CN102977360B - Triphenylphosphine-polyethyleneglycol 1000 vitamin E succinate (TPGS 1000-TPP) conjugated compound, and preparation method and application thereof - Google Patents
Triphenylphosphine-polyethyleneglycol 1000 vitamin E succinate (TPGS 1000-TPP) conjugated compound, and preparation method and application thereof Download PDFInfo
- Publication number
- CN102977360B CN102977360B CN201210521312.8A CN201210521312A CN102977360B CN 102977360 B CN102977360 B CN 102977360B CN 201210521312 A CN201210521312 A CN 201210521312A CN 102977360 B CN102977360 B CN 102977360B
- Authority
- CN
- China
- Prior art keywords
- formula
- cell
- liposome
- preparation
- conjugated compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a triphenylphosphine-polyethyleneglycol 1000 vitamin E succinate (TPGS 1000-TPP) conjugated compound, and a preparation method and application thereof. The structural formula of the compound is disclosed as Formula I. The preparation method of the conjugated compound comprises the following steps: (1) reacting triphenylphosphine and 6-bromocaproic acid to obtain 5-carboxyamyltriphenylphosphonium bromide disclosed as Formula IV; and (2) carrying out esterification reaction on the 5-carboxyamyltriphenylphosphonium bromide disclosed as Formula IV and polyethyleneglycol 1000 vitamin E succinate to obtain the conjugated compound disclosed as Formula I. The invention also discloses a mitochondrion targeted taxol liposome comprising the conjugated compound. The drug effect test proves that the mitochondrion targeted taxol liposome has strong cell toxicant action in the in-vitro cell experiment and in-vivo transplantation tumor model of human lung adenocarcinoma A549 cells and drug-resistant A549/cDDP cells thereof and can enhance the antitumor effect of taxol on drug-resistant A549/cDDP cells.
Description
Technical field
The present invention relates to a kind of triphenylphosphine-polyethylene glycol 1000 vitamin E succinic acid ester (TPGS
1000-TPP) conjugated compound and preparation method thereof and application, be specifically related to this conjugated compound in the application building in Paclitaxel liposome.
Background technology
The medicament transport system that functional material is modified provides a kind of potential strategy for intractable cancer.Tumour intractable except relating to patient's physiological pathology state, also relevant with medicament transport.Chemotherapeutic is carried out administration with the form of injection free drug conventionally, and this administering mode can cause medicine at tumor focus position accumulation volume less and pharmacy in the non-targeted distribution of body tissue's organ.
In addition, the multidrug resistance of tumour is the major cause of chemotherapy failure, can cause resistance, recurrence and the transfer of tumour cell to treatment, especially endogenic multidrug resistance.Tumour cell can cause the change of heredity and epigenetic by the function of the protein of apoptosis and apoptogene coding before changing (as the Caspase albumen in Bcl-2 family protein, apoptotic signal path etc.), thereby causes endogenic multidrug resistance.
The chemotherapy of cancer mainly takes two kinds of basic modes to eliminate tumour cell: the one, and by being directly acted on to tumour cell, chemotherapeutic killed, for example, by causing DNA structure damage cell killing; The 2nd, the suicide of inducing cell, i.e. apoptosis.Apoptosis is a kind of a kind of apoptosis occurring by the regulation and control of gene in cell and product thereof.These two kinds of modes are all relevant with plastosome.
The endogenous path of apoptosis originates in plastosome, can be mediated by three kinds of factors: the Caspase direct activation factor, as cytochrome C (cytochrome C), the Caspase indirect activation factor, as Smac/Diablo, and Caspase dependent/non-dependent programmed cell death factor of influence, as AIF.Cancer therapy drug is by crosslinked with the part of death receptor, thus the Caspase of activation upstream, and by triggering various forms of cellular stress, the release apoptosis factor.
Plastosome is the organoid of the membrane closure that all exists in a kind of most eukaryotic cell, is the energy factory of cell.Plastosome, except supplying cellular energy, is also the regulation and control center of cell fate, participates in cytodifferentiation, necrocytosis, is controlling cycle and the growth of cell.Due to its importance in process of cell death, plastosome is considered to a kind of Effective target site of tumor-targeting medicament transport.
Triphenylphosphine is a kind of have crossover track Mitochondria Membrane positive small molecules in plastosome inner accumulation function.Triphenylphosphine contains three benzene ring structures, has caused the electron delocalization effect of positive charge in three phenyl ring groups on high hydrophobicity and phosphorus atom.The charge distribution of this molecular surface has been in the news and can have reduced the required energy of triphenylphosphine leap lipid film.
Taxol (paclitaxel) is from Chinese yew genus plants, to extract and a kind of crude substance with highly effective antineoplastic activity of obtaining of purifying, is had significant curative effect to permitting eurypalynous cancer.But, water-soluble low due to taxol, the curative effect of its free medicine in oncotherapy is greatly diminished.
Summary of the invention
The object of this invention is to provide a kind of triphenylphosphine-polyethylene glycol 1000 vitamin E succinic acid ester (TPGS
1000-TPP) conjugated compound and preparation method thereof and application.
Triphenylphosphine-polyethylene glycol 1000 vitamin E succinic acid ester (TPGS provided by the invention
1000-TPP) conjugated compound, its structural formula is suc as formula shown in I,
Formula I
In formula I, n is 23.
The preparation method who the invention provides conjugated compound shown in formula I, comprises the steps:
(1) triphenylphosphine reacts with 6-bromocaproic acid and obtains the amyl group of 5-carboxylic shown in formula IV triphenyl bromide phosphine;
Formula IV
The structural formula of triphenylphosphine is suc as formula shown in II, and the structural formula of 6-bromocaproic acid is suc as formula shown in III;
Formula II formula III
(2) the amyl group triphenyl bromide phosphine of 5-carboxylic shown in formula IV and polyethylene glycol 1000 vitamin E succinic acid ester carry out esterification and obtain conjugated compound shown in formula I;
Wherein, the structural formula of polyethylene glycol 1000 vitamin E succinic acid ester is suc as formula shown in V, and in formula, n is 23,
In above-mentioned preparation method, described in step (1), the temperature of reaction can be 80 ~ 85 DEG C, and as 81 DEG C, the time can be 14 ~ 17 hours, and as 16 hours, solvent can be acetonitrile.
