CN107281165A - A kind of triptolide folate-targeted Nano medication and its preparation method and application - Google Patents
A kind of triptolide folate-targeted Nano medication and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to a kind of triptolide folate-targeted Nano medication and its preparation method and application, preparation method is as follows：(1) Mal PEG PLGA dissolvings in a solvent, configure A liquid；(2) dissolving of sulfydryl folic acid in a solvent, configures B liquid；(3) above-mentioned A liquid and B liquid are mixed, obtains FA PEG PLGA；(4) by FA PEG PLGA dissolvings in a solvent, C liquid is configured；(5) triptolide dissolving in a solvent, configures D liquid；(6) C liquid and D liquid are mixed as organic phase, using deionized water as aqueous phase, by organic phase and aqueous phase be placed in it is micro-fluidic it is middle reacted, collect reaction solution, dialysis both must.Triptolide is prepared into targeted nano material by the present invention, triptolide mediation is entered by the special cutaneous lesion of psoriasis by folic acid folacin receptor system, the toxic side effect of tripterygium wilfordii is reduced while playing its pharmacological action, can direct external curing psoriatic lesion.
Description
Technical field
It is a kind of triptolide-folate-targeted Nano medication specifically the present invention relates to biomedicine technical field
And its preparation method and application.
Background technology
Chinese herb triperygium wilfordii is Celastraceae plant tripterygium wilfordii Tripterygium wilfordii Hook.f. root or root
Xylem.It is promoting blood circulation and removing obstruction in channels with dispelling wind and eliminating dampness, swelling and pain relieving, effect of desinsection removing toxic substances.Modern pharmacology research shows that it has
Obvious anti-inflammatory, immunosupress, the bioactivity pharmacological action such as antitumor and anti-male fertility is used for treatment wet in clinic
Rash, pruigo, the disease such as rheumatoid arthritis.However, clinical conventional tripterygium wilfordii and its oral formulations, with many bad anti-
Should, such as indigestion, liver renal toxicity suppress hematological system and mucocutaneous lesions etc., particularly its genotoxicity, serious shadow
The use in child-bearing period patient has been rung, it has been significantly limit in clinical extensive utilization.
Folic acid is a kind of small molecule vitamin, and it can specifically bind with folacin receptor, the α carboxyls in folate molecule
It is coupled by covalent bond and a variety of exogenous molecules, and in folacin coupled compound, folate molecule is remained and folacin receptor
Between high-affinity combine property.Have and show folacin receptor at the Hyperproliferative disease skin damaged such as studies have shown that psoriasis
High the characteristics of, and normal skin does not express (or low expression), and folic acid-conjugate can be realized based on folacin receptor differential expression
Active targeting is conveyed.Compared with monoclonal antibody target system, folic acid small molecule penetration power is strong, reach the target spot time it is short, stably,
Price is low, can carry targeted drug and enter cell interior by cell receptor-mediated endocytosis, with targeting and application
The wide advantage of scope.Triptolide is one of main active in triperygium wilfordii extractive, is also Tripterygium wilfordii Polyglycosidium Tablets
Important composition, but there is triptolide certain toxicity to cause it not use directly, therefore how by triptolide system
While for into targeted nano material, making triptolide directly play pharmacological action, its secondary work of poison to normal cell is reduced
With being wherein difficult point.Chinese patent 2014100393814 discloses a kind of folic acid-nano-TiO2Composite photo-catalyst and preparation side
Method and application, it is the TiO of surface modification folic acid2/SiO2Nano-particle, its structural formula is FA-CONH-TiO2/SiO2, will receive
Rice TiO2/SiO2Composite is coupled with folic acid by amination increases the stability of compound, while strengthen its targeting,
Nano-TiO2Surface coating SiO2Nano-photo catalytic efficiency and heat endurance are improved, killing-efficiency is high and targeting is strong.But it is existing
In technology, on triptolide of the present invention-folate-targeted medicine, report yet there are no.
