CN106880848A - Biodegradable poly HPMA Gd magnetic resonance imaging probes and preparation method thereof - Google Patents
Biodegradable poly HPMA Gd magnetic resonance imaging probes 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 invention provides a kind of biodegradable poly HPMA Gd magnetic resonance imaging probes, by the sensitive polypeptide GFLG insertion poly HPMA backbone structures of enzyme；The polymer molecular weight of preparation is substantially controllable, and molecular weight polydispersity coefficient (PDI) is low；And it is coupled the HPMA DOTA Gd for being linearized by with Gd (III).The probe is the nanoscale MRI magnetic resonance imaging probes of efficient tool biocompatibility, can be used for clinical tumor diagnosis and auxiliary treatment.
Description
Technical field
The invention belongs to medical imaging technology field, it is 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 probes.
Background technology
Because Magnetic resonance imaging (MRI) has high spatial resolution, 3D rendering information and the advantage such as "dead", into
It is current effective tumour Non-Invasive Method.According to incompletely statistics, clinically more than 50% MRI diagnosing tumors need to use
MRI magnetic resonance imaging probes.The conventional MRI magnetic resonance imagings probe of clinic includes that Magnevist Solution (Magnevist) and gadolinium are special
Sour Portugal's amine (Dotarem).However, they belong to small molecule magnetic resonance imaging probe, there is sensitiveness low, non-specific, very
Soon the shortcomings of removing in vivo；And in mainly by Passive diffusion to mesenchyma stroma of tumors or tissue interstitial, cause to be extremely difficult to
Satisfied imaging effect.Therefore, MRI magnetic resonance imagings probe that is more excellent, can overcoming disadvantage mentioned above has research very high
Value, and the nano-complex for being based on Gd (III) illustrates huge potentiality in the research of novel magnetic resonance image probe.
The content of the invention
Based on above-mentioned technical problem, the present invention constructs biodegradable poly HPMA-Gd magnetic resonance imaging probes, is
A kind of macromolecule MRI magnetic resonance imaging probes of efficient tool biocompatibility, can be used for clinical tumor diagnosis and aid in
Treatment.
Technical scheme is as follows：
A kind of biodegradable poly HPMA-Gd magnetic resonance imaging probes, chain structure is as follows：
Wherein：m:n:O=35~45:410~450:65~70；
Present invention design is simultaneously characterized even with higher molecular weight, biodegradable poly HPMA polymer-Gd (III)
The nanometer system of compound, 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- hydroxypropyls) Methacrylamide) can combine multiple small-molecular-weights Gd (III) conjugate (DOTA) so as to
Relaxivity is greatly improved, and with disimmune, nontoxicity, water-soluble 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, so as 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 carries out surface modification by introducing the R1 groups with cRGDyK to material, makes it have active targeting function.
CRGDyK can be by αvβ3The specific identification of integrin and combination, and αvβ3In kinds of tumor cells and tumor neogenetic blood vessels
Expression quantity in chrotoplast is significantly raised, but is expressed in normal tissue cell and relatively low do not express even.
Although the HPMA copolymers of HMW can improve aggregation of the magnetic resonance imaging probe in tumor locus, kidney
Dirty excretion threshold value requires that it is necessarily less than 50kDa again, will otherwise cause Gd to be poisoned.Because HPMA main chains are without biodegradation
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 it to be degraded in the presence of Lysosomal cathepsin B small less than 50kDa
Fragment.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 systems of the present invention show
Antitumor efficiency higher and biological safety.
The mean molecule quantity of the magnetic resonance imaging probe is 85-94KDa.The molecular weight is degraded under tumor microenvironment
Fragment of the molecular weight less than 50kDa, can take into account antitumous effect and biological safety, it is ensured that body can be removed out after degraded
It is interior.
Preferably, wherein, m:n:O=40:443:66.In order to ensure that water miscible macromolecule has certain gadolinium ion
Content (weight/mass percentage composition is generally 3-10%), it is ensured that effective magnetic resonance imaging.Meanwhile, adjust o values, it is ensured that cRGDyK
Content (weight/mass percentage composition be more than 10%), to improve its tumor-targeting.
The present invention is polymerized by reversible addion-fragmentation chain transfer (RAFT) first, by the sensitive polypeptide glycine-phenylpropyl alcohol of enzyme
In propylhomoserin-leucine-glycine (GFLG) insertion poly HPMA backbone structures；And be coupled by with Gd (III), synthesizing linear
HPMA-DOTA-Gd.Herein on basis, introduce 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 probes of the present invention, as R1=SH, comprises the following steps：
The synthesis of poly HPMA-Gd conjugates (pHPMA-DOTA-Gd)：
1) HPMA, MA-DOTA, pTEMA and CTA-GFLGK-CTA are dissolved in into the deionized water containing VA044/methyl alcohol to mix
Solution, is placed in exuberant 40-60 minutes in argon gas, then stirring reaction；Solution liquid nitrogen be quenched after through acetone precipitation, separate out pyridine
Two sulphur modification product pHPMA-DOTA.
