CN105664176A - Mitochondria-targeted polysaccharide nano preparation and preparing method thereof - Google Patents
Mitochondria-targeted polysaccharide nano preparation and preparing method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
Abstract
The invention relates to a mitochondria-targeted polysaccharide nano preparation. The mitochondria-targeted polysaccharide nano preparation comprises water-soluble polysaccharide, triphenylphosphine and antitumor medicine containing primary amine or hydroxyl groups, wherein the triphenylphosphine modifies the antitumor medicine through an amido bond or an ester bond and is connected to the water-soluble polysaccharide through an ionic bond manner; or the triphenylphosphine modifies the water-soluble polysaccharide through an amido bond, and the antitumor medicine is combined to the water-soluble polysaccharide in an amido bond or hydrazone bond manner. The invention further relates to a preparing method of the mitochondria-targeted polysaccharide nano preparation. The polysaccharide nano preparation has the advantage of acid-sensitive drug releasing in a tumor microenvironment and is simple in process. Compared with the single solution of traditional antitumor medicine, the preparation has the advantages that the preparation' toxicity to tumor cells is increased greatly, and the preparation is widely applicable to the field of antitumor tolerance researches.
Description
Technical field
The present invention relates to polysaccharide nanometer formulation and preparation thereof, be specifically related to a kind of Mitochondrially targeted polysaccharide nanometer formulation and preparation method thereof.
Background technology
In the clinical treatment of tumor, operative treatment, radiotherapy, chemotherapy are 3 kinds of traditional therapeutic schemes, and wherein chemotherapy is in occupation of irreplaceable critical role. But multidrug resistance (multidrugresistance, MDR) produced by chemotherapeutics is the major reason causing its clinical treatment failed by tumor cell. MDR is the tumor cell important cells defense mechanism from drug attack, be tumor cell to a kind of chemotherapeutics drug resistance while, also various structures is different, other antitumor drug mechanism of action is different produce the phenomenon of crossing drug resistant, and its generation mechanism is sufficiently complex.
Mitochondrion is apoptotic regulation and control center. The stimulation of apoptotic signal can increase mitochondrial membrane permeability, reduces mitochondrial transmembrane potentials, and causes the opening in Mitochondrial Permeability conversion hole and the release of cytochrome C and finally activate caspase-3, inducing cell apoptosis. Therefore, Mitochondrially targeted administration promotes that apoptosis is thus overcoming drug resistance of tumor, is a new direction of current antitumor research.
The entrance mechanism of mitochondrial transmembrane potential and organelle albumen, it is be used to targeted drug to mitochondrial main path. Triphenylphosphine (triphenylphosphine, TPP) is with its higher electropositive and fat-soluble is used to modified medicaments or polysaccharide, to realize Mitochondrially targeted effect. The preparation of the open a kind of novel living cells mitochondrial probe based on carbon point (CDs) of Chinese invention patent (publication number CN104694116A), first with citric acid and carbamide for the raw water full-boiled process synthetic surface CDs with amino, then pass through end in amidation process grafting and, with triphenylphosphine (TPP) the little molecule of carboxyl, obtain product TPP-CDs with water system film and glucosan G-25 gel column separating purification. Chinese invention patent (publication number CN104523723A) discloses the Mitochondrially targeted micelle delivery system of a kind of reversible tumor drug resistance, this delivery system is polymer micelle, obtained by block copolymer folic acid-PEG-PASP self assembly, simultaneously synthesizing Mitochondrially targeted amycin-triphenylphosphine complex (TPP-DOX), and amycin and amycin-triphenylphosphine (DOX/TPP-DOX) are wrapped in polymer micelle jointly obtain carrier micelle.But in this technical scheme, owing to the TPP-DOX in mode that carries at medicine has certain amphipathic (i.e. having both hydrophilic and lipotropy), therefore physics bag load TPP-DOX mode in carrier is adopted, it is possible to generation drug leakage in depositing process can be caused.
Summary of the invention
Present invention aims to the deficiencies in the prior art, a kind of Mitochondrially targeted polysaccharide nanometer formulation and preparation method thereof is provided, utilize the advantages such as water solublity, biocompatibility and tumor-targeting that polysaccharide is good, antitumor drug is connected in the way of chemical bond and prepares into Mitochondrially targeted nanometer formulation.
