG-PGA-Fe of FITC-
3O
4
With the poly-basic amino acid in the poly-basic amino acid of FITC--PEG-PGA conjugate replacement embodiment 20-PEG-PGA conjugate, other operates with embodiment 20.Direct method prepares the poly-basic amino acid of FITC--PEG-PGA conjugate parcel Fe
3O
4(FITC-gathers basic amino acid-PEG-PGA-Fe to the magnetic composite of particle
3O
4) aqueous solution.
Embodiment 23: the poly-basic amino acid-PEG-PGMA-Fe of direct method preparation
3O
4With the poly-basic amino acid-PEG-PGMA-Fe of FITC-
3O
4
With the poly-basic amino acid in poly-basic amino acid-PEG-PGMA conjugate and the poly-basic amino acid of FITC--PEG-PGMA conjugate replacement embodiment 20-PEG-PGA conjugate, other operates with embodiment 20 respectively.Direct method prepares respectively poly-basic amino acid-PEG-PGMA conjugate parcel Fe
3O
4The magnetic composite of particle (poly-basic amino acid-PEG-PGMA-Fe
3O
4) aqueous solution and the poly-basic amino acid of FITC--PEG-PGMA conjugate parcel Fe
3O
4(FITC-gathers basic amino acid-PEG-PGMA-Fe to the magnetic composite of particle
3O
4) aqueous solution.
Embodiment 24:mPEG-PGA and poly-basic amino acid-PEG-PGA conjugate combination parcel Fe
3O
4The preparation of the magnetic composite of particle
MPEG-PGA and poly-basic amino acid-PEG-PGA conjugate combination parcel Fe
3O
4The synthetic of the preparation of the magnetic composite of particle: mPEG-PGA (mean molecule quantity 2000 of mPEG) can reference: S.Wan, et al.J.Mater.Chem., 2005,15,3424-3430.With mPEG-PGA and poly-basic amino acid-PEG-PGA conjugate respectively in molar ratio from the various ratios of 50: 1 to 1: 50, combination parcel Fe
3O
4Particle, operation is with embodiment 20.Prepare respectively mPEG-PGA and poly-basic amino acid-PEG-PGA conjugate combination parcel Fe
3O
4The magnetic composite of particle.
Embodiment 25: the mPEG-PGA of other molecular weight and poly-basic amino acid-PEG-PGA conjugate combination parcel Fe
3O
4The preparation of the magnetic composite of particle
Be that 200 and 5000 mPEG-PGA replaces the mPEG-PGA in embodiment 24 with the mPEG mean molecule quantity respectively, other operates with embodiment 24.Prepare respectively mPEG-PGA and the poly-basic amino acid-PEG-PGA conjugate combination parcel Fe of corresponding molecular weight
3O
4The magnetic composite of particle.
Embodiment 26:mPEG-PGMA and poly-basic amino acid-PEG-PGMA conjugate combination parcel Fe
3O
4The preparation of the magnetic composite of particle
Use respectively mPEG-PGA and poly-basic amino acid-PEG-PGA conjugate in mPEG-PGMA (mean molecule quantity of mPEG is 2000) and poly-basic amino acid-PEG-PGMA conjugate replacement embodiment 24, other operates with embodiment 24.Prepare respectively mPEG-PGMA and poly-basic amino acid-PEG-PGMA conjugate combination parcel Fe
3O
4The magnetic composite of particle.
Embodiment 27: the mPEG-PGMA of other molecular weight and poly-basic amino acid-PEG-PGMA conjugate combination parcel Fe
3O
4The preparation of the magnetic composite of particle
Be that 200 and 5000 mPEG-PGMA replaces the mPEG-PGMA in embodiment 26 with the mPEG mean molecule quantity respectively, other operates with embodiment 26.Prepare respectively mPEG-PGMA and the poly-basic amino acid-PEG-PGMA conjugate combination parcel Fe of corresponding molecular weight
3O
4The magnetic composite of particle.
Embodiment 28:MTX-PEG-PGA conjugate and poly-basic amino acid-PEG-PGA conjugate combination parcel Fe
3O
4The preparation of the magnetic composite of particle
MTX-PEG-PGA conjugate and poly-basic amino acid-PEG-PGA conjugate combination parcel Fe
3O
4The preparation of the magnetic composite of particle: MTX-PEG-PGA conjugate (mean molecule quantity 200 of PEG) synthetic, can reference: Q.Zhang, et al.J.Mater.Chem., 2009,19,8393-8402.With MTX-PEG-PGA conjugate and poly-basic amino acid-PEG-PGA conjugate respectively in molar ratio from the various ratios of 50: 1 to 1: 50, combination parcel Fe
3O
4Particle, operation is with embodiment 20.Prepare respectively MTX-PEG-PGA conjugate and poly-basic amino acid-PEG-PGA conjugate combination parcel Fe
3O
4The magnetic composite of particle.
