CN106916318A - A kind of biodegradable polymer of core crosslinking containing Gd coordination compound and its production and use - Google Patents
A kind of biodegradable polymer of core crosslinking containing Gd coordination compound and its production and use Download PDFInfo
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- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/12—Macromolecular compounds
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- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
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- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/58—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing oxygen in addition to the carbonamido oxygen, e.g. N-methylolacrylamide, N-(meth)acryloylmorpholine
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- C08F2438/00—Living radical polymerisation
- C08F2438/03—Use of a di- or tri-thiocarbonylthio compound, e.g. di- or tri-thioester, di- or tri-thiocarbamate, or a xanthate as chain transfer agent, e.g . Reversible Addition Fragmentation chain Transfer [RAFT] or Macromolecular Design via Interchange of Xanthates [MADIX]
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- C08G2230/00—Compositions for preparing biodegradable polymers
Abstract
The invention discloses a kind of biodegradable polymer of core crosslinking containing Gd coordination compound and its production and use.The invention also discloses biodegradable linear HPMA copolymers of one kind and its production and use.The biodegradable polymer containing Gd coordination compound of the present invention has relaxation rate high, and the excellent effect used as magnetic resonance imaging contrast agent is degradable, and nontoxic, good biocompatibility, potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to a kind of biodegradable polymer of core crosslinking containing Gd coordination compound and its production and use.
Background technology
In clinical practice, imageological examination plays very important in the qualitative and level diagnosis of most of diseases
Effect.In these imageological examination means, magnetic resonance imaging as non-invasive diagnostic instrument have high-resolution imaging and
Three-dimensional soft tissue imaging etc. advantage, wherein, Contrast enhanced magnetic resonance imaging by contrast medium can further enhance normal structure with
Contrast between diseased tissue, obtains clear accurately image.At present, DTPA-Gd, DTPA-Gd and other gadolinium chelate compounds are contrasted
Agent is just being widely used in magnetic resonance Enhanced Imaging, but the effect of these contrast medium, susceptibility and circulatory system relaxation time have
There are many restrictions, such as, these contrast medium can quickly be eliminated and can not obtain prolonged body-internal-circulation.Therefore, in order to reach
To more preferable effect have to duplicate injection and high dose injection, but this mode can cause some ill effects.Preferably
Contrast medium, should have high relaxation rate, should also have biological safety.Contrast medium for diagnosing tumor also requires it swollen
There is aggregation high at knurl position, realizes tumour targeting high etc..
With the development of nearest materials chemistry and nanometer technology, researchers attempt the coupling of the small molecules such as DTPA-Gd or
It is embedded into macromolecule magnetic resonance contrast medium of the formation with nano-scale in macromolecule system.The carrier of these nano-scales includes
Liposome, micella and other polymers, by strengthening, infiltration retention effect (EPR effects) increases relaxivity and tumour is poly- for they
Intensity.In these materials, the polymer of nano-scale can effectively overcome the shortcoming of convention control's agent, and possess well
Clinical landscapes.
HPMA HPMA is nontoxic, non-immunogenicity, can in blood there is good circulation.Existing report
HPMA copolymers possess good biocompatibility, without immunogenicity, neutral charge and good water soluble characteristic, usually as swollen
Knurl carrier is used, but it there is a problem of effectively degrading, if carrying the materials such as gadolinium, the side effect to human body is larger, can
The disease that kidney can be caused systemic fibrosing etc..
The content of the invention
In order to solve the above problems, the invention provides a kind of biodegradable polymer and its system containing Gd coordination compound
Preparation Method and purposes.
Present invention firstly provides the biodegradable linear HPMA polymer of one kind, its structural formula such as following formula (I)：
Wherein, m=57~170, n=380~1150.
Preferably, in the structural formula of the polymer, m=100, n=672.
Preferably, the weight-average molecular weight of the polymer is 10~40kDa, preferably 28kDa.
Preferably, the percentage by weight of Gd is 4~10%, preferably 6.6% in the polymer.
Preferably, the PDI of the polymer<2.5, preferably 1.09.
Present invention also offers a kind of polymer containing Gd coordination compound of biodegradable core crosslinking, its structural formula is as follows
Formula (II)：
Wherein, m=57~170, n=380~1150, p=16~50, q=16~50；
Represent following structure (III) (of the pHPMA containing Gd coordination compound is single-stranded)：
Preferably, in the structural formula of the polymer, m=100, n=672, p=28, q=28.
Preferably, the weight-average molecular weight of the polymer is 100~300kDa, preferably 181kDa.
Preferably, the percentage by weight of Gd is 4~10%, preferably 6.1% in the polymer.
Preferably, the PDI of the polymer<2.5, preferably 1.95.
Aforementioned linear polymer of the present invention can be prepared as follows, and step is as follows：
(1) synthesis of linear copolymer pHPMA-DOTA：HPMA, 4-CTA, MA-DOTA and VA044 are taken, water/methyl alcohol is added
Solution, reaction is settled out polymer in acetone after being cooled down through liquid nitrogen, obtains pink solid, is freezed after dialysis, obtains containing two sulphur
For the HPMA copolymers of benzoate group；HPMA copolymers containing dithiobenzoic acid ester group are dissolved in methyl alcohol with V501,
Heating, reaction is freezed after dialysis, obtains final product pHPMA-DOTA；
(2) synthesis of the linear copolymers of HPMA containing gadolinium：By pHPMA-DOTA and GdCl3·6H2O is dissolved in water, and then adjusts
Section pH to 5.2-5.4, reaction is freezed after dialysis, obtains final product pHPMA-DOTA-Gd.
In step (1), the mol ratio of described HPMA, 4-CTA, MA-DOTA and VA044 is：9.75mmol：79μmol：
6.5mmol：26.3μmol；In the water/methanol solution, water is 5 with the volume ratio of methyl alcohol：1；Per 9.75mmol HPMA, add
22mL water/methanol solution；The reaction is that solution full of argon gas 40 minutes, then rotates 8h at 44 DEG C at 0 DEG C；The dialysis
Time be 12h.
In step (1), white solid is to rotate 24 hours at room temperature with the reaction of V501；The time of dialysis is 24h.
In step (2), the pHPMA-DOTA and GdCl3·6H2The mass ratio of O is 800：928mg, per 800mg
The water that pHPMA-DOTA is used is 25mL；The time of dialysis is 24h.