Preferably, deionized water and methyl alcohol are with 4：1 volume ratio is mixed to get step 1) in mixed solution.
It is further preferred that the concentration of VA044 containing initiator in the mixed solution, VA044 is chain-transferring agent CTA-
The 1/3-1/5 equivalents of GFLGK-CTA.During for 1/3 equivalent, polymerisation can be rapidly completed, and molecular weight and PDI can be obtained effectively
Control.By RAFT polymerizations (RAFTpolymerization；Reversible addion-fragmentation chain transfer be polymerized) prepare polymer molecule
Amount is substantially controllable, and molecular weight polydispersity coefficient (PDI) is low.
2) under nitrogen protective condition, the sulphur modification product pHPMA-DOTA of pyridine two is dissolved in deionized water, adds DTT
Stirring reaction, obtains intermediate product；By intermediate product and GdCl3·6H2O is dissolved in distilled water, after stirring reaction, adjusts pH value,
After reaction is stirred at room temperature again, pHPMA-DOTA-Gd products are obtained.
Proposed by FPLC chromatographic column, eluant, eluent is water and methyl alcohol with 7：3 mixed liquor, is dialysed by deionized water
Intermediate product is obtained with freeze-drying；
Preferably, the step 2) adjust pH value to 5.2-5.4, it is ensured that the coordination of gadolinium ion, while ensureing the steady of small peptide
It is qualitative.
Present invention also offers another preparation method of poly HPMA-Gd magnetic resonance imaging probes, when R1 is not SH,
The synthesis of pHPMA-DOTA-Gd-cRGD is comprised the following steps：
Take pHPMA-DOTA-Gd to be dissolved in deionized water, while adding the cRGD of the sulphur modification of pyridine two modification, lead to after reaction
Cross FPLC post separation and dialysis is purified, obtain pHPMA-DOTA-Gd-cRGD.
Advantage of the invention is that：
1st, the present invention is designed and characterized with higher molecular weight, biodegradable poly HPMA polymer-Gd (III)
The nanometer system of conjugates, main polymer chain is made up of hydrophilic and hydrophobic block, and introduces 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 combine multiple small-molecular-weights Gd (III) conjugate (DOTA) so as to greatly improve relaxation
Henan efficiency, and with disimmune, nontoxicity, water-soluble and good bio-compatibility；Meanwhile, the nano material of macromolecule
Circulation time in blood can be extended, increase the ability of EPR passive targets, improve magnetic resonance imaging probe in tumor locus
Concentration class, so as to increase the imaging effect and therapeutic effect of tumor locus.
2nd, the present invention carries out surface modification by introducing the R1 groups with cRGDyK to material, makes it have active targeting work(
Energy；Biodegradable GFLGK peptide groups are introduced in main chain, the feelings for alloing it to exist in Lysosomal cathepsin B
The small fragment less than 50kDa is degraded under condition, the nanoscale MRI magnetic of efficient tumor-targeting and biocompatibility is had concurrently
Resonance image-forming probe, it is adaptable to clinical tumor diagnosis and auxiliary treatment etc..
3rd, preparation method of the invention is designed and by RAFT and " click " chemical building pHPMA-DOTA- of functionalization
Gd and pHPMA-DOTA-Gd-cRGD conjugates.It is polymerized by reversible addion-fragmentation chain transfer (RAFT) first, enzyme is sensitive
In polypeptide Gly-Phe-leucine-glycine (GFLG) insertion poly HPMA backbone structures；The polymer of preparation point
Son amount is substantially controllable, and molecular weight polydispersity coefficient (PDI) is low；And be coupled by with Gd (III), the HPMA- of synthesizing linear
DOTA-Gd.Herein on basis, introduce polypeptide cRGDyK and become the MRI magnetic resonance with potential active tumor-targeting
Image probe HPMA-DOTA-Gd-cRGD.HPMA-Gd (III) conjugate of the invention (>Can 90kDa) be degraded to less than kidney
Dirty threshold value low molecular weight product (<44kDa).With clinically diethyl pentetic acid-Gd (DTPA-Gd) magnetic resonance
Image probe is compared, and longitudinal relaxation efficiency (T1) is the former many (15.16mM of three times-1·s-1/Gd)。
4th, the raising relaxivity of poly HPMA-Gd magnetic resonance imagings probe, is significantly higher than the 3 of clinical reagent DTPA-Gd
More than times, clinical reagent is significantly higher than in the magnetic resonance signal intensity of tumor locus；Meanwhile, it is degradable under tumor microenvironment
It is the product of low-molecular-weight, is easy to it quickly to excrete, reduces toxic and side effect.
Brief description of the drawings
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 spectrograms.
Fig. 3 is A.T1Weighted mri imaging results；B. the longitudinal relaxation rate (1/T of various concentrations1) (solvent：PBS, 1.5T).
Fig. 4 is that (0.08mmolGd (III)/kg is small for A.DTPA-Gd, pHPMA-DOTA-Gd or pHPMA-DOTA-Gd-cRGD
Mouse body weight) administration after different time points tumour and bladder axle 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 cells (A) and U87 cells (B).
