CN106474486B - A kind of polymer micelle and its application - Google Patents
A kind of polymer micelle and its application Download PDFInfo
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- CN106474486B CN106474486B CN201610901264.3A CN201610901264A CN106474486B CN 106474486 B CN106474486 B CN 106474486B CN 201610901264 A CN201610901264 A CN 201610901264A CN 106474486 B CN106474486 B CN 106474486B
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- 229920000642 polymer Polymers 0.000 title claims abstract description 81
- 239000000693 micelle Substances 0.000 title claims abstract description 65
- 239000003814 drug Substances 0.000 claims abstract description 40
- 229940079593 drug Drugs 0.000 claims abstract description 30
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 230000002500 effect on skin Effects 0.000 claims abstract description 5
- 229920000469 amphiphilic block copolymer Polymers 0.000 claims abstract description 3
- 238000001338 self-assembly Methods 0.000 claims abstract description 3
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- -1 poly- amino ester Chemical class 0.000 claims description 10
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- 239000000126 substance Substances 0.000 claims description 5
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 abstract description 16
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- GGHDAUPFEBTORZ-UHFFFAOYSA-N propane-1,1-diamine Chemical compound CCC(N)N GGHDAUPFEBTORZ-UHFFFAOYSA-N 0.000 description 3
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- AXTGDCSMTYGJND-UHFFFAOYSA-N 1-dodecylazepan-2-one Chemical compound CCCCCCCCCCCCN1CCCCCC1=O AXTGDCSMTYGJND-UHFFFAOYSA-N 0.000 description 1
- CPKVUHPKYQGHMW-UHFFFAOYSA-N 1-ethenylpyrrolidin-2-one;molecular iodine Chemical compound II.C=CN1CCCC1=O CPKVUHPKYQGHMW-UHFFFAOYSA-N 0.000 description 1
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 1
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- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
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- DCOPUUMXTXDBNB-UHFFFAOYSA-N diclofenac Chemical compound OC(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl DCOPUUMXTXDBNB-UHFFFAOYSA-N 0.000 description 1
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- 229930182833 estradiol Natural products 0.000 description 1
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- 238000010579 first pass effect Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 150000004040 pyrrolidinones Chemical class 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a kind of polymer micelle and its applications, are related to pharmaceutical technology field, and the polymer micelle is nucleocapsid structure, are self-assembly of in water by amphiphilic block copolymer；The present invention is using hyaluronic acid as main chain, by connecting hydrophobic polymer R on main chain1With hydrophilic polypeptide R2It prepares with the polymer micelle for carrying medicine function, drug can be promoted to enter dermal layer of the skin, and under the acidic pH environment of skin, with good stability and biocompatibility can carry out the local target administration of skin in conjunction with genomic medicine.
Description
Technical field
The present invention relates to pharmaceutical technology field more particularly to a kind of polymer micelle and its applications.
Background technique
Transdermal drug delivery system (transdermal therapeutic system, TTS) refers to be administered in skin surface, medicine
Object successively penetrates cuticula, epidermis, skin corium with constant speed, eventually enters into body circulation, generates whole body or local treatment is made
Emerging preparation.Cutaneous penetration has following 4 advantages:
(1) first pass effect and drug that transdermal drug delivery system can avoid liver are in the inactivation of gastrointestinal tract, and the absorption of drug is not
The individual difference that reduces medication is influenced by gastrointestinal factors.
(2) constant effective blood drug concentration or physiological effect are maintained, blood concentration peak valley phenomenon caused by being administered orally is avoided,
Reduce toxicity.
(3) administration number of times is reduced, therapeutic efficacy is improved, extends action time, avoids multiple dose administration, make most patient
It is easy to receive.
(4) easy to use, patient can autonomous medication, medication can also be cancelled at any time.
Skin is the natural barrier of human body, drug can be hindered to enter internal；Most drugs, even dosage is low, treats
High some drugs are imitated, percutaneous rate is also difficult to meet treatment needs, this becomes the biggest obstacle of research and development TTS.How to protect
The drugs through skin of card sufficient amount, which enters, reaches therapeutic dose in vivo, is the emphasis that TTS is studied, and new and high technology and method are answered
With being the important channel solved the problems, such as.
Currently, for promoting the technology of Cutaneous permeation that can be divided into chemical method and physical method.