Embodiment 29: the MTX-PEG-PGA conjugate of other molecular weight and poly-basic amino acid-PEG-PGA conjugate combination parcel Fe
3O
4The preparation of the magnetic composite of particle
Be that 2000 and 5000 MTX-PEG-PGA conjugate replaces the MTX-PEG-PGA conjugate in embodiment 28 with the PEG mean molecule quantity respectively, other operates with embodiment 28.Prepare respectively MTX-PEG-PGA conjugate and the poly-basic amino acid-PEG-PGA conjugate combination parcel Fe of corresponding molecular weight
3O
4The magnetic composite of particle.
Embodiment 30:MTX-PEG-PGMA conjugate and poly-basic amino acid-PEG-PGMA conjugate combination parcel Fe
3O
4The preparation of the magnetic composite of particle
Use respectively MTX-PEG-PGA conjugate and poly-basic amino acid-PEG-PGA conjugate in MTX-PEG-PGMA conjugate (mean molecule quantity of PEG is 200) and poly-basic amino acid-PEG-PGMA conjugate replacement embodiment 28, other operates with embodiment 28.Prepare respectively MTX-PEG-PGMA conjugate and poly-basic amino acid-PEG-PGMA conjugate combination parcel Fe
3O
4The magnetic composite of particle.
Embodiment 31: the MTX-PEG-PGMA conjugate of other molecular weight and poly-basic amino acid-PEG-PGMA conjugate combination parcel Fe
3O
4The preparation of the magnetic composite of particle
Be that 2000 and 5000 MTX-PEG-PGMA conjugate replaces the MTX-PEG-PGMA conjugate in embodiment 30 with the PEG mean molecule quantity respectively, other operates with embodiment 30.Prepare respectively MTX-PEG-PGMA conjugate and the poly-basic amino acid-PEG-PGMA conjugate combination parcel Fe of corresponding molecular weight
3O
4The magnetic composite of particle.
Embodiment 32: the Cytotoxic experiment of magnetic composite
Poly-basic amino acid-PEG-PGA-Fe with gained in embodiment 20
3O
4With the MAL-PEG-PGA-Fe that mentions in embodiment 21
3O
4Join respectively and carry out cell culture experiments in culture fluid, then measure the relative survival rate of cell with the MTS method, concrete experimental technique can reference: S.Wan, et al.J.Biomed.Mater.Res.A, 2007,80,946-954.Fig. 1 is MAL-PEG-PGA-Fe
3O
4With poly-basic amino acid-PEG-PGA-Fe
3O
4Cytotoxic comparison.Magnetic composite and HeLa Cells were hatched 24 hours jointly, and cell survival rate is higher than 90%.
Similarly, verified the magnetic composite of the present invention for preparing in other embodiment, result shows, the survival rate of cell is higher, shows that the toxicity of these magnetic composites is very little or there is no toxicity.
Embodiment 33: the experiment that staining proof magnetic composite passes cell membrane
Poly-basic amino acid-the PEG-PGA-Fe of gained in embodiment 20
3O
4Join and carry out cell culture experiments in culture fluid, then judge with the prussian blue staining method in cell, whether ferrum exists, and safranine T is redyed nucleus.Fig. 2 (A～B) for gathering basic amino acid-PEG-PGA-Fe
3O
4Enter nuclear dyeing photo, can find out, the magnetic composite of the poly-basic amino acid of finishing can carry Fe
3O
4Particle enters nucleus.
Similarly, verified the magnetic composite of the present invention for preparing in other embodiment, the result demonstration, they all can pass cell membrane.
Embodiment 34: fluorescent marker method proof FITC-PEG-PGA-Fe
3O
4Can not pass the experiment of Caco-2 cell membrane
Adopt and embodiment 20 or the synthetic FITC-PEG-PGA-Fe of 21 similar methods
3O
4, replace with FITC except gathering basic amino acid, then it is joined and carry out cell culture experiments in culture fluid, then judge the existence of fluorescent material in cell by the fluorescence co-focusing light spectrum image-forming.Fig. 3 (the proof of A～D) FITC-PEG-PGA-Fe
3O
4Can not pass the Caco-2 cell membrane.
