CN105778127A - Polylactic acid-polypeptide polymeric micelle and preparation method thereof - Google Patents
Polylactic acid-polypeptide polymeric micelle and preparation method thereof Download PDFInfo
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Abstract
The invention provides a polylactic acid-polypeptide polymeric micelle and a preparation method thereof.The polylactic acid-polypeptide polymeric micelle is formed by assembling amphipathic block polymers PPLA-b-poly[Phe-stat-Lys], a core of the polylactic acid-polypeptide polymeric micelle is formed by PLLA, and a shell of the polylactic acid-polypeptide polymeric micelle is formed by Phe and Lys.The preparation method of the polylactic acid-polypeptide polymeric micelle comprises the steps that PPLA-b-poly[Phe-stat-Lys] is dissolved in a solvent with the concentration of 1-20 mg/mL, deionized water is added dropwise at the speed of 10-50 d/min under the stirring condition, the volume of the dropwise added deionized water finally is 2-3 times that of the solvent, dialysis is carried out to remove the solvent, and a solution containing the polylactic acid-polypeptide polymeric micelle is obtained.The polylactic acid-polypeptide polymeric micelle has narrow grain size distribution and carries a large quantity of positive charges, a very good bacterium resisting and killing effect can be achieved without adding antibiotics, and meanwhile the probability of generating drug-resistant bacteria is reduced.
Description
Technical field
The invention belongs to polymeric material field, relate to a kind of polymer micelle and preparation method thereof.
Background technology
At present, antibiotics is frequently used to bacterial-infection resisting, but, a large amount of use antibiotics easily make bacterial resistance occurred, become Resistant strain, and along with the propagation of self entails the next generation, gradually form and be capable of withstanding " superbacteria " by antibiotics, thus the health of the mankind is brought adverse influence.
In order to prevent the generation of above-mentioned situation, usually carrying out substitute antibiotics class medicine with anti-microbial type polymer (such as many peptides), it adopts the mode of physical damage bacterial cell membrane to sterilize.Although this sterilization mechanism can effectively reduce the probability that drug tolerant bacteria produces, but often extract from the culture fluid of poly-viscosity bacillus or aerogenesis spore-bearing bacillus due to many peptides and prepare, extraction process is complex, and extraction cost is higher, and these all limit it and further apply.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, main purpose is in that to provide a kind of polylactic acid poltpeptides micelle not only having good antibiotic property but also not easily make antibacterial produce drug resistance.
Further object is that the preparation method that a kind of above-mentioned polylactic acid poltpeptides micelle is provided.
For reaching above-mentioned purpose, the solution of the present invention is:
A kind of polylactic acid poltpeptides micelle, it is formed by Amphipathilic block polymer PLLA b poly [Phe stat Lys] self assembly, PLLA forms the kernel of polylactic acid poltpeptides micelle, and Phe and Lys forms the shell of polylactic acid poltpeptides micelle.
Wherein, the mean diameter of polylactic acid poltpeptides micelle can be 20 50nm.
The molecular weight of PLLA b poly [Phe stat Lys] can be 6,000 25000.
The preparation method of a kind of polylactic acid poltpeptides micelle, it comprises the steps:
Amphipathilic block polymer PLLA b poly [Phe stat Lys] is dissolved in the first solvent with the concentration of 1 20mg/mL, when stirring, the speed with 10 50d/min drips deionized water, make 23 times of the volume that volume is the first solvent of the deionized water of final dropping, dialysis, to remove the first solvent, obtains the solution containing polylactic acid poltpeptides micelle.
Wherein, the first above-mentioned solvent can be any one or a few in ether, dichloromethane, oxolane, methanol, dimethyl sulfoxide or dimethylformamide.
The preparation method of PLLA b poly [Phe stat Lys] comprises the steps:
(1), by PLLA NH2, Z Lys NCA monomer and Phe NCA monomer be dissolved in the second solvent with the mol ratio of 1 (1.5 6) (1 4), stirring reaction 24 36h under room temperature, rotation is steamed, is washed and obtain PLLA b poly [Phe stat Z Lys] after lyophilizing；
(2), step (1) gained PLLA b poly [Phe stat Z Lys] is dissolved in excessive hydrobromic acetum, stirring reaction 2 24h under room temperature, first precipitant precipitates, then sucking filtration, vacuum drying, in sodium bicarbonate aqueous solution dialyse 48h, obtain PLLA b poly [Phe stat Lys] after lyophilizing.
Wherein, PLLA b poly [Phe stat Lys] is PLLAx‐b‐poly[Phey‐stat‐Lysz] abbreviation, PLLA represents polylactic acid, x represents the number of lactic acid in Amphipathilic block polymer, b represents that the type of polymer is block polymer, Phe represents that phenylalanine, y represent the number of phenylalanine in Amphipathilic block polymer, and stat represents that phenylalanine and lysine are to be polymerized in the way of random copolymerization, Lys represents that lysine, z represent the number of lysine in Amphipathilic block polymer.X, y and z value can be controlled by by controlling the factors such as the inventory of every kind of monomer respectively.Once it is determined that the value of x, y and z, so that it may obtain that there is the Amphipathilic block polymer PLLA determining molecular weightx‐b‐poly[Phey‐stat‐Lysz]。
PLLAx‐b‐poly[Phey‐stat‐Lysz] molecular weight (number average molecular, as follows) in 6000 to 25000 scopes, namely for a certain PLLAx‐b‐poly[Phey‐stat‐Lysz], its molecular weight is any one numerical value in 6000 to 25000 scopes.
The second above-mentioned solvent can be any one or a few in ether, dichloromethane, oxolane, methanol, dimethyl sulfoxide or dimethylformamide, and the kind of the second solvent should be different from the kind of the first solvent.
The first above-mentioned precipitant is ether.
The concentration of above-mentioned hydrobromic acetum is 33wt%.
