CN106589042B - Hepatoma-targeting carbohydrate ligands molecule and preparation method thereof and drug-loading system - Google Patents
Hepatoma-targeting carbohydrate ligands molecule and preparation method thereof and drug-loading system Download PDFInfo
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Abstract
The present invention relates to the preparations of a kind of hepatoma-targeting carbohydrate ligands molecule and its liposome by master of the present invention, more particularly to the targeting carbohydrate ligands molecule of liver ASGPR receptor binding site, and the liposome of application targeting carbohydrate ligands molecule preparation.The carbohydrate ligands molecule is made of three parts: Liver targeting glycosyl group, lipophilicity " anchor " connect the substituted or non-substituted with-(CH2) of the two with by ester bond8Straight chain spacerarm.The hepatoma-targeting carbohydrate ligands molecule enables to the anti-tumor drug in the drug-loading system to be concentrated on tumor cell of liver to greatest extent, improves drug therapeutic indices, reduces whole body toxic side effect, improves the life quality of patient.
Description
Technical field
The present invention relates to the preparations of a kind of hepatoma-targeting carbohydrate ligands molecule and its liposome, more particularly to liver ASGPR
The targeting carbohydrate ligands molecule of receptor binding site, and the liposome of application targeting carbohydrate ligands molecule preparation.
Background technique
Primary carcinoma of liver (primary liver cancer, PLC) be clinically the most common malignant tumor of digestive tract it
One, wherein 90% is hepatocellular carcinoma (hepatocellular carcinoma, HCC).China is the high-incidence state of HCC, accounts for about generation
The 70% of sum occurs for boundary HCC, accounts for [Chen, W., et al., Cancer the statistics in of domestic tumor mortality rate the 3rd
China,2015.CA Cancer J Clin,2016.66(2):p.115-32].Early stage HCC first choice treatment method includes operation
Surgical intervention including excision and liver transfer operation.It but has been middle and advanced stage when Most patients are made a definite diagnosis, and it is viral often to merge second/third type
Hepatitis, cirrhosis etc. have lost surgical operation chance, and chemotherapy is often the only effective treatment means at this time.However chemotherapy
The shortcomings such as the generally existing curative effect of drug is low, side effect is more.In addition to chemotherapeutics pharmacological action itself is still not ideal enough, master
It wants reason that drug exactly cannot be accurately and effectively transported to liver lesion position, causes drug bioavailability low；Even if
Drug, which can target, transports liver, and is caused again by liver rapid metabolization, escalated dose and shortening administration time interval to it
The serious toxic side effect of his normal organ, greatly reduces the useful effect of drug.Because to improve the drug concentration of cancer site, prolong
Novel nano target controlling and releasing system for the purpose of long drug treating time and reduction poisonous side effect of medicine is ground in liver cancer drug therapy
Application in studying carefully is increasingly becoming the hot spot of liver cancer treatment research.
There are a large amount of receptors, such as asialoglycoprotein receptor on hepatic parenchymal cells surface
(asialoglycoprotein receptor, ASGPR), TfR (transferrin receptor, TfR), height
Density lipoprotein receptor (high density lipoprotein receptor, HDLR), LDL receptor (low
Density lipoprotein receptor, LDLR), growth factor receptors, insulin receptor etc., wherein ASGPR and TfR are
Two kinds of efficient endocytic receptors, TfR is present on the cell membrane of various kinds of cell perhaps, and ASGPR exists only in hepatic parenchymal cells, because
This ASGPR becomes best receptor [Huang Yuanyu, Liang Zicai, the asialoglycoprotein receptor and its in drug of liver orientation transhipment
Progress in Biochemistry and Biophysics, 2015 (06): the 501-510 pages are applied in Liver targeting delivering].