Technical scheme provided by the present invention is:
A kind of Mitochondrially targeted polysaccharide nanometer formulation, including component: water soluble polysaccharide, triphenylphosphine and the antitumor drug containing primary amine or oh group;
Described triphenylphosphine modifies antitumor drug by amido link or ester bond, and described triphenylphosphine is connected to water soluble polysaccharide by the mode of ionic bond;
Or, described triphenylphosphine modifies water soluble polysaccharide by amido link, and described antitumor drug is attached to water soluble polysaccharide with the form of amido link or hydrazone key.
Owing to water soluble polysaccharide is difficult to bag medicine carrying thing, and technique scheme adopts the mode of the ionic bond of novelty be connected on water soluble polysaccharide by the antitumor drug having modified Mitochondrially targeted molecule triphenylphosphine, make Mitochondrially targeted nanometer formulation, there is the advantage of acid-sensitive release in tumor microenvironment, and preparation process is simple, compared with the solution of traditional anti-tumor medicine monomer, the toxicity of tumor cell is greatly increased by said preparation; Secondly, the mode of chemical bonds is adopted, it is possible to make medicine more stablize, not easily leak.
Additionally, Mitochondrially targeted molecule triphenylphosphine also can directly be modified on water soluble polysaccharide by technique scheme, antitumor drug is attached on water soluble polysaccharide with the form of amido link or hydrazone key, the nanometer formulation prepared, compared with the solution of traditional anti-tumor medicine monomer, the intracellular accumulation of energy significantly increasing medicament, can be applicable to antitumor drug resistance research field.
As preferably, described water soluble polysaccharide is selected from the one in hyaluronic acid, sodium alginate or heparin sodium.
As preferably, the molecular weight of described water soluble polysaccharide is 10000Da～1000000Da. The molecular weight of the water soluble polysaccharide selected is relatively big, and the particle diameter of the nanometer formulation ultimately formed will increase, and therefore to select applicable molecular weight, to control the particle diameter of nanometer formulation.
It is preferred that, described water soluble polysaccharide is selected from hyaluronic acid. Hyaluronic acid (hyaluronicacid, HA) it is a kind of natural linear glycosaminoglycan, being widely distributed in mammal medullary cell epimatrix and loose connective tissue, it has plurality of advantages, as good in water solublity and biocompatibility, cause inflammatory etc. without challeng, nothing. It addition, hyaluronic specific receptor CD44 is in the equal overexpression of Several Kinds of Malignancy cell surface. The endocytosis that specific receptor mediates can be utilized to be selectively entered tumor cell by hyaluronic acid carrying anti-tumor medicine, improve its targeting in vivo, reduce the medicine accumulation in normal structure.
As preferably, described antitumor drug is selected from the one in doxorubicin hydrochloride, bleomycin, zorubicin, epirubicin, daunorubicin, camptothecine or paclitaxel.
As preferably, described water soluble polysaccharide is hyaluronic acid, and antitumor drug is doxorubicin hydrochloride;The mass ratio of described hyaluronic acid and doxorubicin hydrochloride is 1:0.1～1. Antitumor drug is linked on water soluble polysaccharide with the form of amido link or ionic bond respectively, and amido link and ionic bond can be hydrolyzed gradually to discharge medicine in cell. Nanoparticle can be spontaneously formed when water soluble polysaccharide connects upper hydrophobic antitumor drug; Antitumor drug grafting more many, the hydrophobic parts in carrier is more many, and the water solublity of whole nanometer formulation will reduce, and therefore to control the dosage of antitumor drug.