Described in step (2), the temperature of esterification can be 20 ~ 25 DEG C, and as 20 DEG C, the time can be 18 ~ 24 hours, and as 18 hours, solvent can be DMSO or methylene dichloride.
In above-mentioned preparation method, described in step (2), the condensing agent of esterification can be dicyclohexylcarbodiimide, and catalyzer can be DMAP.
A further object of the present invention is to provide a kind of Mitochondrially targeted property Paclitaxel liposome and preparation method thereof.
Mitochondrially targeted property Paclitaxel liposome provided by the present invention, it is made up of taxol, fat material and targeting material;
Described fat material is made up of Ovum Gallus domesticus Flavus lecithin and cholesterol;
Described targeting material is conjugated compound shown in formula I;
Described Ovum Gallus domesticus Flavus lecithin, cholesterol are (85-90) with the molfraction ratio of targeting material: (2.5-10): (3-8.5), as 88:3.5:8.5, the ratio of quality and the number of copies of described taxol and fat material is 1:(20 ~ 40), as 1:35.
The preparation method of Mitochondrially targeted property Paclitaxel liposome provided by the present invention, comprises the steps:
Described taxol, fat material and targeting material are dissolved in organic solvent, steam except after described organic solvent and obtain adipose membrane;
Described adipose membrane is obtained to suspension by PBS damping fluid aquation, described in described suspension is obtained by filtering with microporous membrane after homogeneous processing, state Mitochondrially targeted property Paclitaxel liposome again.
In above-mentioned preparation method, described organic solvent can be the mixture (both can be 1:3 at volume ratio) of methyl alcohol or methyl alcohol and chloroform; Can be by the ultrasonic or homogeneous processing of high pressure inlet wire.
Another object of the present invention is to provide the application of above-mentioned Mitochondrially targeted property Paclitaxel liposome.
Mitochondrially targeted property Paclitaxel liposome provided by the invention can be used for preparing the medicine that prevents and/or treats tumour, and described tumour can be cancer, and described cancer is preferably lung cancer, more preferably endogenous resistance lung cancer.
Mitochondrially targeted property Paclitaxel liposome provided by the invention can be used for preparing the inhibitor of eukaryote tumor cell proliferation, and described eukaryote can be Mammals; Described tumour cell can be cancer cells; Described cancer cells can be lung carcinoma cell, preferably human lung adenocarcinoma A549 cell or multidrug resistance lung cancer A549/cDDP cell.
The present invention has synthesized a kind of triphenylphosphine-polyethylene glycol 1000 vitamin E succinic acid ester (TPGS
1000-TPP) conjugated compound, and set it as plastosome guidance quality molecular modification and overcome to Mitochondrially targeted property taxusol-lipid surface the resistance of tumour cell.Utilize human lung adenocarcinoma A549 cell and resistance A549/cDDP cell thereof, A549 and A549/cDDP tumour ball, resistance A549/cDDP cell Nude Mouse Model to carry out effect experiment.Mitochondrially targeted property Paclitaxel liposome particle diameter is about 80nm.Mitochondrially targeted property liposome has significantly improved the picked-up of cell to medicine, and being accumulated in plastosome drug selectivity.Mitochondrially targeted property Paclitaxel liposome can and activate Caspase 3 and induced expression sensitivity and the resistance cancer cell-apoptosis of Caspase 9 and adjusting Bcl-2 protein family by induction plastosome release cells pigment C.Mitochondrially targeted property Paclitaxel liposome all has very strong cytotoxicity in transplanted tumor model in the In vitro cell experiment of two kinds of clones and medicine-resistant cell line body.
Brief description of the drawings
Fig. 1 is TPGS of the present invention
1000the synthetic route of-TPP conjugated compound.
Fig. 2 is TPGS
1000the TPGS preparing with embodiment 1
1000the MALDI-TOF-MS collection of illustrative plates of-TPP, wherein, Fig. 2 (A) and Fig. 2 (B) are respectively TPGS
1000and TPGS
1000-TPP collection of illustrative plates.
Fig. 3 is 5-carboxylic amyl group triphenyl bromide phosphine, TPGS
1000the TPGS preparing with embodiment 1
1000-TPP's
1hNMR collection of illustrative plates; Proton peak shown in shade is the signal of the indicated proton (or being connected in the proton on the carbon atom shown in arrow) of the arrow of same color, is respectively fragrant hydrogen and TPGS on TPP molecule
1000polyoxyethylene glycol on-TPP molecule.
Fig. 4 is Electronic Speculum transmission electron microscope photo and the atomic force microscopy of Paclitaxel liposome, and wherein, Fig. 4 (A) and Fig. 4 (B) are respectively Electronic Speculum transmission electron microscope photo and atomic force microscopy.
Fig. 5 is the Cumulative release amount (%) of Paclitaxel liposome Chinese traditional medicine, and release medium is the pH7.4PBS solution that contains 10% foetal calf serum.
Fig. 6 is the survival rate (%) of 48hA549 cell and A549/cDDP cell after administration, and wherein, Fig. 6 (A) and Fig. 6 (B) are respectively the survival rate of A549 cell and A549/cDDP cell.
Fig. 7 is the apoptosis induction rate (%) of 24hA549 cell and A549/cDDP cell after administration.
Fig. 8 is the result that rear cellular uptake and plastosome picked-up are processed in A549 cell and the administration of A549/cDDP cell, wherein Fig. 8 (A) and Fig. 8 (B) are respectively the result of cellular uptake and plastosome picked-up, the medicament contg of cells were tested by flow cytometry for the representative of the fluorescence intensity of ordinate zou in figure.
Fig. 9 is the common location of A549 cell (Fig. 9 (A)) and A549/cDDP cell (Fig. 9 (B)) Chinese traditional medicine and cell Mitochondria, wherein the red fluorescent probe mark of plastosome for the plastosome of cell; And the plastosome fragment Chinese traditional medicine of A549 cell (Fig. 9 (C)) and A549/cDDP cell (Fig. 9 (D)) extraction and mitochondrial location altogether.
Figure 10 is the impact that each formulation for paclitaxel group discharges A549/cDDP cell pigment C.