ASGPR is a kind of quantity endocytosis receptor of heteoro-oligomers abundant, exists only in liver parenchymal cell direction
The cell membrane surface of sinusoid side can be identified specifically and be combined with D- galactolipin (D-galactose, Gal) or N-
Acetylgalactosamine (N-acetylgalactosamine, GalNAc) as terminal saccharide ligand molecular [Franssen,
E.J.F.,et al.,Hepatic and intrahepatic targeting of an anti-inflammatory
agent with human serum albumin and neoglycoproteins as carrier
molecules.Biochemical Pharmacology,1993.45(6):p.1215-26].Literature research discovery, ASGPR by
The compatibility of body and ligand is mainly influenced by following factor: 1) ligand end sugar subtype.End is matching for Gal or GalNAc
Body molecule can identify by ASGPR, GalNAc in conjunction with ASGPR 10~50 times of compatibility ratio Gal high [Rensen, P.C.,
et al.,Determination of the upper size limit for uptake and processing of
ligands by the asialoglycoprotein receptor on hepatocytes in vitro and in
Vivo.J Biol Chem, 2001.276 (40): p.37577-84], and GalNAc ligand molecular is easier the Kupffer that escapes
The identification of cell and more target tumor cell of liver [D'Souza, A.A.and P.V.Devarajan,
Asialoglycoprotein receptor mediated hepatocyte targeting-strategies and
applications.J Control Release,2015.203:p.126-39]；2) glycan molecule replaces site and ASGPR receptor
Compatibility has following rule: 1-OH, 2-OH (or 2-amino), 3-OH, 4-OH and 5-CH2- on Gal (or GalNAc) join
And the combination of ASGPR receptor；5-CH2- is connected with after electronegative group or 4-OH, 3-OH, 2-OH (or 2-amino) be substituted, with
Receptor affinity sharply declines；When glycosidation substitution occurs for 1-OH, when glycosidic bond is α conformation, weaken with receptor affinity, β structure
As when and substituent group be linear chain structure when, unaffected [Lee, R.T., Binding site of the rabbit of affinity
liver lectin specific for galactose/N-acetylgalactosamine.Biochemistry,
1982.21(5):p.1045-50]；Only 6-OH is not involved in receptor and combines and be directed toward solvent area, thus is suitable as connection site
With [Stokmaier, D., et al., Design, synthesis and the evaluation of that is connected such as carrier, drug
monovalent ligands for the asialoglycoprotein receptor(ASGP-R).Bioorg Med
Chem,2009.17(20):p.7254-64]；However, using different terminal moleculars, between the length and affinity of spacerarm
Relationship there is no more detailed data disclosures.
Therefore, according to above-mentioned HCC current treatment status, the present invention provides a kind of in conjunction with liver cell ASGPR receptor-specific
Carbohydrate ligands molecule is targeted, meanwhile, which can modify specific drug-loading system, make anti-tumor drug by the drug-loading system
It is concentrated on tumor cell of liver to greatest extent, reduces drug and is distributed in other normal tissues, significantly improve drug therapeutic indices, reduce
Whole body toxic side effect improves the life quality of patient.
In addition, according to the literature, glycosyl ligand molecular is synthesized using chemical method and is needed because there is a large amount of sugared hydroxyl
Measure is accurately protected and be deprotected to the hydroxyl of non-reaction site, so that reaction condition is complicated, step is tediously long, low efficiency
Under.And biological enzyme is a kind of protein with catalysis, compared with chemical catalyst, have efficient, high regioselectivity,
The advantages that less toxic, is referred to as green catalyst, especially has unique advantage in the regioselectivity esterification of glycan molecule.
Therefore, the present invention provides a kind of method of biological enzymatic synthesis glycosyl ligand molecular that efficient specificity is strong.
Summary of the invention
The present invention provides a kind of Liver targeting carbohydrate ligands molecule, and the carbohydrate ligands molecule particular load is in nano medicament carrying system, energy
Enough so that the anti-tumor drug in the drug-loading system is concentrated on tumor cell of liver to greatest extent, work improves drug therapeutic indices, drops
Low whole body toxic side effect, improves the life quality of patient.The Liver targeting carbohydrate ligands molecule is made of three parts: Liver targeting glycosyl
Group, lipophilicity " anchor " structure connect the substituted or non-substituted with-(CH2) of the two with by ester bond8Straight chain spacerarm.
