CN106880848B - Biodegradable poly HPMA-Gd magnetic resonance imaging probe and preparation method thereof - Google Patents
Biodegradable poly HPMA-Gd magnetic resonance imaging probe and preparation method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
- A61K49/128—Linear polymers, e.g. dextran, inulin, PEG comprising multiple complex or complex-forming groups, being either part of the linear polymeric backbone or being pending groups covalently linked to the linear polymeric backbone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61K49/14—Peptides, e.g. proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
Abstract
The present invention provides a kind of biodegradable poly HPMA-Gd magnetic resonance imaging probes, will be in the polypeptide GFLG insertion poly HPMA backbone structure of enzyme sensitivity；The polymer molecular weight of preparation is substantially controllable, and molecular weight polydispersity coefficient (PDI) is low；And by being coupled the HPMA-DOTA-Gd linearized with Gd (III).The probe is the nanoscale MRI magnetic resonance imaging probe of efficient tool biocompatibility, can be used for clinical tumor diagnosis and adjuvant treatment.
Description
Technical field
The invention belongs to medical imaging technology fields, are related to a kind of high molecule magnetic resonance image-forming probe, and in particular to a kind of
Biodegradable poly HPMA-Gd magnetic resonance imaging probe.
Background technique
Since Magnetic resonance imaging (MRI) has high spatial resolution, 3D rendering information and the advantages such as "dead", have become
For current effective tumour Non-Invasive Method.According to incompletely statistics, clinically 50% or more MRI diagnosing tumor need using
MRI magnetic resonance imaging probe.Clinically used MRI magnetic resonance imaging probe includes that Magnevist Solution (Magnevist) and gadolinium are special
Sour Portugal's amine (Dotarem).However, they belong to small molecule magnetic resonance imaging probe, that there are sensibility is low, non-specific, very
Fastly from internal the disadvantages of removing；And mainly by causing to be extremely difficult in Passive diffusion to mesenchyma stroma of tumors or tissue interstitial
Satisfied imaging effect.Therefore, MRI magnetic resonance imaging probe that is superior, can overcoming disadvantage mentioned above has very high research
Value, and the nano-complex based on Gd (III) illustrates huge potentiality in the research of novel magnetic resonance image probe.
Summary of the invention
Based on above-mentioned technical problem, the present invention constructs biodegradable poly HPMA-Gd magnetic resonance imaging probe, is
A kind of macromolecule MRI magnetic resonance imaging probe of efficient tool biocompatibility, can be used for clinical tumor diagnosis and auxiliary
Treatment.
Technical scheme is as follows:
A kind of biodegradable poly HPMA-Gd magnetic resonance imaging probe, chain structure are as follows:
Wherein: m:n:o=35~45:410~450:65~70；
The present invention is designed and is characterized even with higher molecular weight, biodegradable poly HPMA polymer-Gd (III)
The nanometer system for closing object, C, B and A group put in order indefinite in system, and R group can connect any one in C, B and A.
Poly HPMA (N- (2- hydroxypropyl) Methacrylamide) can in conjunction with multiple small-molecular-weight Gd (III) conjugates (DOTA) to
Relaxivity is greatly improved, and there is disimmune, nontoxicity, water solubility and good bio-compatibility；Meanwhile macromolecule
Nano material can extend circulation time in blood, increase the ability of EPR (infiltration retention effect) passive target, improve
Magnetic resonance imaging probe tumor locus concentration class, to increase the imaging effect and therapeutic effect of tumor locus.
The cyclic peptide cRGDyK of arginine-glycine-aspartic acid (Arg-Gly-Asp RGD) is a kind of specific tumour target
To small peptide, the present invention is surface modified material by introducing the R1 group with cRGDyK, makes it have active targeting function.
CRGDyK can be by αvβ3The identification and combination of integrin specificity, and αvβ3In kinds of tumor cells and tumor neogenetic blood vessels
Expression quantity in chrotoplast is significantly raised, but expresses lower in normal tissue cell or even do not express.
Although magnetic resonance imaging probe can be improved in the aggregation of tumor locus, kidney in the HPMA copolymer of high molecular weight
Dirty excretion threshold value requires it to be necessarily less than 50kDa again, otherwise Gd will be caused to be poisoned.Since HPMA main chain does not have biodegrade
Property, therefore biodegradable group must be introduced in main chain.Design construction of the present invention insertion cathepsin B substrate GFLG
The HPMA polymer of polypeptide allows to be degraded in the presence of Lysosomal cathepsin B small less than 50kDa
Segment.And Lysosomal cathepsin B as a kind of lysosomal cysteine protease in kinds of tumors and tumor vascular endothelium
Cell has high expression.Relative to traditional HPMA polymer, the degradable HPMA Nano medication delivery system of the present invention is shown
Higher antitumor efficiency and biological safety.
The average molecular weight of the magnetic resonance imaging probe is 85-94KDa.The molecular weight is degraded under tumor microenvironment
Molecular weight is lower than the segment of 50kDa, can take into account antitumous effect and biological safety, can be removed out body after guaranteeing degradation
It is interior.
Preferably, wherein m:n:o=40:443:66.In order to guarantee that water-soluble macromolecule has certain gadolinium ion
Content (mass percentage is generally 3-10%), it is ensured that effective magnetic resonance imaging.Meanwhile o value is adjusted, guarantee cRGDyK
Content (mass percentage be greater than 10%), to improve its tumor-targeting.