Embodiment 35: the poly-basic amino acid-PEG-PGA-Fe of fluorescent marker method proof FITC-
3O
4Pass the experiment of cell membrane
Poly-basic amino acid-the PEG-PGA-Fe of the FITC-of gained in embodiment 22
3O
4Join and carry out cell culture experiments in culture fluid, then judge the existence of fluorescent material in cell by the fluorescence co-focusing light spectrum image-forming.(A～D) is the poly-basic amino acid-PEG-PGA-Fe of FITC-to Fig. 4
3O
4Enter the intracellular fluorescence co-focusing photo of Caco-2, can find out, the magnetic composite of the poly-basic amino acid of finishing FITC labelling can carry Fe
3O
4Particle enters in cell.
Similarly, verified the magnetic composite of the present invention for preparing in other embodiment, the result demonstration, they all can pass cell membrane.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Sequence table
＜110〉Inst of Toxic Medicinal Materials, P.L.A. Academy of Military Medical Sciences
Yiping Medicine Science ﹠ Tech. Development Co., Ltd., Chengdu
＜120〉a kind of magnetic composite and its production and use
<130>IDC100077
<160>10
<170>PatentIn version 3.2
<210>1
<211>9
<212>PRT
＜213〉artificial sequence
<400>1
Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5
<210>2
<211>10
<212>PRT
＜213〉artificial sequence
<400>2
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
1 5 10
<210>3
<211>9
<212>PRT
＜213〉artificial sequence
<400>3
Arg Arg Arg Arg Arg Ala Ala Gly Gly
1 5
<210>4
<211>12
<212>PRT
＜213〉artificial sequence
<400>4
Arg Arg Arg Arg Arg Ala Ala Gly Gly Lys Lys Lys
1 5 10
<210>5
<211>13
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＜213〉artificial sequence
<400>5
Arg Arg Arg Arg Arg Ala Ala Gly Gly Lys Arg Arg Arg
1 5 10
<210>6
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＜213〉artificial sequence
<400>6
Arg Arg Arg Arg Arg Ala Ala Gly Lys Cys Cys
1 5 10
<210>7
<211>13
<212>PRT
＜213〉artificial sequence
<400>7
Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Cys Gly
1 5 10
<210>8
<211>13
<212>PRT
＜213〉artificial sequence
<400>8
Gly Arg Arg Arg Gln Arg Arg Lys Lys Arg Gly Cys Gly
1 5 10
<210>9
<211>16
<212>PRT
＜213〉artificial sequence
<400>9
Gly Cys Gly Gly Gly Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10 15
<210>10
<211>18
<212>PRT
＜213〉artificial sequence
<400>10
Arg Arg Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp
1 5 10 15
Lys Lys
Claims (15)
1. magnetic composite, it is with nano level Fe
3O
4Particle is kernel, and skin is the compound shown in following formula 1,
Formula 1
In top formula 1,
Z is selected from following one or more identical or different chemical functional group or function fragment: poly-basic amino acid and the poly-basic amino acid that is connected with fluorescent marker; The content that described poly-basic amino acid is the basic amino acid number is greater than 50% amino acid fragment; Described basic amino acid is selected from one or more in histidine, arginine and lysine, and the length of described poly-basic amino acid is 3-39 aminoacid;
B is the connecting key that is selected from following biological function group and block copolymer: ester bond (COO-), amido link, disulfide bond (S-S-), ehter bond (O-), carbonnitrogen bond, 1,3-triazole ring
And
The structure that comprises block copolymer in formula 1, the first block are Polyethylene Glycol, and its average degree of polymerization x is 1-300;
The second block is polyacrylic acid glycerol monoesters or polymethylacrylic acid monoglyceride, y=1-500, r=H or CH
3,
Wherein the second block and Fe
3O
4The combination of particle core surface, the outermost layer of Z-shaped one-tenth magnetic composite, the mean diameter of described magnetic composite is in the scope of nanometer to tens micron.
2. magnetic composite according to claim 1, wherein, Z also is selected from following one or more identical or different chemical functional group or function fragment: cancer therapy drug, biotin, transferrins and methyl.
3. magnetic composite according to claim 1, wherein, described amido link is-CONR
1-, R wherein
1=H, CH
3, or-CH
2-.
4. magnetic composite according to claim 1, wherein, described carbonnitrogen bond is-C-N (R
2)-, be R wherein
2=H, CH
3, or CH
3CH
2
5. the described magnetic composite of any one according to claim 1 to 4, is characterized in that, the mean diameter of described magnetic composite is 3～300nm.
6. magnetic composite according to claim 1, described poly-basic amino acid is selected from RRRRRRRRR, KKKKKKKKKK, RRRRRAAGG, RRRRRAAGGKKK, RRRRRAAGGKRRR, RRRRRAAGKCC, GRKKRRQRRRGCG, GRRRQRRKKRGCG, GCGGGYGRKKRRQRRR and RRRQIKIWFQNRRMKWKK.