Further, herein described Liver targeting group, including but not limited to GalNAc, lipophilicity " anchor " include but is not limited to
Cholesterol (cholesterol, Chol) and its analogue；
Further, the surprised discovery of applicant, when spacerarm be straight chain it is non-substituted-(CH2)8It is more suitable for loading on and carries medicine system
System.
Specifically, the present invention provides a kind of acetylamino galactosamine (N-acetylgalactosamine, GalNAc) and matches
Body molecule, the ligand molecular can be specific in conjunction with ASGPR in liver cell.The ligand molecular structure is as follows:
The present invention also provides a kind of methods for preparing the carbohydrate ligands molecule, i.e., pass through catalyzed by biological enzyme structure in nonaqueous phase
Carbohydrate ligands molecule is built, is building GalNAc ligand molecular specifically.
Specific experiment method is, with cholesterol-decanedioic acid monoene ester (CHS-SE) for starting material, in lipase TL IM
Under catalysis, C on highly selective single-minded and GalNAc6- OH esterification is coupled to arrive Liver targeting ligand material GalNAc-C8-Chol。
Specific test procedure is as follows: 10mL tool plug bottle taken, CHS-SE (0.1mmol), GalNAc (0.05mmol) is added,
Add 5mL dehydration acetone, enzyme TL IM 20mg, oscillation in air bath constant temperature oscillator (45 DEG C, 250rmin-1), reaction
24h.To after reaction, filtering is dezymotized, recycling design is to get product, and reaction equation is as shown in Figure 1.Product passes through quick
Silica gel column chromatography purifying, purified product using infrared, ESI,1H NMR、13C NMR carries out Structural Identification.
Inventor has found by many experiments, and lipase TL IM can C on single-minded catalysis GalNAc in Examples of non-aqueous solvents6-
OH esterification, and reaction condition is mild, high conversion rate, purification step is easy, and reaction reagent low toxicity easily removal used synthesizes material
Expect cheap and easy to get.
The present invention also provides a kind of drug-loading system of Liver targeting ligand molecular, the lipophilicity of the Liver targeting ligand molecular
" anchor " damascene is in load medicine body system, by substituted or non-substituted with-(CH2)8Straight chain spacerarm is sugared by Liver targeting
Group, which is exposed to, carries medicine body system surface.
Further, the drug-loading system includes but is not limited to nano liposomes drug-loading system.Drug-loading system is nano-lipid
When body drug-loading system, lipophilicity " anchor " damascene is in the phospholipid bilayer of the nano liposomes drug-loading system.
It specifically, is the drug-loading system of GalNAc ligand molecular modification, then the GalNAc ligand molecular is exposed to load medicine
Decorum surface.
Inventor surprisingly it has been found that, when spacerarm be straight chain it is non-substituted-(CH2)8The carbohydrate ligands molecule being more suitable for, specifically
For, when being GalNAc ligand molecular, the liposome drug-loading system modified is more stable, with ASGPR affinity highest.Described
GalNAc ligand modified nano liposomes drug-loading system is as shown in Figure 9.
The present invention also provides a kind of catalyzed by biological enzyme, and the nano liposomes liver that GalNAc modification is constructed in nonaqueous phase is swollen
Tumor targeting drug delivery system.
Specifically the preparation method comprises the following steps: weighing phosphatide (PC), cholesterol (CHS), DSPG-Na, GalNAc ligand by a certain percentage
The mixed dissolutions such as molecule, drug 55 DEG C in eggplant-shape bottle, 40rpm revolving removing chloroform, form uniform adipose membrane, add in chloroform
Enter pH 7.4PBS buffer, then 55 DEG C of aquation 1h successively squeeze filtration with 100nm, 50nm filter membrane to get uniform nanoparticle.
The experimental results showed that the liposome uptake ratio of GalNAc modification is higher.
