CN105031657A - Cross-linkable mitochondrial targeting pegylated phospholipid medicinal material and preparation method and application thereof - Google Patents
Cross-linkable mitochondrial targeting pegylated phospholipid medicinal material and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a cross-linkable mitochondrial targeting pegylated phospholipid medicinal material. Please see the structural formula of the cross-linkable mitochondrial targeting pegylated phospholipid medicinal material in the specification, wherein n is equal to 10, 12, 14 and 16. The preparation method includes the steps that fatty acid acyl phosphatidylethanolamine-polyethylene glycol(peg)-maleimide, polypeptide D- (KLAKLAK)2-C5 and organic base serve as raw materials, and the molecular ratio of the polypeptide to the organic base to the fatty acid acyl phosphatidylethanolamine-polyethylene glycol(peg)-maleimide is (1-3): (1-3):1; under protection of nitrogen, the fatty acid acyl phosphatidylethanolamine-polyethylene glycol(peg)-maleimide, the organic base and a first solvent are mixed so that a first solution can be obtained, the polypeptide is dissolves in a second solvent so that a second solution can be obtained, the first solution and the second solution are mixed, and a mixed solution is stirred to react for 12-8 h at the temperature of 20-40 DEG C. Medicine carrying liposome prepared from the medicinal material can further improve the tumor treatment effect, the toxic and side effects are reduced, and long-time preservation is facilitated.
Description
Technical field
The invention belongs to medicine and preparing technical field thereof, be specifically related to a kind of phospholipid medicinal materials and preparation method thereof and application.
Background technology
Chemotherapy is one of Main Means of Therapeutic cancer, but the mortality rate after current chemotherapy still remains high.Multidrug resistance (multidrugresistance, MDR) is the one of the main reasons of chemotherapy failure clinically.The drug resistance of tumor cell is divided into congenital class large with the day after tomorrow acquired two.Research shows, tumor cell gene or gene phenotype change and cause apoptosis path to change, and finally cause the appearance of the congenital drug resistance of tumor.Mitochondrion is cell " power factory ", is also simultaneously " suicide arm store ", can active cell apoptosis pathway, and thus tumor cell natural resistance is closely related with mitochondrial function exception.Target spot using mitochondrion as cancer therapy drug transmission system, by classic chemotherapy drug delivery to mitochondrion, impel mitochondrion start apoptosis pathway, release cells pigment C, activate caspase9/3, thus it is dead to realize tumor cell " suicide " formula, this will be overcome tumor natural resistance, improve a kind of effective means of oncotherapy effect.Cancer therapy drug is delivered in tumor cell mitochondrial processes, needs to face multiple physiological barrier, comprise and penetrate blood vessel and enter the vascular barrier in tumor gap, the plasma membrane barrier penetrating tumor extracellular matrix and tumor cell membrane and mitochondrial membrane barrier.Mitochondrially targeted treatment obtains successful key and is to deliver drugs into tumor cell mitochondrion, and avoid the drug delivery system when crossing over physiologic barrier to disintegrate, medicine misses the target.
Prior art reports DSPE (DSPE) and 3-maleimidoproprionic acid hydroxysuccinimide eater Reactive Synthesis DSPE maleimide (DSPE-mal), DSPE-mal again with D-(KLAKLAK)
2-C is obtained by reacting DSPE-(KLAKLAK)
2, DSPE-(KLAKLAK)
2to be generated by amidation process with dimethyl maleic anhydride and a kind of there is new modified phospholipid DSPE-(KLAKLAK) that Mitochondrially targeted and pH responds dual-use function
2-dimethylmaleimide (DSPE-KLA-DMA, abbreviation DKD), DKD is mixed with soybean phospholipid, cholesterol, anti-cancer medicine paclitaxel, film dispersion method is adopted to prepare medicament-carried nano DKD liposome, and confirm that this liposome has the absorption of reduction nonspecific proteins and Mitochondrially targeted delivering drugs function, mitochondrial apoptotic pathway can be impelled to start, tumor natural resistance can be overcome to a certain extent, improve oncotherapy effect (L.Jiangetal.Biomaterials52 (2015) 126-139).But DKD also has the following disadvantages: 1) DKD does not have long circulating function, cause the DKD liposome prepared to remove at Ink vessel transfusing fast, be unfavorable for that vascular barrier delivering drugs crossed over by cancer therapy drug; 2) the medicament-carried nano liposome utilizing DKD to prepare is assembled by molecule the Micelle-like Nano-structure of Two formed by non-covalent bonding force, unstable in blood circulation and cross-film process, easy disintegration, medicine is caused to discharge before reaching target spot prerequisite, do not reach therapeutic effect, and medicine miss the target after enter normal structure and very easily cause serious toxic and side effects; 3) owing to not there is hydrophilic protective layer in the molecular structure of DKD; easily produce reunion after the DKD liposome prepared places a period of time, particle diameter becomes large, is unfavorable for that liposome enters tissue from blood vessel; thus reduce tumor tissues targeting, there is the potential safety hazards such as blocking blood capillary simultaneously.Therefore the long-time stability of DKD liposome are bad, are unfavorable for long-term storage.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, Mitochondrially targeted pegylated phospholipids medicinal materials of a kind of crosslinkable and preparation method thereof and application are provided, this medicinal materials has good long circulating function, cross linkable and Mitochondrially targeted property, the drug-loaded liposome utilizing this medicinal materials to prepare has better stability in blood circulation and cross-film process, and there is better tumor tissues targeting, larger cellular uptake amount and Mitochondrially targeted property, thus oncotherapy effect can be improved further, reduce toxic and side effects simultaneously, and can long-term storage.
The Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable of the present invention, its structural formula is as follows:
In structure above, n=10,12,14,16.
The preparation method of the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable of the present invention, with fatty acid acyl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol-maleimide, polypeptide D-(KLAKLAK)
2-C
5be raw material with organic base, polypeptide D-(KLAKLAK)
2-C
5, organic base, fatty acid acyl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol-maleimide mol ratio be (1 ~ 3): (1 ~ 3): 1, described polypeptide D-(KLAKLAK)
2-C
5aminoacid sequence for described in SEQ ID NO:1, processing step is as follows:
Under nitrogen protection, fatty acid acyl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol-maleimide, organic base and the first solvent mix homogeneously are obtained the first solution, and the consumption of described first solvent is make the concentration of fatty acid acyl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol-maleimide in the first solution be 100 ~ 300mmol/L;
Under nitrogen protection, by polypeptide D-(KLAKLAK)
2-C
5be dissolved in the second solvent and obtain the second solution, the consumption of the second solvent is for making polypeptide D-(KLAKLAK) in the second solution
2-C
5concentration be 100 ~ 900mol/L;
By the first solution and the mixing of the second solution, in 20 ~ 40 DEG C of reactions 12 ~ 48 hours under lucifuge, stirring, except desolventizing obtains solid matter after reaction terminates, insoluble matter is filtered out after gained solid matter chloroform or dimethyl sulfoxine being dissolved, collect filtrate, namely the solvent in removing filtrate obtains the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable.
The preparation method of the Mitochondrially targeted pegylated phospholipids medicinal materials of above-mentioned crosslinkable, described fatty acid acyl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol-maleimide is the one in PEG-DSPE 2000-maleic amide, DMPEA-Macrogol 2000-maleic amide, PEG2000-DSPE-maleic amide, two lauroyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleic amides.
The preparation method of the Mitochondrially targeted pegylated phospholipids medicinal materials of above-mentioned crosslinkable, described organic base is any one in triethylamine, DMAP or N, N-diisopropylethylamine.
The preparation method of the Mitochondrially targeted pegylated phospholipids medicinal materials of above-mentioned crosslinkable, described first solvent is dichloromethane or chloroform, any one in the second solvent methanol, DMF, dimethyl sulfoxine.
Present invention also offers the Mitochondrially targeted pegylated phospholipids medicinal materials of above-mentioned crosslinkable and prepare the application in drug-loaded liposome, described medicinal materials can mix obtained liposome with cancer therapy drug, phospholipid, cholesterol, cancer therapy drug is amycin, paclitaxel etc.Described liposome can form disulfide bond crosslinking structure, to improve the stability of drug-loaded liposome in blood circulation and cross-film process with the form of covalent bond adding under dithioglycol and logical Oxygen Condition after preparing.
Compared with prior art, the present invention has following beneficial effect:
1, the present invention is that the preparation of antitumor drug-loaded liposome provides a kind of new medicinal materials.
2, the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable of the present invention is owing to having Polyethylene Glycol segment, can extend circulation time in vivo, and with polypeptide D-(KLAKLAK)
2-C
5for Mitochondrially targeted molecule, and there is crosslinkable structure sulfydryl, therefore there is good long circulating function, cross linkable and Mitochondrially targeted property.
3, the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable of the present invention is owing to having crosslinkable structure, the drug-loaded liposome utilizing this medicinal materials to prepare is adding under dithioglycol and logical Oxygen Condition with the form of covalent bond formation disulfide bond crosslinking structure, there is good stability in blood circulation, liposome can be avoided to disintegrate causes medicine to discharge in advance, tool good better tumor tissues, Mitochondrially targeted ability simultaneously, therefore the toxic and side effects of normal tissue is very little.