Figure 11 is the body weight change (Figure 11 (B)) of tumor bearing nude mice during the result for the treatment of to transplanted tumor model (Figure 11 (A)) and administration after the administration of each formulation for paclitaxel group.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Embodiment material used is as follows:
Triphenylphosphine (TPP) is purchased from J & K company, and lot identification mark is 110246.
6-bromocaproic acid is purchased from Sigma company of the U.S., and product article No. is 150452
Dicyclohexylcarbodiimide (DCC) is purchased from Sigma company of the U.S., and product article No. is D80002.
DMAP (DMAP) is purchased from Sigma company of the U.S., and product article No. is 522805.
Taxol dry powder is purchased from Nanjing celestial worthy Ze Zhong chemical reagents corporation, and lot identification mark is 080315.
PTX injection is purchased from Haikou City Pharmaceutical Factory, and authentication code is the accurate word H20043045 of traditional Chinese medicines.
Ovum Gallus domesticus Flavus lecithin (EPC) is purchased from NOF Corp, and catalog number is 108057-3.
Cholesterol is purchased from Haidian District, Beijing City microbiological culture media products factory, lot number 20020106.
The molecular formula of taxol is C
47h
51nO
14, molecular structural formula:
Synthetic route chart as shown in Figure 1.
5mmol TPP and 5.25mmol 6-bromocaproic acid are mixed in 5ml anhydrous acetonitrile to reflux 16 hours under nitrogen protection.Mix products recrystallization is obtained to white solid powder 5-carboxylic amyl group triphenyl bromide phosphine.
TPGS1000-TPP is by carboxyl and TPGS on 5-carboxylic amyl group triphenyl bromide phosphine
1000on hydroxyl by esterification condensation, with DCC as reaction condensing agent.By 0.33mmol 5-carboxylic amyl group triphenyl bromide phosphine, 0.066mmolTPGS
1000, 0.40mmol DCC and 0.006mmol DMAP be dissolved in 3ml DMSO solution; by reaction solution mixture under room temperature nitrogen protection condition; stir gently 18h with magnetic stirring apparatus and obtain crude product; then by filtration of crude product; filtrate is transferred to regenerated cellulose dialysis tubing (molecular weight cut-off 1000); the 24h that dialyses in DMSO solution, then the 24h that dialyses in deionized water, remove unreacted 5-carboxylic amyl group triphenyl bromide phosphine, DMAP, DCC and by product DCU.Next by reaction solution freeze-drying, obtain dry product mixtures white powder, and use proton nmr spectra (
1h NMR), laser desorption ionisation flight time mass spectrum (MALDI-TOF-MS) detects.
Fig. 2 is TPGS
1000and TPGS
1000the MALDI-TOF-MS collection of illustrative plates of-TPP.In collection of illustrative plates, show, in reaction product, the average molecular mass of TPGS1000-TPP is 1902.9(Fig. 2 (B)); TPGS
1000signal in mass spectrum shows as and adds He JiaK+ peak, Na+ peak, the peak value of average molecular mass be respectively its molecular mass be 1565.8 and 1581.8(Fig. 2 (A)), TPGS
1000-TPP and TPGS
1000between of poor quality and molecular mass 5-carboxylic amyl group triphenyl bromide phosphine match.
Fig. 3 is 5-carboxylic amyl group triphenyl bromide phosphine, TPGS
1000and TPGS
1000-TPP molecule
1h NMR spectrogram.As shown in Figure 5,5-carboxylic amyl group triphenyl bromide phosphine and TPGS
1000the characteristic peak of molecule all can be at reaction product TPGS
1000-TPP's
1in H NMR spectrogram, find.After esterification, TPGS
1000-TPP molecule has retained the fragrant hydrogen signal on TPP (7.70 ~ 7.80ppm uses arrow mark).
The preparation of embodiment 2, liposome and sign
1) preparation of liposome:
By film dispersion method and pushed film preparation targeting Paclitaxel liposome, in addition, not the targeted hposome of medicine carrying, common Paclitaxel liposome, common and targeting tonka bean camphor liposome respectively in contrast, cellulotoxic preparation and fluorescent probe.
Liposome is by fat material (Ovum Gallus domesticus Flavus lecithin (EPC) and cholesterol) and targeting material TPGS
1000-TPP forms, and three's mol ratio is 88:3.5:8.5.Fat material and taxol ratio are fat material: taxol=35:1(mass/mass), fat material, targeting material and taxol are placed in to eggplant-shape bottle with after dissolve with methanol, rotary evaporation is removed methyl alcohol, PBS damping fluid aquation for adipose membrane, Probe Ultrasonic Searching 10 minutes (110W), the extruding polycarbonate membrane by 400nm and 200nm three times respectively, makes targeting Paclitaxel liposome.
Common Paclitaxel liposome is prepared by same procedure, the TPGS in liposome
1000-TPP TPGS
1000replace.The preparation method of targeting tonka bean camphor liposome and tonka bean camphor liposome is identical with aforesaid method, and wherein the ratio of fat material and tonka bean camphor is 500:1(mass/mass).
2) sign of liposome:
Targeting Paclitaxel liposome, common Paclitaxel liposome, targeting tonka bean camphor liposome and the common tonka bean camphor liposome of preparation are crossed respectively to Sephadex G-50 post, the medicine of separated free, moving phase is PBS damping fluid.The liposome suspension of crossing post front and back destroys with 9 times of methyl alcohol respectively, the high-performance liquid chromatogram determination encapsulation rate of UV-detector for taxol, tonka bean camphor fluorimetric detector.The condition determination of taxol: (5.0 μ m) for Nucleodur 100-5C18column, 250 × 4.6mm for ODS C18 post; 25 ° of C; Detect wavelength 227nm; Flow velocity 1.0ml/min; Moving phase acetonitrile/water (60/40, volume ratio).The testing conditions of tonka bean camphor: (5.0 μ m) for Nucleodur 100-5C18column, 250 × 4.6mm for ODS C18 post; 25 ° of C; Excite with emission wavelength and be respectively 467nm and 502nm; Flow velocity is 1.0ml/min; Mobile phase methanol/water (95/5, volume ratio).The encapsulation rate (EE) of taxol, tonka bean camphor calculates as follows: EE=(W
i/ W
total) × 100%, wherein, Wi was the quality of the lipidosome Chinese traditional medicine thing that destroyed by methyl alcohol after Sephadex G-50 post.W
totalthe cross drug quality in the liposome that through methyl alcohol destroy post before identical with the liposome volume of crossing post.