The present invention passes through reversible addion-fragmentation chain transfer (RAFT) polymerization first, by polypeptide glycine-phenylpropyl alcohol of enzyme sensitivity
In propylhomoserin-leucine-glycine (GFLG) insertion poly HPMA backbone structure；And by being coupled with Gd (III), synthesizing linear
HPMA-DOTA-Gd.Herein on basis, introduces polypeptide cRGDyK and become the MRI with potential active tumor-targeting
Magnetic resonance imaging probe HPMA-DOTA-Gd-cRGD.
The preparation method of poly HPMA-Gd magnetic resonance imaging probe of the present invention, as R1=SH, comprising the following steps:
The synthesis of poly HPMA-Gd conjugate (pHPMA-DOTA-Gd):
1) HPMA, MA-DOTA, pTEMA and CTA-GFLGK-CTA the deionized water containing VA044/methanol is dissolved in mix
Solution, is placed in 40-60 minutes exuberant in argon gas, is then stirred to react；Solution be quenched with liquid nitrogen after through acetone precipitation, pyridine is precipitated
Two sulphur modification product pHPMA-DOTA.
Preferably, deionized water and methanol are mixed to get the mixed solution in step 1) with the volume ratio of 4:1.
It is further preferred that the concentration of VA044 containing initiator in the mixed solution, VA044 are chain-transferring agent CTA-
The 1/3-1/5 equivalent of GFLGK-CTA.When for 1/3 equivalent, polymerization reaction can be rapidly completed, and molecular weight and PDI can get it is effective
Control.It polymerize (RAFTpolymerization by RAFT；Reversible addion-fragmentation chain transfer polymerization) preparation polymer molecule
Amount is substantially controllable, and molecular weight polydispersity coefficient (PDI) is low.
2) under the conditions of nitrogen protection, two sulphur modification product pHPMA-DOTA of pyridine is dissolved in deionized water, DTT is added
It is stirred to react, obtains intermediate product；By intermediate product and GdCl3·6H2O is dissolved in distilled water, after being stirred to react, adjusts pH value,
After reaction is stirred at room temperature again, pHPMA-DOTA-Gd product is obtained.
It is proposed by fast protein chromatographic column, eluant, eluent is water and methanol with the mixed liquor of 7:3, passes through deionized water dialysis
Intermediate product is obtained with freeze-drying；
Preferably, step 2) the adjusting pH value guarantees the coordination of gadolinium ion to 5.2-5.4, while guaranteeing the steady of small peptide
It is qualitative.
The present invention also provides another preparation methods of poly HPMA-Gd magnetic resonance imaging probe, when R1 is not SH,
The synthesis of pHPMA-DOTA-Gd-cRGD the following steps are included:
It takes pHPMA-DOTA-Gd to be dissolved in deionized water, while the cRGD of two sulphur modification of pyridine modification is added, lead to after reaction
Fast protein post separation and dialysis purification are crossed, pHPMA-DOTA-Gd-cRGD is obtained.
Present invention has an advantage that
1, the present invention is designed and is characterized with higher molecular weight, biodegradable poly HPMA polymer-Gd (III)
The nanometer system of conjugates, main polymer chain are made of hydrophilic and hydrophobic block, and introduce enzyme sensitivity small peptide at main chain center
GFLGK.Gd (III) is self-assembly of nanometer by way of degradable amphipathic block HPMA polymer-Gd (III) conjugate
Particle is delivered.Poly HPMA can greatly improve relaxation in conjunction with multiple small-molecular-weight Gd (III) conjugates (DOTA)
Henan efficiency, and there is disimmune, nontoxicity, water solubility and good bio-compatibility；Meanwhile the nano material of macromolecule
Circulation time in blood can be extended, increase the ability of EPR passive target, improve magnetic resonance imaging probe in tumor locus
Concentration class, to increase the imaging effect and therapeutic effect of tumor locus.
2, the present invention is surface modified material by introducing the R1 group with cRGDyK, makes it have active targeting function
Energy；Biodegradable GFLGK peptide group is introduced in main chain, allows to the feelings existing for Lysosomal cathepsin B
It is degraded to the small fragment less than 50kDa under condition, has both the nanoscale MRI magnetic of efficient tumor-targeting and biocompatibility
Resonance image-forming probe, be suitable for clinical tumor diagnosis and assist in the treatment of etc..
3, preparation method of the invention designs and passes through RAFT and " click " chemical building pHPMA-DOTA- of functionalization
Gd and pHPMA-DOTA-Gd-cRGD conjugate.It is polymerize first by reversible addion-fragmentation chain transfer (RAFT), enzyme is sensitive
In polypeptide Gly-Phe-leucine-glycine (GFLG) insertion poly HPMA backbone structure；The polymer of preparation point
Son amount is substantially controllable, and molecular weight polydispersity coefficient (PDI) is low；And by being coupled with Gd (III), the HPMA- of synthesizing linear
DOTA-Gd.Herein on basis, introduces polypeptide cRGDyK and become the MRI magnetic resonance with potential active tumor-targeting
Image probe HPMA-DOTA-Gd-cRGD.HPMA-Gd (III) conjugate (> 90kDa) of the invention can be degraded to lower than kidney
The low molecular weight product (< 44kDa) of dirty threshold value.With clinically diethyl pentetic acid-Gd (DTPA-Gd) magnetic resonance
Image probe is compared, and longitudinal relaxation efficiency (T1) is the former more (15.16mM of three times-1·s-1/Gd)。
4, the raising relaxivity of poly HPMA-Gd magnetic resonance imaging probe, is significantly higher than the 3 of clinical reagent DTPA-Gd
Times or more, it is significantly higher than clinical reagent in the magnetic resonance signal intensity of tumor locus；Meanwhile it is degradable under tumor microenvironment
It for the product of low molecular weight, is quickly excreted convenient for it, reduces toxic side effect.