The polymer containing Gd coordination compound of the foregoing core crosslinking of the present invention can be prepared as follows, and step is as follows：
1) synthesis of linear HPMA copolymers pHPMA-DOTA：Take HPMA, 4-CTA, MA-DOTA and VA044, add water/
Methanol solution, reaction is settled out polymer in acetone after being cooled down through liquid nitrogen, obtains pink solid, is thio containing two
The HPMA copolymers of benzoate group；
2) synthesis of the pHPMA-DOTA copolymers of the thio group of pyridine two modification:Take containing dithiobenzoic acid ester group
Polymer (pHPMA-DOTA with dithiobenzoate group) and PTEMA, add water/methanol solution and VA044, instead
Should, polymer is settled out in acetone after being cooled down through liquid nitrogen, obtain pink solid；Pink solid and V501 are dissolved in first
Alcohol, heating response is freezed after dialysis, obtains the thio group of product pyridine two modification HPMA copolymers；
3) core is crosslinked the synthesis of pHPMA-DOTA polymer：Under nitrogen protection, the pHPMA- of the thio group of pyridine two modification
DOTA is dissolved in deionized water/methanol solution, after being sufficiently stirred for, the trimethylolpropane tris (3- of ethyl acetate/acetone solution
Mercaptopropionic acid ester) be slowly added dropwise then proceed to stirring；Acetone sloughs impurity, then by SEC instrumentsSystem is further purified；Collect useful component, dialysis, it is lyophilized after obtain white product core and be crosslinked pHPMA-
DOTA polymer；
4) core is crosslinked the synthesis of pHPMA-DOTA-Gd polymer：Core is crosslinked pHPMA-DOTA polymer and GdCl3·
6H2O is dissolved in water, and then adjusts pH to 5.2-5.4, and reaction is freezed after dialysis, is obtained final product core crosslinking pHPMA-DOTA-Gd and is gathered
Compound.
Step 1) in, the mol ratio of described HPMA, 4-CTA, MA-DOTA and VA044 is：9.75mmol：79μmol：
6.5mmol：26.3μmol；In the water/methanol solution, water is 5 with the volume ratio of methyl alcohol：1；Per 9.75mmol HPMA, add
22mL water/methanol solution；The reaction is that solution full of argon gas 40 minutes, then rotates 8h at 44 DEG C at 0 DEG C；The dialysis
Time be 12h.
Step 2) in, polymer (the pHPMA-DOTA with containing dithiobenzoic acid ester group
Dithiobenzoate group) with the mass ratio of PTEMA it is 6:1；In the water/methanol solution added during reaction, water and methyl alcohol
Volume ratio be 3：1；Per 1.2g pHPMA-DOTA, 1.8mL water/methanol solution and 3.2mg VA044 are added；The reaction is 0
Solution is full of argon gas 40 minutes at DEG C, then rotates 8 hours at 44 DEG C；When white solid is dissolved in water/methanol solution, institute
The water and the volume ratio of methyl alcohol stated in water/methanol solution are 4：1, it is 1.2g with the starting material polymer pHPMA-DOTA for reacting
Meter, the consumption of water/methanol solution is 8mL；
Step 2) in, the pink solid is reacted 3 hours with 10 times of V501 of amount at 70 DEG C.
Step 3) in, it is dissolved in 20mL water/methanol solution per the pHPMA-DOTA of the thio group modification of 900mg nitrogen pyridine two
In；In the water/methanol solution, water is 6 with the volume ratio of methyl alcohol：1；In the ethyl acetate/acetone, the two volume ratio is 1:
1；Trimethylolpropane tris (3-thiopropionate) are added in the ethyl acetate/acetone of 1mL per 20mg；The trimethylolpropane
Time for adding of three (3-thiopropionates) per 20mg is 1 hour；React for stirring reaction 6 hours；SEC is used during the purifying
InstrumentSystem is purified.
Step 4) in, the pHPMA-DOTA and GdCl3·6H2The mass ratio of O is 800：928mg, per 800mg
The water that pHPMA-DOTA is used is 25mL；The time of dialysis is 24h.
Present invention also offers purposes of the aforementioned polymer in the contrast-enhancing agent for preparing magnetic resonance imaging.
Advantage of the invention is that：
1. preparation method of the invention is designed and is polymerized by reversible addion-fragmentation chain transfer (RAFT) and " click " chemistry
Construct the linear polymer pHPMA-DOTA-Gd containing Gd coordination compound of functionalization, and the polymer that degradable core is crosslinked
Core-crosslinked pHPMA-DOTA-Gd.First by reversible addion-fragmentation chain transfer (RAFT) be polymerized, preparation point
Son measures the substantially controllable, polymer that molecular weight polydispersity coefficient (PDI) is low；And be coupled by with Gd (III), synthesizing linear
HPMA-DOTA-Gd.Herein on basis, the polymer of core crosslinking is synthesized, tumor microenvironment has specially been responded degradable two
Sulfide linkage is introduced into kernel, makes it have the molecular characterization of branching molecule, greatly improves relaxivity.With clinically
Diethyl pentetic acid-Gd (DTPA-Gd) magnetic resonance imaging probe is compared, longitudinal relaxation efficiency (T1) be the former three times with
Upper (10.49mM-1·s-1/Gd).Meanwhile, the amount of polymers of crosslinking compares with linear polymer and clinical reagent, can extend
Circulation time in blood, increase the ability of EPR passive targets, improve aggregation of the magnetic resonance imaging probe in tumor locus
Degree, so as to increase the imaging effect and therapeutic effect of tumor locus.
2. the polymer containing Gd coordination compound of kernel crosslinking of the invention can combine multiple small-molecular-weight Gd (III) conjugation
Thing (DOTA), molecular weight is improved (Mw>180kDa), its can be degraded to less than kidney threshold value low molecular weight product (<
50kDa), and with disimmune, nontoxicity, water-soluble and good biocompatibility.
Present invention also offers purposes of the aforementioned polymer in the contrast medium for preparing enhancing magnetic resonance imaging.
The polymer Core-crosslinked pHPMA- containing Gd coordination compound of the biodegradable core crosslinking of the present invention
DOTA-Gd has relaxation rate high, for the excellent effect of in-vivo imaging, while nontoxic, good biocompatibility, degradable, makees
It is the excellent effect that the contrast medium of magnetic resonance imaging is used, potential applicability in clinical practice is excellent.
Obviously, the above of the invention, according to the ordinary technical knowledge and customary means of this area, is not departing from
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification of other diversified forms can also be made, is replaced or is changed.
The specific embodiment of form, remakes further specifically to the above of the invention by the following examples
It is bright.But this scope for being interpreted as above-mentioned theme of the invention should not be only limitted to following example.It is all based on the above of the present invention
The technology realized belongs to the scope of the present invention.
Brief description of the drawings
Fig. 1 synthetic line figures
Fig. 2 polymer nuclear magnetic spectrograms.
Fig. 3 pHPMA-DOTA-Gd, Core-crosslinked pHPMA-DOTA-Gd and the DTPA-Gd aqueous solution are in 3.0T
T in magnetic resonance1- weighted scanning image (A) and longitudinal relaxation rate (1/T1) vs.Gd (III) (B).