The particle diameter of liposome and Zeta potential are measured with Nano Series Zen 4003Zeta Sizer (Malvern instruments, Ltd, UK).
Surface of liposome and form are used transmission electron microscope (TEM, Tecnai G220ST, FEI Co., Japan) and atomic force microscope (AFM, SPI3800N series SPA-40, Japan) to observe.
External liposome release experiment is carried out in the release liquid containing serum (containing the PBS of the PH7.4 of 10% foetal calf serum).1ml liposome and 1ml deionized water are sealed in dialysis tubing, and dialysis tubing is immersed in 40ml and discharges in liquid, is placed in immediately vibrator, 37 ° of C, sustained oscillation 24 hours under the condition of 100 beats/min.Get respectively 0.5ml at 0.25,0.5,1,2,4,6,8,12 and 24 hour and discharge liquid, and add the fresh release liquid of same volume at once.Discharge the measuring method of taxol concentration in liquid with the mensuration of encapsulation rate.The calculation formula of release rate (RR, %): RR=(W
i/ W
total) × 100%, wherein W
ithe quality of taxol in the release liquid taking out at i time point, W
totalit is the quality with the identical liposome taxol before dialysis of volume of dialysing.Every experiment is in triplicate parallel.
3) result
The sign of table 1 liposome
Table 1 is depicted as medicine carrying conventional liposome not, not encapsulation rate, median size, polydispersity index and the Zeta potential of particle diameter of medicine carrying targeted hposome, common Paclitaxel liposome, targeting Paclitaxel liposome, common tonka bean camphor liposome and targeting tonka bean camphor liposome.Result demonstration, the median size of above-mentioned liposome is 80nm left and right, distribution homogeneous.The encapsulation rate of taxol in targeted hposome and conventional liposome is all greater than 85%, and tonka bean camphor encapsulation rate in two kinds of liposomes is all greater than 90%.Except the slight positive polarity of targeting Paclitaxel liposome band, other all liposomes are all with slight electronegativity.
Fig. 4 (A) is the images of transmissive electron microscope of two kinds of Paclitaxel liposomes (common Paclitaxel liposome and targeting Paclitaxel liposome), 4(B) be the atomic force microscope images of two kinds of Paclitaxel liposomes (common Paclitaxel liposome and targeting Paclitaxel liposome).As can be seen from the figure, two kinds of Paclitaxel liposome particle diameters are 80 ~ 100nm left and right, conform to particle instrument detected result.Can find out that by transmission electron microscope the common Paclitaxel liposome of targeting taxusol-lipid body surface ratio is finer and close.
Fig. 5 is the release rate of taxol from liposome, and in initial 2h, release rate is all lower than 30%, and at the 24th hour, preparation was all lower than 50%.
The effect experiment of embodiment 3, Mitochondrially targeted property Paclitaxel liposome
(1) cell cultures
RPMI-1640 culture medium culturing containing 10% foetal calf serum and 1% dual anti-(100U/ml penicillin, 100 μ g/ml Streptomycin sulphates) for human lung adenocarcinoma A549 cell (purchased from Tumar Inst of Tumoor Hospital, Chinese Academy of Medical Sciences); Multidrug resistance lung cancer A549/cDDP cell (purchased from Tumar Inst of Tumoor Hospital, Chinese Academy of Medical Sciences) is with containing 10% foetal calf serum and 1% dual anti-(100U/ml penicillin, 100 μ g/ml Streptomycin sulphates) RPMI-1640 culture medium culturing, in order to keep cells resistance, in nutrient solution, add the cis-platinum of final concentration 4 μ M, and do not contain the above-mentioned culture medium culturing of cis-platinum in experiment use the last week.The culture condition of two kinds of cells is as 5%CO
2, 37 ° of C.
(2) cytotoxicity
By A549 cell or A549/cDDP cell with 5.0 × 10
3the concentration in individual/hole is inoculated in 96 porocyte culture plates, at 5%CO
2, 37 ° of C condition under cultivate after 24h, by nutrient solution sucking-off, add respectively the fresh culture of the not medicine carrying targeted hposome, PTX, common Paclitaxel liposome or the targeting Paclitaxel liposome that contain different concns gradient.The final concentration of taxol is 0 ~ 10M, and the concentration of medicine carrying targeted hposome is not identical with drug-loaded liposome concentration.With blank culture medium culturing as a control group.Administration is hatched after 48h, by acetyl rhodamine B staining evaluation cytotoxicity, the absorbing wavelength of microplate reader is 540nm, and inhibiting rate uses the calculating of following formula: inhibiting rate %=100%-(dosing group is the absorbance at 540nm place in absorbance/blank group at 540nm place) × 100%.In triplicate, each concentration is established five multiple holes in each experiment, and acquired results is drawn beneficial effect curve.
Fig. 6 is targeting Paclitaxel liposome and the restraining effect of other control formulation groups to A549 cell and A549/cDDP cell, wherein, significant difference a, p<0.05, vs is medicine carrying targeted hposome not; B, p<0.05, vs PTX; C, p<0.05, the common Paclitaxel liposome of vs, result shows, is 1, when 5and 10 μ M in each administration concentration, targeting Paclitaxel liposome has the strongest inhibition A549 cell and the effect of A549/cDDP cell proliferation.In addition, common Paclitaxel liposome is greater than PTX to the cytotoxicity of A549 cell and A549/cDDP cell.The Mitochondrially targeted property of medicine carrying liposome does not only have certain cytotoxicity for cell in the time that concentration is greater than 1 μ M, and concentration is while being 10 μ M, and the kill rate of cell is not exceeded to 30%.
(3) apoptosis-induced effect
Annexin V-FITC apoptosis probe reagent box (Beijing Bao Sai Bioisystech Co., Ltd, production code member CX1001) dyeing for the apoptosis of cell, flow cytometer detects.By A549 cell or A549/cDDP cell with 4 × 10
5the concentration in individual/hole is inoculated in 6 porocyte culture plates, and nutrient solution volume is 2ml, and at 5%CO
2under the condition of 37 ° of C of at, cultivate 24h.Next, with PTX, common Paclitaxel liposome, targeting Paclitaxel liposome incubated cell 24h, taxol final concentration is 10 μ M, cultivates as a control group containing blood serum medium.Next by cell harvesting, be resuspended in the damping fluid providing in test kit, add 10 μ l Annexin V-FITC probes, mix, under room temperature, lucifuge is hatched 15min, adds afterwards 5 μ l PI, uses immediately flow cytometry analysis, and collecting number is each sample 1 × 10
4individual cell.Each experiment in triplicate.