Detailed description of the invention
Fig. 1 is the synthetic route chart of poly HPMA-Gd (III) conjugate of the present invention.
Fig. 2 is pHPMA-DOTA-Gd's and pHPMA-DOTA-Gd-cRGD1HNMR spectrogram.
Fig. 3 is A.T1Weighted mri imaging results；B. the longitudinal relaxation rate (1/T of various concentration1) (solvent: PBS, 1.5T).
Fig. 4 is that (0.08mmolGd (III)/kg is small by A.DTPA-Gd, pHPMA-DOTA-Gd or pHPMA-DOTA-Gd-cRGD
Mouse weight) administration after different time points tumour and bladder axis MR imaging Typical Representative.B. mouse tumor tissue is with respect to water mould
Signal enhancing ratio (* P ﹤ 0.05, * * P ﹤ 0.01).C. signal enhancing ratio (* * P the ﹤ 0.001) (n of mouse bladder with respect to water mould
=5).
Fig. 5 is the quantitative analysis results (n=of Gd (III) content in U87 Transplanted tumor model BALB/c nude mice different tissues
5)。
Fig. 6 is the cytotoxicity analysis of L02 cell (A) and U87 cell (B).
Fig. 7 is erythrocyte splitting situation after different materials processing: A.pHPMA-DOTA-Gd；B.pHPMA-DOTA-Gd-
cRGD；C. the opposite hemolysis rate (mean ± SD) of red blood cell after various concentration is handled.
Fig. 8 is the influence that different materials processing is detected by SEM to erythrocyte aggregation and form: A.PBS is compareed；
B.pHPMA-DOTA-Gd；C.pHPMA-DOTA-Gd-cRGD.
Fig. 9 is influence of the different materials processing to APTT and PT: A.pHPMA-DOTA-Gd；B.pHPMA-DOTA-Gd-
cRGD。
Figure 10 is after giving PBS (A), pHPMA-DOTA-Gd (B) and pHPMA-DOTA-Gd-cRGD (C) respectively, and difference is small
Mouse organ histology analyzes result (100 ×).
Specific embodiment
Property content is described in further detail for the essence of the present invention With reference to embodiment.
Embodiment 1
A kind of biodegradable poly HPMA-Gd magnetic resonance imaging probe, chain structure are as follows:
Wherein: m:n:o=35~45:410~450:65~70；
C, B and A group put in order indefinite in system, and R group can connect any one in C, B and A.
Embodiment 2
On the basis of embodiment 1:
Wherein, R1 SH, m:n:o=35:410:65.
The average molecular weight of the magnetic resonance imaging probe is 85KDa.
Embodiment 3
On the basis of embodiment 1:
Wherein, R1 is not SH, m:n:o=45:450:70.
The average molecular weight of the magnetic resonance imaging probe is 94KDa.
Embodiment 4
On the basis of embodiment 1:
Wherein, R1 is not SH, m:n:o=40:443:66.
The average molecular weight of the magnetic resonance imaging probe is 90KDa.
Embodiment 5
The preparation method of the magnetic resonance imaging probe of embodiment 2, comprising the following steps:
1) HPMA, MA-DOTA, pTEMA and CTA-GFLGK-CTA the deionized water containing VA044/methanol is dissolved in mix
Solution, is placed in 40-60 minutes exuberant in argon gas, is then stirred to react；Solution be quenched with liquid nitrogen after through acetone precipitation, pyridine is precipitated
Two sulphur modification product pHPMA-DOTA；
2) under the conditions of nitrogen protection, two sulphur modification product pHPMA-DOTA of pyridine is dissolved in deionized water, DTT is added
It is stirred to react, obtains intermediate product；By intermediate product and GdCl3·6H2O is dissolved in distilled water, after being stirred to react, adjusts pH value,
After reaction is stirred at room temperature again, pHPMA-DOTA-Gd product is obtained.
Embodiment 6
The preparation method of the magnetic resonance imaging probe of embodiment 2, comprising the following steps:
1) HPMA, MA-DOTA, pTEMA and CTA-GFLGK-CTA the deionized water containing VA044/methanol is dissolved in mix
Solution, is placed in 40-60 minutes exuberant in argon gas, is then stirred to react；Solution be quenched with liquid nitrogen after through acetone precipitation, pyridine is precipitated
Two sulphur modification product pHPMA-DOTA.
Deionized water and methanol are mixed to get the mixed solution in step 1) with the volume ratio of 4:1；All polymerization reactions are divided
The solvent used from purification is three classes solvent.
The concentration of VA044 containing initiator in the mixed solution, VA044 are the 1/3- of chain-transferring agent CTA-GFLGK-CTA
1/5 equivalent.When for 1/3 equivalent, polymerization reaction can be rapidly completed, and molecular weight and PDI can get effectively control.
2) under the conditions of nitrogen protection, two sulphur modification product pHPMA-DOTA of pyridine is dissolved in deionized water, DTT is added
It is stirred to react, obtains intermediate product；It is proposed by fast protein chromatographic column, eluant, eluent is water and methanol with the mixed liquor of 7:3, is led to
It crosses deionized water dialysis and freeze-drying obtains intermediate product；The intermediate product and GdCl3·6H2O is dissolved in distilled water, stirring
After reaction, pH value is adjusted to 5.2-5.4, after reaction is stirred at room temperature, obtains pHPMA-DOTA-Gd product.