The preparation method that the present invention provides a kind of Mitochondrially targeted polysaccharide nanometer formulation, comprises the steps:
1) 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide solution and N-hydroxy-succinamide solution are added separately in triphenylphosphine solution, regulate PH5～6 stirring reaction; Continuously add n-BOC-1,6-diamino hexane HCI solution, regulate PH7～8 stirring reaction; It is scattered in organic solvent after lyophilizing, adds trifluoroacetic acid; Then rotation is evaporated, extracts, and obtains amido modified triphenylphosphine;
2) 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide solution and N-hydroxy-succinamide solution are added separately in water soluble polysaccharide solution, regulate PH5～6 stirring reaction; Continuously add the antitumor drug containing primary amine or oh group, regulate PH7～8 stirring reaction, obtain the antitumor drug that water soluble polysaccharide is modified;
3) 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide solution and N-hydroxy-succinamide solution are added separately to the antitumor drug that water soluble polysaccharide is modified, regulate PH5～6 stirring reaction; It is subsequently adding amido modified triphenylphosphine, regulates PH7～8 stirring reaction, obtain Mitochondrially targeted polysaccharide nanometer formulation.
As preferably, described water soluble polysaccharide is hyaluronic acid, and antitumor drug is doxorubicin hydrochloride; The mass ratio of described hyaluronic acid, triphenylphosphine and doxorubicin hydrochloride is 1:0.1～1:0.1～1.
The preparation method that the present invention also provides for a kind of Mitochondrially targeted polysaccharide nanometer formulation, comprises the steps:
1) triphenylphosphine is dissolved in DMF, adds dicyclohexylcarbodiimide and N-hydroxy-succinamide, stirring reaction, obtain the triphenylphosphine after activation;
2) antitumor drug containing primary amine or oh group is dissolved in DMF, adds anhydrous triethylamine; Then, after being added dropwise over the triphenylphosphine ice bath stirring after activation, continue room temperature reaction, obtain the antitumor drug that triphenylphosphine is modified;
3) preparation water soluble polysaccharide solution, regulates PH7～8, adds silver nitrate stirring reaction, dialysis; Continuously add the antitumor drug that triphenylphosphine is modified, stirring reaction, obtain Mitochondrially targeted polysaccharide nanometer formulation.
As preferably, described antitumor drug is doxorubicin hydrochloride, and the mass ratio of described triphenylphosphine and doxorubicin hydrochloride is 1:1～1.35.
As preferably, described water soluble polysaccharide is hyaluronic acid, and antitumor drug is doxorubicin hydrochloride; Described hyaluronic acid, the antitumor drug of triphenylphosphine modification are 1:1～2.5:0.2～0.5 with the mass ratio of silver nitrate.
Compared with the existing technology, beneficial effects of the present invention is embodied in:
(1) present invention adopts the mode of the ionic bond of novelty to be connected on water soluble polysaccharide by the antitumor drug having modified Mitochondrially targeted molecule triphenylphosphine, there is the advantage of acid-sensitive release in tumor microenvironment, and preparation process is simple, compared with the solution of traditional anti-tumor medicine monomer, the toxicity of tumor cell is greatly increased by said preparation.
(2) Mitochondrially targeted molecule triphenylphosphine also can directly be modified on water soluble polysaccharide by the present invention, antitumor drug is attached on water soluble polysaccharide with the form of amido link or hydrazone key, the nanometer formulation prepared, compared with the solution of traditional anti-tumor medicine monomer, the intracellular accumulation of energy significantly increasing medicament, can be applicable to antitumor drug resistance research field.
(3) present invention adopts the mode of chemical bonds, it is possible to make medicine more stablize, not easily leak; Preparation method is reasonable in design simultaneously, has good application prospect.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope picture of the TPP-HA-amide-DOX polysaccharide nanometer formulation of embodiment 1 preparation;
Fig. 2 is the transmission electron microscope picture of the HA-ionic-TPP-DOX polysaccharide nanometer formulation of embodiment 4 preparation;
Fig. 3 is the grain size distribution of the HA-ionic-TPP-DOX polysaccharide nanometer formulation of embodiment 4 preparation;
Fig. 4 is the nuclear magnetic resonance map of the TPP-HA-amide-DOX polysaccharide nanometer formulation of embodiment 1 preparation;
Fig. 5 is the different HA-ionic-TPP-DOX polysaccharide nanometer formulation of doxorubicin concentration, amycin aqueous solution and the TPP-DOX cytotoxicity detection figure to MCF-7/ADR cell;
Fig. 6 is medicine carrying TPP-HA-amide-DOX polysaccharide nanometer formulation and the amycin aqueous solution intake detection figure in MCF-7/ADR cell of different doxorubicin concentration;
Fig. 7 is that TPP-HA-amide-DOX polysaccharide nanometer formulation enters under the effect of born of the same parents' inhibitor in difference, and the intake at MCF-7/ADR cell detects figure;
Fig. 8 is concentration is that the TPP-DOX aqueous solution of 4 μ g/mL acts on mdr cell laser co-focusing picture after MCF-7/ADR4 hour.