In step (1), PLLA NH2Preparation method comprise the steps:
(a), by the Boc ethanolamine protected and lactide monomer with the mixed in molar ratio of 1 (20 60); add catalyst; 24 36h are reacted in 130 ± 10 DEG C under the protection of noble gas; add the 3rd solvent to dissolve; second precipitant precipitates; sucking filtration and vacuum drying, obtain PLLA NH Boc polymer；
(b), PLLA NH Boc polymer is dissolved in the 3rd solvent, add trifluoroacetic acid, the mol ratio of PLLA NH Boc polymer and trifluoroacetic acid is 1 (10 18), stirring reaction 2 24h at normal temperatures, second precipitant precipitates, sucking filtration and vacuum drying, obtain PLLA NH2。
Wherein, in step (a), catalyst is stannous octoate, and noble gas is argon.
In step (a) and (b), 3rd solvent is any one or a few in ether, dichloromethane, oxolane, methanol, dimethyl sulfoxide or dimethylformamide, and the kind of the 3rd solvent, the second solvent and the first solvent should be different.
In step (a) and (b), the second precipitant is methanol.
In step (b), PLLA NH2Molecular weight be 1,800 4000.
In step (1), the preparation method of Z Lys NCA monomer comprises the steps:
N ε benzyloxycarbonyl group L lysine monomer, α pinene and triphosgene are dissolved in oxolane according to the ratio that mol ratio is 1 (4 10) (1 2), 4 8h are reacted at 50 70 DEG C, then precipitate in methanol, sucking filtration and vacuum drying, obtain Z Lys NCA monomer.
In step (1), the preparation method of Phe NCA monomer comprises the steps:
L phenylalanine monomer, α pinene and triphosgene are dissolved in oxolane according to the ratio that mol ratio is 1 (4 10) (0.5 2), 4 8h are reacted at 50 70 DEG C, then precipitation, sucking filtration and vacuum drying in methanol, obtain Phe NCA monomer.
Owing to adopting such scheme, the invention has the beneficial effects as follows:
The first, the many peptides raw material for preparing polylactic acid poltpeptides micelle of the present invention adopts synthetic method to make, and not extracts from bacterium solution, and therefore its production process is simple and less costly.
The second, the polylactic acid poltpeptides micelle of the present invention contains substantial amounts of lysine, containing exposed amino in lysine, amino can occur protonation to form the ammonium radical ion of positively charged in water, therefore, the surface of this polylactic acid poltpeptides micelle with substantial amounts of positive charge, can have the function of stronger bactericidal；Additionally, the polylactic acid poltpeptides micelle of the present invention carries out bactericidal by destroying the integrity of bacterial organisms structure and cell membrane with physical method, therefore the probability producing drug-resistant bacteria can be reduced.
3rd, in the polylactic acid poltpeptides micelle of the present invention, polylactic acid (PLLA) segment can be degraded by lipase, and poly-polypeptide chain can be easily degraded by proteases, so having good biocompatibility and biodegradability.
4th, the mean diameter of the polylactic acid poltpeptides micelle of the present invention is only 20 50nm, much smaller than the particle diameter of vesicle, it is easier to transfer, bactericidal effect is better.
Accompanying drawing explanation
Fig. 1 is the PLLA in the embodiment of the present invention one31‐b‐poly[Phe24‐stat‐Lys36] nuclear magnetic resonance map.
Fig. 2 is the transmission electron microscope picture of the polylactic acid poltpeptides micelle in the embodiment of the present invention one.
Fig. 3 is that the polylactic acid poltpeptides micelle in the embodiment of the present invention one is to colibacillary anti-microbial property rectangular histogram.
Fig. 4 is the anti-microbial property rectangular histogram to staphylococcus aureus of the polylactic acid poltpeptides micelle in the embodiment of the present invention one.
Detailed description of the invention
Below in conjunction with accompanying drawing illustrated embodiment, the present invention is further illustrated.
The invention provides a kind of polylactic acid poltpeptides micelle and preparation method thereof.
<polylactic acid poltpeptides micelle>
The polylactic acid poltpeptides micelle of the present invention is by Amphipathilic block polymer PLLAx‐b‐poly[Phey‐stat‐Lysz] self assembly forms.
Wherein, PLLA represents polylactic acid, x represents the number of lactic acid in Amphipathilic block polymer, b represents that this polymer is block polymer, Phe represents that phenylalanine, y represent the number of phenylalanine in Amphipathilic block polymer, and stat represents that phenylalanine and lysine are to be polymerized in the way of random copolymerization, Lys represents that lysine, z represent the number of lysine in Amphipathilic block polymer.X, y and z value can pass through to control respectively the factor such as inventory of every kind of monomer and be controlled by.Once it is determined that the value of x, y and z, so that it may obtain that there is the Amphipathilic block polymer PLLA determining molecular weightx‐b‐poly[Phey‐stat‐Lysz]。
Amphipathilic block polymer PLLAx‐b‐poly[Phey‐stat‐Lysz] molecular weight (in the present invention, unless stated otherwise, the otherwise equal number average molecular of molecular weight that can be 6,000 25000.).Every kind of PLLAx‐b‐poly[Phey‐stat‐Lysz] molecular weight be corresponding with the polylactic acid poltpeptides micelle formed by it, namely, for certain polylactic acid poltpeptides micelle, it is only by the PLLA of a kind of molecular weight (any one numerical value in 6000 to 25000 scopes)x‐b‐poly[Phey‐stat‐Lysz] make, and be not by the PLLA of different molecular weightx‐b‐poly[Phey‐stat‐Lysz] mix.
Work as PLLAx‐b‐poly[Phey‐stat‐Lysz] be self-assembled into polylactic acid poltpeptides micelle after, PLLA forms the kernel of this polylactic acid poltpeptides micelle, Phe and Lys formed polylactic acid poltpeptides micelle shell.Character due to polypeptide chain, namely containing exposed amino in lysine, amino can occur protonation to form the ammonium radical ion of positively charged in water, and therefore the surface of this polylactic acid poltpeptides micelle can with substantial amounts of positive charge, and these positive charges can give the function of its bactericidal.
The particle diameter of the polylactic acid poltpeptides micelle of the present invention is 20 50nm, i.e. the particle diameter of polylactic acid poltpeptides micelle and PLLAx‐b‐poly[Phey‐stat‐Lysz] etc. factor relevant.PLLA for some molecular weightx‐b‐poly[Phey‐stat‐Lysz], what its method according to the invention was made is the polylactic acid poltpeptides micelle with some particle diameter (any one particle diameter in 20nm to 50nm scope), and not make the polylactic acid poltpeptides micelle with different-grain diameter, that is, by the PLLA of some molecular weightx‐b‐poly[Phey‐stat‐Lysz] made by containing polylactic acid poltpeptides micelle solution in, the particle diameter of this polylactic acid poltpeptides micelle is homogeneous.