4, the drug-loaded liposome utilizing this medicinal materials to prepare has longer circulation time (being good at circulation experiment) in vivo, the cross linkable of medicinal materials makes drug-loaded liposome add 1, 2-dithioglycol also forms disulfide bond crosslinking structure with the form of covalent bond under logical Oxygen Condition, after entering human body, there is good stability in blood, drug release extremely slowly (see extracorporeal releasing experiment), be conducive to liposome and cross over vascular barrier delivering drugs, simultaneously because tumor vessel wall epithelial cell gap is larger than the epithelial cell gap of normal blood vessels wall, stable drug-loaded liposome circulation time length is in vivo conducive to drug-loaded liposome and oozes out from tumor vessel, more drug-loaded liposome is made to enter tumor tissues by strengthening infiltration with retention effect (EPR effect), therefore there is better tumor tissues targeting (see the comparative experiments of tumor tissues targeting), after drug-loaded liposome enters tumor tissues gap, because this drug-loaded liposome zeta current potential is just, the tumor cell picked-up of aobvious negative charge is attracted more easily by electrostatic interaction, in addition drug-loaded liposome has the cross-linked structure with disulfide formation, there is good stability, liposome can be avoided to disintegrate, avoid medicine to discharge in advance, the plasma membrane barrier of tumor extracellular matrix and tumor cell membrane can be penetrated smoothly, by drug delivery in tumor cell, after drug-loaded liposome enters tumor cell, because the glutathione concentrations in tumor cell is higher than blood and interstice, liposome ruptures because of the cross-linked structure of disulfide formation under glutathione reduction effect, be conducive to liposome and enter drug release after mitochondrion, in addition mitochondrion intake large (see line grain picked-up test), has more drug release in mitochondrion.In sum, the drug-loaded liposome utilizing crosslinkable of the present invention Mitochondrially targeted pegylated phospholipids medicinal materials to prepare has better tumor tissues targeting, larger cellular uptake amount, better Mitochondrially targeted property, medicine just starts a large amount of release after arriving mitochondrion, thus more can the suicide death of effectively start tumor cell, overcome the congenital drug resistance of tumor cell, improve drug-resistant tumor therapeutic effect (see the experiment of oncotherapy Contrast on effect) further.
5, drug-loaded liposome prepared by the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable of the present invention is utilized, because its Polyethylene Glycol segment can form hydrophilic layer outward at the bilayer of liposome, liposome is hindered to reunite by space steric effect, particle diameter is avoided to become large, thus maintain the stability of liposomal particle size, its stability is better than DKD liposome (see stability comparative experiments), thus be beneficial to long-term storage, ensure tumor tissues targeting and the safety in utilization of depositing rear use.
6, the preparation method of crosslinkable of the present invention Mitochondrially targeted pegylated phospholipids medicinal materials is simple to operate, and process conditions are gentle, adopts conventional equipment to realize.
Accompanying drawing explanation
Fig. 1 is the mass spectrum of the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable prepared by embodiment 1.
Fig. 2 is the hydrogen nuclear magnetic spectrogram of the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable prepared by embodiment 1.
Fig. 3 is the cytotoxicity test result of the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable prepared by embodiment 1 ~ 4.
Fig. 4 is the crosslinked Mitochondrially targeted liposome drug release experiment result figure outer with the DKD Via Liposomes carrying paclitaxel carrying paclitaxel.
Fig. 5 is that the crosslinked Mitochondrially targeted liposome of paclitaxel and the tumor-bearing mice gross tumor volume change curve (* * represents P<0.01) of the DKD liposome of year paclitaxel are carried in injection.
Fig. 6 is the scattergram of fluorescence molecule in mouse tissue and tumor (wherein scheme A for crosslinked Mitochondrially targeted fluorescent lipid group, figure B is DKD fluorescent lipid group) in crosslinked Mitochondrially targeted fluorescent lipid and DKD fluorescent lipid.
Fig. 7 is the cellular uptake experimental result picture of crosslinked Mitochondrially targeted fluorescent lipid and DKD fluorescent lipid.
Fig. 8 is the mitochondrion picked-up experimental result picture of crosslinked Mitochondrially targeted fluorescent lipid and DKD fluorescent lipid.
Fig. 9 carries the crosslinked Mitochondrially targeted liposome structure schematic diagram of paclitaxel.
Detailed description of the invention
Below by embodiment, Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable of the present invention and preparation method thereof is described further with application.