Fig. 4 pHPMA-DOTA-Gd, Core-crosslinked pHPMA-DOTA-Gd and the DTPA-Gd aqueous solution are in 1.5T
T in magnetic resonance1- weighted scanning image (A) and longitudinal relaxation rate (1/T1) vs.Gd (III) (B).
Fig. 5 injection concentrations are pHPMA-DOTA-Gd, core-crosslinked- of 0.08mmol Gd (III)/kg
The mouse of pHPMA-DOTA-Gd and DTPA-Gd scan image (A) mouse interior tumors position signal phase under different time points
To enhancing percentage (B), every group of 5 mouse.
Fig. 6 injection concentrations are pHPMA-DOTA-Gd, core-crosslinked- of 0.08mmol Gd (III)/kg
The mouse of pHPMA-DOTA-Gd and DTPA-Gd bladder position signal phase in scan image (A) Mice Bodies under different time points
To enhancing percentage (B), every group of 5 mouse.
Fig. 7 injection concentrations are the pHPMA-DOTA-Gd and Core-crosslinked of 0.08mmol Gd (III)/kg
The residual volume of internal vitals after pHPMA-DOTA-Gd24 hours；Every group of 5 mouse.
Fig. 8 pHPMA-DOTA-Gd, Core-crosslinked pHPMA-DOTA-Gd and DTPA-Gd are in 4T1And LO (A)2
(B) cytotoxicity experiment in cell line.
Fig. 9 red blood cells hemolytic experiment at different conditions:(A) and Core- in pHPMA-DOTA-Gd solution
In crosslinked pHPMA-DOTA-Gd solution (B)；Red blood cell is neutralized in the pHPMA-DOTA-Gd solution of various concentrations
(C) in Core-crosslinked pHPMA-DOTA-Gd solution
Figure 10 various concentrations (2mg/mL and 5mg/mL) pHPMA-DOTA-Gd (A) and Core-crosslinked
Influence of pHPMA-DOTA-Gd (B) solution for normal person's blood APTT and PT, PBS is control group.
Figure 11 observes pHPMA-DOTA-Gd (3mg/mL, A), Core-crosslinked pHPMA-DOTA-Gd by SEM
The influence of (3mg/mL, B) and PBS solution (C) for red cell morphology and concentration class.
The normal BALB/c mouses of Figure 12 inject the DTPA-Gd of 0.08mmol Gd (III)/kg, pHPMA-DOTA-Gd,
Core-crosslinked pHPMA-DOTA-Gd and physiological saline.Mouse Weight change (A) in 21 days, mouse after injecting 20 days
The residual volume (B) of vitals gadolinium；Every group of 5 mouse.
The normal BALB/c mouses of Figure 13 inject the DTPA-Gd of 0.08mmol Gd (III)/kg, pHPMA-DOTA-Gd,
Core-crosslinked pHPMA-DOTA-Gd and physiological saline.Mouse blood routine testing result after injecting 20 days；Every group 7
Mouse.
The normal BALB/c mouses of Figure 14 inject the DTPA-Gd of 0.08mmol Gd (III)/kg, pHPMA-DOTA-Gd,
Core-crosslinked pHPMA-DOTA-Gd and physiological saline.Mouse biochemistry detection result after injecting 20 days；Every group 7 small
Mouse.
Figure 15 injected Core-crosslinked pHPMA-DOTA-Gd, pHPMA-DOTA-Gd and physiological saline after 20 days
The tissue HE stained slices of internal vitals.
Specific embodiment
The preparation of the biodegradable core crosslinked, branched macromolecular gadolinium polymer of the invention of embodiment 1
1st, preparation method
The isobutyl imidazoline hydrochloride (VA044) of azo two, the thio phenyl of ring azo amidine class initiator V501,4- cyanopentanoic acid two
Formic acid (4-CTA) and trimethylolpropane tris (3-thiopropionate) are bought from sigma-Adrich companies.
Monomer HPMA (Eur.Polym.J., 1973,9,7-14), MA-DOTA (ACS Appl.Mater.Interfaces,
2016,8,10499-10512), PTEMA (Bioconjug.Chem., 1998,9,749-757) is prepared by existing document.
The preparation method of HPMA, PTEMA and MA-DOTA is as follows：
HPMA：Its structural formula isAccording to J,H.Poly[N-
(2-hydroxypropyl)methacrylamide]—I.Radical polymerization and
copolymerization.Eur Polym J.1973；9:It is prepared by HPMA preparation methods disclosed in 7-14.
MA-DOTA：Its structural formula isAccording to Ling Sunetal., Stimuli-
Responsive Biodegradable Hyperbranched Polymer-Gadolinium Conjugates as
Efficient and Biocompatible Nanoscale Magnetic Resonance Imaging Contrast
Agents, ACS Appl.Mater.Interfaces, 2016,8 (16), MA- disclosed in the accessories section of pp 10499-10512
It is prepared by DOTA methods.
PTEMA：Its structural formula isAccording to Wang, L., J.Kristensen, and
D.E.Ruffner,Delivery of antisense oligonucleotides using HPMA polymer:
Synthesis of A thiol polymer and its conjugation to water-soluble molecules.,
《Bioconjugate Chemistry》,1998,9(6):It is prepared by method disclosed in 749-757 methods part.
The mean molecule quantity (Mw) and polydispersity of synthetic material are detected using size exclusion chromatography (SEC)
(GE companies,System), using the sodium acetate solution containing 30% methyl alcohol as mobile phase.
Synthetic line figure of the invention is as shown in Figure 1.
The synthesis of linear HPMA copolymers (pHPMA-DOTA)：HPMA (1.4g, 9.75mmol), 4-CTA (12mg, 79 μ
Mol), MA-DOTA (3.34g, 6.5mmol) and VA044 (8.5mg, 26.3 μm of ol) inputs reaction bulb, adds deionized water/first
Alcoholic solution (5：1,22mL).Solution is full of argon gas 40 minutes at 0 DEG C, then rotates 8 hours at 44 DEG C.After solution is cooled down through liquid nitrogen
Polymer is settled out in acetone, obtains pink solid.Dialysis is freezed after 12 hours, obtains the thio phenyl first of pink product two
Perester radical reunion compound (pHPMA-DOTA with dithiobenzoate group, 45% yield, 2.15g).The polymer
's1H NMR spectra figures are shown in Fig. 2A, secondly the Hydrogen Proton peak of thiobenzoate ester group is 7.51-7.80ppm.Two thio phenyl first
Perester radical reunion compound (pHPMA-DOTA with dithiobenzoate group, 1.2g) is dissolved in V501 (10 times of amounts)
50ml methyl alcohol, reacts 3 hours in 70 DEG C of condensing refluxes, and accessory substance of dialysing away can obtain polymer by freeze-drying
(pHPMA-DOTA), yield 90%, 1.08g.The 1H NMR spectra figures of pHPMA-DOTA are shown in Fig. 2 B, secondly thiobenzoate ester group
Group is substituted, and 7.51-7.80ppmDE Hydrogen Protons peak disappears.