<preparation method of polylactic acid poltpeptides micelle>
The polylactic acid poltpeptides micelle of the present invention is by Amphipathilic block polymer PLLAx‐b‐poly[Phey‐stat‐Lysz] formed by solvent exchange method self assembly, specifically include following steps: by Amphipathilic block polymer PLLAx‐b‐poly[Phey‐stat‐Lysz] be dissolved in the first solvent with the concentration of 1 20mg/mL, deionized water is dripped with the speed of 10 50d/min when being stirred continuously, make 23 times of the volume that volume is the first solvent of the deionized water of final dropping, dialysis, to remove the first solvent, obtains the solution containing polylactic acid poltpeptides micelle.
Wherein, the first above-mentioned solvent is any one or a few in ether, dichloromethane, oxolane, methanol, dimethyl sulfoxide or dimethylformamide.
In the solution containing polylactic acid poltpeptides micelle, the mean diameter of polylactic acid poltpeptides micelle is homogeneous, for any number in 20nm to 50nm.In the present invention, it is possible to controlled the particle diameter of polylactic acid poltpeptides micelle by concentration when regulating and controlling the number-average molecular weight of Amphipathilic block polymer, hydrophobe segment composition and ratio and self assembly.
Above-mentioned Amphipathilic block polymer PLLAx‐b‐poly[Phey‐stat‐Lysz] molecular weight be 6,000 25000, its preparation method comprises the steps:
(1), by PLLA NH2, Z Lys NCA monomer and Phe NCA monomer be dissolved in the second solvent with the mol ratio of 1 (1.5 6) (1 4), stirring reaction 24 36h under room temperature, rotation is steamed, is washed and obtain PLLA b poly [Phe stat Z Lys], i.e. PLLA after lyophilizingx‐b‐poly[Phey‐stat‐Z‐Lysz]；
(2), step (1) gained PLLA b poly [Phe stat Z Lys] is dissolved in excessive hydrobromic acetum, stirring reaction 2 24h under room temperature, first precipitant precipitates, then sucking filtration, vacuum drying, in sodium bicarbonate aqueous solution dialyse 48h, PLLA b poly [Phe stat Lys], i.e. PLLA is obtained after lyophilizingx‐b‐poly[Phey‐stat‐Lysz]。
Wherein, the Z in step (1) represents that benzyloxycarbonyl group, Z lys are a kind of monomers, represents the lysine protected by benzyloxycarbonyl group.
The second solvent in step (1) is any one or a few in ether, dichloromethane, oxolane, methanol, dimethyl sulfoxide or dimethylformamide.Need to ensure that the second solvent is different from and does not comprise the first solvent, such as, when the first solvent is dimethyl sulfoxide, the second solvent is any one or a few in ether, dichloromethane, oxolane, methanol or dimethylformamide, namely the second solvent can get rid of the use to the first solvent.
In step (1), by controlling to put into the amount of monomer, it is possible to control the numerical value of x, y and z, and make the molecular weight of PLLA b poly [Phe stat Lys] that step (2) finally gives between 6000 and 25000.
In step (2), in hydrobromic acetum, hydrobromic concentration is 33wt%.1mol hydrobromic acid molecule reacts with the Z lys of 1mol in theory, to take off benzyloxycarbonyl group therein (Z) protection, generates lysine.And be actually added into hydrobromic amount and should be 5 to 10 times of theoretical addition value, namely hydrobromic molal quantity is 5 to 10 times of the molal quantity of Z lys.
In step (2), the first precipitant is ether.First precipitant should be different from the second solvent.Such as, when the first precipitant is methanol, the second solvent should get rid of the use to methanol.
[about PLLA NH2Synthesis]
In step (1), PLLA NH2Preparation method comprise the steps:
(a). the ethanolamine protect tertbutyloxycarbonyl (Boc) and lactide monomer are with the mixed in molar ratio of 1 (20 60); add catalyst; under the protection of noble gas, react 24 36h in 130 ± 10 DEG C, add the 3rd solvent and dissolve.Precipitate in the second precipitant afterwards, sucking filtration and after vacuum drying, obtain PLLA NH Boc polymer (for white powdery solids)；
(b). PLLA NH Boc polymer is dissolved in the 3rd solvent again, add trifluoroacetic acid, affiliated PLLA NH Boc polymer and the mol ratio of trifluoroacetic acid are 1 (10 18), stirring reaction 2 24h at normal temperatures, second precipitant precipitates, sucking filtration and vacuum drying, obtain PLLA NH2。
In step (a), catalyst includes stannous octoate.
In step (a), the 3rd solvent is any one or a few in ether, dichloromethane, oxolane, methanol, dimethyl sulfoxide or dimethylformamide.First solvent, the second solvent and the 3rd solvent are answered different and do not comprise mutually, such as, when the first solvent is dimethyl sulfoxide, when second solvent is dimethylformamide, 3rd solvent is any one or a few in ether, dichloromethane, oxolane or methanol, namely eliminates the use to the first solvent and the second solvent.The addition of the 3rd solvent is so that lactide is substantially dissolved in the 3rd solvent is advisable.
In step (b), made PLLA NH2Molecular weight be 1,800 4000.
In step (b), the second precipitant is methanol.Second precipitant should be different from the 3rd solvent.Such as, when the second precipitant is methanol, the 3rd solvent should get rid of the use to methanol.
[synthesis about Z Lys NCA monomer]
Wherein, in step (1), the preparation method of Z Lys NCA monomer comprises the steps:
N ε benzyloxycarbonyl group L lysine monomer, α pinene and triphosgene are dissolved in oxolane according to the ratio that mol ratio is 1 (4 10) (1 2),.Reacting 4 8h at 50 70 DEG C, then precipitation, sucking filtration and vacuum drying in methanol, obtain Z Lys NCA monomer (for Powdered).