In following examples, PEG-DSPE 2000-maleic amide (DSPE-PEG2000-Mal), DMPEA-Macrogol 2000-maleic amide (DMPE-PEG2000-Mal), PEG2000-DSPE-maleic amide (DPPE-PEG2000-Mal), two lauroyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleic amide (DLPE-PEG2000-Mal) purchased from American Nanocs company.D-(KLAKLAK)
2-C
5polypeptide is purchased from Zhong Keya Micron Technology Co., Ltd.Paclitaxel is purchased from Dalian Mei Lun Bioisystech Co., Ltd.Ovum Gallus domesticus Flavus lecithin, hydrolecithin, cholesterol are purchased from Rui Xi bio tech ltd, Xi'an.
Embodiment 1
The Mitochondrially targeted Pegylation DSPE (DSPE-PEG2000-KLA) of crosslinkable described in the present embodiment, its structural formula is:
Preparation method is as follows:
With PEG-DSPE 2000-maleimide (DSPE-PEG2000-Mal), polypeptide D-(KLAKLAK)
2-C
5be raw material with triethylamine, polypeptide D-(KLAKLAK)
2-C
5, triethylamine, DSPE-PEG2000-Mal mol ratio be 1:1:1;
Under nitrogen protection, DSPE-PEG2000-Mal, triethylamine and chloroform mix homogeneously are obtained the first solution, the consumption of described chloroform is make the concentration of DSPE-PEG2000-Mal in the first solution be 100mmol/L, and the concentration of triethylamine is 100mmol/L;
Under nitrogen protection, by polypeptide D-(KLAKLAK)
2-C
5be dissolved in methanol and obtain the second solution, the consumption of methanol is for making polypeptide D-(KLAKLAK) in the second solution
2-C
5concentration is 100mol/L;
By the first solution and the mixing of the second solution, stirring reaction 48 hours under lucifuge, 20 DEG C of conditions, reaction terminates the steaming of rear employing decompression spin drying method and desolventizes, gained solid matter chloroform is dissolved, and falls insoluble matter with filter paper filtering, collect filtrate, decompression spin drying method is adopted to steam except the solvent in filtrate, namely obtain DSPE-PEG2000-KLA, its mass spectrum is shown in Fig. 1, and hydrogen nuclear magnetic spectrogram is shown in Fig. 2.
Embodiment 2
The Mitochondrially targeted Pegylation DMPEA (DMPE-PEG2000-KLA) of crosslinkable described in the present embodiment, its structural formula is:
Preparation method is as follows:
With DMPEA-Macrogol 2000-maleimide (DMPE-PEG2000-Mal), polypeptide D-(KLAKLAK)
2-C
5be raw material with DMAP, polypeptide D-(KLAKLAK)
2-C
5, DMAP, DMPE-PEG2000-Mal mol ratio be 2:2:1;
Under nitrogen protection, DMPE-PEG2000-Mal, DMAP, dichloromethane mix homogeneously are obtained the first solution, the consumption of dichloromethane is make the concentration of DMPE-PEG2000-Mal in the first solution be 200mmol/L, and the concentration of DMAP is 400mmol/L;
Under nitrogen protection, by polypeptide D-(KLAKLAK)
2-C
5be dissolved in DMF and obtain the second solution, the consumption of DMF is for making polypeptide D-(KLAKLAK) in the second solution
2-C
5concentration is 400mol/L;
By the first solution and the mixing of the second solution, stirring reaction 24 hours under lucifuge, 40 DEG C of conditions, reaction terminates the steaming of rear employing decompression spin drying method and desolventizes, gained solid matter dimethyl sulfoxine is dissolved, and fall insoluble matter with filter paper filtering, collect filtrate, adopt decompression spin drying method to steam except the solvent in filtrate, namely obtain DMPE-PEG2000-KLA.
Embodiment 3
The Mitochondrially targeted Pegylation DPPE (DPPE-PEG2000-KLA) of crosslinkable described in the present embodiment, its structural formula is:
Preparation method is as follows:
With PEG2000-DSPE-maleimide (DPPE-PEG2000-Mal), polypeptide D-(KLAKLAK)
2-C
5be raw material with DIPEA, polypeptide D-(KLAKLAK)
2-C
5, DIPEA, DPPE-PEG2000-Mal mol ratio be 3:3:1;
Under nitrogen protection, by DPPE-PEG2000-Mal, N, N-diisopropylethylamine, dichloromethane mix homogeneously obtain the first solution, the consumption of described dichloromethane is make the concentration of DPPE-PEG2000-Mal in the first solution be 300mmol/L, the concentration of DIPEA is 900mmol/L;
Under nitrogen protection, by polypeptide D-(KLAKLAK)
2-C
5be dissolved in DMF and obtain the second solution, the consumption of DMF is for making polypeptide D-(KLAKLAK) in the second solution
2-C
5concentration is 900mol/L;
By the first solution and the mixing of the second solution, stirring reaction 12 hours under lucifuge, 25 DEG C of conditions, reaction terminates the steaming of rear employing decompression spin drying method and desolventizes, gained solid matter dimethyl sulfoxine is dissolved, and fall insoluble matter with filter paper filtering, collect filtrate, adopt decompression spin drying method to steam except the solvent in filtrate, namely obtain DPPE-PEG2000-KLA.