Fig. 7 is erythrocyte splitting situation after different materials treatment：A.pHPMA-DOTA-Gd；B.pHPMA-DOTA-Gd-
cRGD；C. the relative hemolysis rate (mean ± SD) of red blood cell after various concentrations are processed.
Fig. 8 is to detect that different materials process the influence to erythrocyte aggregation and form by SEM：A.PBS is compareed；
B.pHPMA-DOTA-Gd；C.pHPMA-DOTA-Gd-cRGD.
Fig. 9 is influence of the different materials treatment to APTT and PT：A.pHPMA-DOTA-Gd；B.pHPMA-DOTA-Gd-
cRGD。
Figure 10 is for after giving PBS (A), pHPMA-DOTA-Gd (B) and pHPMA-DOTA-Gd-cRGD (C) respectively, difference is small
Mouse organ histology analysis result (100 ×).
Specific embodiment
Essentiality content of the invention is described in further detail with reference to specific embodiment.
Embodiment 1
A kind of biodegradable poly HPMA-Gd magnetic resonance imaging probes, chain structure is 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 is SH, m:n:O=35:410:65.
The mean molecule quantity 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 mean molecule quantity 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 mean molecule quantity of the magnetic resonance imaging probe is 90KDa.
Embodiment 5
The preparation method of the magnetic resonance imaging probe of embodiment 2, comprises the following steps：
1) HPMA, MA-DOTA, pTEMA and CTA-GFLGK-CTA are dissolved in into the deionized water containing VA044/methyl alcohol to mix
Solution, is placed in exuberant 40-60 minutes in argon gas, then stirring reaction；Solution liquid nitrogen be quenched after through acetone precipitation, separate out pyridine
Two sulphur modification product pHPMA-DOTA；
2) under nitrogen protective condition, the sulphur modification product pHPMA-DOTA of pyridine two is dissolved in deionized water, adds DTT
Stirring reaction, obtains intermediate product；By intermediate product and GdCl3·6H2O is dissolved in distilled water, after stirring reaction, adjusts pH value,
After reaction is stirred at room temperature again, pHPMA-DOTA-Gd products are obtained.
Embodiment 6
The preparation method of the magnetic resonance imaging probe of embodiment 2, comprises the following steps：
1) HPMA, MA-DOTA, pTEMA and CTA-GFLGK-CTA are dissolved in into the deionized water containing VA044/methyl alcohol to mix
Solution, is placed in exuberant 40-60 minutes in argon gas, then stirring reaction；Solution liquid nitrogen be quenched after through acetone precipitation, separate out pyridine
Two sulphur modification product pHPMA-DOTA.
Deionized water is with methyl alcohol with 4：1 volume ratio is mixed to get step 1) in mixed solution；All polymerisations, point
The solvent used from purification is three class solvents.
The concentration of VA044 containing initiator in the mixed solution, VA044 is the 1/3- of chain-transferring agent CTA-GFLGK-CTA
1/5 equivalent.During for 1/3 equivalent, polymerisation can be rapidly completed, and molecular weight and PDI can obtain effectively control.
2) under nitrogen protective condition, the sulphur modification product pHPMA-DOTA of pyridine two is dissolved in deionized water, adds DTT
Stirring reaction, obtains intermediate product；Proposed by FPLC chromatographic column, eluant, eluent is water and methyl alcohol with 7：3 mixed liquor, leads to
Cross deionized water dialysis and freeze-drying obtains intermediate product；The intermediate product and GdCl3·6H2O is dissolved in distilled water, stirring
After reaction, regulation pH value after reaction is stirred at room temperature, obtains pHPMA-DOTA-Gd products to 5.2-5.4.
Embodiment 7
The preparation method of the magnetic resonance imaging probe of embodiment 3 and 4, synthetic route is shown in Fig. 1, in the preparation method of embodiment 5
On the basis of：
3) pHPMA-DOTA-Gd is taken to be dissolved in deionized water, while the cRGD of the sulphur modification of pyridine two modification is added, after reaction
Purified by FPLC post separation and dialysis, obtain pHPMA-DOTA-Gd-cRGD.
Embodiment 8
The preparation method of the magnetic resonance imaging probe of embodiment 3 and 4, on the basis of the preparation method of embodiment 6：
3) under nitrogen protective condition, the sulphur modification product pHPMA-DOTA-Gd of pyridine two is dissolved in deionized water, is added
The sulphur modification cRGD stirring reactions of pyridine two；Proposed by FPLC chromatographic column, eluant, eluent is water and methyl alcohol with 7：3 mixing
Liquid, is dialysed and freeze-drying product pHPMA-DOTA-Gd-cRGD by deionized water.
Experimental technique
Material and method
The isobutyl imidazoline hydrochloride (VA044) of azo two 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) prepared by existing document.The synthetic method of the sulphur modification cRGD of pyridine two is shown in document
(ACSAppl.Mater.Interfaces,2016,8,10499-10512).The number-average molecular weight (Mn) of synthetic material, divide equally again
Son amount (Mw) and polydispersity detected using size exclusion chromatography (SEC) (GE companies,System
System), using the sodium acetate solution containing 30% methyl alcohol as mobile phase.Zeta potential is detected by nano particle size and potentiometric analyzer
(Malvern instruments, Wu Site prefectures, Britain), and data processing is carried out using DTS softwares, as a result shown with frequency curve chart.