Further, the nano liposomes liver tumour targeting drug delivery system package-contained drug is anti-cancer chemical small-molecule drug
And/or traditional Chinese medicine monomer drug and/or bioavilability reinforcing agent, including but not limited to drug taxol (paclitaxel, PTX)
And/or bioavilability reinforcing agent piperine.
Detailed description of the invention
Fig. 1 GalNAc-C8- Chol mass spectrogram
Fig. 2 GalNAc-C8- Chol13C NMR spectra
Fig. 3 GalNAc-C8- Chol1H NMR spectra
Fig. 4 GalNAc (A) and GalNAc-C8- Chol glycosyl part (B) 13C-NMR spectrogram compares
Fig. 5 GalNAc-C8The part-Chol HMBC spectrogram
Fig. 6 GalNAc modified liposome (A) and common nano liposomes (B) particle diameter distribution
Fig. 7 GalNAc modified liposome (A) and the distribution of common nano liposomes (B) Zeta potential
Fig. 8 HepG2 cell compares (n=3) to FL-LP, GAL-FL, GalNAc-LP-FL uptake ratio.(**p<0.01).
Fig. 9 GalNAc ligand modified nano liposomes drug-loading system.
Below by specific embodiment, the invention will be further described.
Specific embodiment
A part of specific embodiment is set forth below, and the present invention will be described, it is necessary to which indicated herein is real in detail below
It applies example and is served only for that the invention will be further described, do not represent limiting the scope of the invention.Other people are according to the present invention
The some nonessential modifications and adjustment made still fall within protection scope of the present invention.
The synthesis of 1 GalNAc-C8-Chol of embodiment
Method: 10mL tool plug bottle is taken, CHS-SE (0.1mmol), GalNAc (0.05mmol) is added, it is de- to add 5mL
Water acetone, enzyme TL IM 20mg, air bath constant temperature oscillator is interior to vibrate (45 DEG C, 250rmin-1), and reaction is for 24 hours.Wait react
After, filtering is dezymotized, and recycling design is to get product, and reaction equation is as shown in Figure 1.Product is chromatographed by Flash silica column
Purifying, purified product carry out Structural Identification using infrared, ESI, 1H NMR, 13C NMR.
Synthetic product identification
Product structure is accredited as target product through MS, NMR, and specific data are as follows:
MS condition: it after product is dissolved with proper amount of methanol, is analyzed with mass spectrometer (MS), mass spectrometry parameters: triple level four bars
LC-MS/MS: electron spray positively ionized (ESI+) detection, scanning range be m/z150-m/z 1000, [M+Na]+: 796.50；
1H NMR (500MHz, C5D5N) δ: 5.99 (H-1' α, d, J=3.65,3.65Hz, 1H), 5.43 (H-2, d, J=
5.19Hz, 1H), 5.33 (H-1' β, m, 1H), 4.93 (H-3, t, J=4.91,4.91Hz, 1H), 4.91 (H-6' α, d, J=
2.13Hz, 1H), 4.90 (H-2', s, 1H), 4.89 (H-5', s, 1H), 4.86 (H-6' β, m, 1H), 4.64 (H-3', d, J=
10.74Hz, 1H), 4.50 (H-4', s, 1H), 2.54 (H-7, m, 2H), 2.41 (H-28, t, J=7.43,7.43Hz, 2H),
2.33 (H-35, td, J=1.59,7.36,7.25Hz, 2H), 2.13 (H-38, s, 3H), 2.03-1.07 (m, 38H), 1.03 (H-
24, s, 3H), 0.99 (H-25, d, J=6.52Hz, 3H), 0.91 (H-22*2, dd, J=1.14,6.61,6H), 0.68 (H-26,
S, 3H)；13C NMR (125MHz, C5D5N): δ: 173.94 (C-36), 173.46 (C-27), 171.34 (C-37), 140.43
(C-1), 123.25 (C-2), 93.30 (C-1'), 74.27 (C-3), 70.84 (C-4'), 69.93 (C-5'), 69.56 (C-3'),
65.68 (C-6'), 57.17 (C-4), 56.74 (C-5), 52.59 (C-2'), 50.64 (C-6), 42.88 (C-8), 40.32 (C-
9), 40.13 (C-10), 39.02 (C-7), 37.64 (C-11), 37.23 (C-12), 36.88 (C-13), 36.44 (C-14),
35.15 (C-28), 34.73 (C-35), 32.55 (C-15), 32.42 (C-16), 29.72 (C-31,32), 29.68 (C-30,
33), 28.91 (C-18), 28.65 (C-19), 28.61 (C-17), 25.75 (C-29), 25.56 (C-34), 24.89 (C-20),
24.55 (C-21), 23.67 (C-38), 23.35 (C-22), 23.09 (C-22), 21.67 (C-23), 19.78 (C-24), 19.35
(C-25), 12.39 (C-26).