Embodiment 4
The Mitochondrially targeted Pegylation two of crosslinkable described in the present embodiment lauroyl PHOSPHATIDYL ETHANOLAMINE (DLPE-PEG2000-KLA), its structural formula is:
Preparation method is as follows:
With two lauroyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleimides (DLPE-PEG2000-Mal), polypeptide D-(KLAKLAK)
2-C
5be raw material with triethylamine, polypeptide D-(KLAKLAK)
2-C
5, triethylamine, DLPE-PEG2000-Mal mol ratio be 3:3:1;
Under nitrogen protection, DLPE-PEG2000-Mal, triethylamine, chloroform mix homogeneously are obtained the first solution, the consumption of described chloroform is make the concentration of DLPE-PEG2000-Mal in the first solution be 100mmol/L, and the concentration of triethylamine is 300mmol/L;
Under nitrogen protection, by polypeptide D-(KLAKLAK)
2-C
5be dissolved in methanol and obtain the second solution, the consumption of methanol is for making polypeptide D-(KLAKLAK) in the second solution
2-C
5concentration is 100mol/L;
By the first solution and the mixing of the second solution, stirring reaction 48 hours under lucifuge, 20 DEG C of conditions, reaction terminates the steaming of rear employing decompression spin drying method and desolventizes, gained solid matter chloroform is dissolved, and fall insoluble matter with filter paper filtering, collect filtrate, adopt decompression spin drying method to steam except the solvent in filtrate, namely obtain DLPE-PEG2000-KLA.
Embodiment 5: Materials Cell toxicity test
Be 1 × 10 by density
5l929 cell (purchased from Chinese Academy of Sciences's cell bank) suspension inoculation of individual/mL is in 96 well culture plates, every hole inoculation 0.1mL, and 0.1mL complete medium (DMEM culture medium+10% hyclone+100g/mL streptomycin) is added to every hole, be then placed in 37 DEG C, 5%CO
2, saturated humidity cell constant temperature incubator in hatch 24h.Dilute the crosslinkable Mitochondrially targeted pegylated phospholipids medicinal materials complete medium prepared by embodiment 1 ~ 4 as test group respectively, final concentration is respectively 0.01mg/mL, 0.05mg/mL, 0.30mg/mL, 1.0mg/mL.Take complete medium as negative control group.Each test group and negative control group all establish parallel sample 3.Each test group and negative control group are placed in 37 DEG C, 5%CO
2, saturated humidity cell constant temperature incubator in continue to cultivate, cultivate the supernatant discarded after 48h in every hole, with PBS buffer (135mMNaCl, 2.7mMKCl, 1.5mMKH
2pO
4, 8mMK
2hPO
4pH=7.4) wash 2 times, then every hole adds 200LPBS buffer, add the MTT solution (be dissolved in by 0.5gMTT in 100mLPBS buffer and obtain) of 20L5mg/mL again, continue cultivate 4h, continue after exhaust supernatant, add 100L dimethyl sulfoxide (DMSO), vibration 10min, measures the optical density value (OD) that wavelength is 570nm place, by comparing with the OD value of negative control group the cell survival rate obtaining each test group with enzyme-linked immunosorbent assay instrument.
Cell toxicity test the results are shown in Figure 3, and as can be seen from the figure, crosslinkable Mitochondrially targeted pegylated phospholipids medicinal materials prepared by embodiment 1 ~ 4 is all without obvious cytotoxicity.