Chemical method includes the chemical reagent such as Laurocapram, organic acid and its ester type compound, pyrrolidones analog derivative,
Its mechanism of action is dissolution skin lipid or is denaturalized skin protein, increases the randomness of lipoids skeleton, to promote drug
It is spread in cuticula, increases the solubility of drug in skin, increase Drug Percutaneous Absorption.
Physical method includes that iontophoresis promotees saturating method, ultrasonic wave promotees saturating method, electroporation promotees saturating method etc., mainly logical
The physical means such as electromagnetic field, ultrasonic wave, laser are crossed to promote the Transdermal absorption of drug, physics promotees saturating technology and is most appropriate to albumen
The Transdermal absorption of matter, peptides and certain macromolecular drugs.
But above two method has their limitation, surface texture of the chemical method due to that can destroy skin, so
It will lead to the irritative response of some skins；And physical method relies primarily on extraneous effect, generally requires some special equipment,
So limiting the application of such methods.
In recent years, research of the nano-medicament carrier in percutaneous dosing field increases sharply.Some nano-drug preparations into
Enter clinical investigation phase, certain formulations product such as heparin liposome, Diclofenac liposome, povidone iodine liposome, estradiol
Nano-emulsion, Indomethacin nano-emulsion etc. have listed application.Nano-medicament carrier can effectively facilitate the Transdermal absorption of small-molecule drug,
And good prospect is also presented in terms of macromolecular drug percutaneous dosing research.
A kind of structure of the micellar material as Nano grade, the modifiability on surface, biocompatibility become
A kind of pharmaceutical carrier to receive much attention is widely used in drug delivery, antitumor and targeted therapy etc., but is at present
Only, the design of micella is based on tumor microenvironment, and micella is used in transdermal aspect and is had not been reported.
Summary of the invention
In view of the above deficiencies, the present invention provides a kind of polymer micelle and its application, which can be carried
Drug promotes drug to enter dermal layer of the skin, and the acidic pH environment of skin is with good stability and biocompatibility,
The local target administration of skin can be carried out in conjunction with drug.
In order to achieve the above object, the technical solution adopted in the present invention is as follows: a kind of polymer micelle, is core-shell structure copolymer knot
Structure is self-assembly of in water by amphiphilic block copolymer, shown in the general structure of the block copolymer such as formula (I),
Wherein, R1For poly lactide-glycolide acid, polylactic acid, polystyrene, poly- amino ester or their derivative
Object；R2For transdermal peptide, amino acid sequence ACTGSTQHQCG, RRRRRRR, ACSSSPSKHCG, ACKTGSHNQCG or
HIITDPNMAEYL；M, n, z are the integer greater than 2.
In above-mentioned polymer micelle, R1Part is hydrophobic section；Hyaluronic acid and R2Part is hydrophilic section；M refers to polymer latex
R is connected in beam1Hyaluronic acid structural unit unit number；N refers to the hyaluronic acid structure being connected in polymer micelle
The unit number of unit；Z refers to not connected R1And R2Hyaluronic acid structural unit unit number.
R1The height and R of grafting rate1Molecular size range have an impact to the targeting of drug, preferably, the R1
Grafting rate in the monomer of place is 1~20%.
Preferably, in polymer micelle, the R1Total molecular weight be 1000~50000.
R2The height of grafting rate has an impact to the transdermal effect of entire polymer micelle, preferably, the R2In place list
Grafting rate in body is 1~20%.
Preferably, the R1For poly- amino ester, R2Amino acid sequence be ACTGSTQHQCG, m:n=1:1, the program
Not only target-oriented drug is good, and transdermal effect is also best, can preferably play the effect of drug.
The present invention also provides the polymer micelles to prepare the application in dermal drug carrier.
The present invention also provides a kind of polymer micelle compound of carrying medicaments, including drug and carrier, the carriers
For the polymer micelle.
Specifically, the drug is DNA, siRNA, miRNA or hydrophobic class chemicals.
Compared with prior art, the invention has the following advantages:
(1) present invention is using hyaluronic acid as main chain, by connecting hydrophobic polymer R on main chain1With hydrophilic polypeptide R2
It prepares with the polymer micelle for carrying medicine function, drug can be promoted to enter dermal layer of the skin, and in the acid pH of skin
Under environment, with good stability and biocompatibility can carry out the local target administration of skin in conjunction with genomic medicine.
(2) Inventive polymers micella can efficient carrying gene drug, and act on skin, realize the local target of skin
To administration.