Embodiment 7
The preparation method of the magnetic resonance imaging probe of embodiment 3 and 4, synthetic route are shown in Fig. 1, in 5 preparation method of embodiment
On the basis of:
3) it takes pHPMA-DOTA-Gd to be dissolved in deionized water, while the cRGD of two sulphur modification of pyridine modification is added, after reaction
By fast protein post separation and dialysis purification, pHPMA-DOTA-Gd-cRGD is obtained.
Embodiment 8
The preparation method of the magnetic resonance imaging probe of embodiment 3 and 4, on the basis of 6 preparation method of embodiment:
3) under the conditions of nitrogen protection, two sulphur modification product pHPMA-DOTA-Gd of pyridine is dissolved in deionized water, is added
Two sulphur modification cRGD of pyridine is stirred to react；It is proposed by fast protein chromatographic column, eluant, eluent is water and methanol with the mixing of 7:3
Liquid passes through deionized water dialysis and freeze-drying product pHPMA-DOTA-Gd-cRGD.
Experimental method
Material and method
Two isobutyl imidazoline hydrochloride (VA044) of azo is bought from sigma-Adrich.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)、CTA-GFLGK-CTA(Biomaterials 2013,
34 (33), 8430-8443) by existing document preparation.The synthetic method of two sulphur modification cRGD of pyridine is shown in document
(ACSAppl.Mater.Interfaces,2016,8,10499-10512).The number-average molecular weight (Mn) of synthetic material is divided equally again
Son amount (Mw) and polydispersity be all made of size exclusion chromatography (SEC) detected (GE company,System
System), use the sodium acetate solution containing 30% methanol as mobile phase.Zeta potential is detected by nano particle size and potentiometric analyzer
(Malvern instrument, the prefecture Wu Site, Britain), and data processing is carried out using DTS software, as a result shown with frequency curve chart.
The degradable HPMA copolymer-Gd conjugate of diblock formula main chain and as MRI magnetic resonance imaging probe, can be with
Increase the aggregation in tumor locus by EPR effect to improve tumour MR imaging effect.In addition, relaxivity is similarly dependent on
The structure and molecular weight (MW) of magnetic resonance imaging probe, but the main chain of HPMA copolymer cannot be degraded, and easily cause Gd accumulation poison
Property.In order to improve biological safety, the present invention is by the RAFT polymerization of HPMA monomer, MA-DOTA and PTEMA by a kind of enzymatic
The oligopeptides (GFLGK) of degradation is introduced into linear backbone (such as Fig. 1).
The synthesis of pHPMA-DOTA-Gd
By HPMA (1.16g, 8.13mmol), MA-DOTA (1.61g, 3.13mmol), PTEMA (635mg, 2.5mmol) and
CTA-GFLGK-CTA (23.7mg, 22.7 μm of ol) be dissolved in containing VA044 (5.0mg, 15.4 μm of ol) deionized water/methanol (4:
1,10mL) solution is placed in 0 DEG C of argon gas exuberant 50 minutes, is then gone to 44 DEG C and is stirred 12 hours.Solution passes through after being quenched with liquid nitrogen
Acetone precipitation obtains the solid for having some pink.It is freeze-dried, is obtained with slight pink after being dialysed 24 hours with water
The two sulphur modification product pHPMA-DOTA (60% purity, 2.06g) of pyridine of color.
Under the conditions of nitrogen protection, pink solid (2.0g) is dissolved in 20mL deionized water, addition DTT (200mg,
1.3mmol) stir 8 hours.Crude product by exclusion chromatography purifying (System, Superose 6HR10/30；
Mobile phase is buffer/methanol=7:3, pH 6.5；Flow velocity: 2.5mL/ minutes).Then, water is dialysed 24 hours and is freeze-dried,
It obtains white product (1.69g), mercapto content is detected (ACS by having method reported in the literature
Appl.Mater.Interfaces,2016,8,10499-10512)。
In RAFT polymerization, present invention uses the chain-transferring agent (CTA-GFLGK- of enzyme response polypeptide GFLGK functionalization
CTA), the MW and PDI of copolymer are well controlled.Two sulphur modification product pHPMA-DOTA of pyridine is reacted with DTT, is made
It has thiol group, and the mercapto content of every kind of product is 0.66mmol/g (table 2).Coloured product is pink.In 1H core
The peak value 7.31-7.71ppm of Magnetic Resonance Spectrum.
By 1.5g white product and GdCl3·6H2O (1.48g, 4.0mmol) is dissolved in 30mL distilled water, is stirred 15 hours
And pH value is adjusted until within the scope of 5.2-5.4 using 0.1MNaOH.It is stirred at room temperature 15 hours.Then, water is dialysed 20 hours and cold
Dry, acquisition white pHPMA-DOTA-Gd product (1.46g) is lyophilized.GFLGK polypeptide is detected by aforementioned amino acid analysis method
Content (ACS Appl.Mater.Interfaces, 2016,8,10499-10512), and pass through inductivity coupled plasma mass spectrometry
Analysis (ICP-MS) show that the content of Gd in product (III) is 6.5%, pHPMA-DOTA-Gd's1H NMR spectra figure is shown in Fig. 2A.