Detailed description of the invention
Embodiment 1
Take 40mg triphenylphosphine TPP and be dissolved in 15ml ultra-pure water, 19mg1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC, 12mgN-N-Hydroxysuccinimide NHS are dissolved in 1ml ultra-pure water respectively, successively it is added dropwise in TPP solution, regulate PH5.5, this solution is placed on magnetic stirring apparatus and stirs 40min. Take 22mgn-BOC-1,6-diamino hexane hydrochlorate to be dissolved in 5ml water, be added dropwise in the TPP solution after activation, regulate PH7.5, reaction stirring 4 hours, the TPP-C after lyophilizing6-BOC is dissolved in 20ml methanol, adds 2ml trifluoroacetic acid, reaction stirring 1 hour, is transferred in eggplant-shape bottle, and rotation steaming is evaporated, and yellow oily material occurs, adds saturated NaHCO3Solution, chloroform extracts, combined chloroform layer, and rotation steaming is evaporated and obtains TPP-C6-NH2。
Take 40mg hyaluronic acid HA (molecular weight 150000Da) and be dissolved in 10ml ultra-pure water, 19mgEDC, 12mgNHS are dissolved in 1ml ultra-pure water respectively, are successively added dropwise in HA solution, regulate PH5.5, reaction stirring 40min, takes 10mg doxorubicin hydrochloride DOX and is dissolved in 5ml water. Being added dropwise in the HA of activation, regulate PH7.5, reaction stirring 4 hours, dialyse 24 hours with 8000-14000WM bag filter after collecting product, dialysis medium is ultra-pure water.
HA-amide-DOX is centrifugal goes precipitation, takes supernatant, and 15mgEDC, 9mgNHS are dissolved in 1ml ultra-pure water respectively, are successively added dropwise in HA-amide-DOX solution, regulate PH5.5, stir 40 minutes, add TPP-C6-NH2, regulate PH7.5, room temperature reaction 4h. Dialysing 24 hours with 8000-14000WM bag filter after collecting product TPP-HA-amide-DOX, dialysis medium is ultra-pure water.
Embodiment 2
Take 40mg triphenylphosphine TPP and be dissolved in 15ml ultra-pure water, 19mg1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC, 12mgN-N-Hydroxysuccinimide NHS are dissolved in 1ml ultra-pure water respectively, successively it is added dropwise in TPP solution, regulate PH5.5, this solution is placed on magnetic stirring apparatus and stirs 40min.Take 22mgn-BOC-1,6-diamino hexane hydrochlorate to be dissolved in 5ml water, be added dropwise in the TPP solution after activation, regulate PH7.5, reaction stirring 4 hours, the TPP-C after lyophilizing6-BOC is dissolved in 20ml methanol, adds 2ml trifluoroacetic acid, reaction stirring 1 hour, is transferred in eggplant-shape bottle, and rotation steaming is evaporated, and yellow oily material occurs, adds saturated NaHCO3Solution, chloroform extracts, combined chloroform layer, and rotation steaming is evaporated and obtains TPP-C6-NH2。
Take 40mg hyaluronic acid HA (molecular weight 150000Da) and be dissolved in 10ml ultra-pure water, 19mgEDC, 12mgNHS are dissolved in 1ml ultra-pure water respectively, are successively added dropwise in HA solution, regulate PH5.5, reaction stirring 40min, takes 20mg doxorubicin hydrochloride DOX and is dissolved in 5ml water. Being added dropwise in the HA of activation, regulate PH7.5, reaction stirring 4 hours, dialyse 24 hours with 8000-14000WM bag filter after collecting product, dialysis medium is ultra-pure water.