Wherein, the addition of oxolane only need to meet fully dissolving monomer and triphosgene, not easily too much addition.
[synthesis about Phe NCA monomer]
Wherein, in step (1), the preparation method of Phe NCA monomer comprises the steps:
L phenylalanine monomer, α pinene and triphosgene are dissolved in oxolane according to the ratio that mol ratio is 1 (4 10) (0.5 2), 4 8h are reacted at 50 70 DEG C, then precipitation, sucking filtration and vacuum drying in methanol, obtain Phe NCA monomer.
Below in conjunction with accompanying drawing illustrated embodiment, the present invention is further illustrated.
Embodiment one
The preparation method of the polylactic acid poltpeptides micelle of the present embodiment comprises the steps:
a、PLLA31‐NH2Synthesis step:
(a‐1)、PLLA31The synthesis of NH Boc:
The 1gBoc ethanolamine protected and 33gl lactide monomer (l LA) are joined (namely the ethanolamine of Boc protection and the mol ratio of 1 lactide monomer are 1 37) in round-bottomed flask; pass into argon to get rid of oxygen; a small amount of octoate catalyst Asia stannum (3) is added again in round-bottomed flask; 36h is reacted in 130 DEG C; add dichloromethane (as the 3rd solvent) to dissolve; precipitation in methanol (as the second precipitant); sucking filtration in 30 DEG C of vacuum dryings, obtains white powdery solids PLLA31NH Boc polymer；
(a‐2)、PLLA31‐NH2Synthesis:
By 6gPLLA31NH Boc polymer is dissolved in 15mL dichloromethane (as the 3rd solvent), is subsequently adding 15mL trifluoroacetic acid (PLLA31The mol ratio of NH Boc polymer and trifluoroacetic acid is 1 16), stirring reaction 12h at normal temperatures, using gained solution precipitation in the low-temp methanol (as the second precipitant, temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtain PLLA31‐NH2。
The synthesis step of b, Z Lys NCA monomer:
Respectively 5gN ε benzyloxycarbonyl group L lysine monomer (Z Lys), 15g α pinene and 5g triphosgene are joined (mol ratio of N ε benzyloxycarbonyl group L lysine monomer, α pinene and triphosgene is 1 6.4 1) in flask, add 150mL oxolane, stirring reaction 4h at 50 DEG C, then precipitation in low-temp methanol (temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtains Powdered Z Lys NCA monomer.
The synthesis step of c, Phe NCA monomer:
Respectively 5gL phenylalanine monomer (L Phe), 25g α pinene and 8g triphosgene are joined (mol ratio of L phenylalanine monomer, α pinene and triphosgene is 1 6.1 0.9) in flask, add 150mL oxolane, stirring reaction 4h at 50 DEG C, then precipitation in low-temp methanol (temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtains Powdered Phe NCA monomer.NCA full name is N carboxyanhydrides, and Chinese name is alpha amino acid N carboxy acid anhydride.
d、PLLA31‐b‐poly[Phe24‐stat‐Lys36] synthesis step:
(d‐1)、PLLA31‐b‐poly[Phe24‐stat‐Z‐Lys36] synthesis:
By 0.25gPLLA31‐NH2, 1gZ Lys NCA monomer and 0.5gPhe NCA monomer join (PLLA in round-bottomed flask31‐NH2, Z Lys NCA monomer and Phe NCA monomer mol ratio be 13 2.4), add appropriate (10mL) dimethylformamide (DMF, as the second solvent) dissolve, under room temperature, stirring reaction 24h is to carry out ring opening polymerisation process, by products therefrom rotation steam, with substantial amounts of water washing to remove dimethylformamide, obtain white powder PLLA after lyophilizing31‐b‐poly[Phe24‐stat‐Z‐Lys36]；
(d‐2)、PLLA31‐b‐poly[Phe24‐stat‐Lys36] synthesis:
By step (d 1) gained PLLA31‐b‐poly[Phe24‐stat‐Z‐Lys36] to be dissolved in the hydrobromic acetum of 15mL (excessive, concentration is 33wt%) in, stirring reaction 12h under room temperature, using the solution of gained precipitation in ether (as the first precipitant), then sucking filtration in 30 DEG C of vacuum dryings, dialysis 48h in sodium bicarbonate aqueous solution (mass concentration is 5%), obtains PLLA after lyophilizing 48h again31‐b‐poly[Phe24‐stat‐Lys36]。
E, polylactic acid poltpeptides micelle synthesis step:
By Amphipathilic block polymer PLLA31‐b‐poly[Phe24‐stat‐Lys36] it is dissolved in dimethyl sulfoxide (DMSO with the concentration of 1mg/mL, as the first solvent) in, deionized water is dripped with the speed of 15d/min when being stirred vigorously, make 2 times of the volume that volume is dimethyl sulfoxide of the deionized water of final dropping, dialysis, to remove dimethyl sulfoxide, obtains the solution containing polylactic acid poltpeptides micelle.
The PLLA NH of the present embodiment gained2The molecular weight being calculated gained by nucleus magnetic hydrogen spectrum is 2293, but the molecular weight of polymer is a distribution, the dispersion (PDI) being measured polymer by gel permeation chromatography (GPC) is 1.22, it can thus be appreciated that the molecular weight distribution of synthesized polymer controls a less scope.PLLA31‐b‐poly[Phe24‐stat‐Lys36] to calculate the molecular weight of gained by nucleus magnetic hydrogen spectrum be 10790, owing to this polymer is insoluble in tetrahydrofuran solvent, so the molecular weight distribution of its reality cannot be recorded by GPC.
The PLLA of the present embodiment31‐b‐poly[Phe24‐stat‐Lys36] nuclear magnetic resonance map as shown in Figure 1.The mean diameter of the polylactic acid poltpeptides micelle of the present embodiment is 22.5nm, and its transmission electron microscope picture is as shown in Figure 2.
The anti-microbial property method of testing of the polylactic acid poltpeptides micelle of the present embodiment is as follows:
Arranging two groups of culture medium of A and B, A group is inoculated colibacillary LB culture medium, and B group is the LB culture medium of inoculated staphylococcus aureus, and often group arranges 6 references, by the solution containing polylactic acid poltpeptides micelle according to 60 μ g mL‐1、50μg·mL‐1、40μg·mL‐1、30μg·mL‐1、20μg·mL‐1With 0 μ g mL‐1(as a control group) concentration is separately added in 6 references of A group and B group, and after cultivating 24 hours, turbidity according to culture medium judges the anti-microbial property of micelle.