The content of the invention
First purpose of the present invention is that there is provided a kind of triptolide-folate-targeted for deficiency of the prior art
Nano medication preparation method.
There is provided prepared by method as described above for deficiency of the prior art for second object of the present invention
Triptolide-folate-targeted Nano medication.
Third object of the present invention is that there is provided triptolide-folic acid as described above for deficiency of the prior art
The purposes of targeted nano medicine.
To realize above-mentioned first purpose, the present invention is adopted the technical scheme that：
A kind of triptolide-folate-targeted Nano medication preparation method, the triptolide-folate-targeted nanometer medicine
Thing preparation method is as follows：
(1) Mal-PEG-PLGA dissolvings in a solvent, configure A liquid；
(2) dissolving of sulfydryl folic acid in a solvent, configures B liquid；
(3) above-mentioned A liquid and B liquid are mixed, unnecessary sulfydryl folic acid is removed after reaction, FA-PEG-PLGA is obtained；
(4) by FA-PEG-PLGA dissolvings in a solvent, C liquid is configured；
(5) triptolide dissolving in a solvent, configures D liquid；
(6) C liquid and D liquid are mixed as organic phase, using deionized water as aqueous phase, organic phase and aqueous phase is placed in miniflow
Reacted in control, collect reaction solution, dialysis was both obtained.
As the preferred embodiment of the present invention, PEG molecular weight is 5000- in the Mal-PEG-PLGA
PLGA molecular weight is 5000-20000Da in 20000Da, the Mal-PEG-PLGA.
As the preferred embodiment of the present invention, PEG molecular weight is 5000Da, institute in the Mal-PEG-PLGA
The molecular weight for stating PLGA in Mal-PEG-PLGA is 20000Da.
As the preferred embodiment of the present invention, sulfydryl folic acid and Mal-PEG-PLGA mass ratioes in the step 3
For 2：1.
As the preferred embodiment of the present invention, the FA-PEG-PLGA is 8-30 with triptolide mass ratio：
3。
As the preferred embodiment of the present invention, the FA-PEG-PLGA is 20 with triptolide mass ratio：3.
As the preferred embodiment of the present invention, the solvent of the step 1 and step 4 is acetonitrile, the step 2
Solvent is acetonitrile solution, and the solvent of the step 5 is methanol.
To realize above-mentioned second purpose, the present invention is adopted the technical scheme that：
As above triptolide-folate-targeted Nano medication that any preparation method is prepared.
As the preferred embodiment of the present invention, the medicine also includes the auxiliary material pharmaceutically allowed, the pharmacy
The auxiliary material of upper permission includes but is not limited to：Mannitol, sorbierite, sodium pyrosulfite, sodium hydrogensulfite, sodium thiosulfate, hydrochloric acid
Cysteine, TGA, methionine, injection Vitamin B_6 DTA disodiums, Ethylenediaminetetraacetic Acid Calcium Salt, the carbonate of monovalence alkali metal, acetate,
Phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, sodium chloride, potassium chloride, sodium lactate, xylitol, malt
Sugar, glucose, fructose, dextran, glycine, starch, sucrose, lactose, brown sugar, mannitol, silicon derivative, cellulose and
Its derivative, alginates, gelatin, polyvinylpyrrolidone, glycerine, POLYSORBATE 80, agar, calcium carbonate, calcium bicarbonate, surface-active
Agent, polyethylene glycol, cyclodextrin, beta-schardinger dextrin, phospholipid material, kaolin, talcum powder, calcium stearate, magnesium stearate.
To realize above-mentioned 3rd purpose, the present invention is adopted the technical scheme that：
Application of the triptolide-folate-targeted Nano medication as described above in the medicine for preparing treatment psoriasis.
The invention has the advantages that：
1st, triptolide is prepared into targeted nano material by the present invention, by folic acid-folacin receptor system by tripterygium wilfordii
A prime mediation enters the special cutaneous lesion of psoriasis, and the toxic side effect of tripterygium wilfordii is reduced while playing its pharmacological action, can be straight
Connect external curing psoriatic lesion.