Detailed description of the invention
Fig. 1 is hyaluronic acid in polymer micelle/FITC-DNA compound1H-NMR map (solvent D2O)；
Fig. 2 is transdermal peptide (ACTGSTQHQCG) in polymer micelle/FITC-DNA compound1H-NMR map (solvent
For D2O)；
Fig. 3 is polymer R in polymer micelle/FITC-DNA compound1's1H-NMR map (solvent CDCL3)；
Fig. 4 is hyaluronic acid-polypeptide in polymer micelle/FITC-DNA compound1H-NMR map (solvent D2O)；
Fig. 5 is polymer micelle/FITC-DNA compound1H-NMR map (solvent D2O)；
Fig. 6 is polymer micelle/FITC-DNA compound TEM electron microscope；
Fig. 7 is polymer micelle/FITC-DNA compound DLS particle diameter measurements schematic diagram；
Fig. 8 is the TEM electron microscope after polymer micelle/FITC-DNA compound carrying gene；
The born of the same parents that enter after Fig. 9 is polymer micelle/FITC-DNA compound carrying gene FITC-DNA scheme；In figure A be micella/
FITC-DNA group；B is naked FITC-DNA group；
Figure 10 is transdermal under laser co-focusing after being polymer micelle/FITC-DNA compound carrying gene FITC-DNA
Effect picture；A is micella/FITC-DNA in figure；B is naked FITC-DNA group；
Specific embodiment
Hyaluronic acid derives from Shandong Fu Ruida Pharmaceutical Group in the embodiment of the present invention, and the polypeptide after synthesis is purified,
Purity is 95%, takes 5mg hyaluronic acid or polypeptide sample, is dissolved in D2O, is used Nuclear Magnetic Resonance (Brooker,Switzerland company)
It is analyzed, the 13H NMR spectra of the two is as shown in Figure 1 and Figure 2.
Embodiment 1
Polymer micelle and the preparation of polymer micelle/FITC-DNA compound:
1, high molecular polymer R1The synthesis of (poly- amino ester)
3.73g3- aminopropanol and 10g diacrylate-Isosorbide-5-Nitrae-fourth diester are taken, is placed at 90 DEG C and is stirred and be heated to reflux, into
Row homopolymerization 3 hours；After completion of the reaction, product is dissolved in 20ml chloroform, 10 times of volumes are added passes through pre-cooling
Diethyl ether solution, precipitate product；Three times, filtered product vacuum drying 24 hours obtains for the product for repeating filtering precipitating
To the higher polymer poly amino ester of purity, nuclear magnetic resonance result is as shown in Figure 3.
2, hyaluronic acid-polypeptide synthesis
100mg hyaluronic acid (MW=17000) is taken, stirring and dissolving in 20ml ultrapure water is placed in, 15mg is added thereto
NHS and 20mg EDC is added the transdermal peptide of 1mg (ACTGSTQHQCG) after activation hyaluronic acid 1 hour, continues stirring 0.5 hour
~5 hours to fully reacting carry out；Reaction solution after reaction is fitted into the bag filter that molecular weight is 3500, is dialysed 24 hours,
The impurity and polypeptide on unreacted are removed, freeze drying protectant is added and carries out freeze-drying 24 hours, it is more to obtain dry hyaluronic acid-
Peptide solid, nuclear magnetic resonance result are as shown in Figure 4.
3, the synthetic method of polymer micelle
50mg hyaluronic acid-polypeptide is dissolved in 20ml water, 5mg NHS and 10mg EDC, stirring are added after stirring and dissolving
1h makes remaining part activated carboxylic on hyaluronic acid.
Take 0.1g polymer R1, it is dissolved in 10ml chloroform, 100 μ l propane diamine is added, after stirring 12h, pours into pre-cooling
In the diethyl ether solution crossed, takes out in the vacuum oven that precipitating places it at room temperature, obtain by amido modified polymer
R1。
Polymer R by 0.1g by modification1It is dissolved in the water of pH=3, hyaluronic acid-polypeptide after activation is then added
Solution continues stirring 6 hours, and final product is dialysed 24 hours, removes impurity and unreacted raw material；Add frozen-dried protective
Agent, and be lyophilized 24 hours, dry final product polymer micelle is obtained, nuclear magnetic resonance result is as shown in Figure 5.