The synthesis of pHPMA-DOTA-Gd-cRGD:
It takes 800mgpHPMA-DOTA-Gd to be dissolved in sodium acetate solution (pH 6.5,8mL), while two sulphur of 400mg pyridine is added
The cRGD of modifying and decorating is stirred at room temperature 20 hours.Crude product by SEC method purifying (System, GE medical treatment):
Superose 6HR10/30 prepacked column load containing 30% methanol (pH6.5) sodium acetate solution as mobile phase (turnover rate:
2.5mL/min).Then, it dialyses and is freeze-dried by 24 hours water, obtain 785mg white product.Pass through amino acid analysis
Method detects the content of GFLGK and cRGDyK polypeptide, and mercapto content is 0.10mmol/g, detects through ICP-MS, wherein Gd
(III) content is 4.0% (table 2), pHPMA-DOTA-Gd-cRGD's1H NMR spectra is shown in Fig. 2 B.
The amino acid of table 1.pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD conjugate forms, and relative amino acid contents are pressed
The percentage of shared sample quality indicates.
The composition analysis of table 2.pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD.
The presence (Fig. 2A) for showing aromatic rings proton also implies the two thiobenzoate ester groups and benzene of GFLG polypeptide
Contain aryl in alanine residues.With GdCl3·6H2After O reaction, white product pHPMA-DOTA-Gd is obtained.Gadolinium conjugate is logical
It crosses ICP-MS to be identified, wherein gadolinium concentrations are the 6.5% of gross mass.Glycine, phenylalanine, leucine and lysine contain
Amount is respectively 0.17%, 0.18%, 0.14% and 0.17% (table 1) of gross mass, and molar ratio is in close proximity to 2:1:1:1,
Show that GFLGK is successfully introduced into main chain, and in the whole preparation process of the biodegradable HPMA-Gd conjugate of diblock
In be stabilized.
In order to enhance polymer MRI magnetic resonance imaging probe in the aggregation of tumor locus, the present invention passes through mercapto and sulphur
The cyclic peptide cRGDyK and pHPMA-DOTA copolymer of two sulphur modification of pyridine is coupled by alcohol-disulphide exchange reaction.And
The α that cRGDyK can be overexpressed with activated endothelial cells in specific recognition tumor vesselvβ3Integrin.Therefore, cRGDyK is modified
Conjugate pHPMA-DOTA-Gd-cRGD is by the MRI magnetic resonance imaging probe as a kind of active targeting.Conjugate passes throughFPLC system is purified and is dialysed, and small molecule compound and by-product are effectively removed.The conjugate of functionalization passes through
Mercapto, amino acid analysis and1H NMR spectrum is identified.Compared to pHPMA-DOTA, pHPMA-DOTA-Gd-cRGD1H NMR spectrum contains more peaks (Fig. 2 B) for being located at 4.44,4.55,6.88,7.16 and 7.04 ppm.4.44 with 4.55
The peak ppm shows the presence of chiral hydrogen atom (α-H) in cRGDyK amino acid, aromatic protons representated by 7.16 and 7.04 ppm
It is attributable to aromatic protons contained by tyrosine in cRGDyK.The content of mercapto is 0.10 mmol/g in conjugate, is significantly lower than
pHPMA-DOTA-Gd(0.66 mmol/g).In addition, according to the content (table 1) of amino acid, arginine, aspartic acid and tryptophan
Molar ratio close to 1:1:1, in conjugate, amino acid accounts for the percentage of gross mass is 22.74%.However, pHPMA-DOTA-Gd
Total amino acid content be only 0.66%.The reduction of pHPMA-DOTA-Gd-cRGD mercapto content,1HNMR wave spectrum occurs more
The raising of peak (4.44,4.55,6.88,7.16 and 7.04ppm) and amino acid content shows to hand over by thio-disulfide
It changes, cRGDyK polypeptide is successfully coupled among pHPMA-DOTA-Gd copolymer by the present invention.CRGDyK content accounts for gross mass
22%, gadolinium concentrations account for the 4% of gross mass.
The present invention carries out experiment detection to poly HPMA-Gd magnetic resonance imaging probe:
One, biodegradable Journal of Sex Research
Papain of the present invention using Lysosomal cathepsin B and with shares activity is come the biology of research material
Degradability, the PBS without enzyme is as negative control.PHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD are dissolved in containing 4mM
Papain or McIlvaine's buffer (3mg/mL, the 50mM citrate/0.1M phosphoric acid of 2.4mM cathepsin B
Salt, 2mM EDTA, 2mM glutathione, pH=5.4), 37 DEG C be incubated for 24 hours, then by SEC (System
System, GE medical treatment) degradability of sample is detected, Superose 6HR10/30 prepacked column loads (pH6.5) containing 30% methanol
Sodium acetate solution as mobile phase (turnover rate: 0.4mL/min).
In order to improve the aggregation of relaxivity and tumor locus, need to improve the molecular weight of gadolinium conjugate.pHPMA-DOTA-
The molecular weight of Gd and pHPMA-DOTA-Gd-cRGD is respectively 85kDa and 94kDa.In addition, in order to meet the needs of kidney excretion,
Kidney threshold value requires molecular weight lower again.Therefore, biodegradable polypeptide is introduced in the backbone structure of two kinds of conjugates.?
In the presence of cathepsin B, two kinds of conjugates can be degraded into the segment of low molecular weight.It is incubated altogether with cathepsin B
After educating 8 hours, the degradable copolymer chain of linearisation is low molecular weight product (being less than 43kDa) (table 3), and this is big
It is small to be lower than kidney threshold value (50kDa).This degradability, which is attributed to cathepsin B, can cut GFLGK polypeptide.However, two kinds total
Yoke object can be stabilized (7.4,37 DEG C of pH) in PBS, not find apparent degradability.Without non-degradable under the conditions of enzyme
And quickly biodegrade announcement HPMA conjugate can be stabilized in blood circulation in tumor microenvironment, and once arrive
It completes to degrade as the task of MRI contrast agent up to tumor locus, to guarantee their effective removing and biofacies
Capacitive.