The synthesis of the linear copolymers of HPMA containing gadolinium (pHPMA-DOTA-Gd)：PHPMA-DOTA (800mg) and GdCl3·
6H2O (928mg, 2.5mmol) is dissolved in 25mL deionized waters, then adjusts pH to 5.2-5.4 with 0.1M NaOH solutions, so
Room temperature backspin turns 24 hours afterwards.Dialysis is freezed after 24 hours and obtains end-product (pHPMA-DOTA-Gd, 810mg), by ICP-MS
It is 6.6% to measure load Gd (III) amount, and molecular weight and distribution are shown in Table 1.
The synthesis of the pHPMA-DOTA copolymers of the thio group of pyridine two modification:Dithiobenzoic acid ester group polymer
(pHPMA-DOTA with dithiobenzoate group, 1.2g) and PTEMA (200mg, 0.79mmol) put into reaction bulb,
It is subsequently adding deionized water/methanol solution (3:1,8mL) and VA044 (3.2mg, 10 μm of ol), solution is full of 40 points of argon gas at 0 DEG C
Clock, then rotates 8 hours at 44 DEG C.Solution is settled out polymer in acetone after being cooled down through liquid nitrogen, is dried to obtain pink solid
Body (yield 75%y, 1.05g).Simultaneously the polymer (1.0g) of ester group containing dithiobenzoic acid and the thio group of pyridine two and
V501 (10 times of amounts) is dissolved in 50ml methyl alcohol, is reacted 3 hours in 70 DEG C of condensing refluxes, and accessory substance is removed out in dialysis, by freeze-drying
The polymer (Pyridine disulfide modified pHPMA-DOTA) of the thio group of pyridine two, yield can be obtained
91%, 0.91g.
The synthesis of the polymer (core-corsslinked pHPMA-DOTA) of core crosslinking：Under nitrogen protection, the sulphur of pyridine two
Polymer (Pyridine disulfide modified pHPMA-DOTA, 900mg) for group is dissolved in deionized water/first
Alcoholic solution (6:1,20mL) in, after being sufficiently stirred for, ethyl acetate/acetone (1:1,1mL) trimethylolpropane tris (the 3- mercaptos of dissolving
Base propionic ester) (20mg) be slowly added dropwise (1 hour) then proceed to stirring 6 hours.200mL acetone precipitations remove impurity, so
Afterwards by SEC instrumentsSystem is further purified, dialyses, it is lyophilized after obtain white product core cross-linked polymeric
Thing (Core-crosslinked pHPMA-DOTA, 810mg).
The synthesis of core crosslinking polymer containing gadolinium (core-corsslinked pHPMA-DOTA-Gd):Core cross-linked polymer
(Core-crosslinked pHPMA-DOTA, 800mg) and GdCl36H2O (928mg, 2.5mmol) reaction generation core crosslinkings
Polymer core-crosslinked pHPMA-DOTA-Gd, synthesis step is identical with pHPMA-DOTA-Gd synthesis steps.Pass through
ICP-MS measures containing for core crosslinking pHPMA-DOTA-Gd polymer (Core-crosslinked pHPMA-DOTA-Gd) gadolinium ions
It is 6.1% (weight percentage) to measure.Core crosslinking pHPMA-DOTA-Gd (core-crosslinked pHPMA-DOTA-Gd)
1H NMR spectra figures are shown in Fig. 2 C, and molecular weight characterization is shown in Table 1.
2nd, nature examination
2.1 detection methods
Weight-average molecular weight (Mw) and polydispersity (PDI) are measured by size exclusion chromatography (SEC).Polymer
Nuclear magnetic spectrogram is through HIGH RESOLUTION SUPERCONDUCTING nuclear magnetic resonance chemical analyser (Bruker companies of Switzerland, Bruker AV II-400MHz, Bruker
AV II-600MHz) measure.Gadolinium ion content is through icp mses (ICP-MS, Elan DRC-e, U.S.'s amber
Jin Aiermo) measure.
PHPMA-DOTA and core crosslinking pHPMA-DOTA-Gd (core-crosslinked pHPMA-DOTA- is detected simultaneously
Gd 1H NMR spectra figures).
2.2 testing results
Polymer (the Core- of straight chain polymer (pHPMA-DOTA-Gd) and the core crosslinking containing Gd coordination compound
Crosslinked pHPMA-DOTA-Gd) characterization result such as table 1：
Straight chain polymer (pHPMA-DOTA-Gd) and the polymer (Core- of core crosslinking that table 1. coordinates containing gadolinium
Crosslinked pHPMA-DOTA-Gd) sign：
The 1H NMR spectra figures of pHPMA-DOTA are shown in Fig. 2 B, secondly thiobenzoate ester group is substituted, 7.51-
7.80ppmDE Hydrogen Protons peak disappears.The 1H of core crosslinking pHPMA-DOTA-Gd (core-crosslinked pHPMA-DOTA-Gd)
NMR spectra figure is shown in Fig. 2 C, and molecular weight characterization is shown in Table 1.
Experimental result illustrates that the present invention has prepared linear pHPMA-DOTA-Gd, and its structural formula is：
Wherein, m=100, n=672；
The present invention has also prepared core crosslinking pHPMA-DOTA-Gd (core-crosslinked pHPMA-DOTA-
Gd), its structural formula such as following formula (II)：
Wherein, m=100, n=672, p=28, q=28；
Represent following structure (III) (of the pHPMA containing Gd coordination compound is single-stranded)：
Biology effect of the invention is proved with the mode of experimental example below：
The property of the polymer containing Gd coordination compound of the biodegradable core crosslinking of the invention of experimental example 1 and application
The polymer pHPMA-DOTA-Gd containing Gd coordination compound of core crosslinking prepared by Example 1, is tested as follows,
To detect its property：
1.1 biodegradable Journal of Sex Research
Core-crosslinked pHPMA-DOTA-Gd are dissolved in and contain the McIlvaine's containing glutathione (GSH)
Buffer solution (4mg/mL, 50mM citrate/0.1M phosphate, 2mM EDTA, 4mM glutathione, pH=5.4), 37 DEG C of incubations
12 hours, then by SEC (System, GE medical treatment) degradability of sample is detected, Superose
6HR10/30 prepacked columns load the sodium acetate solution containing 30% methyl alcohol (pH 6.5) as mobile phase (turnover rate：0.4mL/min).