It has been observed that with the rising of the polylactic acid poltpeptides micellar concentration added in inoculum, the culture fluid of inoculated antibacterial is increasingly clarified, it was demonstrated that the concentration of polymer micelle is more high, and antibacterial effect is more good after cultivating 24 hours.The muddy degree of culture fluid is measured by ultraviolet spectrophotometer, and namely absorbance is more low, and bacteria living amount is more low, and antibacterial effect is more good.
The polylactic acid poltpeptides micelle of Fig. 3 the present embodiment is to colibacillary anti-microbial property rectangular histogram, and wherein control group is do not add the matched group of polymer micelle.Control group, 20 μ g mL from left to right it are followed successively by each time group‐1Group, 30 μ g mL‐1Group, 40 μ g mL‐1Group, 50 μ g mL‐1Group and 60 μ g mL‐1Group.
Fig. 4 is the polylactic acid poltpeptides micelle of the present embodiment anti-microbial property rectangular histogram to staphylococcus aureus, and wherein control group is do not add the matched group of polymer micelle.Control group, 20 μ g mL from left to right it are followed successively by each time group‐1Group, 30 μ g mL‐1Group, 40 μ g mL‐1Group, 50 μ g mL‐1Group and 60 μ g mL‐1Group.
Embodiment two
The preparation method of the polylactic acid poltpeptides micelle of the present embodiment comprises the steps:
a、PLLA31‐NH2Synthesis step:
(a‐1)、PLLA31The synthesis of NH Boc:
The 1gBoc ethanolamine protected and 33g1 lactide monomer (1 LA) are joined (namely the ethanolamine of Boc protection and the mol ratio of 1 lactide monomer are 1 37) in round-bottomed flask; pass into argon to get rid of oxygen; a small amount of octoate catalyst Asia stannum (3) is added again in round-bottomed flask; 36h is reacted in 130 DEG C; add dichloromethane (as the 3rd solvent) to dissolve; precipitation in methanol (as the second precipitant); sucking filtration in 30 DEG C of vacuum dryings, obtains white powdery solids PLLA31NH Boc polymer；
(a‐2)、PLLA31‐NH2Synthesis:
By 6gPLLA31NH Boc polymer is dissolved in 15mL dichloromethane (as the 3rd solvent), is subsequently adding 15mL trifluoroacetic acid (PLLA31The mol ratio of NH Boc polymer and trifluoroacetic acid is 1 16), stirring reaction 12h at normal temperatures, using gained solution precipitation in the low-temp methanol (as the second precipitant, temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtain PLLA31‐NH2。
The synthesis step of b, Z Lys NCA monomer:
Respectively 5gN ε benzyloxycarbonyl group L lysine monomer (Z Lys), 15g α pinene and 5g triphosgene are joined (mol ratio of N ε benzyloxycarbonyl group L lysine monomer, α pinene and triphosgene is 1 6.4 1) in flask, add 150mL oxolane, stirring reaction 4h at 50 DEG C, then precipitation in low-temp methanol (temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtains Powdered Z Lys NCA monomer.
The synthesis step of c, Phe NCA monomer:
Respectively 5gL phenylalanine monomer (L Phe), 25g α pinene and 8g triphosgene are joined (mol ratio of L phenylalanine monomer, α pinene and triphosgene is 1 6.1 0.9) in flask, add 150mL oxolane, stirring reaction 4h at 50 DEG C, then precipitation in low-temp methanol (temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtains Powdered Phe NCA monomer.
d、PLLA31‐b‐poly[Phe24‐stat‐Lys45] synthesis step:
(d‐1)、PLLA31‐b‐poly[Phe24‐stat‐Z‐Lys45] synthesis:
By 0.25gPLLA31‐NH2, 1.4gZ Lys NCA monomer and 0.5gPhe NCA monomer join (PLLA in round-bottomed flask31‐NH2, Z Lys NCA monomer and Phe NCA monomer mol ratio be 1 4.2 2.4), add appropriate (15mL) dimethylformamide (DMF, as the second solvent) dissolve, under room temperature, stirring reaction 24h is to carry out ring opening polymerisation process, by products therefrom rotation steam, with substantial amounts of water washing to remove dimethylformamide, obtain white powder PLLA after lyophilizing31‐b‐poly[Phe24‐stat‐Z‐Lys45]；
(d‐2)、PLLA31‐b‐poly[Phe24‐stat‐Lys45] synthesis:
By step (d 1) gained PLLA31‐b‐poly[Phe24‐stat‐Z‐Lys45] to be dissolved in the hydrobromic acetum of 15mL (excessive, concentration is 33wt%) in, stirring reaction 12h under room temperature, using the solution of gained precipitation in ether (as the first precipitant), then sucking filtration in 30 DEG C of vacuum dryings, dialysis 48h in sodium bicarbonate aqueous solution (mass concentration is 5%), obtains PLLA after lyophilizing 48h again31‐b‐poly[Phe24‐stat‐Lys45]。
E, polylactic acid poltpeptides micelle synthesis step:
By Amphipathilic block polymer PLLA31‐b‐poly[Phe24‐stat‐Lys45] it is dissolved in dimethyl sulfoxide (DMSO with the concentration of 1mg/mL, as the first solvent) in, deionized water is dripped with the speed of 15d/min when being stirred vigorously, make 2 times of the volume that volume is dimethyl sulfoxide of the deionized water of final dropping, dialysis, to remove dimethyl sulfoxide, obtains the solution containing polylactic acid poltpeptides micelle.
The PLLA of the present embodiment gained31‐NH2The molecular weight being calculated gained by nucleus magnetic hydrogen spectrum is 2293, but the molecular weight of polymer is a distribution, the dispersion (PDI) being measured polymer by gel permeation chromatography (GPC) is 1.22, it can thus be appreciated that the molecular weight distribution of synthesized polymer controls a less scope.PLLA31‐b‐poly[Phe24‐stat‐Lys45] to calculate the molecular weight of gained by nucleus magnetic hydrogen spectrum be 11942, owing to this polymer is insoluble in tetrahydrofuran solvent, so the molecular weight distribution of its reality cannot be recorded by GPC.The mean diameter of the polylactic acid poltpeptides micelle in the present embodiment is 25nm.