The degradable HPMA copolymer-Gd conjugates of diblock formula main chain and as MRI magnetic resonance imaging probes, it can be with
Increased in the aggregation of tumor locus so as to improve tumour MR imaging effects by EPR effects.Additionally, relaxivity is similarly dependent on
The structure and molecular weight (MW) of magnetic resonance imaging probe, but the main chain of HPMA copolymers can not be degraded, and easily cause Gd accumulation poison
Property.In order to improve biological safety, the present invention is by the RAFT polymerizations of HPMA monomers, MA-DOTA and PTEMA by a kind of enzymatic
The oligopeptides (GFLGK) of degraded 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) is dissolved in the deionized water/methyl alcohol (4 containing VA044 (5.0mg, 15.4 μm of ol):
1,10mL) solution, is placed in exuberant 50 minutes in 0 DEG C of argon gas, then goes to 44 DEG C and stirs 12 hours.Solution liquid nitrogen is passed through after being quenched
Acetone precipitation, obtains and carries some pink solids.Freeze-drying after being dialysed 24 hours with water, obtains with slight pink
The sulphur modification product pHPMA-DOTA (60% purity, 2.06g) of pyridine two of color.
Under nitrogen protective condition, pink solid (2.0g) is dissolved in 20mL deionized waters, addition DTT (200mg,
1.3mmol) stir 8 hours.Crude product by exclusion chromatography purify (System, Superose 6HR10/30；
Mobile phase is buffer solution/methyl alcohol=7:3, pH 6.5；Flow velocity：2.5mL/ minutes).Then, water dialysis 24 hours and freeze-drying,
White product (1.69g) is obtained, mercapto content is detected (ACS by the method for existing document report
Appl.Mater.Interfaces,2016,8,10499-10512)。
In RAFT polymerizations, present invention uses the chain-transferring agent (CTA-GFLGK- that enzyme responds polypeptide GFLGK functionalization
CTA), the MW and PDI of copolymer are well controlled.The sulphur modification product pHPMA-DOTA of pyridine two and DTT is reacted, is made
It carries thiol group, and the mercapto content of every kind of product is 0.66mmol/g (table 2).Coloured product is pink.In 1H cores
The peak value 7.31-7.71ppm of Magnetic Resonance Spectrum.
By 1.5g white products and GdCl3·6H2O (1.48g, 4.0mmol) is dissolved in 30mL distilled water, is stirred 15 hours
And pH value is adjusted until in the range of 5.2-5.4 using 0.1MNaOH.It is stirred at room temperature 15 hours.Then, water is dialysed 20 hours and cold
It is lyophilized dry, obtain white pHPMA-DOTA-Gd products (1.46g).GFLGK polypeptides are detected by aforementioned amino acid analysis method
Content (ACS Appl.Mater.Interfaces, 2016,8,10499-10512), and by inductivity coupled plasma mass spectrometry
Analysis (ICP-MS) show that the content of Gd (III) in product is 6.5%, pHPMA-DOTA-Gd's1H NMR spectra figures are shown in Fig. 2A.
The synthesis of pHPMA-DOTA-Gd-cRGD:
Take 800mgpHPMA-DOTA-Gd and be dissolved in sodium acetate solution (pH 6.5,8mL), while adding the sulphur of 400mg pyridines two
The cRGD of modifying and decorating, is stirred at room temperature 20 hours.Crude product by SEC methods purify (System, GE medical treatment):
Superose 6HR10/30 prepacked columns load the sodium acetate solution containing 30% methyl alcohol (pH6.5) as mobile phase (turnover rate：
2.5mL/min).Then, dialysed and freeze-drying by the water of 24 hours, obtain 785mg white products.By amino acid analysis
Method detects the content of GFLGK and cRGDyK polypeptides, and mercapto content is 0.10mmol/g, is detected through ICP-MS, wherein Gd
(III) content is 4.0% (table 2), pHPMA-DOTA-Gd-cRGD's1H NMR spectras are shown in Fig. 2 B.Table 1.pHPMA-DOTA-Gd
Amino acid with pHPMA-DOTA-Gd-cRGD conjugates is constituted, and relative amino acid contents press the percentage table of shared sample quality
Show.
The composition analysis of table 2.pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD.
Show the presence (Fig. 2A) of aromatic rings proton, also imply that two thiobenzoate ester groups and benzene of GFLG polypeptides
Contain aryl in alanine residues.With GdCl3·6H2After O reactions, white product pHPMA-DOTA-Gd is obtained.Gadolinium conjugate leads to
Cross 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 mol ratio is in close proximity to 2：1：1：1,
Show that GFLGK is successfully introduced into the middle of main chain, and in the whole preparation process of the biodegradable HPMA-Gd conjugates of diblock
Middle stable existence.