Embodiment 6: long circulating is tested
The DSPE-PEG2000-KLA prepare soybean phospholipid, cholesterol, embodiment 1 and paclitaxel are dissolved in (volume ratio of chloroform and methanol is 3:1) in chloroform-methanol mixed solvent, wherein soybean phospholipid, cholesterol and DSPE-PEG-KLA mol ratio are 90:7:3, the mass ratio of paclitaxel and soybean phospholipid is 1:10, and the amount of described mixed solvent amount is 0.1mmol/L for making soybean phospholipid molar concentration.Decompression rotary evaporation, except desolventizing, obtains dry adipose membrane.In gained adipose membrane, add phosphate buffer (PBS), the amount of described PBS makes the molar concentration of soybean phospholipid be 1mM.Then be placed in 50 DEG C of ultrasonic 15min of water-bath, continue with Probe Ultrasonic Searching 5min, obtain Liposomal suspensions.The dithioglycol of DSPE-PEG2000-KLA bis-times moles is added in Liposomal suspensions, and continue logical oxygen 10 minutes, again with the centrifugal 3min of the rotating speed of 3500r/min to remove free paclitaxel, gained supernatant is and carries the crosslinked Mitochondrially targeted liposome of paclitaxel, and structure is shown in Fig. 9.
Soybean phospholipid, cholesterol, DSPE-PEG2000-MAL and paclitaxel are dissolved in (volume ratio of chloroform and methanol is 3:1) in chloroform-methanol mixed solvent, wherein soybean phospholipid, cholesterol and DSPE-PEG-MAL molar fraction are than being 90:7:3, paclitaxel is 1:10 with the mass fraction ratio of matrix material, all the other preparation methoies are the same, obtain common Paclitaxel liposome.
12 Wistar rats (purchased from Sichuan University's animal experimental center) are divided into two groups at random, and often organize tail vein injection respectively and carry the Mitochondrially targeted liposome of paclitaxel and common Paclitaxel liposome, dosage is 5mg/kg.Respectively at 5min, 30min, 1h, 2h, 4h, 8h, 12h, 24h, the 48h after tail vein injection administration by tail venous blood sampling.Adopt high-efficient liquid phase technique detection of drugs concentration, adopt DAS pharmacokinetics computed in software medicine biological half-life (t
1/2).Carry the t of the Mitochondrially targeted liposome of paclitaxel
1/2for 22.5h, the t of common Paclitaxel liposome
1/2for 1.2h, the drug-loaded liposome that as seen prepared by the described Mitochondrially targeted pegylated lipids medicinal materials of this utilization invention has macrocyclic feature.
Embodiment 7: contrast experiment
The DKD liposome of citing document in background technology and the liposome utilizing crosslinkable of the present invention Mitochondrially targeted pegylated phospholipids medicinal materials to prepare are done following contrast.
1, vitro drug release contrast experiment
The crosslinked Mitochondrially targeted liposome of paclitaxel is carried according to the method preparation in embodiment 6.
Paclitaxel DKD liposome is carried according to the described method preparation of 2.3 joint in the article (L.Jiangetal.Biomaterials52 (2015) 126-139) of background technology.
Get the crosslinked Mitochondrially targeted liposome and each 0.5ml of DKD liposome that carry paclitaxel, mix with equivalent Ox blood serum respectively, often kind of liposome repeats one group, totally four groups, being respectively charged into molecular cut off is again in the bag filter of 6000 ~ 7000, the bag filter and the bag filter that DKD liposome is housed that one are equipped with crosslinked Mitochondrially targeted liposome immerse 50ml respectively containing in the phosphate buffer of 0.1% Tween 80, two remaining bag filters immerse 50ml respectively and contain in the phosphate buffer of 10mmoL/L glutathion (GSH) and 0.1% Tween 80, 37 DEG C of constant temperature oscillations.After 0.5,1,2,4,6,12,18,24 hour, get dialysis medium 1ml, supplement the fresh release medium of 1ml simultaneously.According to the drug level in the described high effective liquid chromatography for measuring drug release medium of 2.14 joint in background technology article (L.Jiangetal.Biomaterials52 (2015) 126-139), calculate medicine Cumulative release amount, the results are shown in Figure 4.
As can be seen from Figure 4, carry the crosslinked Mitochondrially targeted liposome of paclitaxel more stable in serum, drug release is slow, adds the disulfide bonds of liposome after GSH, after cross-linked structure destruction, and faster drug release.And DKD liposome stability in serum is poorer, drug release is very fast, and does not affect by GSH concentration.
2, oncotherapy Contrast on effect experiment
The crosslinked Mitochondrially targeted liposome of paclitaxel is carried according to the method preparation of embodiment 6.
Paclitaxel DKD liposome is carried according to the described method preparation of 2.3 joint in the article (L.Jiangetal.Biomaterials52 (2015) 126-139) in background technology.
According to 2.12 in-vivo tumour Experiment on therapy methods in article (L.Jiangetal.Biomaterials52 (2015) 126-139), anti-tumor in vivo effect assessment is carried out to two kinds of drug-loaded liposomes of preparation, the results are shown in Figure 5.