Fig. 7 is each preparation group induction A549 cell and the apoptotic result of A549/cDDP, wherein, in A549 cell, significant difference a, p<0.05, vs blank; B, p<0.05, vs PTX; C, p<0.05, the common Paclitaxel liposome of vs; In A549/cDDP cell, significant difference d, p<0.05, vs blank; E, p<0.05, vs PTX; F, p<0.05, the common Paclitaxel liposome of vs, result demonstration, compared with PTX and common Paclitaxel liposome, targeting Paclitaxel liposome all has the most significant apoptosis induction effect in two kinds of clones.
(4) cellular uptake
Utilize flow cytometer to measure A549 cell or the picked-up of A549/cDDP cell to medicine.By A549 cell or A549/cDDP cell with 4 × 10
5the concentration in individual/hole is inoculated in 6 porocyte culture plates, and nutrient solution volume is 2ml, and at 5%CO
2, under the condition of 37 ° of C, cultivate 24h.Next, with free tonka bean camphor, common tonka bean camphor liposome and targeting tonka bean camphor liposome incubated cell 0.5h, tonka bean camphor final concentration is 1 μ M, with blank culture medium culturing as a control group.After administration is hatched, cell is collected with 0.25% trypsinase, and washed twice with cold PBS.Cellular uptake amount cells were tested by flow cytometry, collecting number is each sample 1 × 10
4individual cell, fluorescence intensity represents the size of cellular uptake amount.Each experiment in triplicate.
Fig. 8 (A) is the fluorescence intensity result of cellular uptake after A549 cell and the administration of A549/cDDP cell are processed, administration is hatched after 0.5h, blank group, free tonka bean camphor, common tonka bean camphor liposome and the geometric mean fluorescence intensity of targeting tonka bean camphor liposome in A549 cell are respectively 5.59 ± 0.12, 173.38 ± 1.26, 792.72 ± 5.55 and 1273.27 ± 14.42, correspondingly be respectively 6.64 ± 0.08 for A549/cDDP cell, 125.67 ± 6.32, 795.59 ± 13.13 and 1208.27 ± 48.32, wherein, in A549 cell, significant difference a, p<0.05, vs blank, b, p<0.05, the vs tonka bean camphor that dissociates, c, p<0.05, the common tonka bean camphor liposome of vs, in A549/cDDP cell, significant difference d, p<0.05, vs blank, e, p<0.05, the vs tonka bean camphor that dissociates, f, p<0.05, the common tonka bean camphor liposome of vs.
(5) plastosome picked-up
Further drug level in plastosome is carried out quantitatively with flow cytometer.Use dissociate tonka bean camphor, tonka bean camphor liposome and Mitochondrially targeted property tonka bean camphor liposome at 5%CO in A549 cell or A549/cDDP cell
2, 37 ° of C condition under hatch 24h, in A549 cell, tonka bean camphor final concentration is 1.0 μ M, in A549/cDDP cell, final concentration is 2.5 μ M, with the culture medium culturing containing serum as a control group.Next by cell harvesting and with cold PBS rinsing twice, extract plastosome, the specification sheets that its step is extracted test kit (green skies biotechnology research institute, production code member C3601) according to cell mitochondrial operates.Add plastosome to extract in reagent in cell and react 15 minutes, with glass homogenizer stirring, the centrifugal 10min of centrifugal force 600g for the suspension obtaining, collects supernatant, and with the centrifugal 10min of centrifugal force 11000g, its precipitation is cell mitochondrial again.Mitochondrial ingestion of medicines amount cells were tested by flow cytometry, collecting number is each sample 1 × 10
4individual plastosome, fluorescence intensity represents the size of intake.Each experiment in triplicate.
Fig. 8 (B) is the mitochondrial fluorescence intensity of A549 cell and A549/cDDP cell extraction after cells were tested by flow cytometry administration.Administration is hatched after 24h, the geometric mean fluorescence intensity of blank group, free tonka bean camphor group, common tonka bean camphor liposome group and targeting tonka bean camphor liposome group A549 cell is respectively 8.90 ± 1.94,165.79 ± 36.52,121.94 ± 24.18 and 388.09 ± 70.92, correspondingly be respectively 11.49 ± 2.51 for A549/cDDP cell, 311.79 ± 45.68 and 895.04 ± 17.96.The plastosome of targeting tonka bean camphor liposome group has the strongest fluorescence intensity.
(6) Mitochondrially targeted property
A, intracellular plastochondria are located altogether
Utilize the mitochondrial effect of the Mitochondrially targeted property of confocal laser scanning microscope liposome target.By A549 cell or A549/cDDP cell with 1 × 10
5the concentration in individual/hole is inoculated at the bottom of glass in ware, and nutrient solution volume is 2ml, and at 5%CO
2, 37 ° of C condition under cultivate 24h.Next, with free tonka bean camphor, common tonka bean camphor liposome and targeting tonka bean camphor liposome incubated cell 24h, in A549 cell, tonka bean camphor final concentration is 1.0 μ M, and in A549/cDDP cell, final concentration is 2.5 μ M, with containing the culture medium culturing of serum as a control group.Next cell is used to PBS rinsing, with the red fluorescent probe labeled cell of plastosome plastosome, at 5%CO
2, 37 ° of C condition under hatch 30min, the red fluorescent probe concentration of plastosome of A549 cell is 0.25 μ M, the red fluorescent probe concentration of plastosome of A549/cDDP cell is 0.5 μ M, after PBS rinsing, carries out image analysis with laser confocal microscope.
Cell mitochondrial is dyed redness by the red fluorescent probe of plastosome, green tonka bean camphor is as the fluorescent probe of each preparation group, show its Subcellular Localization, Fig. 9 (A) (A549 cell) and Fig. 9 (B) yellow fluorescence in (A549/cDDP) is the combination of green and red fluorescence, represents preparation and mitochondrial common location.As shown in the figure, Mitochondrially targeted property tonka bean camphor liposome has specific distribution, and selectivity concentrates on plastosome, has the most obvious yellow fluorescence.But, in the cell of free tonka bean camphor group and common tonka bean camphor liposome group, do not show yellow fluorescence.