4, the method for polymer micelle loaded gene
10mg polymer micelle is taken, is dissolved in 1ml water, pH to 3 is adjusted, completely dissolves in water polymer micelle.
2 μ g FITC-DNA are taken, the polymer micelle solution of 10 μ l is added thereto, are then diluted to 30 μ l, 15 points are incubated at 37 DEG C
Polymer micelle/FITC-DNA compound is made in clock.
Embodiment 2
Polymer micelle and the preparation of polymer micelle/FITC-DNA compound:
1, high molecular polymer R1The synthesis of (poly- amino ester)
3.73g3- aminopropanol and 10g diacrylate-Isosorbide-5-Nitrae-fourth diester are taken, is placed at 90 DEG C and is stirred and be heated to reflux, into
Row homopolymerization 3 hours；After completion of the reaction, product is dissolved in 20ml chloroform, 10 times of volumes are added passes through pre-cooling
Diethyl ether solution, precipitate product；Three times, filtered product vacuum drying 24 hours obtains for the product for repeating filtering precipitating
To the higher polymer poly amino ester of purity, nuclear magnetic resonance result is as shown in Figure 3.
2, hyaluronic acid-polypeptide synthesis
100mg hyaluronic acid (MW=17000) is taken, stirring and dissolving in 20ml ultrapure water is placed in, 15mg is added thereto
NHS and 20mg EDC is added the transdermal peptide of 50mg (ACTGSTQHQCG) after activation hyaluronic acid 1 hour, continues stirring 0.5 hour
~5 hours to fully reacting carry out；Reaction solution after reaction is fitted into the bag filter that molecular weight is 3500, is dialysed 24 hours,
The impurity and polypeptide on unreacted are removed, freeze drying protectant is added and carries out freeze-drying 24 hours, it is more to obtain dry hyaluronic acid-
Peptide solid, nuclear magnetic resonance result are as shown in Figure 4.
3, the synthetic method of polymer micelle
50mg hyaluronic acid-polypeptide is dissolved in 20ml water, 5mg NHS and 10mg EDC, stirring are added after stirring and dissolving
1h makes remaining part activated carboxylic on hyaluronic acid.
Take 1g polymer R1, it is dissolved in 10ml chloroform, 100 μ l propane diamine is added, after stirring 12h, pours into precooled
Diethyl ether solution in, take out precipitating and place it in vacuum oven at room temperature, obtain by amido modified polymer R1。
Polymer R by 1g by modification1It is dissolved in the water of pH=3, hyaluronic acid-polypeptide after activation is then added is molten
Liquid continues stirring 6 hours, and final product is dialysed 24 hours, removes impurity and unreacted raw material；Freeze drying protectant is added,
And be lyophilized 24 hours, dry final product polymer micelle is obtained, nuclear magnetic resonance result is as shown in Figure 5.
4, the method for polymer micelle loaded gene
10mg polymer micelle is taken, is dissolved in 1ml water, pH to 3 is adjusted, completely dissolves in water polymer micelle.
2 μ g FITC-DNA are taken, the polymer micelle solution of 10 μ l is added thereto, are then diluted to 30 μ l, 15 points are incubated at 37 DEG C
Polymer micelle/FITC-DNA compound is made in clock.
Embodiment 3
100mg hyaluronic acid (MW=17000) is taken, stirring and dissolving in 20ml ultrapure water is placed in, 15mg is added thereto
NHS and 20mg EDC is added the transdermal peptide of 10mg (ACTGSTQHQCG) after activation hyaluronic acid 1 hour, continues stirring 4 hours extremely
Fully reacting carries out；Reaction solution after reaction is fitted into the bag filter that molecular weight is 3500, is dialysed 24 hours, removes unreacted
On impurity and polypeptide, add freeze drying protectant carry out freeze-drying 24 hours, obtain dry hyaluronic acid-polypeptide solid.
50mg hyaluronic acid-polypeptide is dissolved in 20ml water, 5mg NHS and 10mg EDC, stirring are added after stirring and dissolving
1h makes remaining part activated carboxylic on hyaluronic acid.
Take 0.5g polymer R1, it is dissolved in 10ml chloroform, 100 μ l propane diamine is added, after stirring 12h, pours into pre-cooling
In the diethyl ether solution crossed, takes out in the vacuum oven that precipitating places it at room temperature, obtain by amido modified polymer
R1。
Polymer R by 0.5g by modification1It is dissolved in the water of pH=3, hyaluronic acid-polypeptide after activation is then added
Solution continues stirring 6 hours, and final product is dialysed 24 hours, removes impurity and unreacted raw material；Add frozen-dried protective
Agent, and be lyophilized 24 hours, dry final product polymer micelle is obtained, this product is resulting glue under optimal experiment condition
Beam.