As can be seen from Figure 5, the crosslinked Mitochondrially targeted lipid physical ability adopting crosslinkable of the present invention Mitochondrially targeted pegylated phospholipids medicinal materials to prepare obviously reduces gross tumor volume, at the 25th day part tumor bearing nude mice tumor disappearance, and the gross tumor volume of the tumor bearing nude mice of the DKD liposome of injection year paclitaxel is 1.4 times before treatment, gross tumor volume has slight increase.The crosslinked Mitochondrially targeted liposome carrying paclitaxel prepared by visible employing crosslinkable of the present invention Mitochondrially targeted pegylated phospholipids medicinal materials has better oncotherapy effect.
3, tumor tissues targeting compares
According to the method in embodiment 6, paclitaxel is replaced by DiR, the crosslinked Mitochondrially targeted fluorescent lipid of preparation.
According to the 127th page of 2.3 described method of joint of the article (L.Jiangetal.Biomaterials52 (2015) 126-139) in background technology, fluorescence molecule DiL is replaced by DiR and prepares DKD fluorescent lipid.
To 18 ~ 20gBALB/c nude mice by subcutaneous inoculation 1 × 10
6individual drug resistance human lung carcinoma cell, after inoculating 2 weeks, is divided into two groups by nude mice, often organize 3, each group Mitochondrially targeted fluorescent lipid of tail vein injection and DKD fluorescent lipid respectively.After 6 hours, each group nude mice abdominal cavity injection chloral hydrate injection, treat that nude mice anesthesia is placed in small animal living body imager and observe the distribution situation of fluorescence molecule DiR in Mice Body, result as shown in Figure 6.
As can be seen from Figure 6, the tumor locus fluorescence intensity of the nude mice of the crosslinked Mitochondrially targeted fluorescent lipid of injection is higher, illustrates that it has stronger tumor tissues targeting.
4, cellular uptake amount compares
According to the method in embodiment 6, paclitaxel is replaced by DiL, the crosslinked Mitochondrially targeted fluorescent lipid of preparation.
DKD fluorescent lipid is prepared according to the 127th page of 2.3 described method of joint of the article (L.Jiangetal.Biomaterials52 (2015) 126-139) in background technology.
Detect the picked-up situation of drug resistance lung cell A549 to two kinds of liposomees of preparation according to the 128th page of 2.7 described cellular uptake experimental technique of joint in the article (L.Jiangetal.Biomaterials52 (2015) 126-139) in background technology, the results are shown in Figure 7.
As can be seen from Figure 7, the intake of drug resistance lung cell A549 to the crosslinked Mitochondrially targeted fluorescent lipid adopting crosslinkable of the present invention Mitochondrially targeted pegylated phospholipids medicinal materials to prepare is significantly higher than DKD fluorescent lipid.
5, mitochondrion intake contrast experiment
According to the method in embodiment 6, paclitaxel is replaced by DiL, the crosslinked Mitochondrially targeted fluorescent lipid of preparation.
DKD fluorescent lipid is prepared according to the 127th page of 2.3 described method of joint of the article (L.Jiangetal.Biomaterials52 (2015) 126-139) in background technology.
Save described mitochondrion picked-up experimental technique according to the 128th page of 2.10.2 in the article (L.Jiangetal.Biomaterials52 (2015) 126-139) in background technology and detect the drug resistance lung cell A549 mitochondrion of separation to the picked-up situation of two kinds of liposomees of preparation, the results are shown in Figure 8.
As can be seen from Figure 8, be separated the intake of drug resistance lung cell A549 mitochondrion to the crosslinked Mitochondrially targeted fluorescent lipid adopting crosslinkable of the present invention Mitochondrially targeted pegylated phospholipids medicinal materials to prepare and be significantly higher than DKD fluorescent lipid.
6, stability compares
The crosslinked Mitochondrially targeted liposome of paclitaxel is carried according to the method preparation in embodiment 6.
According to the DKD liposome of the 127th page of 2.3 described method preparation of joint year paclitaxel in the article (L.Jiangetal.Biomaterials52 (2015) 126-139) in background technology.
Two kinds of drug-loaded liposomes of preparation are left standstill storage 2 weeks in 4 DEG C of cold preservations, respectively at before storage, store and within 1 week, 2 weeks, get two kinds of liposome solutions and adopt dynamic laser light scattering experimental instrument detection liposomal particle size to change.Testing result is in table 1.
As known from Table 1, carry paclitaxel DKD liposomal particle size and become very large, the particle diameter carrying the crosslinked Mitochondrially targeted liposome of paclitaxel adopting crosslinkable of the present invention Mitochondrially targeted pegylated phospholipids medicinal materials to prepare is almost unchanged, thus there is better stability, be beneficial to long-term storage.