The molecular weight and PDI of table 3.pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD and its degradation become at any time
The analysis of change.
Two, external relaxation effect research
DTPA-Gd, pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD are dissolved separately in 0.1M PBS, prepared
At the solution (0.1,0.15,0.2,0.25,0.3,0.4,0.5,0.6and 0.7mM) of different Gd (III) concentration.Then, it uses
1.5T clinical magnetic resonance scanner (Siemens) carries out-dot matrix relaxation time (T that spins to sample1) weighted mri detection.Parameter is set
It sets: TE=8.7ms；TR=25,30,50,70,90,110,150,170,190,210,250,300,400,600,700 and
800ms；The visual field (FOV)=200mm；Slice thickness=2.0mm；Matrix size=256 × 256.With various concentration gradient sample
Longitudinal relaxation rate [1/T1(s-1)] map with Gd (III) concentration, slope represents r1 value.
Various concentration Gd (III) conjugate of the acquisition T on 1.5T MR scanner is shown in Fig. 3 A1Weight MR imaging
As a result.Compared with the DTPA-Gd clinically used, conjugate prepared by the present invention has higher under identical Gd (III) concentration
Uniform enhancing signal.Fig. 3 B is according to 1/T1 (s-1) with Gd (III) concentration production curve.PHPMA-DOTA-Gd and
The r of pHPMA-DOTA-Gd-cRGD conjugate1Value is 13.91mM respectively-1·s-1/ Gd (III) and 15.16mM-1·s-1/Gd
(III), it is above DTPA-Gd (3.98mM-1·s-1/ Gd (III)) 3 times or more.According to Solomon-Bloembergen-
Morgan theory can be very good to explain this result: due to spin correlation time τRIt is directly related to size, by by low point
Paramagnetism Gd (III) conjugate of son amount can obtain higher relaxation rate in conjunction with copolymer.Two material 1/T1 (s-1) value
It is linear to illustrate material of the invention dissolubility and stability with higher in aqueous solution.In addition, after cRGD modification
Conjugate shows higher r compared with pHPMA-DOTA-Gd1Value, this may be point because of pHPMA-DOTA-Gd-cRGD
Son amount is higher.The above characteristic and higher relaxation rate imply that material prepared by the present invention can become effective MRI magnetic in vivo
Resonance image-forming probe.
Three, cell culture and experimental animal
U87 cell (people's Malignant glioma cells) and L02 cell (Human normal hepatocyte) are bought from Chinese Academy of Sciences's cell
Library (Shanghai).Two kinds of cells are using containing 10% fetal calf serum (Hyclone) and 1% penicillin/streptomycin (Hyclone)
RPMI1640 culture medium is placed in 37 DEG C, the saturated humidity incubator culture containing 5%CO2.Female BAl BIc/c nude mice buys self-contained
All reach large Biotechnology Co., Ltd, 6-8 week old, weight about 20g.All zooperies are entrusted according to Sichuan University's the care of animal
The requirement of member's meeting executes.
1, internal MRI imaging research
The present invention carries out Contrast enhanced in Mice Body using a 3.0T imaging system (Siemens's Sonata medical system)
MRI research.In short, by U87 cell (1 × 107A cell is dissolved in 200 μ L PBS) it is inoculated in the back bottom right of BALB/c nude mice
Side is subcutaneous.U87 people's malignant glioma diameter to be transplanted reaches 3-5mm, and mouse is randomly divided into three groups, every group 5, is distinguished
It injects DTPA-Gd, pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD (0.08mmol Gd (III)/kg mouse weight).Penta
Barbital sodium anesthetized mice places it in and carries out signal transmitting and detection in the mouse coil of MRI scanner customization.T1It is weighted to
As variable is provided that TR/TE=450/11ms, level=11, three-dimensional size=0.2 × 0.2 × 1.5mm3, FOV=51mm.
Acquire different time points respectively (before injection, 10 minutes, 30 minutes, 1 hour, 3 hours, 19 hours and 24 hours after injection)
The enhancing variation of destination region signal correlation.As a result (△ SNR) is indicated using signal-to-noise ratio: △ SNRT=SI (tumour)/SI (water mould),
△SNRB=SI (bladder)/SI (water mould)；Wherein, SI (tumour), SI (bladder) and SI (water mould) respectively represent tumour, bladder and
The signal strength of water mould.
Different time points (by 24 hours after injection before injection) are taken to be detected (Fig. 4 A) with 3.0T MR scanner.Injection
Afterwards 10 minutes when, only have slight signal compared with normal tissue and water mould using the tumor tissues of DTPA-Gd radiography and change, and
And signal enhancing will fail less than 30 minutes.On the contrary, carrying out the mouse of radiography using HPMA-Gd conjugate prepared by the present invention
Tumor tissues have higher signal enhancing, always can be to 24 hours 10 minutes after injection.In addition, conjugate
Radiography can preferably define the boundary between normal tissue and diseased tissue, and this point is for tumor diagnosis and therapy to Guan Chong
It wants.