Mass spectrogram, nuclear-magnetism figure are shown in Fig. 1,2,3
It is verifying CHS-SE and GalNAc in the site of the lipase-catalyzed lower esterification of TL IM, we compared13C-NMR spectrum
The change in displacement of GalNAc and GalNAc-C8-Chol glycosyl part carbon signal on figure find GalNAc-C8-Chol glycosyl part
C-6 signal is to low field displacement about 2.4ppm, and C-5 signal is displaced about 2.5ppm to High-Field, and the displacement of other carbon signals does not have substantially
Variation, is shown in Fig. 4.Its alkane methyl carbon (α-C) signal can be made to low field displacement (+2~+4ppm) in general, saccharide part OH is acetylation,
Its ortho position carbon (β-C) signal is displaced (- 2~-6ppm) to High-Field, that is, there is glycosidation offset phenomena.It is regular using this, we
Determine that esterification only occurs on the position GalNAc C-6.
Further to confirm the site that CHS-SE and GalNAc are esterified under TL IM catalysis, we use NMR spectra technology
(HMBC) analysis of heteronuclear Long-Range Correlation, two hydrogen of discovery C-6 are carried out to C-6 hydrogen of sugar in structure and carbonyl (C-36)
(H-6'a, H-6'b) has long-range related (see Fig. 5) to C-36 carbonyl carbons, further demonstrates esterification and exists only in glycosyl
C-6 hydroxyls.
The preparation and characterization of 2 GalNAc of embodiment modification nano liposomes
Preparation method: phosphatide (PC), cholesterol (CHS), DSPG-Na, GalNAc ligand molecular, medicine are weighed by a certain percentage
Object etc. presses (40:20:1:4:1) quality than mixed dissolution in chloroform, 55 DEG C in eggplant-shape bottle, 40rpm revolving removing chloroform, shape
At uniform adipose membrane, pH 7.4PBS buffer is added, then 55 DEG C of aquation 1h successively squeeze filtration with 100nm, 50nm filter membrane, i.e.,
Obtain uniform nanoparticle.
And the partial size of the two (Fig. 6), zeta current potential (Fig. 7), encapsulation rate etc. are characterized.As a result it is made and is received using the method
Grain of rice diameter is smaller, and particle diameter distribution is uniform, and quality is stablized, and encapsulation rate height (being shown in Table 1) meets follow-on test requirement.
1 conventional liposome of table (n=3) compared with galactose modification nano liposomes
3 GalNAc of embodiment modifies nano liposomes targeting Journal of Sex Research
Experimental method: taking the human hepatoma HepG2 cell in logarithmic growth phase to be inoculated in 24 orifice plates, to cell fusion degree
Up to 80% or so and cellular morphology it is full after, be separately added into appropriate common fluorescent liposome (FL-LP) and GalNAc-LP-FL and (use
It is 1mmolmL that culture medium, which adjusts lipid concentration,-1).In 37 DEG C, 5%CO2It is incubated for 1h in incubator, then uses 1mL PBS (pH
7.4) the fluorescence nano liposome not by cellular uptake is washed off, 1%TriotnX-100 PBS lytic cell is measured with microplate reader
The fluorescence intensity of HepG2 cellular uptake FL-LP and GalNAc-LP-FL carry out liver tumour and target in-vitro evaluation.