2nd, the present invention is optimized by experiment screening to preparation method, and prepared triptolide-folate-targeted is received
Rice medicine is significantly reduced to normal humanization HaCaT cytotoxicities, and to the Hacat cell killing rates of folacin receptor height expression
Significantly increase.
Brief description of the drawings
Accompanying drawing 1 is targeted nano particle UV absorption spectrogram of the present invention.
Accompanying drawing 2 is non-targeted nano particle transmission electron microscope picture.
Accompanying drawing 3 is targeted nano particle transmission electron microscope picture.
Accompanying drawing 4 is tripterygium wilfordii nano target action principle schematic diagram.
Accompanying drawing 5 is that the immunofluorescence of psoriatic lesion and folacin receptor at tissue by skin damaged is analyzed with mRNA.
Accompanying drawing 6 is the mouse Animal Models of Psoriasis epidermis folacin receptor expression enhancing that imiquimod is induced.
Accompanying drawing 7 is the skin histology chemical analysis before and after psoriasis model mouse medication.
Accompanying drawing 8 is that plasmid transfection is overexpressed folacin receptor, and display cell secretion folacin receptor albumen increases (NC：Normally；
OE：It is overexpressed).
Accompanying drawing 9 is that tripterygium wilfordii nano target intervenes cell in vitro situation.
Accompanying drawing 10 is that different HaCaT cell models change over time curve to wavelength 450nm absorptivity.With OD450
The quantity of the great-hearted cell of reflection tool.Wherein OE is that folacin receptor is overexpressed, and sgRNA is that folacin receptor is knocked out.Experimental result
Show, the growth that OE folacin receptors are overexpressed HaCaT cells breeds more vigorous compared with normal HaCaT cells.
Accompanying drawing 11 is FA-PEG-PLGA targeting confirmatory experiment.Using Ce6 as hydrophobic model medicine, as a result show：With
After cell incubation 24h, the average fluorescent strength of targeting group cell is 2.5 times of non-targeted group and targeted inhibition group, is molecule medicine
7 times of thing group (Ce6).In 12h, targeting group is 1.5-2 times of non-targeted group.The above results show after folic acid is incubated,
Targeting triptolide is being added, its efficiency declines, and also explanation relies on the correctness of the targeting of folacin receptor.
Embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
Bright rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention recorded has been read, art technology
Personnel can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Fixed scope.
The preparation (one) of 1 triptolide of embodiment-folate-targeted medicine
First, raw material and equipment
1.1 raw material
Mal-PEG-PLGA：(molecular weight：PEG：PLGA=5000:20000) (sigma, Aldrich-900339,
100MG) purity：99%.
PEG-PLGA：(molecular weight：PEG：PLGA=5000:20000) (sigma) purity：99%.
Sulfydryl folic acid：Entrust the biochemistry synthesis of Shanghai gill.
Triptolide：Shanghai Tauto Biotechnology Co., Ltd..
Acetonitrile：Analysis is pure, purchased from sigma.
Methanol：Analysis is pure, purchased from sigma.
1.2 equipment
Microfluidics, ultraviolet/visible spectrophotometer (Cry50, VARIAN), laser particle analyzer-Zeta potential meter
(Nano-ZS90, Malvern), high-efficient liquid phase chromatogram HPLC (LC-2030), bag filter (base star is biological), transmission electron microscope
(Hitachi H7100)
2nd, the synthesis of Nano medication
The synthesis of 2.1 non-targeted Nano medications
1) 20mg PEG-PLGA are weighed, is dissolved in 2mL acetonitriles, is configured to 10mg/mL solution；
2) 3mg triptolides are weighed, is dissolved in 1mL methanol, is configured to 3mg/mL solution；
3) both the above solution is uniformly mixed in 25mL vials, is used as organic phase；A 25mL vials separately are taken, are fallen
Enter 20mL deionized waters, be used as aqueous phase.