In B, plastosome fragment, locate altogether
The mitochondrial effect that utilizes the further observation line plastochondria of laser confocal microscope targeted hposome target extraction to go out.Fig. 9 (C) and Fig. 9 (D) are respectively the laser co-focusing micro-image of each preparation group in the plastosome being gone out by A549 cell and A549/cDDP cell extraction.As shown in the figure, targeted hposome can be assembled in plastosome inside through mitochondrial membrane, instead of only around plastosome, accumulates or surface adsorption.By contrast, in the plastosome of free tonka bean camphor group and common tonka bean camphor liposome group, there is no to show the yellow fluorescence of location altogether.Its result is with in cell, positioning image is consistent altogether.
(7) induction line grain cylinder cell pigment C release effects
Streptavidin-peroxidase (streptavidin-peroxidase for release of cytochrome C in rear plastosome and tenuigenin is processed in cell administration, SP) ImmunohistochemistryMethods Methods is investigated, adopt SP immunohistochemical methods test kit to test (bio tech ltd of Zhong Shan Golden Bridge, production code member SP9003).By A549/cDDP cell 1 × 10
5the concentration in individual/hole is hatched 24h, and with PTX, common Paclitaxel liposome and targeting Paclitaxel liposome incubated cell 12h, taxol final concentration is 10 μ M, cultivates as a control group containing blood serum medium.After administration finishes, cell is fixed to 10 minutes with 4% paraformaldehyde, after be sequentially added into following reagent: 0.3%Tritonx-100,3%H
2o
2(v/v) and 10% lowlenthal serum.Then add cytochrome C primary antibodie (Kai Ji bio tech ltd, production code member KG22230), 4 ° of C spend the night.Add two anti-and HRP-association reaction liquid to react with it.Hatch, after rinsing, sample utilizes DAB test kit (bio tech ltd of Zhong Shan Golden Bridge, production code member ZLI-9033) to develop the color, observation by light microscope.
The results are shown in Figure 10.A
1, A
2, A
3and A
4represent respectively the rear blank group of A549/cDDP cell administration processing, PTX group, common Paclitaxel liposome group and targeting Paclitaxel liposome group cytochrome C release conditions.In figure, brown area represents the release of cytochrome C.As shown in the figure, after three kinds of Paclitaxel liposomes, tenuigenin inner cell pigment C discharges increase, illustrates that Intramitochondrial cytochrome C has been discharged in tenuigenin by drug-induced.Wherein, targeting Paclitaxel liposome group cytochrome C discharges the most obvious.PTX and common Paclitaxel liposome equally can inducing cell pigment C release, inductive effect is lower than Mitochondrially targeted property Paclitaxel liposome.
(8) Caspase is active detects
Caspase 3 after administration and the activity of Caspase 9 are measured by the protein substrate test kit (Kai Ji bio tech ltd, production code member KGA201, KGA401) that discharges fluorescence after special proteolytic enzyme (being the material in test kit) cracking.A549/cDDP cell is hatched to 24h, and with PTX, common Paclitaxel liposome and targeting Paclitaxel liposome incubated cell 12h, taxol final concentration is 10 μ M, cultivates as a control group containing blood serum medium.By cell harvesting, cracking, cell pyrolysis liquid is 10000 revs/min of centrifugal 1min under 4 ° of C, retain supernatant liquor, hatch with the specific substrate of Caspase 3 and Caspase 9 respectively, measure its absorbance under 405nm by microplate reader again, be the concentration of Caspase 3 and Caspase 9, and calculate its active ratio by method shown in test kit.Each experiment in triplicate.
The results are shown in Table 2.In table 2, the capable and B of A is capable is respectively after administration caspase 3 and the activity of caspase 9 and the ratio of blank group in A549/cDDP cell.For A549/cDDP cell, give after PTX, common Paclitaxel liposome and targeting Paclitaxel liposome, in cell the specific activity of caspase 3 be respectively 1.00 ± 0.02,1.32 ± 0.30,1.37 ± 0.40 and the specific activity of 2.48 ± 0.85, caspase 9 be respectively 1.00 ± 0.03,1.19 ± 0.07,1.16 ± 0.11 and 2.51 ± 0.64.From result, targeting Paclitaxel liposome has the effect of inducing the most significantly caspase 9 and caspase 3 to activate.
(9) expression of Bcl-2 apoptotic proteins family
Bax, Bid, Bcl-2 and Bcl-xl protein-active adopt the detection of elisa test kit, and (Bax and Bcl-2 test kit are purchased from Wuhan magnificent biotechnology company; Bid & Bcl-xl test kit is purchased from Beijing agency of R & D company of the U.S.).A549/cDDP cell is hatched to 24h, and with PTX, common Paclitaxel liposome and targeting Paclitaxel liposome incubated cell 24h, taxol final concentration is 10 μ M, cultivates as a control group containing blood serum medium.By cell harvesting, cracking, antibody incubation 30 minutes in use test kit.After rinsing with damping fluid (providing in test kit), add HRP-association reaction liquid to hatch 30 minutes.Develop the color according to specification sheets in test kit, then measure its absorbance under 405nm by microplate reader, be the concentration of Bcl-2 apoptotic proteins, then calculate its active ratio.
The results are shown in Table 2.In table 2, C walks to that F is capable is respectively after administration Bax, Bid, Bcl-2 and the activity of Bcl-xl albumen and the ratio of blank group in A549/cDDP cell.For A549/cDDP cell, give after PTX, common Paclitaxel liposome and targeting Paclitaxel liposome, in cell, the specific activity of Bax is respectively 1.00 ± 0.08,1.12 ± 0.36,0.64 ± 0.28 and 1.42 ± 0.23, the specific activity of Bid is respectively 1.00 ± 0.02,0.96 ± 0.09,1.17 ± 0.27 and 1.37 ± 0.39, the specific activity of Bcl-2 be respectively 1.00 ± 0.08,0.26 ± 0.09,0.21 ± 0.09 and the specific activity of 0.23 ± 0.12, Bcl-xl be respectively 1.00 ± 0.02,0.99 ± 0.12,0.99 ± 0.13 and 0.78 ± 0.20.