7. magnetic composite according to claim 1, wherein, described fluorescent marker is fluorescein series, the serial fluorescent dye of Cy or Alexa Fluor series fluorescent dye.
8. magnetic composite according to claim 7, wherein, described fluorescein series is selected from FITC, TRITC or FAM, and described Cy series fluorescent dye is selected from Cy5, Cy5.5 or Cy7.
9. the described magnetic composite of any one according to claim 2 to 4, wherein, described Z is selected from following two or more than different chemical functional group or the function fragment of two: poly-basic amino acid, be connected with poly-basic amino acid, cancer therapy drug, biotin, transferrins, the methyl of fluorescent marker; Wherein, described poly-basic amino acid and cancer therapy drug or with biotin or with the mol ratio of methyl be 50: 1 to 1: 50.
10. magnetic composite according to claim 2, wherein, described cancer therapy drug is methotrexate and/or cyclophosphamide.
11. the preparation method of magnetic composite claimed in claim 1 comprises the steps:
1) prepare with mineral acid or the stable Fe of organic acid
3O
4Magnetic fluid,
2) Fe of the magnetic fluid of preparation the block copolymer parcel 1 shown in the block copolymer structure in use formula 1)
3O
4Particle,
3) will gather basic amino acid and link parcel Fe by the active reactive group key on above-mentioned block copolymer
3O
4On the surface of the block copolymer of particle, reaction temperature is 0 ℃ to 90 ℃, and the response time is 1 minute to 50 hours.
12. method according to claim 11, wherein, described mineral acid is perchloric acid, hydrochloric acid, nitric acid or sulphuric acid; Described organic acid is formic acid, acetic acid or trifluoracetic acid.
13. in claim 1-10, the described magnetic composite of any one is for the preparation of the purposes of pharmaceutical carrier, RNA carrier or DNA vector.
14. purposes according to claim 13, wherein, described medicine is the medicine of cancer therapy drug, antiviral drugs, treatment diabetes or the medicine for the treatment of cardiovascular and cerebrovascular disease.
15. purposes according to claim 14, wherein, described cancer therapy drug is methotrexate and/or cyclophosphamide.
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| CN2010101910097A CN102266566B (en) | 2010-06-03 | 2010-06-03 | Magnetic compound, and preparation method and purpose thereof |
| PCT/CN2011/000892 WO2011150671A1 (en) | 2010-06-03 | 2011-05-24 | Magnetic composite, preparation method and use thereof |
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| CN103665118B (en) * | 2013-12-03 | 2015-11-25 | 南昌大学 | The method of purifying water soluble ferric oxide nano particles Streptavidin conjugate |
| CN107189058A (en) * | 2017-07-07 | 2017-09-22 | 湖南华腾制药有限公司 | A kind of preparation method of amino-polyethyleneglycols hydroxyl |
| CN108524949B (en) * | 2018-04-03 | 2021-05-25 | 江苏大学 | Composite prodrug nano-carrier for reversing drug resistance of tumor drug and preparation method thereof |
| CN112378837B (en) * | 2020-09-15 | 2021-12-28 | 深圳市华中生物药械有限公司 | Cervical exfoliated cell detection method and related device |
| CN113616807B (en) * | 2021-07-26 | 2023-07-21 | 中山大学 | Mitochondrion-targeted polypeptide and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5200270A (en) * | 1986-02-25 | 1993-04-06 | Toyo Soda Manufacturing Co., Ltd. | Carrier for a biologically active component for immunoassay or enzymatic reaction |
| CN1706865A (en) * | 2004-06-11 | 2005-12-14 | 北京键凯科技有限公司 | Branched polyglycol-amino acid oligopeptide and its active derivative and medicinal composition |
| CN1737956A (en) * | 2004-08-19 | 2006-02-22 | 中国人民解放军军事医学科学院毒物药物研究所 | Magnetic Nano material |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01179401A (en) * | 1988-01-07 | 1989-07-17 | Canon Inc | Magnetic fluid |
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2010
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2011
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5200270A (en) * | 1986-02-25 | 1993-04-06 | Toyo Soda Manufacturing Co., Ltd. | Carrier for a biologically active component for immunoassay or enzymatic reaction |
| CN1706865A (en) * | 2004-06-11 | 2005-12-14 | 北京键凯科技有限公司 | Branched polyglycol-amino acid oligopeptide and its active derivative and medicinal composition |
| CN1737956A (en) * | 2004-08-19 | 2006-02-22 | 中国人民解放军军事医学科学院毒物药物研究所 | Magnetic Nano material |
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