4) organic phase and aqueous phase are respectively placed in two micro-fluidic air pumps, adjust air pump pressure, make organic phase and water
Phase velocity ratio is 40:320 (units：μL/min)
5) after the completion of reacting, reaction solution liquid, particle instrument detection particle size, surface potential are collected
6) with 8000-14000 molecular weight bag filter dialysis 48h, freeze.
7) certain mass dried frozen aquatic products is weighed, is dissolved in acetonitrile, HPLC detects its carrying drug ratio.
The synthesis of 2.2 targeted nano medicines
1) 20mg Mal-PEG-PLGA are weighed, is dissolved in 2mL acetonitriles, is configured to 10mg/mL, be designated as solution A；
2) 3.52mg sulfydryl folic acid is weighed, 1mL acetonitrile/waters (v% is dissolved in:V%=1:1) in mixed liquor, it is configured to
3.52mg/mL, is designated as solution B.
3) the μ L of solution B 200 2) configured are taken, (reaction ratio M is added drop-wise in AFA:MPEG-PLGA=2：1), react at room temperature
12h, dialyses 48h to remove unreacted sulfydryl folic acid, with the qualitative folic acid of ultraviolet specrophotometer with 3000 molecular weight bag filters
Whether it is connected with Mal groups, quantitatively calculates folic acid concentration.
4) 20mg FA-PEG-PLGA are weighed, is dissolved in 2mL acetonitriles, is configured to 10mg/mL solution；
5) 3mg triptolides are weighed, is dissolved in 1mL methanol, is configured to 3mg/mL solution；
6) step 4,5 solution prepared are uniformly mixed in 25mL vials, are used as organic phase；Separately take a 25mL glass
Bottle, pours into 20mL deionized waters, is used as aqueous phase；
7) organic phase and aqueous phase are respectively placed in two micro-fluidic air pumps, adjust air pump pressure, make organic phase and water
Phase velocity ratio is 40:320 (units：μL/min)；
8) after the completion of reacting, reaction solution, particle instrument detection particle size, surface potential are collected；
9) with 8000-14000 molecular weight bag filter dialysis 48h, freeze.
10) certain mass dried frozen aquatic products is weighed, is dissolved in acetonitrile, HPLC detects its carrying drug ratio.
2.3 transmission electron microscope observing Nano medication macro morphologies
The synthetic Nano medication of both the above is diluted to 1mg/mL.Tweezers grip copper mesh, to towards 20- is added dropwise above
After 30 μ L material liquid, copper mesh absorption 120s, waste liquid on copper mesh is drawn with filter paper, then 4% acetic acid uranium 15-20 μ L are added dropwise on copper mesh
After dyeing, 30-60s, filter paper, which is inhaled, abandons unnecessary acetic acid uranium solution.
3rd, experimental result
3.1 non-targeted Nano medications：PEG-PLGA (load triptolide)
The non-targeted Nano medication particle diameter of table 1, current potential and carrying drug ratio
3.2 targeted nano medicines：FA-PEG-PLGA (load triptolide)：
The targeted nano diameter of aspirin particle of table 2, current potential and carrying drug ratio
3.3FA is ultraviolet qualitative
As shown in figure 1, occurring folic acid characteristic absorption peak (280nm) in FA-PEG-PLGA confirms that folic acid is successfully linked：
3.4 nano particle transmission electron microscope photos
Non-targeted nano particle transmission electron microscope picture is as shown in Fig. 2 targeted nano particle transmission electron microscope picture such as Fig. 3 institutes
Show.Nanoparticle size is less than hydration particle diameter under Electronic Speculum, is because nano particle there occurs that contraction is caused in the drying process.
Fig. 4 is tripterygium wilfordii nano target action principle figure of the present invention.