HA-amide-DOX is centrifugal goes precipitation, takes supernatant, and 15mgEDC, 9mgNHS are dissolved in 1ml ultra-pure water respectively, are successively added dropwise in HA-amide-DOX solution, regulate PH5.5, stir 40 minutes, add TPP-C6-NH2, regulate PH7.5, room temperature reaction 4h. Dialysing 24 hours with 8000-14000WM bag filter after collecting product TPP-HA-amide-DOX, dialysis medium is ultra-pure water.
Embodiment 3
Take 40mg triphenylphosphine TPP and be dissolved in 15ml ultra-pure water, 19mg1-(3-dimethylamino-propyl)-3-ethyl carbodiimide EDC, 12mgN-N-Hydroxysuccinimide NHS are dissolved in 1ml ultra-pure water respectively, successively it is added dropwise in TPP solution, regulate PH5.5, this solution is placed on magnetic stirring apparatus and stirs 40min. Take 22mgn-BOC-1,6-diamino hexane hydrochlorate to be dissolved in 5ml water, be added dropwise in the TPP solution after activation, regulate PH7.5, reaction stirring 4 hours, lyophilizing. TPP-C after lyophilizing6-BOC is dissolved in 20ml methanol, adds 2ml trifluoroacetic acid, reaction stirring 1 hour, is transferred in eggplant-shape bottle, and rotation steaming is evaporated, and yellow oily material occurs, adds saturated NaHCO3Solution, chloroform extracts, combined chloroform layer, and rotation steaming is evaporated and obtains TPP-C6-NH2。
Take 40mg hyaluronic acid HA (molecular weight 150000Da) and be dissolved in 10ml ultra-pure water, 19mgEDC, 12mgNHS are dissolved in 1ml ultra-pure water respectively, are successively added dropwise in HA solution, regulate PH5.5, reaction stirring 40min, takes 35mg doxorubicin hydrochloride DOX and is dissolved in 5ml water. Being added dropwise in the HA of activation, regulate PH7.5, reaction stirring 4 hours, dialyse 24 hours with 8000-14000WM bag filter after collecting product, dialysis medium is ultra-pure water.
HA-amide-DOX is centrifugal goes precipitation, takes supernatant, and 15mgEDC, 9mgNHS are dissolved in 1ml ultra-pure water respectively, are successively added dropwise in HA-amide-DOX solution, regulate PH5.5, stir 40 minutes, add TPP-C6-NH2, regulate PH7.5, room temperature reaction 4h. Dialysing 24 hours with 8000-14000WM bag filter after collecting product TPP-HA-amide-DOX, dialysis medium is ultra-pure water.
Embodiment 4
Take 44.8mg triphenylphosphine TPP, be dissolved in the anhydrous DMF DMF of 10mL, add 24.2mgDCC dicyclohexylcarbodiimide, add N-hydroxy-succinamide NHS13.8mg after two minutes, 3h is stirred at room temperature, centrifugal 15min, take supernatant, take 50mg doxorubicin hydrochloride DOX simultaneously and be dissolved in dry DMF, add 13 μ l anhydrous triethylamines, and be added dropwise over the TPP supernatant after activation, after ice bath stirring 45min, change stirred overnight at room temperature into.Product adds 250ml ether, and centrifuging and taking precipitates, and adds chloroform ultrasonic disperse, is repeatedly centrifuged, and collects chloroform rotation steaming and is evaporated. Being subsequently adding deionized water 20ml, transition into vial after dispersion, centrifuging and taking supernatant is collected, repeatedly resuspended pelleting centrifugation, collects aqueous solution, and lyophilizing obtains TPP-DOX.
Take 35mg hyaluronic acid HA (molecular weight 48000Da) and be dissolved in 10ml ultra-pure water, regulate PH7.5. Take 8mg silver nitrate to be dissolved in 1ml ultra-pure water, and be added dropwise in HA aqueous solution, dialyse after reacting 2 hours. Taking 40mgTPP-DOX to be dissolved in 5ml ultra-pure water, be added dropwise over above-mentioned solution, reaction stirring is overnight. Collecting product HA-ionic-TPP-DOX to dialyse 24 hours with 8000-14000WM bag filter, dialysis medium is ultra-pure water.