The anti-microbial property method of testing of the polylactic acid poltpeptides micelle of the present embodiment is as follows:
Arranging two groups of culture medium of A and B, A group is inoculated colibacillary LB culture medium, and B group is the LB culture medium of inoculated staphylococcus aureus, and often group arranges 6 references, by the solution containing polylactic acid poltpeptides micelle according to 60 μ g mL‐1、50μg·mL‐1、40μg·mL‐1、30μg·mL‐1、20μg·mL‐1With 0 μ g mL‐1(as a control group) concentration is separately added in 6 references of A group and B group, and after cultivating 24 hours, turbidity according to culture medium judges the anti-microbial property of micelle.
It has been observed that with the rising of the polylactic acid poltpeptides micellar concentration added in inoculum, the culture fluid of inoculated antibacterial is increasingly clarified, it was demonstrated that the concentration of polymer micelle is more high, and antibacterial effect is more good after cultivating 24 hours.
Embodiment three
The preparation method of the polylactic acid poltpeptides micelle of the present embodiment comprises the steps:
a、PLLA31‐NH2Synthesis step:
(a‐1)、PLLA31The synthesis of NH Boc:
The 1gBoc ethanolamine protected and 33gl lactide monomer (l LA) are joined (namely the ethanolamine of Boc protection and the mol ratio of l lactide monomer are 1 37) in round-bottomed flask; pass into argon to get rid of oxygen; a small amount of octoate catalyst Asia stannum (3) is added again in round-bottomed flask; 36h is reacted in 130 DEG C; add dichloromethane (as solvent) to dissolve; precipitation in methanol (as the second precipitant); sucking filtration in 30 DEG C of vacuum dryings, obtains white powdery solids PLLA31NH Boc polymer；
(a‐2)、PLLA31‐NH2Synthesis:
By 6gPLLA31NH Boc polymer is dissolved in 15mL dichloromethane (as the 3rd solvent), is subsequently adding 15mL trifluoroacetic acid (PLLA31The mol ratio of NH Boc polymer and trifluoroacetic acid is 1 16), stirring reaction 12h at normal temperatures, using gained solution precipitation in the low-temp methanol (as the second precipitant, temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtain PLLA31‐NH2。
The synthesis step of b, Z Lys NCA monomer:
Respectively 5gN ε benzyloxycarbonyl group L lysine monomer (Z Lys), 15g α pinene and 5g triphosgene are joined (mol ratio of N ε benzyloxycarbonyl group L lysine monomer, α pinene and triphosgene is 1 6.4 1) in flask, add 150mL oxolane, stirring reaction 4h at 50 DEG C, then precipitation in low-temp methanol (temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtains Powdered Z Lys NCA monomer.
The synthesis step of c, Phe NCA monomer:
Respectively 5gL phenylalanine monomer (L Phe), 25g α pinene and 8g triphosgene are joined (mol ratio of L phenylalanine monomer, α pinene and triphosgene is 1 6.1 0.9) in flask, add 150mL oxolane, stirring reaction 4h at 50 DEG C, then precipitation in low-temp methanol (temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtains Powdered Phe NCA monomer.
d、PLLA31‐b‐poly[Phe15‐stat‐Lys30] synthesis step:
(d‐1)、PLLA31‐b‐poly[Phe15‐stat‐Z‐Lys30] synthesis:
By 0.25gPLLA31‐NH2, 0.8gZ Lys NCA monomer and 0.4gPhe NCA monomer join (PLLA in round-bottomed flask31‐NH2, Z Lys NCA monomer and Phe NCA monomer mol ratio be 1 2.4 1.92), add appropriate (10mL) dimethylformamide (DMF, as the second solvent) dissolve, under room temperature, stirring reaction 24h is to carry out ring opening polymerisation process, by products therefrom rotation steam, with substantial amounts of water washing to remove dimethylformamide, obtain white powder PLLA after lyophilizing31‐b‐poly[Phe15‐stat‐Z‐Lys26]；
(d‐2)、PLLA31‐b‐poly[Phe15‐stat‐Lys30] synthesis:
By step (d 1) gained PLLA31‐b‐poly[Phe15‐stat‐Z‐Lys30] to be dissolved in the hydrobromic acetum of 15mL (excessive, concentration is 33wt%) in, stirring reaction 12h under room temperature, using the solution of gained precipitation in ether (as the first precipitant), then sucking filtration in 30 DEG C of vacuum dryings, dialysis 48h in sodium bicarbonate aqueous solution (mass concentration is 5%), obtains PLLA after lyophilizing 48h again31‐b‐poly[Phe15‐stat‐Lys30]。
E, polylactic acid poltpeptides micelle synthesis step:
By Amphipathilic block polymer PLLA31‐b‐poly[Phe15‐stat‐Lys30] it is dissolved in dimethyl sulfoxide (DMSO with the concentration of 1mg/mL, as the first solvent) in, deionized water is dripped with the speed of 15d/min when being stirred vigorously, make 2 times of the volume that volume is dimethyl sulfoxide of the deionized water of final dropping, dialysis, to remove dimethyl sulfoxide, obtains the solution containing polylactic acid poltpeptides micelle.
The PLLA NH of the present embodiment gained2The molecular weight being calculated gained by nucleus magnetic hydrogen spectrum is 2293, but the molecular weight of polymer is a distribution, the dispersion (PDI) being measured polymer by gel permeation chromatography (GPC) is 1.22, it can thus be appreciated that the molecular weight distribution of synthesized polymer controls a less scope.PLLA31‐b‐poly[Phe15‐stat‐Lys30] to calculate the molecular weight of gained by nucleus magnetic hydrogen spectrum be 8564, owing to this polymer is insoluble in tetrahydrofuran solvent, so the molecular weight distribution of its reality cannot be recorded by GPC.
The mean diameter of the polylactic acid poltpeptides micelle of the present embodiment is 16nm.