1.2 external T1Water phase relaxation experiments
PH7.4PBS dissolved materials at room temperature, material concentration be correspondence Gd (III) solubility (0.1,0.15,0.2,0.25,
0.3,0.35,0.4,0.45,0.5mM), clinic is control group with Gd-DTPA, and 1.5T magnetic resonance imagings, T1 weighted magnetic resonances are swept
Retouch sequential parameter as follows：TE=8.7ms, TR=25,30,50,70,90,110,150,170,190,210,250,300,400,
600,700, and 800ms, Fov=200mm, slice thickness=2.0mm, matrix dimensions=256 ×
256.Relaxivity value is drawn by the slope of 1/T1 and Gd (III) ion linear relationship.
Magnetic resonance imaging in 1.3 bodies
Tested by internal magnetic resonance imaging, aggregation of the observation material in tumor locus.Using 4T1Cell mouse back skin
Mouse breast cancer subcutaneous model is set up in lower inoculation.First 7 × 105Individual 4T1Cell is accurately inoculated into the 6-7 week old BALB/c mouse back of the body
Portion, tumour growth to about 15mm3Mouse is randomly divided into 3 groups behind left and right, every group includes 5 mouse.Three groups of mouse difference tails are quiet
Arteries and veins injects pHPMA-DOTA-Gd, Core-crosslinked pHPMA-DOTA-Gd and clinical DTPA-Gd ((0.08mmol
Gd(III)/kg).Concentration class of the different time points material in tumor locus is obtained by 3.0T magnetic resonance scanners.Used before scanning
Phenobarbital anesthetized mice, is then fixed on customization coil by mouse.T1 weighted scanning sequences are as follows：TR=450ms, TE=
11ms, slices=11, voxel size=0.2 × 0.2 × 1.5mm, Fov=51mm.Before injection, 10 after injection, 30,45,
60th, scanned respectively after 180 minutes, obtain tumor locus focused image, then take tumour using magnetic resonance imaging system instrument circle
Position obtains concrete numerical value.The relative enhancing of signal is than (Δ SNR) computing formula below figure：
SI (tumor), SI (bladder) and SI (water) are respectively the signal intensity of tumour, bladder and moisture film.
1.4 distributions are tested
15 subcutaneous breast cancer model mouse are randomly divided into 3 groups, respectively by tail vein injection pHPMA-DOTA-Gd,
Core-crosslinked pHPMA-DOTA-Gd and DTPA-Gd (0.08mM Gd (III)/kg), mouse is put to death after 24 hours,
Important organ and tissue (heart, liver, spleen, lung, kidney, tumour) are taken out, 4% formalin is fixed.Organ and tissue PBS one
It is secondary, weigh.Use H2O2(1mL) and HNO3(3mL) dissolve organ, be heated to 120 DEG C until tissue digest completely, then spend from
Sub- water is diluted to 4mL.Final sample inductivity coupled plasma mass spectrometry (ICP-MS) measures remaining in each organ or tissue
The concentration of Gd.The content of internal residue Gd can be calculated by formula below：
1.5 Materials Cell toxicity tests
Its toxicity is evaluated by observing influence of the material to tumour cell and the cytoactive of normal cell.Experiment is used
4T1Cell (breast cancer cell) and LO2Cell (normal liver cell), two kinds of cells are inoculated into (5 × 10 in 96 orifice plates respectively3
Individual/hole, 100 μ L DMEM culture mediums after cultivating 24h in the incubator of 5%CO2, discard the culture in 96 well culture plates at 37 DEG C
Base.It is subsequently adding pHPMA-DOTA-Gd, Core-crosslinked containing various concentrations (25,50,100,200 μ g/mL)
The culture medium of pHPMA-DOTA-Gd and DTPA-Gd continues to be incubated 24 hours.24 as a child washed three cells with PBS, then per hole
Add Cytotoxic evaluation kit CCK-8 (Dojindo, Japan).After being incubated 2 hours, with ELIASA (Thermo
Fisher SCIENTIFIC) survey 450nm at absorbance.For the cytoactive for the treatment of is denoted as 100%, according to horizontal line and longitudinal direction
Comparative evaluation its cytotoxic effect.
1.6 erythrocyte hemolysis are tested
The experiment is used for evaluating whether material has an impact for red blood cell.Extracted with the anticoagulant tube containing sodium citrate normal
It is in a short time the volunteer's new blood for taking any medicine, 1000g is centrifuged 5min, and PBS is washed 3 times, discards supernatant liquor, is taken red
Cell and PBS are made into 16% red cell suspension.Then to being separately added into concentration respectively pHPMA- in 50uL cell suspensions
DOTA-Gd and Core-crosslinked pHPMA-DOTA-Gd to final concentration of 1,3,5mg/mL, it is right to set up deionized water
According to group, 37 DEG C be incubated 12 hours after 1000g centrifugation 5min, take the μ L of supernatant liquor 200 and be transferred in 96 orifice plates, ELIASA determine sample
OD value of the product in 540nm.Each concentration is set up three parallel groups and is tested.Percentage of hemolysis is counted by following formula
Calculate：
Hemolysis rate (%)=(A-B)/(C-B) × 100
A:The absorbance of nanoparticle sample solution test, B:The absorbance of PBS sample test, C:Aqueous sample
The absorbance of test.
1.7 activated partial thromboplastin times (APTT), prothrombin time (PT)
Healthy human body new blood (in anti-sodium citrate pipe) is extracted, 1000g centrifugation 5min take upper plasma.To
In the EP pipes of 1.5mL, add pHPMA-DOTA-Gd the and Core-crosslinked pHPMA-DOTA-Gd materials of 40 μ L molten
Liquid, is subsequently adding the blood plasma of 360 μ L, sets up same volume PBS for negative control group, is well mixed in whirlpool concussion instrument.Using
Tested on automatic coagulation analyzer, each sample sets up 3 Duplicate Samples.
1.8 red blood cell patterns and aggregation
Healthy human body new blood (in anti-sodium citrate pipe) is extracted, PBS, 1000g centrifugation 5min is added, upper strata is discarded
Clear liquid, is repeated twice, and collects red blood cell.To in the EP pipes of 1.5mL, the pHPMA-DOTA-Gd and Core- of 100 μ L are added
Crosslinked pHPMA-DOTA-Gd material PBS solutions, set up same volume PBS solution for control group, are subsequently adding 20 μ L's
Red blood cell, piping and druming is mixed.Incubation at room temperature 15min, centrifugation, abandoning supernatant adds the PBS paraformaldehyde solutions of 0.5mL 4%,
Piping and druming, mixes, fixed at least 1h.Fixed red blood cell is resuspended, draw 20 μ L uniform suspensions and be coated in 24 orifice plate bottoms, 5min
Secondary afterwards to use 75%, 85%, 95%, 100% ethanol water is dehydrated.Finally dried naturally under 25 degree of constant temperatures,
SEM scannings cell morphology and aggregation.