The verifying of polymer micelle/FITC-DNA compound:
(1) it to unloaded polymer micelle and carries polymer micelle/FITC-DNA compound after DNA and carries out Appearance View
It examines
It takes unloaded polymer micelle and carries polymer micelle/FITC-DNA compound after DNA, be added in respectively dedicated
On copper mesh, the size and form of particle are observed directly under transmission electron microscope, as a result as shown in Figure 6 and Figure 8.
(2) intake experiment of the polymer micelle/FITC-DNA compound in melanoma cells
By B16F10 cell (1 × 105/ hole) 12 orifice plates are inoculated in, it is incubated overnight, is separately added into polymer micelle/FITC-
DNA compound and blank FITC-DNA solution wash cell three times using PBS (PH=7.4) after cultivating 3h in incubator, eventually
Only intake of the cell to compound or solution is added collects cell, and cell is placed in flow cytometer to the sun for detecting cell
Property rate, as a result as shown in Figure 9.
The results show that carried by micella can significantly improve compound after FITC-DNA enter born of the same parents' efficiency, only after 3 hours
Micella/FITC-DNA compound can be entered proximal to 80% it is intracellular, and individually naked FITC-DNA almost can not be into
Enter cell.
(3) polymer micelle/FITC-DNA compound
Rat skin is taken, absorbent cotton carefully removes the subcutaneous tissue and fat of skin, with physiological saline clearing skin, by skin
It is fixed between the supply chamber and receiving chamber of Franz Transdermal diffusion cell, upward, receiving chamber fills PBS solution (PH=to cuticula
7.4), the mixed solution containing polymer micelle and FITC-DNA that 1ml is prepared is added in supply chamber, is sealed, is kept away with sealed membrane
Exempt from supply chamber liquid evaporation.Diffusion cell is placed in 32 DEG C of waters bath with thermostatic control and persistently stirs, and revolving speed 300rmp stops after 24 hours
It only tests, removes diffusion cell, it is gently that skin wiping is clean with cotton swab, it is placed on glass slide, it is burnt aobvious to be placed in copolymerization at once
Micro- microscopic observation polymer micelle/distribution of the FITC-DNA compound in skin.
Using vertical skin cuticula as z-axis, using keratoderma as sweep starting point, successively sweeping for skin is carried out along z-axis
It retouches, at interval of 10 μm of run-downs.With 488 for FITC excitation wavelength, using the FITC-DNA solution of blank as control group, skin is observed
Fluorescence in skin, the results are shown in Figure 10.
Claims (8)
1. a kind of polymer micelle is nucleocapsid structure, is self-assembly of in water by amphiphilic block copolymer, feature
It is, shown in the general structure of the block copolymer such as formula (I),
Wherein, R1For poly lactide-glycolide acid, polylactic acid, polystyrene, poly- amino ester or their derivative；R2For
Transdermal peptide, amino acid sequence ACTGSTQHQCG, RRRRRRR, ACSSSPSKHCG, ACKTGSHNQCG or
HIITDPNMAEYL；M, n, z are the integer greater than 2.
2. polymer micelle as described in claim 1, which is characterized in that the R1Grafting rate in the monomer of place is 1~
20%.
3. polymer micelle as described in claim 1, which is characterized in that in polymer micelle, the R1Total molecular weight be
1000~50000.
4. polymer micelle as described in claim 1, which is characterized in that the R2Grafting rate in the monomer of place is 1~
20%.
5. polymer micelle as described in claim 1, which is characterized in that the R1For poly- amino ester, R2Amino acid sequence be
ACTGSTQHQCG, m:n=1:1.
6. the polymer micelle as described in Claims 1 to 5 is any is preparing the application in dermal drug carrier.
7. a kind of polymer micelle compound of carrying medicaments, including drug and carrier, which is characterized in that the carrier is right
It is required that 1~5 any polymer micelle.
8. the polymer micelle compound of carrying medicaments as claimed in claim 7, which is characterized in that the drug be DNA, siRNA,
MiRNA or hydrophobic class chemicals.
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