(10) anti-drug resistance lung cancer transplanted tumor effect in body
Female BALB/c nude mice (body weight 18-20g) is purchased from Department Of Medicine, Peking University's Experimental Animal Center, and animal is raised under aseptic condition, under aseptic condition, operates.By 1 × 10
7a549/cDDP cell is suspended in 200 μ l serum free mediums, is subcutaneously injected into nude mice oxter.Observe the oxter tumour growing state of nude mice every day, and record the volume of tumour.When gross tumor volume reaches 210 ~ 230mm
3time, nude mice is divided into 4 groups at random, 5 every group are carried out drug treatment.At the 15th, 18,20,22 and 24 days that give after nude inoculation A549/cDDP cell, respectively by tail vein injection saline, PTX, common Paclitaxel liposome and targeting Paclitaxel liposome, wherein dose of paclitaxel is 10mg/kg, and physiological saline group is blank group.Measure the size of tumour every day.Formula calculating below for gross tumor volume (V): V=is long × wide
2/ 2(mm
3).The tumor bearing nude mice of each treatment group is put to death for the 26th day after tumor inoculation afterwards, and the length of measurement tumour and wide, adopts following formula to calculate the tumor control rate after administration: tumor control rate (%)=100%-(administration group gross tumor volume/blank group gross tumor volume) × 100%.
Anti-drug resistance lung cancer A549/cDDP transplanted tumor effect in the body that Figure 11 (A) is medicine, significant difference a, p<0.05, vs physiological saline group; B, p<0.05, vs PTX; C, p<0.05, the common Paclitaxel liposome of vs; Result shows, compared with PTX and common Paclitaxel liposome, targeting Paclitaxel liposome has the most significant anti-tumor in vivo effect, and after inoculation the 26th day, gross tumor volume inhibiting rate was respectively 36.35 ± 21.93%, 54.76 ± 13.33% and 73.14 ± 5.89%.During administration, the body weight change of tumor bearing nude mice is as shown in Figure 11 (B).The body weight of three administration group nude mices is all lower than blank group, but there is no significant difference.Illustrate that targeting Paclitaxel liposome may exist certain toxicity being caused by chemotherapeutic taxol, but lower at nude mice toxicity in vivo.
The effect of Caspase 3, Caspase 9, Bax, Bid, Bcl-2 and the Bcl-xl protein-active of the each preparation group of table 2 to A549/cDDP cell
Claims (10)
1. triphenylphosphine-polyethylene glycol 1000 vitamin E succinic acid ester conjugated compound, its structural formula is suc as formula shown in I,
In formula I, n is 23.
2. the preparation method of conjugated compound shown in formula I, comprises the steps:
(1) triphenylphosphine reacts with 6-bromocaproic acid and obtains the amyl group of 5-carboxylic shown in formula IV triphenyl bromide phosphine;
(2) the amyl group triphenyl bromide phosphine of 5-carboxylic shown in formula IV and polyethylene glycol 1000 vitamin E succinic acid ester carry out esterification and obtain conjugated compound shown in formula I;
In formula I, n is 23.
3. preparation method according to claim 2, is characterized in that: described in step (1), the temperature of reaction is 80～85 DEG C, and the time is 14～17 hours, and solvent is acetonitrile;
Described in step (2), the temperature of esterification is 20～25 DEG C, and the time is 18～24 hours, and solvent is DMSO or methylene dichloride.
4. according to the preparation method described in claim 2 or 3, it is characterized in that: described in step (2), the condensing agent of esterification is dicyclohexylcarbodiimide, catalyzer is DMAP.
5. a Mitochondrially targeted property Paclitaxel liposome, it is made up of taxol, fat material and targeting material;
Described fat material is made up of Ovum Gallus domesticus Flavus lecithin and cholesterol;
Described targeting material is conjugated compound shown in formula I;
Described Ovum Gallus domesticus Flavus lecithin, cholesterol are (85-90) with the molfraction ratio of targeting material: (2.5-10): (3-8.5), the ratio of quality and the number of copies of described taxol and fat material is 1:(20-40);
In formula I, n is 23.
6. the preparation method of Mitochondrially targeted property Paclitaxel liposome described in claim 5, comprises the steps:
Described taxol, fat material and targeting material are dissolved in organic solvent, steam except after described organic solvent and obtain adipose membrane;
Described adipose membrane is obtained to suspension by PBS damping fluid aquation, and described suspension obtains described Mitochondrially targeted property Paclitaxel liposome by filtering with microporous membrane again after homogeneous processing.
7. described in claim 5, Mitochondrially targeted property Paclitaxel liposome prevents and/or treats the application in tumour medicine in preparation.
8. application according to claim 7, is characterized in that: described tumour is cancer, and described cancer is lung cancer.
9. the application of Mitochondrially targeted property Paclitaxel liposome in the inhibitor of preparation eukaryote tumor cell proliferation described in claim 5.