1.9 toxicity in vivo are tested
28 normal female BALB/c mouses (20 ± 2g), are randomly divided into 4 groups, weigh, and (experiment passes through zoopery to mark
The examination of Ethics Committee).Tail vein gives the DTPA-Gd of 0.08mmol Gd (III)/kg concentration respectively for wherein three groups mouse,
PHPMA-DOTA-Gd and Core-crosslinked pHPMA-DOTA-Gd, another set gives the physiological saline of same volume
As negative control, it is administered once within every 4 days, totally 3 times, every 2 days mouse weights once and are recorded after administration.After 20 days, small rathole
Ball send blood routine and biochemistry detection after taking blood, wherein after death taking out vitals (heart, liver, spleen, lung, kidney) at 5, is used after digestion
Inductivity coupled plasma mass spectrometry (ICP-MS) measures the concentration of remaining Gd in each organ or tissue, and calculates residue Gd in vivo
Content.Vitals (heart, liver, spleen, lung, kidney) are taken out after remaining 2 sacrifices, is fixed with 4% paraformaldehyde solution
48 hours, histotomy analysis was made in FFPE, HE dyeing.
1.10 data analyses
Experimental data means standard deviation, variance analysis and double tail T- check analyses, p<0.05 is considered as result has
Significant difference.
2. result and discussion
The degraded of 2.1 core cross-linked polymers
The effective ways for improving the aggregation of the relaxivity and tumor locus of polymer magnetic resonance imaging contrast agent are exactly to carry
Its molecular weight high, while the molecular structure regulation of polymer is arrived into branched structure.The polymer containing Gd coordination compound of core crosslinking
Polymer (C pHPMA- containing Gd coordination compound of the molecular weight of (Core-crosslinked pHPMA-DOTA-Gd) compared with its straight chain
DOTA-Gd, Mw=28kDa) molecular weight be significantly improved, reach 1-2 times.Due to the crosslinking of kernel, its molecular structure is adjusted
It is branched structure to save.Additionally, the need in order to meet RE, kidney threshold value requires that molecular weight is lower again.Therefore, biological can drop
The disulfide bond of solution is introduced in the kernel of cross-linked polymer.In the feelings that the glutathione (GSH) of tumour cell expression high is present
Under condition, the polymer of core crosslinking can be degraded into the fragment of low-molecular-weight.After being incubated 8 hours altogether with GSH, the polymer of crosslinking is
Degradable is low molecular weight product (being less than 30kDa) (table 2), and this size is less than kidney threshold value (50kDa).This degradability
Being attributed to GSH can cut disulfide bond.Result of study represents the polymer of this core crosslinking and completes to make once reaching tumor locus
For the task of mr contrast agent will degrade, so as to ensure their effective removing and biocompatibility.The core of table 2. is handed over
The molecular weight and PDI of the polymer (core-crosslinked pHPMA-DOTA-Gd) containing Gd coordination compound of connection, and its drop
The analysis that solution is changed over time.
2.2 external magnetic resonance imagings
As shown in figure 3, T under 3T magnetic resonance1Weighting MR imaging be displayed under identical Gd concentration, pHPMA-DOTA-Gd and
Core-crosslinked pHPMA-DOTA-Gd are in tumor locus signal intensity apparently higher than clinic DTPA-Gd, it is meant that
Two kinds of polymer tumor locus concentration class apparently higher than clinic DTPA-Gd.Simultaneous quantitative r1Relaxation rate shows pHPMA-
The r of DOTA-Gd and Core-crosslinked pHPMA-DOTA-Gd1Value is respectively 6.42 and 10.49mM-1·s-1, it is clinical
With DTPA-Gd [2.54mM-1·s-1, per gadolinium (III)] 3-4 times.Also same knot is obtained in 1.5T magnetic resonance
Really (see Fig. 4).Also, compared with pHPMA-DOTA-Gd, the former has more preferably Core-crosslinked pHPMA-DOTA-Gd
Magnetic resonance enhancing effect.
Magnetic resonance imaging in 2.3 bodies
Based on external T1Imaging effect, we have further probed into magnetic resonance imaging in mouse model body, and observation material exists
The enhancing effect of tumor locus.As shown in figure 5, by internal magnetic resonance imaging picture this it appears that injection clinic contrast
After agent DTPA-Gd, mouse tumor position intensity was first raised before 10 minutes to 180 minutes and declined afterwards, and injected two kinds of materials
Afterwards, tumor locus signal intensity substantially increases, and downward trend does not occur.
In order to more accurately compare, we have done quantitative analysis to tumor locus reinforcing, by the tumour of different time points
The signal intensity (SI) at position quantifies enhancing effect.As shown in fig. 6, the clinical mouse with DTPA-Gd of tail vein injection, with water
Film is compared, and signal enhancing is obvious (about 220%) after 10 minutes, but signal intensity begins to decline after 30 minutes, after 180 minutes
Return to level before injection.And apply the mouse of pHPMA-DOTA-Gd and Core-crosslinked pHPMA-DOTA-Gd to swell
Knurl position signal strengthens after 10 minutes, then proceedes to increase, and signal intensity is apparently higher than (about 240- before injection after 180 minutes
260%).Both compare, and the enhancing effect of different time points Core-crosslinked pHPMA-DOTA-Gd compares pHPMA-
DOTA-Gd is more obvious.The EPR effect each side factors of entity tumor performance are relevant, Core-crosslinked pHPMA-
DOTA-Gd possesses the size being more suitable for than pHPMA-DOTA-Gd through tumor neogenetic blood vessels gap, and aggregation tumor locus reach
Enhanced effect.
It is worth noting that, the change of the bladder signal intensity of mouse model is just with tumor locus conversely, such as Fig. 6 institutes
Show.The clinical mouse with DTPA-Gd of injection is above injection Core-crosslinked pHPMA-DOTA- in different time points
The mouse of Gd and pHPMA-DOTA-Gd.This also reflects two kinds of materials circulation time in vivo than clinic from another point of view
It is long with DTPA-Gd, the reason for molecular weight may be also due to.Mouse bladder is more prone to filter the DTPA-Gd of small molecule, and big
Molecular medicine is then exhausted from external after the effect of internal enzyme is degraded after being circulated throughout in vivo.Such characteristic also ensure that material
Biological safety, be conducive to future utilization.