Then using the MR imaging signal intensity (SI) of opposite enhancing, i.e., around tumor locus and the water mould for being placed in side
Signal difference, the further quantitative analysis Contrast enhanced of tumor tissues.Average value and time with the opposite enhancing of MRI SI
It maps (Fig. 4 B).The peak (183%) of DTPA-Gd group appears in that treated 10 minutes, is gradually reduced later, until processing
It reverts within 3 hours afterwards and (149%) suitable before injection.The rapid decline of SI may be shorter with it circulation time in vivo and by
It quickly removes related.On the contrary, being infused using the tumor tissues that pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD carries out radiography
There is higher SI value, and SI value still will continue to increase, and respectively reach 200% and 240% when 10 minutes after penetrating.pHPMA-
The high SI value of DOTA-Gd group can be continued until within 10 minutes after injection injection after 19 hours, pHPMA-DOTA-Gd-cRGD
Holding time for group is longer to after injecting 24 hours.
The reason of 24 hours can achieve for the signal enhancing of poly conjugate, it may be possible to due to the extension of circulation time
With higher two aspect reason of tumor accumulation.Result of study for mouse bladder SI signal enhancing is that this conclusion has strong evidence
According to.The highest point of DTPA-Gd group bladder SI enhancing occurs 10 minutes after injection, and then at 10 to 30 minutes, decline (was schemed rapidly
4C).Therefore, it is possible to determine that the renal excretion of DTPA-Gd is begun within a few minutes after injection, and this explains why swell
The contrasting effects of tumor imaging are very poor.On the contrary, the bladder SI signal enhancing of conjugate group can remain small to 3 in 30 minutes after injection
When, slower kidney excretion and longer circulation time are imply, tracing it to its cause is that the EPR effect that high molecular weight mediates makes
Conjugate is gathered in tumor locus.It is interesting that pHPMA-DOTA-Gd-cRGD is relative to pHPMA-DOTA-Gd and DTPA-Gd
Enhance with higher and the longer time SI, this phenomenon may be due to cRGD polypeptide and αvβ3The specific binding of integrin
It is caused, and this combination is more stable, further promotes aggregation of the conjugate in tumor tissues to extend circulation
Time.
2, the Tissue distribution of conjugate nanosystems
U87 tumor-bearing mice is randomly divided into 3 groups (every group 7), injects DTPA-Gd, pHPMA-DOTA-Gd and pHPMA- respectively
DOTA-Gd-cRGD (0.08mmolGd (III)/kg mouse weight) carries out MRI Contrast enhanced imaging research, 24 hours after injection
Put to death mouse.Coring is dirty, liver, spleen, lung, kidney and tumor tissues are weighed, and with 1mL H2O2With 3mL HNO3Disappear
Change, sample is handled 48 hours at 120 DEG C.Gd (III) content of sample is measured by ICP-MS, and data use shared every gram
The percentage of the injection dosage of mouse indicates.
Fig. 5 shows that nanosystems prepared by the present invention have higher Gd (III) in tumor tissues compared to DTPA-Gd
Hold-up (p < 0.01).This result is consistent with internal MR imaging results.
The tumor tissues accumulation of pHPMA-DOTA-GdP and HPMA-DOTA-Gd-cRGD is higher by 13 than DTPA-Gd group respectively
Times and 44 times.It is not difficult to find out that the ligand specificity that cRGD polypeptide mediates is incorporated in the tumor tissues aggregation and residual of conjugate
Important function is played.In addition, in liver and spleen there are the gadolinium of higher concentration also confirm copolymer in vivo and have it is longer
Circulation time.This also prompts the vent path of magnetic resonance imaging probe and may cause the target organ of toxicity.
3, cytotoxicity analysis
The vitro cytotoxicity analysis of three kinds of materials is carried out using CCK-8 (colleague's chemistry, Japan) kit.By U87 and
L02 cell is inoculated with 96 orifice plates (5 × 10 respectively3A/hole), culture 24 hours after, change into respectively containing various concentration DTPA-Gd,
Culture medium (Gd (III) concentration: 50,100,250 and 500nmol/ of pHPMA-DOTA-Gd or pHPMA-DOTA-Gd-cRGD
ML), continue culture 48 hours.PBS is washed 3 times, and 100 μ L CCK-8 reagents (10 μ L stostes: 90 μ L RPMI1640 training are added in every hole
Support base).After being incubated for 2 hours, pass through VarioscanFlash microplate reader (Thermo Fisher science and technology, the U.S.) detection 450nm's
Absorbance.Cell without drug-treated is compareed as 100% competent cell.
Fig. 6 A is shown, by various concentration DTPA-Gd, pHPMA-DOTA-Gd or pHPMA-DOTA-Gd-cRGD processing 48
L02 cell activity after hour does not substantially change (Gd (III) content: 50-500nmol/mL).However, pHPMA-DOTA-Gd
It both shows to act on (Fig. 6 B) to the dose-dependent inhibition of U87 cell activity with pHPMA-DOTA-Gd-cRGD.This can
Can and HPMA copolymer it is related with the intercellular interaction of U87.The high express alpha of U87 cellvβ3And αvβ5Integrin, and cRGD target
To αvβ3And αvβ5Integrin improves cell to the phagocytosis amount of magnetic resonance imaging probe, since toxicity of gadolinium ion etc. causes
The apoptosis of cancer cell.It is worth noting that, conjugate (Gd (III) ﹤ 50nmol/mL) the processing U87 cell of low concentration does not have
Show cytotoxicity.