10. application according to claim 9, is characterized in that: described eukaryote is Mammals; Described tumour cell is cancer cells; Described cancer cells is lung carcinoma cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210521312.8A CN102977360B (en) | 2012-12-06 | 2012-12-06 | Triphenylphosphine-polyethyleneglycol 1000 vitamin E succinate (TPGS 1000-TPP) conjugated compound, and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210521312.8A CN102977360B (en) | 2012-12-06 | 2012-12-06 | Triphenylphosphine-polyethyleneglycol 1000 vitamin E succinate (TPGS 1000-TPP) conjugated compound, and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102977360A CN102977360A (en) | 2013-03-20 |
| CN102977360B true CN102977360B (en) | 2014-07-02 |
Family
ID=47851728
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210521312.8A Expired - Fee Related CN102977360B (en) | 2012-12-06 | 2012-12-06 | Triphenylphosphine-polyethyleneglycol 1000 vitamin E succinate (TPGS 1000-TPP) conjugated compound, and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102977360B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104398493B (en) * | 2014-12-08 | 2017-06-30 | 中国人民解放军第四军医大学 | A kind of tumor-targeting nanoscale medicine delivery system of reversible tumor drug resistance |
| CN105924492B (en) * | 2016-04-28 | 2017-05-10 | 山东大学 | Mitochondrion-targeted antitumor pentacyclic triterpene derivatives, and preparation method and application thereof |
| CN106344513A (en) * | 2016-10-27 | 2017-01-25 | 烟台大学 | Mitochondrion-targeted micelle material and curcumin micelle preparation prepared from material |
| CN107936058B (en) * | 2017-11-20 | 2020-05-19 | 沈阳药科大学 | Docetaxel derivative and preparation method and application thereof |
| CN108420794B (en) * | 2018-03-12 | 2020-07-28 | 中国药科大学 | Programming targeted liposomal cascade delivery of single drugs for chemical combination therapy |
| CN111603571A (en) * | 2020-05-28 | 2020-09-01 | 中国药科大学 | Mitochondrion targeting lipid-like carrier Mito-Alisposome, compound thereof, preparation method and application |
| CN111920957A (en) * | 2020-08-04 | 2020-11-13 | 河南中医药大学 | Preparation method of mitochondrion targeted tocopherol-oleanolic acid double-drug nanoparticles |
| CN113637049A (en) * | 2021-08-11 | 2021-11-12 | 复旦大学附属中山医院 | Preparation and application of ischemic myocardium targeted mitochondrion targeting peptide compound |
| CN113786386B (en) * | 2021-09-15 | 2023-03-21 | 烟台大学 | ROS (reactive oxygen species) -responsive mitochondrial targeting quercetin liposome as well as preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101032620A (en) * | 2007-03-08 | 2007-09-12 | 上海凯昭医药科技有限公司 | Cyclosporine emulsion and the preparing method |
| CN101084880A (en) * | 2006-06-09 | 2007-12-12 | 中国人民解放军军事医学科学院毒物药物研究所 | Biological solid dispersion of vitamin E esters derivatives and preparation method thereof |
-
2012
- 2012-12-06 CN CN201210521312.8A patent/CN102977360B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101084880A (en) * | 2006-06-09 | 2007-12-12 | 中国人民解放军军事医学科学院毒物药物研究所 | Biological solid dispersion of vitamin E esters derivatives and preparation method thereof |
| CN101032620A (en) * | 2007-03-08 | 2007-09-12 | 上海凯昭医药科技有限公司 | Cyclosporine emulsion and the preparing method |
Non-Patent Citations (4)
| Title |
|---|
| Michael P. Murphy.Targeting lipophilic cations to mitochondria.《Biochimica et Biophysica Acta》.2008,第1777卷第1028-1031页. |
| Targeting lipophilic cations to mitochondria;Michael P. Murphy;《Biochimica et Biophysica Acta》;20080408;第1777卷;第1028-1031页 * |
| 维生素E 唬拍酸聚乙二醇醋在紫杉醇聚酸醉微球中的应用研究;赵亮等;《中国药师》;20071231;第10卷(第5期);第433-435页 * |
| 赵亮等.维生素E 唬拍酸聚乙二醇醋在紫杉醇聚酸醉微球中的应用研究.《中国药师》.2007,第10卷(第5期),第433-435页. |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102977360A (en) | 2013-03-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102977360B (en) | Triphenylphosphine-polyethyleneglycol 1000 vitamin E succinate (TPGS 1000-TPP) conjugated compound, and preparation method and application thereof | |
| CN107812197B (en) | A kind of inflammation targeted neutrophil leucocyte delivery system and its application | |
| CN102757555B (en) | Dequalinium chloride-polyethylene glycol-distearoyl phosphatidyl ethanolamine conjugated compound and resveratrol liposome modified thereby | |
| Saraydin et al. | Antitumoral effects of Melissa officinalis on breast cancer in vitro and in vivo | |
| CN107936058B (en) | Docetaxel derivative and preparation method and application thereof | |
| CN109821024A (en) | ROS producing agent and oxidation response anti-tumor predrug carry micella and its application altogether | |
| Xie et al. | Hybrid-cell membrane-coated nanocomplex-loaded chikusetsusaponin IVa methyl ester for a combinational therapy against breast cancer assisted by Ce6 | |
| CN112521388B (en) | Perylene bisimide derivative and application thereof | |
| CN110433292A (en) | A kind of double targeting materials and its application in drug delivery | |
| CN109745326B (en) | Pharmaceutical composition containing gefitinib and histone deacetylase inhibitor, liposome preparation of pharmaceutical composition and pharmaceutical application of liposome preparation | |
| Liu et al. | Enhanced treatment for cerebral ischemia-reperfusion injury of puerarin loading liposomes through neutrophils-mediated targeted delivery | |
| JP2024508128A (en) | Preparation method and uses of novel abiraterone derivatives for injection | |
| CN114469865B (en) | Liposome drug carrier combined with blood cell membrane and preparation method and application thereof | |
| CN102846551A (en) | Liver-targeting high-density lipoprotein analogue nano-particles, preparation method thereof, and application thereof | |
| CN111001006A (en) | Curbitacin B and oxidation response antitumor prodrug co-carried bionic nanoparticle | |
| Shao et al. | Plant-derived extracellular vesicles-a novel clinical anti-inflammatory drug carrier worthy of investigation | |
| CN104208704A (en) | Preparation method for pH-sensitive carbon nano tube-targeted drug delivery system | |
| CN110123754A (en) | A kind of targeting is in the Xanthatin nano-micelle and preparation method and application of dendritic cells | |
| CN105944108A (en) | Liposome pH-sensitivity modifier containing menthone 1,2-glycerol ketal and paclitaxel-curcumin compound liposome preparation | |
| WO2021226762A1 (en) | Tumor microenvironment response-type nano-composite drug loading system, and preparation method therefor and use thereof | |
| CN105287612B (en) | Salinomycin Sodium and adriamycin nano liposome and the preparation method and application thereof are carried altogether | |
| CN110898231B (en) | Functionalized Lalotxel liposome and preparation method and application thereof | |
| CN110917139A (en) | Preparation and application of multi-branch biotin modified breast cancer targeted liposome | |
| CN110522923A (en) | The matrix material of fructose and the co-modified dual-target triple negative breast cancer of RGD peptide | |
| CN113398276B (en) | Preparation and application of brain glioma targeted berberine and folic acid modified lipid material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140702 Termination date: 20141206 |
|
| EXPY | Termination of patent right or utility model |