Embodiment 5
Take 44.8mg triphenylphosphine TPP, be dissolved in the anhydrous DMF DMF of 10mL, add 24.2mg dicyclohexylcarbodiimide DCC, add N-hydroxy-succinamide NHS13.8mg after two minutes, 3h is stirred at room temperature, centrifugal 15min, take supernatant, take 50mg doxorubicin hydrochloride DOX simultaneously and be dissolved in dry DMF, add 13 μ l anhydrous triethylamines, and be added dropwise over the TPP supernatant after activation, after ice bath stirring 45min, change stirred overnight at room temperature into. Product adds 250ml ether, and centrifuging and taking precipitates, and adds chloroform ultrasonic disperse, is repeatedly centrifuged, and collects chloroform rotation steaming and is evaporated. Being subsequently adding deionized water 20ml, transition into vial after dispersion, centrifuging and taking supernatant is collected, repeatedly resuspended pelleting centrifugation, collects aqueous solution, and lyophilizing obtains TPP-DOX.
Take 35mg hyaluronic acid HA (molecular weight 48000Da) and be dissolved in 10ml ultra-pure water, regulate PH7.5. Take 8mg silver nitrate to be dissolved in 1ml ultra-pure water, and be added dropwise in HA aqueous solution, dialyse after reacting 2 hours. Taking 80mgTPP-DOX to be dissolved in 5ml ultra-pure water, be added dropwise over above-mentioned solution, reaction stirring is overnight. Collecting product HA-ionic-TPP-DOX to dialyse 24 hours with 8000-14000WM bag filter, dialysis medium is ultra-pure water.
Characterize and performance evaluation
The polysaccharide nanometer formulation of the present invention and existing preparation are carried out form, nuclear magnetic resonance, NMR, cytotoxicity, picked-up in mdr cell, enter born of the same parents' approach and the distribution in mdr cell is analyzed:
1, Mitochondrially targeted polysaccharide nanometer formulation TPP-HA-amide-DOX and HA-ionic-TPP-DOX is carried out morphologic observation
Take the TPP-HA-amide-DOX that appropriate embodiment 1 prepares and the HA-ionic-TPP-DOX that embodiment 4 prepares, add to special purpose copper respectively online, under transmission electron microscope, observe size and the form of particle respectively, respectively such as Fig. 1 and Fig. 2; Fig. 3 is the grain size distribution of the HA-ionic-TPP-DOX that embodiment 4 prepares.
Conclusion: under polysaccharide nanometer formulation TPP-HA-amide-DOX Electronic Speculum, form is that class is spherical, and particle diameter is less homogeneous; Under polysaccharide nanometer formulation HA-ionic-TPP-DOX Electronic Speculum, form is spherical, and particle diameter is 257nm.
2, polysaccharide nanometer formulation TPP-HA-amide-DOX is carried out nuclear magnetic resonance spectroscopy
After polysaccharide nanometer formulation TPP-HA-amide-DOX lyophilizing prepared by embodiment 1, it is dissolved in deuterated water and carries out nuclear magnetic resonance spectroscopy, referring to Fig. 4.
Result shows: contrasted by the nuclear magnetic spectrum of the nuclear magnetic resonance map of synthetic product TPP-HA-amide-DOX Yu monomer TPP, DOX, HA, it has been found that the stronger peak occurred at a place is-CH in DOX3Peak, the methylene peak that peak is monomer HA sugar chain of b place comparatively dense, between 7.5-8.0, the peak for c place is produced peak by TPP phenyl ring, and TPP-HA-amide-DOX described above successfully synthesizes.
3, the polysaccharide nanometer formulation HA-ionic-TPP-DOX toxicity test to mdr cell
MCF-7/ADR cell (1 × 104Individual/hole) it is inoculated in 96 well culture plates, incubated overnight, it is separately added into the different polysaccharide nanometer formulation HA-ionic-TPP-DOX of the present invention of doxorubicin concentration, amycin aqueous solution and TPP-DOX aqueous solution as experimental group, blank group adds isopyknic cell culture fluid, incubator is cultivated after 48h, every hole adds MTT (tetramethyl azo azoles salt) the PBS buffer solution that 20 μ l concentration are 5mg/ml, culture fluid is sucked after 4h, every hole adds 150 μ l dimethyl sulfoxide (DMSO) again, jolting 10min, in microplate reader, measure A value in 570nm.