The anti-microbial property method of testing of the polylactic acid poltpeptides micelle of the present embodiment is as follows:
Arranging two groups of culture medium of A and B, A group is inoculated colibacillary LB culture medium, and B group is the LB culture medium of inoculated staphylococcus aureus, and often group arranges 6 references, by the solution containing polylactic acid poltpeptides micelle according to 60 μ g mL‐1、50μg·mL‐1、40μg·mL‐1、30μg·mL‐1、20μg·mL‐1With 0 μ g mL‐1(as a control group) concentration is separately added in 6 references of A group and B group, and after cultivating 24 hours, turbidity according to culture medium judges the anti-microbial property of micelle.
It has been observed that with the rising of the polylactic acid poltpeptides micellar concentration added in inoculum, the culture fluid of inoculated antibacterial is increasingly clarified, it was demonstrated that the concentration of polymer micelle is more high, and antibacterial effect is more good after cultivating 24 hours.The muddy degree of culture fluid is more low by ultraviolet spectrophotometer mensuration and absorbance, and bacteria living amount is more low, and antibacterial effect is more good.
Implement four
The preparation method of the polylactic acid poltpeptides micelle of the present embodiment comprises the steps:
a、PLLA31‐NH2Synthesis step:
(a‐1)、PLLA31The synthesis of NH Boc:
The 1gBoc ethanolamine protected and 33g1 lactide monomer (1 LA) are joined (namely the ethanolamine of Boc protection and the mol ratio of 1 lactide monomer are 1 37) in round-bottomed flask; pass into argon to get rid of oxygen; a small amount of octoate catalyst Asia stannum (3) is added again in round-bottomed flask; 36h is reacted in 130 DEG C; add dichloromethane (as the 3rd solvent) to dissolve; precipitation in methanol (as the second precipitant); sucking filtration in 30 DEG C of vacuum dryings, obtains white powdery solids PLLA31NH Boc polymer；
(a‐2)、PLLA31‐NH2Synthesis:
By 6gPLLA31NH Boc polymer is dissolved in 15mL dichloromethane (as the 3rd solvent), is subsequently adding 15mL trifluoroacetic acid (PLLA31The mol ratio of NH Boc polymer and trifluoroacetic acid is 1 16), stirring reaction 12h at normal temperatures, using gained solution precipitation in the low-temp methanol (as the second precipitant, temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtain PLLA31‐NH2。
The synthesis step of b, Z Lys NCA monomer:
Respectively 5gN ε benzyloxycarbonyl group L lysine monomer (Z Lys), 15g α pinene and 5g triphosgene are joined (mol ratio of N ε benzyloxycarbonyl group L lysine monomer, α pinene and triphosgene is 1 6.4 1) in flask, add 150mL oxolane, stirring reaction 4h at 50 DEG C, then precipitation in low-temp methanol (temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtains Powdered Z Lys NCA monomer.
The synthesis step of c, Phe NCA monomer:
Respectively 5gL phenylalanine monomer (L Phe), 25g α pinene and 8g triphosgene are joined (mol ratio of L phenylalanine monomer, α pinene and triphosgene is 1 6.1 0.9) in flask, add 150mL oxolane, stirring reaction 4h at 50 DEG C, then precipitation in low-temp methanol (temperature is 4 DEG C), sucking filtration in 30 DEG C of vacuum dryings, obtains Powdered Phe NCA monomer.
d、PLLA31‐b‐poly[Phe24‐stat‐Lys36] synthesis step:
(d‐1)、PLLA31‐b‐poly[Phe24‐stat‐Z‐Lys36] synthesis:
By 0.25gPLLA31‐NH2, 1gZ Lys NCA monomer and 0.5gPhe NCA monomer join (PLLA in round-bottomed flask31‐NH2, Z Lys NCA monomer and Phe NCA monomer mol ratio be 13 2.4), add appropriate (10mL) dimethylformamide (DMF, as the second solvent) dissolve, under room temperature, stirring reaction 24h is to carry out ring opening polymerisation process, by products therefrom rotation steam, with substantial amounts of water washing to remove dimethylformamide, obtain white powder PLLA after lyophilizing31‐b‐poly[Phe24‐stat‐Z‐Lys36]；
(d‐2)、PLLA31‐b‐poly[Phe24‐stat‐Lys36] synthesis:
By step (d 1) gained PLLA31‐b‐poly[Phe24‐stat‐Z‐Lys36] to be dissolved in the hydrobromic acetum of 15mL (excessive, concentration is 33wt%) in, stirring reaction 12h under room temperature, using the solution of gained precipitation in ether (as the first precipitant), then sucking filtration in 30 DEG C of vacuum dryings, dialysis 48h in sodium bicarbonate aqueous solution (mass concentration is 5%), obtains PLLA after lyophilizing 48h again31‐b‐poly[Phe24‐stat‐Lys36]。
E, polylactic acid poltpeptides micelle synthesis step:
By Amphipathilic block polymer PLLA31‐b‐poly[Phe24‐stat‐Lys36] it is dissolved in dimethyl sulfoxide (DMSO with the concentration of 5mg/mL, as the first solvent) in, deionized water is dripped with the speed of 15d/min when being stirred vigorously, make 2 times of the volume that volume is dimethyl sulfoxide of the deionized water of final dropping, dialysis, to remove dimethyl sulfoxide, obtains the solution containing polylactic acid poltpeptides micelle.
The PLLA NH of the present embodiment gained2The molecular weight being calculated gained by nucleus magnetic hydrogen spectrum is 2293, but the molecular weight of polymer is a distribution, the dispersion (PDI) being measured polymer by gel permeation chromatography (GPC) is 1.22, it can thus be appreciated that the molecular weight distribution of synthesized polymer controls a less scope.PLLA31‐b‐poly[Phe15‐stat‐Lys30] to calculate the molecular weight of gained by nucleus magnetic hydrogen spectrum be 10790, owing to this polymer is insoluble in tetrahydrofuran solvent, so the molecular weight distribution of its reality cannot be recorded by GPC.The mean diameter of the polylactic acid poltpeptides micelle of the present embodiment is 32nm.