The histology experiment method of embodiment 2 proves the high expression of psoriatic lesion middle period acid acceptor
1. experimental subjects:The biopsy of clinical psoriatic；
2. reagent:Folic Acid receptor(Santa Cruz Biotechnology,sc-16387)；
3. experimental method：SABC, Realtime PCR methods；
4. experimental result：As seen in figs. 5-6, the Animal Models of Psoriasis mouse skin that folacin receptor is induced in imiquimod
Also high expression in damage.
The zoopery of embodiment 3
1. psoriasis model mouse and the analysis of control mice phenotype before and after medication
1) imiquimod modeling：
Imiquimod induces Pigs with Psoriasis mouse model：Female BAl BIc/c mouse (8-11 week old), by back hair
After rejecting completely, 5% imiquimod cream (Aldara, 3M Products) is smeared, daily dosage is 3.125mg.Control group is adopted
With the vaseline of Isodose.Continuous modeling in 7 days.Give glucocorticoid treatment after modeling success, prevention group is in modeling premise
Give within first 3 days three careless ointment.Skin damaged erythema, the scales of skin that peel off, thickness etc. (0-4 grades) before and after recording medicine, carry out PASI scoring records.
Mouse is put to death within 14th day, skin of back materials are respectively placed in formalin solution and liquid nitrogen and preserved.One is observed in experimentation
As situation and local scytitis situation and record.
2) evaluation method：PASI scores, dermal pathology.
2.1 the skin being grievously injured degree PASI score
Mouse back scytitis severity scale is referred to using domestic generally acknowledged psoriatic lesion area with the order of severity
Number (PASI) scoring：Erythema, the scales of skin that peel off and skin damaged thickness individually score, score value 0-4 points of (0, nothing；1, slightly；2, moderate；3,
Substantially；4, it will be apparent that).The fraction (erythema, the scales of skin that peel off with skin damaged thickness scoring be added) of accumulation as severity of inflammation most
Final review point (0-12 points).
2.2 dermal pathologies are analyzed
Mouse back is drawn materials, and is fixed in 4% formalin solution.Histotomy carries out HE dyeing, then carries out tissue
Learn Baker scorings.Baker methods score：2.0 points of Munro microabscesses meter is found in cuticula；0.5 point of hyperkeratosis meter；Angling is not
Complete 1.0 points of meter.Find that stratum granulosum is thinning or disappears 1.0 points in epidermis；1.0 points of acanthosis；Skin suddenly extends long, fluctuating, according to
Gently, in, weight degree count 0.5 respectively, 1.0,1.5 points.The single core of skin corium and PMN infiltration, according to it is light, in, weight degree
Respectively meter 0.5,1.0,2.0 points；0.5 point is pushed up in mastoid process；0.5 point of telangiectasis.
3) experimental result and analysis：As a result as Figure 7-8, Tripterygium Wilfordii Hook group mouse erythema, the scales of skin that peel off is compared with model pair
Decline according to group, degree mitigates, pointed out Tripterygium Preparations to mitigate local erythema oedema by anti-inflammatory.Pathological examination shows, mould
Type group epidermis is thickened, and skin corium inflammatory cell increases.And treatment group's epidermal hyperplasia is reduced, dermal inflammation Leukopenia.
The cell experiment of embodiment 4
1 cell derived：Humanization HaCaT cells (The American Type Culture Collection are derived from,
Abbreviation ATCC), it is incubated at fresh improvement RPMI-1640 complete mediums (MEM commercialization basic culture solution 445ml, hyclone
50ml and dual anti-5l is mixed), passing on is used for subsequent experimental after well-grown.
2 cell constructions and identification：Keratinocyte (according to laboratory maturation method) at psoriatic lesion is separated, in addition
The healthy patients keratinocyte of section's beauty treatment is control.The preoperative alcohol disinfecting of routine 75%, scalpel takes skin damaged tissue
0.5X0.5CM sizes, 1 × PBS cleans bloodstain repeatedly, is immersed in 0.25% trypsase (Roche) solution.Next day separates table
Corium, abandons corium, and by epidermis, it is cut into the big fractionlets of 5mm × 5mm, adds the clostridiopetidase As of 2ml 0.35%, 37 DEG C of vibration digestion
45min, adds 12.5 μ l DNase and continues to vibrate digestion 15min.Nylon net filter cell, high calcium EMEM is settled to 10ml.