In order to strengthen aggregation of the polymer MRI magnetic resonance imagings probe in tumor locus, the present invention passes through mercapto and sulphur
The exchange reaction of alcohol-disulphide, the cyclic peptide cRGDyK and pHPMA-DOTA copolymers of the sulphur modification of pyridine two are coupled.And
CRGDyK can be with the α of activated endothelial cells overexpression in specific recognition tumor vesselvβ3Integrin.Therefore, cRGDyK is modified
Conjugate pHPMA-DOTA-Gd-cRGD is using as a kind of MRI magnetic resonance imaging probes of active targeting.Conjugate passes throughFPLC systems are purified and dialysed, effectively removal micromolecular compound and accessory substance.The conjugate of functionalization passes through
Mercapto, amino acid analysis and1H NMR spectrums are identified.Compared to pHPMA-DOTA, pHPMA-DOTA-Gd-cRGD1H NMR spectrums contain more peaks (Fig. 2 B) for being located at 4.44,4.55,6.88,7.16 and 7.04 ppm.4.44 and 4.55
Ppm peaks show the presence of chiral hydrogen atom (α-H) in cRGDyK amino acid, the aromatic protons representated by 7.16 and 7.04 ppm
It is attributable to the 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).Additionally, the content (table 1) according to amino acid, arginine, aspartic acid and tryptophan
Mol ratio close to 1：1：1, the percentage that amino acid accounts for gross mass in conjugate is 22.74%.However, pHPMA-DOTA-Gd
Total amino acid content be only 0.66%.The reduction of pHPMA-DOTA-Gd-cRGD mercaptos content,1HNMR wave spectrums occur more
Peak (4.44,4.55,6.88,7.16 and 7.04ppm) and the rising of amino acid content are shown, are handed over by thio-disulfide
Change, successfully be coupled to cRGDyK polypeptides among pHPMA-DOTA-Gd copolymers by the present invention.CRGDyK contents account 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 imagings probe：
First, biodegradable Journal of Sex Research
The present invention is using Lysosomal cathepsin B and the papain with shares activity is come the biology of research material
Degradability, the PBS without enzyme is used as negative control.PHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD are dissolved in and contain 4mM
McIlvaine's buffer solutions (3mg/mL, the 50mM citrate/0.1M phosphoric acid of papain or 2.4mM cathepsin Bs
Salt, 2mM EDTA, 2mM glutathione, pH=5.4), 37 DEG C be incubated 24 hours, then by SEC (System
System, GE medical treatment) degradability of sample is detected, Superose 6HR10/30 prepacked columns load (pH6.5) containing 30% methyl alcohol
Sodium acetate solution as mobile phase (turnover rate：0.4mL/min).
In order to improve the aggregation of relaxivity and tumor locus, it is necessary 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.Additionally, the need in order to meet RE,
Kidney threshold value requires that molecular weight is lower again.Therefore, biodegradable polypeptide is introduced in two kinds of backbone structures of conjugate.
In the presence of cathepsin B, two kinds of conjugates can be degraded into the fragment of low-molecular-weight.Incubated altogether with cathepsin B
After educating 8 hours, it is low molecular weight product (being less than 43kDa) (table 3) that the copolymer chain of linearisation is degradable, and this is big
It is small less than kidney threshold value (50kDa).This degradability is attributed to cathepsin B and can cut GFLGK polypeptides.However, two kinds common
Yoke thing can be with stable existence (7.4,37 DEG C of pH), without the obvious degradability of discovery in PBS.Without non-degradable under the conditions of enzyme
And in tumor microenvironment quickly it is biodegradable disclose HPMA conjugates can in blood circulation stable existence, and once arrive
Complete to be degraded as the task of MRI contrast agent up to tumor locus, so as to ensure their effective removing and biofacies
Capacitive.
The molecular weight and PDI of table 3.pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD, and its anaplasia at any time of degrading
The analysis of change.
2nd, external relaxation effect research
DTPA-Gd, pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD are dissolved separately in 0.1M PBS, are prepared
Into different Gd (III) concentration solution (0.1,0.15,0.2,0.25,0.3,0.4,0.5,0.6and 0.7mM).Then, use
1.5T clinical magnetic resonances scanner (Siemens) carry out-dot matrix relaxation time (T that spins to sample1) weighted mri detection.Parameter sets
Put：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 concentrations gradient sample
Longitudinal relaxation rate [1/T1(s-1)] mapped with Gd (III) concentration, its slope represents r1 values.