Calculate cytotoxicity as follows:
Cytotoxicity (%)=(1-experimental group A value/blank group A value) × 100%
Result shows: under same concentrations, the polysaccharide nanometer formulation HA-ionic-TPP-DOX of the present invention to the toxicity of mdr cell slightly larger than amycin aqueous solution, referring to Fig. 5.
4, polysaccharide nanometer formulation TPP-HA-amide-DOX picked-up experiment in mdr cell
MCF-7/ADR cell (1 × 106Individual/hole) it is inoculated in 6 well culture plates, incubated overnight, adding the nanometer formulation TPP-HA-amide-DOX of different doxorubicin concentration, amycin aqueous solution, as experimental group, is cultivated in incubator after 3h, cell is washed three times with cold PBS (pH7.4), terminating the cell picked-up to medicine, every hole adds ultra-pure water 2ml, scrapes cell with cell scraper, being transferred to 4ml centrifuge tube, Probe Ultrasonic Searching crushes (power: 100W; Working time: 2s; Intermittent time: 3s; Ultrasonic number of times: 30 times), 12,000r/min centrifugal 5min, take the efficient liquid phase instrument detection DOX concentration of supernatant fluorescence, result is shown in Fig. 6.
Liquid phase chromatogram condition: DiamonsilC18 post (5 μm, 250*4.6mm); Mobile phase is acetonitrile: 15mMNaH2PO4 (32:68), and wherein 15mMNaH2PO4 solution is NaH2PO4: triethylamine: phosphoric acid (70:0.1:0.125); DOX excitation wavelength is 488nm, and transmitting wavelength is 570nm.
Result shows: under same concentrations, and the nanometer formulation of the present invention picked-up in mdr cell is more than amycin aqueous solution.
5, polysaccharide nanometer formulation TPP-HA-amide-DOX enters the experiment of born of the same parents' Study of way
MCF-7/ADR cell (1 × 106Individual/hole) it is inoculated in 6 well culture plates, incubated overnight, 20ug/mlHA solution, 10ug/ml chlorpromazine solution, after hatching 30min. Add the nanometer formulation TPP-HA-amide-DOX of 25 μ g/ml doxorubicin concentration, incubator is cultivated after 3h, washes cell three times with cold PBS (pH7.4), add ultra-pure water 2ml, scraping cell with cell scraper, be transferred to 4ml centrifuge tube, Probe Ultrasonic Searching crushes (power: 100W; Working time: 2s; Intermittent time: 3s; Ultrasonic number of times: 30 times), 12,000r/min centrifugal 5min, take the efficient liquid phase instrument detection DOX concentration of supernatant fluorescence, result is shown in Fig. 7.
Result shows: enter born of the same parents inhibitor HA and chlorpromazine solution all can reduce nanometer formulation TPP-HA-amide-DOX picked-up in mdr cell, illustrate preparation be mediated by CD44 and clathrin-mediated endocytosis approach enter cell.
6, Mitochondrially targeted medicine TPP-DOX distribution experiments in mdr cell
MCF-7/ADR cell (2 × 105Individual/hole) it is inoculated in copolymerization Jiao's capsule, overnight incubation, add after TPP-DOX4h, change the culture fluid containing Hoechst33342 and hatch 20min, PBS and wash 3 times;Add Mito-TrackerGreenFM (140nM) dye 40min, PBS to wash 3 times, be inverted copolymerization Jiao and observe, referring to Fig. 8.
Result shows: observe white portion in the drawings, it was shown that TPP-DOX achieves mitochondrion and positions altogether, demonstrates the Mitochondrially targeted function of lipophilic cation TPP simultaneously.
Claims (10)
1. polysaccharide nanometer formulation one kind Mitochondrially targeted, it is characterised in that include component: water soluble polysaccharide, triphenylphosphine and the antitumor drug containing primary amine or oh group;
Described triphenylphosphine modifies antitumor drug by amido link or ester bond, and described triphenylphosphine is connected to water soluble polysaccharide by the mode of ionic bond;
Or, described triphenylphosphine modifies water soluble polysaccharide by amido link, and described antitumor drug is attached to water soluble polysaccharide with the form of amido link or hydrazone key.