800rpm, 4 DEG C of centrifugation 5min, abandons supernatant, 35ml high calcium EMEM solution gently blows and beats resuspension cell.300rpm, 5min, 4 DEG C
Centrifugation cell.Cell is resuspended in 2ml to contain in 9%Ficoll high calcium EMEM solution.Prepare the clean tube of 15ml (to contain
9%Ficoll solution fresh 2.5ml), by the addition of cell suspension gently, 4 DEG C, 400rpm centrifuges 5min, suctions out Ficoll
Cell is resuspended in supernatant, 35ml high calciums EMEM, and 4 DEG C, 500rpm centrifuges 5min, cleans cell.Add Cnt-07 nutrient solutions
(CELL nTEC Products), are positioned over 37 DEG C, 5%CO2Cultivated in incubator.Next day changes fresh medium.
4.3 conclusion：As shown in Figure 9 A, triptolide and nanometer threewingnut A prime intervene normal Hacat cells respectively,
As a result it is shown under 0.003 μm of concentration, triptolide does not have overt toxicity to Hacat cells, cell growth does not have notable shadow
Ring, but cells survival rate is reduced to 40% under 0.01 μm of concentration, shows the cytotoxicity of triptolide.But through nanometer material
The triptolide of material parcel is significantly reduced to the toxicity of cell, and normal Hacat cell survival rates are still up under 0.3 μm of concentration
90%.
We by triptolide and triptolide Nano medication intervene respectively normal Hacat cells (NC) and folic acid by
Body height expression (OE) cell, intervention time is 3h, and then row CKK8 detects cell survival again, as a result as shown in Figure 9 B：1 is
Normal Hacat cells are intervened using DMSO solvents, as negative control, it is found that cell growth is good；2 be normal Hacat cells
Handled using triptolide, cell survival rate is about 65%；3 be that normal Hacat cells are handled using blank nano-carrier, hair
Existing cell growth is unaffected, intervenes with DMSO without significant difference.4 be that normal Hacat cells use nano target tripterygium wilfordii system
Agent is handled, and finds cell survival rate about 80%, cell is suppressed to a certain extent.5 is thin to be overexpressed the Hacat of folacin receptor
Born of the same parents, are handled using nano target Tripterygium Preparations, are as a result shown in this cell, its survival rate is significantly reduced, about 50%.
Accompanying drawing 10 is that different HaCaT cell models change over time curve to wavelength 450nm absorptivity.With OD450
The quantity of the great-hearted cell of reflection tool.Wherein OE is that folacin receptor is overexpressed, and sgRNA is that folacin receptor is knocked out.Experimental result
Show, the growth that OE folacin receptors are overexpressed HaCaT cells breeds more vigorous compared with normal HaCaT cells.
Accompanying drawing 11 is FA-PEG-PLGA targeting confirmatory experiment.Using Ce6 as hydrophobic model medicine, as a result show：With
After cell incubation 24h, the average fluorescent strength of targeting group cell is 2.5 times of non-targeted group and targeted inhibition group, is molecule medicine
7 times of thing group (Ce6).In 12h, targeting group is 1.5-2 times of non-targeted group.The above results show after folic acid is incubated,
Targeting triptolide is being added, its efficiency declines, and also explanation relies on the correctness of the targeting of folacin receptor.