Fig. 3 A are shown various concentrations Gd (III) conjugates of the acquisition T on 1.5T MR scanners1Weighting MR imagings
As a result.Compared with the DTPA-Gd for clinically using, conjugate prepared by the present invention has higher under identical Gd (III) concentration
Uniform enhancing signal.Fig. 3 B are according to 1/T1 (s-1) with Gd (III) concentration make curve.PHPMA-DOTA-Gd and
The r of pHPMA-DOTA-Gd-cRGD conjugates1Value is respectively 13.91mM-1·s-1/ Gd (III) and 15.16mM-1·s-1/Gd
(III) DTPA-Gd (3.98mM, are above-1·s-1/ Gd (III)) more than 3 times.According to Solomon-Bloembergen-
Morgan theories 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) conjugates of son amount are combined with copolymer can obtain relaxation rate higher.Two material 1/T1 (s-1) value
Linear explanation material of the invention has dissolubility and stability higher in aqueous.Additionally, after cRGD modifications
Conjugate shows r higher compared with pHPMA-DOTA-Gd1Value, this is probably because pHPMA-DOTA-Gd-cRGD's divides
Son amount is higher.Above characteristic and relaxation rate higher imply that the material of present invention preparation can turn into effective MRI magnetic in vivo
Resonance image-forming probe.
3rd, cell culture and experimental animal
U87 cells (people's Malignant glioma cells) and L02 cells (Human normal hepatocyte) are bought from Chinese Academy of Sciences's cell
Storehouse (Shanghai).Two kinds of cells are using containing 10% hyclone (Hyclone) and 1% penicillin/streptomycin (Hyclone)
RPMI1640 culture mediums, are positioned over 37 DEG C, the saturated humidity incubator culture containing 5%CO2.The purchase of female BAl BIc/c nude mices from into
All reach large bio tech ltd, 6-8 week old, body weight about 20g.All zooperies are entrusted according to Sichuan University's the care of animal
The requirement of member's meeting is performed.
1st, internal MRI imaging research
The present invention carries out Contrast enhanced in Mice Body using a 3.0T imaging system (Siemens Sonata medical systems)
MRI is studied.In short, by U87 cells (1 × 107Individual cell is dissolved in 200 μ L PBS) it is inoculated in the back bottom right of BALB/c nude mices
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, difference
Injection DTPA-Gd, pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD (0.08mmol Gd (III)/kg Mouse Weights).Penta
Barbital sodium anesthetized mice, be placed on MRI scanner customization mouse coil in carry out signal transmission and detection.T1It is weighted to
As variable sets as follows：TR/TE=450/11ms, aspect=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 related enhancing change of purpose region signal.Result is represented (△ SNR) 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) represent respectively tumour, bladder and
The signal intensity 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 scanners.Injection
Afterwards 10 minutes when, changed using the signal only slight compared with normal structure and water mould of the tumor tissues of DTPA-Gd radiographies, and
And signal enhancing will fail less than 30 minutes.Conversely, carrying out the mouse of radiography using HPMA-Gd conjugates prepared by the present invention
Tumor tissues have signal enhancing higher, always can be to 24 hours since after injection 10 minutes.Additionally, conjugate
Radiography can preferably define the border between normal structure and diseased tissue, and this point is for tumor diagnosis and therapy to closing weight
Will.
Relatively enhanced MR imaging signals intensity (SI) is then used, i.e., around tumor locus and the water mould for being positioned over side
Signal difference, the further quantitative analysis Contrast enhanced of tumor tissues.With the relatively enhanced average values of MRI SI and time
Mapping (Fig. 4 B).There are 10 minutes after treatment in the peak (183%) of DTPA-Gd groups, is gradually reduced afterwards, until treatment
Revert within 3 hours afterwards and suitable (149%) before injection.The rapid decline of SI may be shorter with it circulation time in vivo and quilt
Quickly remove relevant.Conversely, carrying out the tumor tissues of radiography in note using pHPMA-DOTA-Gd and pHPMA-DOTA-Gd-cRGD
With SI values higher when 10 minutes after penetrating, and SI values still may proceed to raise, and respectively reach 200% and 240%.pHPMA-
The SI values high of DOTA-Gd groups can be continued until 19 hours, pHPMA-DOTA-Gd-cRGD after injection for 10 minutes from after injection
Holding time for group is longer to after injecting 24 hours.
The reason for signal enhancing for poly conjugate can reach 24 hours, it may be possible to due to the extension of circulation time
The aspect reason of tumor accumulation two higher.It is that this conclusion has strong evidence for the result of study of mouse bladder SI signal enhancings
According to.The enhanced peaks of DTPA-Gd group bladders SI occur 10 minutes after injection, then rapid at 10 to 30 minutes to decline (figure
4C).Therefore, it can judge that the renal excretion of DTPA-Gd is begun within a few minutes after injection, this explains why swelling
The contrasting effects of knurl imaging are very poor.Conversely, the bladder SI signal enhancings of conjugate group can remain small to 3 in 30 minutes from after injection
When, slower RE and circulation time more long is imply, it is that the EPR effects that molecular weight high is mediated make to trace it to its cause
Conjugate is gathered in tumor locus.It is interesting that pHPMA-DOTA-Gd-cRGD is relative to pHPMA-DOTA-Gd and DTPA-Gd
With higher and the longer time SI enhancings, this phenomenon is probably due to cRGD polypeptides and αvβ3The specific binding of integrin
Cause, and this combination is more stablized, and further promotes conjugate in the aggregation of tumor tissues so as to extend circulation
Time.