The assessment of pHPMA-Gd conjugate biocompatibility in vitro:
1, hemolytic analysis
By healthy human body new blood using PBS dilution after (16%v/v, 300 μ L), respectively with 1mg/mL, 3mg/mL or
The different materials of 5mg/mL are incubated for 12 hours jointly at 37 DEG C, and then 1000g is centrifuged 10min, take 200 μ L upper liquids to 96 holes
Plate measures the absorbance of 540nm by VarioscanFlash microplate reader.Blood sample be dissolved in distilled water and PBS respectively as
100% haemolysis and negative control.
For erythrocyte hemolysis according to American Society for Testing Materials (ASTM) standard, magnetic resonance imaging probe prepared by the present invention is equal
Blood compatibility (erythrocyte hemolysis ﹤ 5%) (Fig. 7) with higher, (5mg/mL) material group and PBS control group under higher concentration
Compared to hemolysis rate still without notable difference.Suitable molecular weight, hydrophilic nmature possessed by conjugate and surface negative electricity among these
The excellent properties such as lotus protect red cell membrane from the destruction of magnetic resonance imaging probe, with blood compatibility.
Erythrocyte aggregation and the form present invention further have evaluated high concentration by SEM method and test magnetic resonance imaging spy
Whether there is or not aggregation and morphologic changes for red blood cell after needle (5mg/mL) processing.Fig. 8 is shown, similar with PBS control group, after conjugate processing
Red blood cell still remains monodispersed normal biconcave form and smooth surface.It is red thin after material processing according to existing document
Haemolysis, aggregation and the morphologic change of born of the same parents usually polymerize with electrostatic interaction, hydrophilic and hydrophobic grouping distribution and high molecular weight
The aggregation of object etc. is related.Fortunately, process is well-designed, and magnetic resonance imaging probe prepared by the present invention has both surface negative electricity
Lotus, hydrophilic radical and suitable molecular weight, to have good biocompatibility to red blood cell.
2, coagulation analysis
Healthy human body new blood is acquired, platelet-poor plasma is collected by centrifugation.Take 360 μ L blood plasma materials different from three kinds respectively
Expect (40 μ L) mixing.Activated partial thromboplastin time (APTT) and prothrombin time (PT) pass through automatic coagulation analyzer
Detection.Each concentration does 3 parallel groups.Isometric PBS is as control.
As shown in figure 9, even if magnetic resonance imaging probe prepared by the present invention still has in higher concentrations (5mg/mL)
There is APTT and PT similar with PBS control group.This shows that material of the invention does not influence blood clotting factor significantly, returns
Because in the hydrophily of HPMA copolymer chain.
3, thrombelastogram (TEG)
Extraction healthy human body new blood, which mixes (blood: material=9:1,1mL) with different materials, makes nano material most
Final concentration of 0.1mg/mL and 1mg/mL.It adds mixture to containing in kaolinic special pipe.Take 340 μ L supernatants and 20
The CaCl of μ L2(0.2M) is added in test sample cup, is tested by thrombelastogram instrument (Haemoscope company).In equal volume
PBS is as control.
The whole blood of different experimental materials processing is similar with control group.R, K value, the angle α and MA are without obvious between different groups
Difference.It is all these to prove that magnetic resonance imaging probe (5mg/mL) of the invention does not influence blood coagulation process, this all attribution
In suitable molecular weight, hydrophily and surface negative charge that well-designed conjugate has.In conclusion preparation can be determined
HPMA-Gd (III) conjugate be safety and blood compatibility.
Four, toxicity in vivo is studied
In vitro toxicity research is only the premise of In vivo study, only after internal security verification, magnetic resonance imaging
Probe just can be applied to clinic.The present invention is studied using the BALB/c mouse of health, and concrete operations are detailed in method part.
The present invention is carried out by toxicity in vivo of behavior, weight, hematology and the histologic analysis to animal to magnetic resonance imaging probe
It fully assesses.By research in 19 days by a definite date, the present invention do not find any dehydration, dyskinesia, muscular atrophy, anorexia or
Other phenomenons related to animal toxicity of person.Meanwhile the mouse after the administration of conjugate nanosystems is with similar to GTPA-Gd group
Changes of weight.Other abnormal phenomenon are not found.
In order to further study other genotoxic potentials or apparent side effect, red blood cell, blood of the present invention to experimental animal
Lactoferrin, hematocrit, blood platelet, mean platelet volume and leucocyte etc. are detected.Compared with the control group, do not have
It discovers a marked discrepancy.This result shows that, conjugate nanosystems are consistent in vitro study in blood, same to have biology
Compatibility.In order to study tissue or organ toxicity, the present invention has carried out histologic analysis to major organs and tissue.Figure 10 is shown
Heart, liver, spleen, lung and the big major organs of kidney five be it is normal, there is no the exception thought in histopathology,
It degenerates or damages.Therefore, it is considered herein that the conjugate of preparation has good biocompatibility and safety, among these
The well-designed structure of pHPMA-Gd (III) conjugate and biodegradable characteristic necessarily play an important role.
Statistical analysis
All data use mean ± SD to indicate.The significance of difference between each group of data uses ANOVA and Student's
Sided t-inspection is for statistical analysis, and P ﹤ 0.05 is statistically significant.
Claims (3)
1. a kind of biodegradable poly HPMA-Gd magnetic resonance imaging probe, which is characterized in that chain structure is as follows:
Wherein: m:n:o=35~45:410~450:65~70；
2. biodegradable poly HPMA-Gd magnetic resonance imaging probe according to claim 1, which is characterized in that should
The average molecular weight of magnetic resonance imaging probe is 85-94KDa.
3. biodegradable poly HPMA-Gd magnetic resonance imaging probe according to claim 2, which is characterized in that its
In, m:n:o=40:443:66.
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