2. Mitochondrially targeted polysaccharide nanometer formulation according to claim 1, it is characterised in that described water soluble polysaccharide one in hyaluronic acid, sodium alginate or heparin sodium.
3. Mitochondrially targeted polysaccharide nanometer formulation according to claim 2, it is characterised in that the molecular weight of described water soluble polysaccharide is 10000Da～1000000Da.
4. Mitochondrially targeted polysaccharide nanometer formulation according to claim 1, it is characterised in that described antitumor drug one in doxorubicin hydrochloride, bleomycin, zorubicin, epirubicin, daunorubicin, camptothecine or paclitaxel.
5. Mitochondrially targeted polysaccharide nanometer formulation according to claim 1, it is characterised in that described water soluble polysaccharide is hyaluronic acid, and antitumor drug is doxorubicin hydrochloride; The mass ratio of described hyaluronic acid and doxorubicin hydrochloride is 1:0.1～1.
6. the preparation method of polysaccharide nanometer formulation one kind Mitochondrially targeted, it is characterised in that comprise the steps:
1) 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide solution and N-hydroxy-succinamide solution are added separately in triphenylphosphine solution, regulate PH5～6 stirring reaction; Continuously add n-BOC-1,6-diamino hexane HCI solution, regulate PH7～8 stirring reaction; It is scattered in organic solvent after lyophilizing, adds trifluoroacetic acid; Then rotation is evaporated, extracts, and obtains amido modified triphenylphosphine;
2) 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide solution and N-hydroxy-succinamide solution are added separately in water soluble polysaccharide solution, regulate PH5～6 stirring reaction; Continuously add the antitumor drug containing primary amine or oh group, regulate PH7～8 stirring reaction, obtain the antitumor drug that water soluble polysaccharide is modified;
3) 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide solution and N-hydroxy-succinamide solution are added separately to the antitumor drug that water soluble polysaccharide is modified, regulate PH5～6 stirring reaction; It is subsequently adding amido modified triphenylphosphine, regulates PH7～8 stirring reaction, obtain Mitochondrially targeted polysaccharide nanometer formulation.
7. the preparation method of Mitochondrially targeted polysaccharide nanometer formulation according to claim 6, it is characterised in that described water soluble polysaccharide is hyaluronic acid, and antitumor drug is doxorubicin hydrochloride; The mass ratio of described hyaluronic acid, triphenylphosphine and doxorubicin hydrochloride is 1:0.1～1:0.1～1.
8. the preparation method of polysaccharide nanometer formulation one kind Mitochondrially targeted, it is characterised in that comprise the steps:
1) triphenylphosphine is dissolved in DMF, adds dicyclohexylcarbodiimide and N-hydroxy-succinamide, stirring reaction, obtain the triphenylphosphine after activation;
2) antitumor drug containing primary amine or oh group is dissolved in DMF, adds anhydrous triethylamine; Then, after being added dropwise over the triphenylphosphine ice bath stirring after activation, continue room temperature reaction, obtain the antitumor drug that triphenylphosphine is modified;
3) preparation water soluble polysaccharide solution, regulates PH7～8, adds silver nitrate stirring reaction, dialysis; Continuously add the antitumor drug that triphenylphosphine is modified, stirring reaction, obtain Mitochondrially targeted polysaccharide nanometer formulation.
9. the mass ratio of the preparation method of Mitochondrially targeted polysaccharide nanometer formulation according to claim 8, it is characterised in that described antitumor drug is doxorubicin hydrochloride, described triphenylphosphine and doxorubicin hydrochloride is 1:1～1.35.
10. the preparation method of Mitochondrially targeted polysaccharide nanometer formulation according to claim 8, it is characterised in that described water soluble polysaccharide is hyaluronic acid, and antitumor drug is doxorubicin hydrochloride; Described hyaluronic acid, the antitumor drug of triphenylphosphine modification are 1:1～2.5:0.2～0.5 with the mass ratio of silver nitrate.
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