2nd, the Tissue distribution of conjugate nanosystems
U87 tumor-bearing mices are randomly divided into 3 groups (every group 7), and DTPA-Gd, pHPMA-DOTA-Gd and pHPMA- are injected respectively
DOTA-Gd-cRGD (0.08mmolGd (III)/kg Mouse Weights) carries out MRI Contrast enhanced imaging research, 24 hours after injection
Put to death mouse.Core dirty, liver, spleen, lung, kidney and tumor tissues are weighed, and use 1mL H2O2With 3mL HNO3Disappear
Change, sample is processed 48 hours at 120 DEG C.Gd (III) content of sample is measured by ICP-MS, and data are using shared every gram
The percentage of the injection dosage of mouse is represented.
Fig. 5 shows that nanosystems prepared by the present invention have Gd (III) higher compared to DTPA-Gd in tumor tissues
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
Again with 44 times.It is not difficult to find out, cRGD how peptide-mediated ligand specificity is combined in the tumor tissues aggregation of conjugate and residual
Important function is played.Additionally, the gadolinium that there is higher concentration in liver and spleen also confirms copolymer in vivo with longer
Circulation time.This also points out the vent path of magnetic resonance imaging probe and may cause the target organ of toxicity.
3rd, cytotoxicity analysis
Three kinds of vitro cytotoxicity analyses of material are carried out using CCK-8 (colleague's chemistry, Japan) kit.By U87 and
L02 cells are inoculated with 96 orifice plates (5 × 10 respectively3Individual/hole), culture 24 hours after, change into respectively containing various concentrations DTPA-Gd,
Culture medium (Gd (III) concentration of pHPMA-DOTA-Gd or pHPMA-DOTA-Gd-cRGD：50th, 100,250 and 500nmol/
ML), continue to cultivate 48 hours.PBS is washed 3 times, and 100 μ L CCK-8 reagents (10 μ L stostes are added per hole：90 μ L RPMI1640 are trained
Support base).After being incubated 2 hours, by VarioscanFlash ELIASAs (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 show, by various concentrations DTPA-Gd, pHPMA-DOTA-Gd or pHPMA-DOTA-Gd-cRGD treatment 48
L02 cytoactives after hour are without substantially change (Gd (III) content：50-500nmol/mL).However, pHPMA-DOTA-Gd
Show to act on (Fig. 6 B) to the dose-dependent inhibition of U87 cytoactives with pHPMA-DOTA-Gd-cRGD both of which.This can
Can be relevant with the intercellular interactions of U87 with HPMA copolymers.U87 cells express alpha highvβ3And αvβ5Integrin, and cRGD targets
To αvβ3And αvβ5Integrin, improves phagocytosis amount of the cell to magnetic resonance imaging probe, because the toxicity etc. of gadolinium ion triggers
The apoptosis of cancer cell.It is worth noting that, conjugate (Gd (III) ﹤ 50nmol/mL) the treatment U87 cells of low concentration do not have
Show cytotoxicity.
PHPMA-Gd conjugates biocompatibility in vitro is assessed：
1st, hemolytic analysis
By healthy human body new blood using PBS dilute after (16%v/v, 300 μ L), respectively with 1mg/mL, 3mg/mL or
The different materials of 5mg/mL are common to be incubated 12 hours at 37 DEG C, and then 1000g centrifugations 10min, takes 200 μ L upper liquids to 96 holes
Plate, the absorbance of 540nm is measured by VarioscanFlash ELIASAs.Blood sample be dissolved in distilled water and PBS respectively as
100% haemolysis and negative control.
Erythrocyte hemolysis are according to American Society for Testing Materials (ASTM) standard, and magnetic resonance imaging probe prepared by the present invention is equal
With blood compatibility (erythrocyte hemolysis ﹤ 5%) (Fig. 7) higher, (5mg/mL) material group and PBS control group under higher concentration
Compared to hemolysis rate still without notable difference.Conjugate has among these suitable molecular weight, hydrophilic nmature and surface negative electricity
The excellent properties such as lotus protect red cell membrane from the destruction of magnetic resonance imaging probe, are allowed to blood compatibility.
Erythrocyte aggregation and the form present invention further have evaluated high concentration and test magnetic resonance imaging spy by SEM methods
Red blood cell whether there is aggregation and morphologic change after pin (5mg/mL) treatment.Fig. 8 shows, similar with PBS control group, after conjugate treatment
Red blood cell still remains monodispersed normal biconcave form and smooth surface.It is red thin after material process according to existing document
The haemolysis of born of the same parents, aggregation and morphologic change are generally polymerized with electrostatic interaction, hydrophilic and hydrophobic grouping distribution and HMW
The aggregation of thing etc. is relevant.Fortunately, by well-designed, magnetic resonance imaging probe prepared by the present invention has surface negative electricity concurrently
Lotus, hydrophilic radical and suitable molecular weight, so as to have good biocompatibility to red blood cell.
2nd, coagulation analysis
Collection healthy human body new blood, is collected by centrifugation platelet-poor plasma.Take 360 μ L blood plasma materials different from three kinds respectively
Material (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 used as control.