The vivo biodistribution distribution of 2.4 materials
A key factor for influenceing signal intensity is exactly the internal distribution of material.As shown in fig. 7, with clinic DTPA-
Gd is compared, Core-crosslinked pHPMA-DOTA-Gd and pHPMA-DOTA-Gd tumor locus and other are heavy after 24 hours
The residual volume of organ gadolinium is wanted to dramatically increase.The gadolinium residual volume of tumor locus also just mutually confirms with magnetic resonance signal intensity, again
Demonstrate two kinds of enhancing effects of material.Secondly, the residual volume of the gadolinium of other organs also substantially increases, and is material the reason for possible
Molecular weight and size it is larger, circulation time is more long in vivo, in organ blood supply enrich, causing the residual volume of gadolinium increases.
The cytotoxicity of 2.5 materials
Tested by CCK-8, we have further probed into material to tumour cell and the cytotoxic effect of normal cell.
As shown in figure 8, compared with clinic is with DTPA-Gd, material is in 4T1The cytoactive of tumour cell is lower and in LO2Normal liver cell
Active (from 25 μ of μ g/mL to 200 g/mL) higher.Biocompatibility and the surface of our material compositions should be mainly due to
The factor such as negatively charged.This also demonstrates the good biological safety of material.
2.6 Hemocompatibility Tests
Influence of the material for erythrocyte hemolysis：Erythrocyte surface can combine to form immune complex with some antibody,
Activating complement causes erythrocyte hemolysis.Material enters in human body, is in contact with cell membrane first, may cause complement activation, institute
Influence with analysis of material to erythrocyte hemolysis is necessary.In our experiment, as shown in figure 9,1.0,2.0,
, by being incubated 12 hours, without haemolysis is found, highest hemolysis rate is again below 1% for the material of 5.0mg/mL concentration.And it is beautiful
Testing of materials association of state standard points out that acceptable hemolysis rate should be less than 5%, so two kinds of materials have good bio-safety
Property.This result is likely to be caused by several factors, surface negative charge and it is degradable the features such as may play important function.
APTT and PT:For normal body, some endogenous and/or exogenous material can stimulate blood coagulation in blood
The activation of the factor, hematoblastic aggregation, the activation of fibrinogen ultimately results in blood coagulation, so the detection reaction blood of APTT and PT
The experiment in liquid clotting time is particularly important.As shown in Figure 10, compare with PBS negative controls, the APTT and PT under different concentration
Time within normal range (NR), illustrate that material does not have too much influence for blood coagulation function.
Influence of 2.7 materials for erythrocyte aggregation and form
According to document report before, the electronic interaction between erythrocyte surface negative electrical charge and positively charged material can
The coherent condition of the form of red blood cell can be had influence on, while partially hydrophobic structure in material also can be to red blood cell shadow
Ring, therefore the form and coherent condition of observation red blood cell are also necessary.As shown in figure 11, the material of various concentrations and
PBS compares, and the form of red blood cell and aggregation all do not change significantly.Under low power, the red blood cell being incubated by material has
There is good dispersiveness, without the performance for exception occur；Under high power, Surface of Erythrocytes is smooth complete, and normal concave-concave is presented
Disc-shaped structure, compares with PBS control group, does not have obvious structure change.It is therefore believed that pattern of the material to red blood cell
Do not negatively affected significantly with coherent condition, blood safety is good.
Toxic action of 2.8 materials for normal mouse
Material for normal mouse whether have toxic action can concentrated expression go out the biological safety of material.Tail vein
After injection material, mouse do not show to be dehydrated, move other animal toxicity correlated characteristics such as discoordination, muscular atrophy, anaemia.
Mouse Weight change is as shown in figure 12, with saline control, material group and DTPA-Gd groups without too big change, locate
Within the scope of normal growth.Changes of weight explanation material does not have obvious toxic and side effect for normal mouse.Blood routine and life
Changing detection can reflect blood status and hepatic and renal function in Mice Body.As shown in figure 13, Core-crosslinked is injected
The mouse routine blood indexes of pHPMA-DOTA-Gd and pHPMA-DOTA-Gd：Red blood cell (RBC), hemoglobin (HGB), red blood cell
Hematocrit (HCT), platelet count (PLT), mean platelet volume (MPV), leukocyte count (WBCs), all indexs and injection
DTPA-Gd group no significant differences.As shown in figure 14, injection Core-crosslinked pHPMA-DOTA-Gd and pHPMA-
The mouse biochemical analysis index of DOTA-Gd：Glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminease (AST), alkaline phosphatase (ALP), paddy
Aminoacyl transferases (GGT), urea nitrogen (BUN), creatinine (CRE), all indexs and injection DTPA-Gd group no significant differences.In order to
Toxicity in vivo is further verified, organ-tissue slice analysis do not find the change (figure such as histologic lesion, inflammation after sacrifice
15)。
3. conclusion
In sum, the relaxation rate of pHPMA-DOTA-Gd and Core-crosslinked pHPMA-DOTA-Gd of the present invention
r1Value is respectively 6.42 and 10.49mM-1·s-1, it is clinic DTPA-Gd [2.54mM-1·s-1,per gadolinium
(III)] 2.5-4.1 times, internal magnetic resonance imaging experiment also further verify they as a comparison agent be used for in-vivo imaging
Effect is very good, while degradable, internal in vitro toxicity experiment shows that material has good biocompatibility and biological peace
Quan Xing.
To sum up, biodegradable core crosslinked, branched macromolecular gadolinium polymer Core-crosslinked pHPMA- of the invention
DOTA-Gd has relaxation rate high, for the excellent effect of in-vivo imaging, while degradable, nontoxic, good biocompatibility, faces
Bed application prospect is excellent.
Claims (20)
1. a kind of biodegradable linear HPMA polymer, it is characterised in that：Its structural formula such as following formula (I)：
Wherein, m=57~170, n=380~1150.
2. linear HPMA polymer according to claim 1, it is characterised in that：In the structural formula of the polymer, m=
100, n=672.
3. polymer according to claim 1 and 2, it is characterised in that：The weight-average molecular weight of the polymer is 10
~40kDa, preferably 28kDa.
4. polymer according to claim 1 and 2, it is characterised in that：In the polymer percentage by weight of Gd be 4~
10%, preferably 6.6%.
5. polymer according to claim 1 and 2, it is characterised in that：The PDI of the polymer<2.5, preferably 1.09.
6. the HPMA polymer that a kind of biodegradable core is crosslinked, it is characterised in that：Its structural formula such as following formula (II)：
Wherein, m=57~170, n=380~1150, p=16~50, q=16~50；
Represent following structure (III)：
7. polymer as requested described in 6, it is characterised in that：In the structural formula of the polymer, m=100, n=672, p=
28, q=28.
8. the polymer according to claim 6 or 7, it is characterised in that：The weight-average molecular weight of the polymer is 100
~300kDa, preferably 181kDa.
9. the polymer according to claim 6 or 7, it is characterised in that：In the polymer percentage by weight of Gd be 4~
10%, preferably 6.1%.