The preparation (two) of 5 triptolides of embodiment-folate-targeted medicine
1) Mal-PEG-PLGA (molecular weight is taken：PEG：PLGA=20000:20000), it is dissolved in acetonitrile, is configured to
10mg/mL, is designated as solution A；
2) sulfydryl folic acid is taken, acetonitrile/water (v% is dissolved in:V%=1:1) in mixed liquor, 3.52mg/mL is configured to, is designated as
Solution B；
3) the μ L of solution B 200 2) configured are taken, (reaction ratio M is added drop-wise in AFA:MPEG-PLGA=3：1), react at room temperature
12h, dialyses 48h to remove unreacted sulfydryl folic acid with 3000 molecular weight bag filters；
4) 10mg FA-PEG-PLGA are weighed, is dissolved in 2mL acetonitriles, is configured to 5mg/mL solution；
5) 3mg triptolides are weighed, is dissolved in 1mL methanol, is configured to 3mg/mL solution；
6) step 4,5 solution prepared are uniformly mixed in 25mL vials, are used as organic phase；Separately take a 25mL glass
Bottle, pours into 20mL deionized waters, is used as aqueous phase；
7) organic phase and aqueous phase are respectively placed in two micro-fluidic air pumps, adjust air pump pressure, make organic phase and water
Phase velocity ratio is 40:320 (units：μL/min)；
8) after the completion of reacting, reaction solution is collected.
The preparation (three) of 6 triptolides of embodiment-folate-targeted medicine
1) Mal-PEG-PLGA (molecular weight is taken：PEG：PLGA=20000:5000), it is dissolved in acetonitrile, is configured to
10mg/mL, is designated as solution A；
2) sulfydryl folic acid is taken, acetonitrile/water (v% is dissolved in:V%=1:1) in mixed liquor, 3.52mg/mL is configured to, is designated as
Solution B；
3) the μ L of solution B 200 2) configured are taken, (reaction ratio M is added drop-wise in AFA:MPEG-PLGA=1：1), react at room temperature
12h, dialyses 48h to remove unreacted sulfydryl folic acid with 3000 molecular weight bag filters；
4) 30mg FA-PEG-PLGA are weighed, is dissolved in 2mL acetonitriles, is configured to 15mg/mL solution；
5) 3mg triptolides are weighed, is dissolved in 1mL methanol, is configured to 3mg/mL solution；
6) step 4,5 solution prepared are uniformly mixed in 25mL vials, are used as organic phase；Separately take a 25mL glass
Bottle, pours into 20mL deionized waters, is used as aqueous phase；
7) organic phase and aqueous phase are respectively placed in two micro-fluidic air pumps, adjust air pump pressure, make organic phase and water
Phase velocity ratio is 40:320 (units：μL/min)；
8) after the completion of reacting, reaction solution is collected.
The preparation (four) of 7 triptolides of embodiment-folate-targeted medicine
1) Mal-PEG-PLGA (molecular weight is taken：PEG：PLGA=10000:5000), it is dissolved in acetonitrile, is configured to
10mg/mL, is designated as solution A；
2) sulfydryl folic acid is taken, acetonitrile/water (v% is dissolved in:V%=1:1) in mixed liquor, 3.52mg/mL is configured to, is designated as
Solution B；
3) the μ L of solution B 200 2) configured are taken, (reaction ratio M is added drop-wise in AFA:MPEG-PLGA=1：2), react at room temperature
12h, dialyses 48h to remove unreacted sulfydryl folic acid with 3000 molecular weight bag filters；
4) 8mg FA-PEG-PLGA are weighed, is dissolved in 2mL acetonitriles, is configured to 4mg/mL solution；
5) 3mg triptolides are weighed, is dissolved in 1mL methanol, is configured to 3mg/mL solution；
6) step 4,5 solution prepared are uniformly mixed in 25mL vials, are used as organic phase；Separately take a 25mL glass
Bottle, pours into 20mL deionized waters, is used as aqueous phase；
7) organic phase and aqueous phase are respectively placed in two micro-fluidic air pumps, adjust air pump pressure, make organic phase and water
Phase velocity ratio is 40:320 (units：μL/min)；
8) after the completion of reacting, reaction solution is collected.
Vitro cytotoxicity of 8 triptolides of the embodiment-folate-targeted medicine to HaCaT cells
Cell derived：Humanization HaCaT cells (The American Type Culture Collection are derived from,
Abbreviation ATCC), it is incubated at fresh improvement RPMI-1640 complete mediums (MEM commercialization basic culture solution 445ml, hyclone
50ml and dual anti-5l is mixed), passing on is used for subsequent experimental after well-grown.