Claims (7)
1. the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable, is characterized in that the structural formula of this medicinal materials is as follows:
in structure above, n=10,12,14,16.
2. the preparation method of the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable described in claim 1, is characterized in that with fatty acid acyl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol-maleimide, polypeptide D-(KLAKLAK)
2-C
5be raw material with organic base, polypeptide D-(KLAKLAK)
2-C
5, organic base, fatty acid acyl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol-maleimide mol ratio be (1 ~ 3): (1 ~ 3): 1, described polypeptide D-(KLAKLAK)
2-C
5aminoacid sequence for described in SEQ ID NO:1, processing step is as follows:
Under nitrogen protection, fatty acid acyl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol-maleimide, organic base and the first solvent mix homogeneously are obtained the first solution, and the consumption of described first solvent is make the concentration of fatty acid acyl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol-maleimide in the first solution be 100 ~ 300mmol/L;
Under nitrogen protection, by polypeptide D-(KLAKLAK)
2-C
5be dissolved in the second solvent and obtain the second solution, the consumption of the second solvent is for making polypeptide D-(KLAKLAK) in the second solution
2-C
5concentration be 100 ~ 900mol/L;
By the first solution and the mixing of the second solution, in 20 ~ 40 DEG C of reactions 12 ~ 48 hours under lucifuge, stirring, except desolventizing obtains solid matter after reaction terminates, insoluble matter is filtered out after gained solid matter chloroform or dimethyl sulfoxine being dissolved, collect filtrate, namely the solvent in removing filtrate obtains the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable.
3. the preparation method of the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable according to claim 2, is characterized in that described fatty acid acyl PHOSPHATIDYL ETHANOLAMINE-Polyethylene Glycol-maleimide is the one in PEG-DSPE 2000-maleic amide, DMPEA-Macrogol 2000-maleic amide, PEG2000-DSPE-maleic amide, two lauroyl PHOSPHATIDYL ETHANOLAMINE-Macrogol 2000-maleic amides.
4. the preparation method of the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable according to Claims 2 or 3, is characterized in that described organic base is the one in triethylamine, DMAP, DIPEA.
5. the preparation method of the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable according to Claims 2 or 3, it is characterized in that described first solvent is dichloromethane or chloroform, second solvent is the one in methanol, DMF, dimethyl sulfoxine.
6. the preparation method of the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable according to claim 4, it is characterized in that described first solvent is dichloromethane or chloroform, the second solvent is the one in methanol, DMF, dimethyl sulfoxine.
7. the Mitochondrially targeted pegylated phospholipids medicinal materials of crosslinkable described in claim 1 is preparing the application in drug-loaded liposome.
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Cited By (3)
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| CN111643453A (en) * | 2020-05-27 | 2020-09-11 | 四川大学华西医院 | Medicinal preparation and preparation method and application thereof |
| CN111793200A (en) * | 2020-06-19 | 2020-10-20 | 上海易全化学有限公司 | Preparation method of recessive immunoliposome modified phospholipid raw material |
| CN114989415A (en) * | 2022-08-04 | 2022-09-02 | 北京鑫开元医药科技有限公司 | Active targeting phospholipid and its synthesis process |
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| CN103371975A (en) * | 2012-04-16 | 2013-10-30 | 上海现代药物制剂工程研究中心有限公司 | Targeted long-circulation liposome preparation and preparation method thereof |
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| CN103371975A (en) * | 2012-04-16 | 2013-10-30 | 上海现代药物制剂工程研究中心有限公司 | Targeted long-circulation liposome preparation and preparation method thereof |
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| LEI JIANG ET AL.: "Overcoming drug-resistant lung cancer by paclitaxel loaded dual-functional liposomes with mitochondria targeting and pH-response", 《BIOMATERIALS》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111643453A (en) * | 2020-05-27 | 2020-09-11 | 四川大学华西医院 | Medicinal preparation and preparation method and application thereof |
| CN111793200A (en) * | 2020-06-19 | 2020-10-20 | 上海易全化学有限公司 | Preparation method of recessive immunoliposome modified phospholipid raw material |
| CN111793200B (en) * | 2020-06-19 | 2023-05-26 | 上海易全化学有限公司 | Preparation method of recessive immunoliposome modified phospholipid raw material |
| CN114989415A (en) * | 2022-08-04 | 2022-09-02 | 北京鑫开元医药科技有限公司 | Active targeting phospholipid and its synthesis process |
| CN114989415B (en) * | 2022-08-04 | 2022-10-21 | 北京鑫开元医药科技有限公司 | Active targeting phospholipid and its synthesis process |
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