GalNAc, which is evaluated, by human hepatoma HepG2 cell modifies fluorescent lipid (GalNAc-LP-FL) hepatic targeting, knot
Fruit shows that HepG2 is significantly higher than conventional liposome (FL-LP) to the liposome uptake ratio after GalNAc is modified.It is previously added
GalNAc can significantly inhibit intake of the HepG2 to GalNAc modified liposome.And we also provide a comparison of early period using galactolipin
(GAL) the uptake ratio difference of modified liposome (GAL-FL) and GalNAc modified liposome, discovery HepG2 cell repair GalNAc
It is higher to adorn liposome uptake ratio, sees Fig. 8.
Claims (5)
1. a kind of nano liposomes drug-loading system, which is characterized in that include Liver targeting ligand molecular, the Liver targeting ligand molecular
It is made of three parts: Liver targeting glycosyl group, lipophilicity " anchor " structure and non-substituted-(CH2) both connected by ester bond8-
Straight chain spacerarm；Wherein the Liver targeting glycosyl group is GalNAc, and lipophilicity " anchor " structure is cholesterol and its structure class
Like object, lipophilicity " anchor " damascene passes through-(CH2) in the drug-loading system8Spacerarm is by Liver targeting glycosyl group
It is exposed to drug-loading system surface.
2. nano liposomes drug-loading system as described in claim 1, which is characterized in that lipophilicity " anchor " damascene in
In the phospholipid bilayer of the nano liposomes drug-loading system.
3. nano liposomes drug-loading system as described in claim 1, which is characterized in that the system of the Liver targeting carbohydrate ligands molecule
Preparation Method are as follows: carbohydrate ligands molecule is constructed by catalyzed by biological enzyme in nonaqueous phase, with cholesterol-decanedioic acid monoene ester CHS-SE
For starting material, under lipase TL IM catalysis, with the C on GalNAc6The esterification coupling of the position-OH.
4. nano liposomes drug-loading system as described in claim 1, which is characterized in that the nano liposomes drug-loading system
The preparation method comprises the following steps: phosphatide, cholesterol, DSPG-Na, the Liver targeting ligand molecular, drug mixed dissolution are weighed in chloroform,
55 DEG C in eggplant-shape bottle, 40rpm revolving remove chloroform, form uniform adipose membrane, be added pH 7.4PBS buffer, 55 DEG C of aquation 1h, so
Afterwards successively with 100nm, 50nm filter membrane squeeze filtration to get.
5. nano liposomes drug-loading system as claimed in claim 4, which is characterized in that the nano liposomes drug-loading system
Zeta potential is -57.7 ± 1.6mV.
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| JP7011080B2 (en) * | 2017-12-26 | 2022-02-10 | クワンチョウ リボバイオ カンパニー リミテッド | Modified oligonucleotides and compounds that can be used to synthesize modified oligonucleotides |
| CN109957566B (en) * | 2017-12-26 | 2023-08-25 | 广州市锐博生物科技有限公司 | Modified oligonucleotides and compounds useful for synthesizing modified oligonucleotides |
| CN112778389B (en) * | 2019-12-31 | 2022-05-06 | 嘉应学院 | Targeting ligand molecule, preparation method thereof and drug loading system |
| CN111514307A (en) * | 2020-04-30 | 2020-08-11 | 杭州勇诚睿生物科技有限公司 | Novel liver targeting drug carrier |
| CN111748003B (en) * | 2020-06-24 | 2021-10-15 | 河北大学 | N-acetamido galactose modified 3-nitrophthalimide derivative, preparation method and application thereof, and liver targeting probe |
| CN112891552B (en) * | 2021-01-28 | 2022-08-09 | 山东大学 | Paclitaxel galactosamine conjugate and nanoparticles targeting hepatocellular carcinoma, and preparation methods and applications thereof |
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