10. the polymer according to claim 6 or 7, it is characterised in that：The PDI of the polymer<2.5, preferably
1.95。
A kind of 11. methods for preparing polymer described in Claims 1 to 5 any one, it is characterised in that：Step is as follows：
(1) synthesis of linear copolymer pHPMA-DOTA：HPMA, 4-CTA, MA-DOTA and VA044 are taken, adds water/methyl alcohol molten
Liquid, reaction is settled out polymer in acetone after being cooled down through liquid nitrogen, obtains pink solid, is freezed after dialysis, obtains two thio phenyls
Carbamate group polymer；Dithiobenzoic acid ester group polymer and V501 are dissolved in methyl alcohol, heating response is frozen after dialysis
It is dry, obtain final product pHPMA-DOTA；
(2) synthesis of the linear copolymers of HPMA containing gadolinium：By pHPMA-DOTA and GdCl3·6H2O is dissolved in water, and then adjusts pH
To 5.2-5.4, reaction is freezed after dialysis, obtains final product pHPMA-DOTA-Gd.
12. methods according to claim 10, it is characterised in that：In step (1), described HPMA, 4-CTA, MA-DOTA and
The mol ratio of VA044 is：9.75mmol：79μmol：6.5mmol：26.3μmol；In the water/methanol solution, water and methyl alcohol
Volume ratio is 5：1；Per 9.75mmolHPMA, 22mL water/methanol solution is added；The reaction is that solution is full of argon gas at 0 DEG C
40 minutes, 8h then was rotated at 44 DEG C；The time of the dialysis is 12h.
13. methods according to claim 10, it is characterised in that：In step (1), the reaction of white solid and V501 be
24h is rotated at room temperature；The time of dialysis is 24h.
14. methods according to claim 10, it is characterised in that：In step (2), the pHPMA-DOTA and GdCl3·
6H2The mass ratio of O is 800：928mg, the water that the pHPMA-DOTA per 800mg is used is 25mL；The time of dialysis is 24h.
A kind of 15. methods for preparing polymer described in claim 6~10 any one, it is characterised in that：Step is as follows：
1) synthesis of dithiobenzoic acid ester group polymer：HPMA, 4-CTA, MA-DOTA and VA044 are taken, water/methyl alcohol is added
Solution, reaction is settled out polymer in acetone after being cooled down through liquid nitrogen, obtains pink solid, is dithiobenzoic acid ester group
Reunion compound (pHPMA-DOTA with dithiobenzoate group)；
2) synthesis of the pHPMA-DOTA copolymers of the thio group of pyridine two modification:Take dithiobenzoic acid ester group polymer
(pHPMA-DOTA with dithiobenzoate group) and PTEMA, add water/methanol solution and VA044, reaction, warp
Polymer is settled out in acetone after liquid nitrogen cooling, obtains pink solid；Pink solid and V501 are dissolved in methyl alcohol, are heated
Reaction, freezes after dialysis, obtains the HPMA copolymers of the thio group of pyridine two modification；
3) core is crosslinked the synthesis of pHPMA-DOTA polymer：Under nitrogen protection, the HPMA copolymers of the thio group of pyridine two modification
It is dissolved in deionized water/methanol solution, after being sufficiently stirred for, by trimethylolpropane tris (the 3- mercaptos of ethyl acetate/acetone solution
Base propionic ester) it is slowly added dropwise, then stirring reaction；Acetone sloughs impurity, then by SEC instruments/FPLC
System is further purified, dialyse, it is lyophilized after obtain white product for core is crosslinked pHPMA-DOTA polymer；
4) core is crosslinked the synthesis of pHPMA-DOTA-Gd polymer：Core is crosslinked pHPMA-DOTA polymer and GdCl3·6H2O is molten
In Xie Shui, pH to 5.2-5.4 is then adjusted, reaction is freezed after dialysis, obtain final product core crosslinking pHPMA-DOTA-Gd polymer.
16. methods according to claim 15, it is characterised in that：Step 1) in, described HPMA, 4-CTA, MA-DOTA and
The mol ratio of VA044 is：9.75mmol：79μmol：6.5mmol：26.3μmol；In the water/methanol solution, water and methyl alcohol
Volume ratio is 5：1；Per 9.75mmolHPMA, 22mL water/methanol solution is added；The reaction is that solution is full of argon gas at 0 DEG C
40 minutes, 8h then was rotated at 44 DEG C；The time of the dialysis is 12h.
17. methods according to claim 15, it is characterised in that：Step 2) in, the ester group containing dithiobenzoic acid
The mass ratio of polymer (pHPMA-DOTA with dithiobenzoate group) and PTEMA be 6:1；Added during reaction
Water/methanol solution in, the volume ratio of water and methyl alcohol is 3：1；Per 1.2g pHPMA-DOTA, 1.8mL water/methanol solution is added
With 3.2mg VA044；The reaction is that solution full of argon gas 40 minutes, is then rotated 8 hours at 44 DEG C at 0 DEG C；White is solid
When body is dissolved in water/methanol solution, water and the volume ratio of methyl alcohol in the water/methanol solution are 4：1, with the starting reacted
Thing polymer pHPMA-DOTA is counted for 1.2g, and the consumption of water/methanol solution is 8mL；
Step 2) in, the pink solid is reacted 3 hours with 10 times of V501 of amount at 70 DEG C.
18. methods according to claim 15, it is characterised in that：Step 3) in, repaiied per the thio group of 900mg nitrogen pyridine two
The pHPMA-DOTA of decorations is dissolved in 20mL water/methanol solution；In the water/methanol solution, water is 6 with the volume ratio of methyl alcohol：
1；In the ethyl acetate/acetone, the two volume ratio is 1:1；Trimethylolpropane tris (3-thiopropionate) are added per 20mg
In the ethyl acetate/acetone of 1mL；Time for adding of the trimethylolpropane tris (3-thiopropionate) per 20mg is 1 hour；
React for stirring reaction 6 hours；Using SEC instruments during the purifying/ FPLC systems are purified.
19. methods according to claim 15, it is characterised in that：Step 4) in, the pHPMA-DOTA and GdCl3·
6H2The mass ratio of O is 800：928mg, the water that the pHPMA-DOTA per 800mg is used is 25mL；The time of dialysis is 24h.
Purposes of the polymer described in 20. claim 1~10 any one in the contrast-enhancing agent for preparing magnetic resonance imaging.
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| CN108478814A (en) * | 2018-03-16 | 2018-09-04 | 甘肃农业大学 | Poly- (HPMA)-DOTA-Gd mri contrast agents and preparation method thereof |
| CN109503770A (en) * | 2018-10-18 | 2019-03-22 | 合肥工业大学 | A kind of water-soluble cross-linked nanometer polymer and its preparation method and use with high relaxation rate |
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