The nanometer threewingnut A prime that not prepared by be the same as Example is respectively adopted and intervenes normal Hacat cells, as a result such as following table
It is shown：
Vitro cytotoxicity of 3 triptolides of the table-folate-targeted medicine to normal HaCaT cells
The nanometer threewingnut A prime that not prepared by be the same as Example is respectively adopted and intervenes folacin receptor height expression (OE) cell respectively,
Intervention time is 3h, and then row CKK8 detects cell survival again, and proliferation inhibition rate of the medicine to cell is calculated using formula,
Calculation formula is as follows：Cell proliferation inhibition rate=1-A administrations/A controls, wherein control group uses RPMI-1640 complete mediums
Processing.Half-inhibition concentration (the IC of cell is calculated according to cell proliferation inhibition rate50).As a result show each administration group to acid acceptor mistake
It is in concentration dependent to express HaCaT cytotoxicities, and is improved with the concentration of nanometer threewingnut A prime, each administration group cell propagation
Inhibiting rate is improved.Embodiment 1, embodiment 5, embodiment 6, the nanometer threewingnut A prime of embodiment 7 are to being overexpressed HaCaT cells
IC50Respectively 0.47 μm ol/L, 1.28 μm of ol/L, 1.46 μm of ol/L, 1.17 μm of ol/L,
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of triptolide-folate-targeted Nano medication preparation method, it is characterised in that the triptolide-folic acid
Targeted nano process for preparing medicine is as follows：
(1) Mal-PEG-PLGA dissolvings in a solvent, configure A liquid；
(2) dissolving of sulfydryl folic acid in a solvent, configures B liquid；
(3) above-mentioned A liquid and B liquid are mixed, obtains FA-PEG-PLGA；
(4) by FA-PEG-PLGA dissolvings in a solvent, C liquid is configured；
(5) triptolide dissolving in a solvent, configures D liquid；
(6) C liquid and D liquid are mixed as organic phase, using deionized water as aqueous phase, by organic phase and aqueous phase be placed in it is micro-fluidic in
Reacted, collect reaction solution, dialysis was both obtained.
2. preparation method according to claim 1, it is characterised in that PEG molecular weight is in the Mal-PEG-PLGA
PLGA molecular weight is 5000-20000Da in 5000-20000Da, the Mal-PEG-PLGA.
3. preparation method according to claim 1, it is characterised in that characterized in that, PEG in the Mal-PEG-PLGA
Molecular weight be 5000Da, PLGA molecular weight is 20000Da in the Mal-PEG-PLGA.
4. preparation method according to claim 1, it is characterised in that characterized in that, in the step 3 sulfydryl folic acid with
Mal-PEG-PLGA mass ratioes are 2：1.
5. preparation method according to claim 1, it is characterised in that the FA-PEG-PLGA and triptolide quality
Than for 8-30：3.
6. preparation method according to claim 1, it is characterised in that the FA-PEG-PLGA and triptolide quality
Than for 20：3.
7. preparation method according to claim 1, it is characterised in that the solvent of the step 1 and step 4 is acetonitrile, institute
The solvent for stating step 2 is acetonitrile solution, and the solvent of the step 5 is methanol.
8. triptolide-folate-targeted Nano medication that any preparation methods of claim 1-7 are prepared.
9. triptolide according to claim 8-folate-targeted Nano medication, it is characterised in that the medicine is also wrapped
Include the auxiliary material pharmaceutically allowed.
10. the answering in the medicine for preparing treatment psoriasis of triptolide-folate-targeted Nano medication described in claim 8
With.
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| PCT/CN2018/086479 WO2019019773A1 (en) | 2017-07-28 | 2018-05-11 | Triptolide-folic acid targeted nano-drug, preparation method therefor and use thereof |
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