As shown in figure 9, the present invention prepare magnetic resonance imaging probe even if (5mg/mL) still has in higher concentrations
There is the APTT and PT similar to PBS control group.This shows that material of the invention does not influence significantly on blood clotting factor, returns
Because in the hydrophily of HPMA copolymer chains.
3rd, thrombelastogram (TEG)
Extract healthy human body new blood and mix (blood with different materials：Material=9：1,1mL) nano material is made most
Final concentration of 0.1mg/mL and 1mg/mL.Add mixture to containing in kaolinic special pipe.Take 340 μ L of supernatant liquid and 20
The CaCl of μ L2(0.2M) is added in test sample cup, is tested by thrombelastogram instrument (Haemoscope companies).In equal volume
PBS is used as control.
The whole blood of different experiments material process is similar with control group.R, K value, α angles and MA are without substantially 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 sum, it may be determined that prepare
HPMA-Gd (III) conjugate be safety and blood compatibility.
4th, toxicity in vivo research
In vitro toxicity research is only the premise of In vivo study, only by after internal security verification, magnetic resonance imaging
Probe just can apply to clinic.The present invention is studied using the BALB/c mouse of health, and concrete operations refer to method part.
The present invention is carried out by the behavior to animal, body weight, hematology and histologic analysis to the toxicity in vivo of magnetic resonance imaging probe
Comprehensive assessment.By the research of 19 days by a definite date, the present invention without find any dehydration, dyskinesia, muscular atrophy, apocleisis or
Person other phenomenons related to animal toxicity.Meanwhile, the mouse after the administration of conjugate nanosystems has similar to GTPA-Gd groups
Changes of weight.Other anomalies are not found.
In order to further study other genotoxic potentials or obvious 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 control group, do not have
Discover a marked discrepancy.This result shows that conjugate nanosystems are consistent with 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 shows
Heart, liver, spleen, lung and the big major organs of kidney five be it is normal, in the absence of think in histopathology it is abnormal,
Degenerate or damage.Therefore, it is considered herein that the conjugate for preparing has good biocompatibility and security, among these
The well-designed structure of pHPMA-Gd (III) conjugate and biodegradable characteristic necessarily play an important role.
Statistical analysis
All data are represented using mean ± SD.The significance of difference between each group of data uses ANOVA and Student's
Sided t-inspection carries out statistical analysis, and P ﹤ 0.05 are statistically significant.
Claims (8)
1. a kind of biodegradable poly HPMA-Gd magnetic resonance imaging probes, it is characterised 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 probes according to claim 1, it is characterised in that should
The mean molecule quantity of magnetic resonance imaging probe is 85-94KDa.
3. biodegradable poly HPMA-Gd magnetic resonance imaging probes according to claim 2, it is characterised in that its
In, m:n:O=40:443:66.
4. the preparation method of biodegradable poly HPMA-Gd magnetic resonance imaging probes, its feature according to claim 1
It is, as R1=SH, to comprise the following steps：
1) HPMA, MA-DOTA, pTEMA and CTA-GFLGK-CTA are dissolved in the deionized water containing VA044/methyl alcohol and mix molten
Liquid, is placed in exuberant 40-60 minutes in argon gas, then stirring reaction；Solution liquid nitrogen be quenched after through acetone precipitation, separate out pyridine two
Sulphur modification product pHPMA-DOTA；
2) under nitrogen protective condition, the sulphur modification product pHPMA-DOTA of pyridine two is dissolved in deionized water, adds DTT stirrings
Reaction, obtains intermediate product；By intermediate product and GdCl3·6H2O is dissolved in distilled water, after stirring reaction, adjusts pH value, then room
After warm stirring reaction, pHPMA-DOTA-Gd products are obtained.
5. the preparation method of biodegradable poly HPMA-Gd magnetic resonance imaging probes, its feature according to claim 1
It is, when R1 is not SH, to comprise the following steps：
On the basis of claim 2 preparation method：
3) take pHPMA-DOTA-Gd to be dissolved in deionized water, while adding the cRGD of the sulphur modification of pyridine two modification, pass through after reaction
FPLC post separation and dialysis are purified, and obtain pHPMA-DOTA-Gd-cRGD.
6. the preparation method of biodegradable poly HPMA-Gd magnetic resonance imaging probes, its feature according to claim 4
It is that deionized water is with methyl alcohol with 4：1 volume ratio is mixed to get step 1) in mixed solution；All polymerisations, separation
The solvent that purification is used is three class solvents.
7. the preparation method of biodegradable poly HPMA-Gd magnetic resonance imaging probes, its feature according to claim 6
It is that the amount of contained initiator VA044 is only 1/3 equivalent of chain-transferring agent CTA-GFLGK-CTA in the mixed solution.
8. the preparation method of poly HPMA-Gd magnetic resonance imagings probe according to claim 4, it is characterised in that step 2)
Adjust pH value to 5.2-5.4.
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| CN113797350A (en) * | 2021-08-13 | 2021-12-17 | 四川大学华西医院 | Glycosyl polymer and preparation method and application thereof |
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