The anti-microbial property method of testing of the polylactic acid poltpeptides micelle of the present embodiment is as follows:
Arranging two groups of culture medium of A and B, A group is inoculated colibacillary LB culture medium, and B group is the LB culture medium of inoculated staphylococcus aureus, and often group arranges 6 references, by the solution containing polylactic acid poltpeptides micelle according to 60 μ g mL‐1、50μg·mL‐1、40μg·mL‐1、30μg·mL‐1、20μg·mL‐1With 0 μ g mL‐1(as a control group) concentration is separately added in 6 references of A group and B group, and after cultivating 24 hours, turbidity according to culture medium judges the anti-microbial property of micelle.
It has been observed that with the rising of the polylactic acid poltpeptides micellar concentration added in inoculum, the culture fluid of inoculated antibacterial is increasingly clarified, it was demonstrated that the concentration of polymer micelle is more high, and antibacterial effect is more good after cultivating 24 hours.The muddy degree of culture fluid is more low by ultraviolet spectrophotometer mensuration and absorbance, and bacteria living amount is more low, and antibacterial effect is more good.
The above-mentioned description to embodiment is to be understood that for ease of those skilled in the art and use the present invention.These embodiments obviously easily can be made various amendment by person skilled in the art, and General Principle described herein is applied in other embodiments without through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art's announcement according to the present invention, the improvement made without departing from scope and amendment all should within protection scope of the present invention.
Claims (10)
1. a polylactic acid poltpeptides micelle, it is characterized in that: formed by Amphipathilic block polymer PLLA b poly [Phe stat Lys] self assembly, PLLA forms the kernel of described polylactic acid poltpeptides micelle, and Phe and Lys forms the shell of described polylactic acid poltpeptides micelle.
2. polylactic acid poltpeptides micelle according to claim 1, it is characterised in that: the mean diameter of described polylactic acid poltpeptides micelle is 20 50nm；And/or,
The molecular weight of PLLA b poly [Phe stat Lys] is 6,000 25000.
3. the method preparing polylactic acid poltpeptides micelle as claimed in claim 1 or 2, it is characterised in that: comprise the steps:
Amphipathilic block polymer PLLA b poly [Phe stat Lys] is dissolved in the first solvent with the concentration of 1 20mg/mL, when stirring, the speed with 10 50d/min drips deionized water, make 23 times of the volume that volume is described first solvent of the deionized water of final dropping, dialysis, to remove described first solvent, obtains the solution containing polylactic acid poltpeptides micelle.
4. the method preparing polylactic acid poltpeptides micelle according to claim 3, it is characterised in that: described first solvent is any one or a few in ether, dichloromethane, oxolane, methanol, dimethyl sulfoxide or dimethylformamide.
5. the method preparing polylactic acid poltpeptides micelle according to claim 3, it is characterised in that: the preparation method of PLLA b poly [Phe stat Lys] comprises the steps:
(1), by PLLA NH2, Z Lys NCA monomer and Phe NCA monomer be dissolved in the second solvent with the mol ratio of 1 (1.5 6) (1 4), stirring reaction 24 36h under room temperature, rotation is steamed, is washed and obtain PLLA b poly [Phe stat Z Lys] after lyophilizing；
(2), step (1) gained PLLA b poly [Phe stat Z Lys] is dissolved in excessive hydrobromic acetum, stirring reaction 2 24h under room temperature, first precipitant precipitates, then sucking filtration, vacuum drying, in sodium bicarbonate aqueous solution dialyse 48h, obtain PLLA b poly [Phe stat Lys] after lyophilizing.
6. the method preparing polylactic acid poltpeptides micelle according to claim 5, it is characterised in that: the molecular weight of PLLA b poly [Phe stat Lys] is 6,000 25000；And/or,
Described second solvent is any one or a few in ether, dichloromethane, oxolane, methanol, dimethyl sulfoxide or dimethylformamide, and described second solvent is different from described first solvent；And/or,
Described first precipitant is ether；And/or,
The concentration of described hydrobromic acetum is 33wt%.
7. the method preparing polylactic acid poltpeptides micelle according to claim 5, it is characterised in that: in step (1), PLLA NH2Preparation method comprise the steps:
By the Boc ethanolamine protected and lactide monomer with the mixed in molar ratio of 1 (20 60); add catalyst; 24 36h are reacted in 130 ± 10 DEG C under the protection of noble gas; add the 3rd solvent to dissolve; second precipitant precipitates; sucking filtration and vacuum drying, obtain PLLA NH Boc polymer；
Being dissolved in the 3rd solvent by PLLA NH Boc polymer, add trifluoroacetic acid, the mol ratio of PLLA NH Boc polymer and trifluoroacetic acid is 1 (10 18), stirring reaction 2 24h at normal temperatures, in the second precipitant, precipitation, sucking filtration and vacuum drying, obtain PLLA NH2。
8. the method preparing polylactic acid poltpeptides micelle according to claim 7, it is characterised in that: described catalyst is stannous octoate；And/or,
Described noble gas is argon；And/or,
Described 3rd solvent is any one or a few in ether, dichloromethane, oxolane, methanol, dimethyl sulfoxide or dimethylformamide；And/or,
Described second precipitant is methanol；And/or,
PLLA‐NH2Molecular weight be 1,800 4000.
9. the method preparing polylactic acid poltpeptides micelle according to claim 5, it is characterised in that: in step (1), the preparation method of Z Lys NCA monomer comprises the steps:
N ε benzyloxycarbonyl group L lysine monomer, α pinene and triphosgene are dissolved in oxolane according to the ratio that mol ratio is 1 (4 10) (1 2), 4 8h are reacted at 50 70 DEG C, then precipitate in methanol, sucking filtration and vacuum drying, obtain Z Lys NCA monomer.
10. the method preparing polylactic acid poltpeptides micelle according to claim 5, it is characterised in that: in step (1), the preparation method of Phe NCA monomer comprises the steps:
L phenylalanine monomer, α pinene and triphosgene are dissolved in oxolane according to the ratio that mol ratio is 1 (4 10) (0.5 2), 4 8h are reacted at 50 70 DEG C, then precipitation, sucking filtration and vacuum drying in methanol, obtain Phe NCA monomer.
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Application publication date: 20160720 |