CN110354079A - A kind of lipid peroxide generator and its preparation method and application - Google Patents
A kind of lipid peroxide generator and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of lipid peroxide generators and its preparation method and application；Lipid peroxide generator includes lipid, source of iron, and source of iron is wrapped in lipid by the preparation method of liposome and forms liposome.The endocytosis intake that the liposome is mediated by caveolin enters in tumour cell, then iron ion is discharged in acid lysosome environment, and then the iron redox couple formed in cytoplasm can be catalyzed unsaturated lipids and lipid peroxidation occurs, cause the accumulation of lipid within endothelial cells peroxide, iron apoptosis occurs for the final tumour cell that causes, and realizes the purpose of oncotherapy.Illustrate that the lipid peroxide generator can generate lipid peroxide under the triggering of iron redox couple by vitro characterization；And pass through cellular level and the evaluation of the antitumor activity of animal level, it was demonstrated that the lipid peroxide generator can generate a large amount of lipid peroxide in tumour cell, effectively realize the oncotherapy of iron apoptosis mediation.
Description
Technical field
The present invention relates to a kind of lipid peroxide generators and its preparation method and application, belong to liposome technology neck
Domain.
Background technique
Cancer is a kind of disease for seriously threatening human health, and " global cancer report in 2018 " data display whole world is newly-increased
Cases of cancer 18,100,000, up to 9,600,000, global cancer burden is further aggravated death toll.Current clinically used treatment hand
Section is operative treatment, radiation treatment, chemotherapy.Wherein, chemotherapy is mostly real by way of apoptosis, necrosis, autophagy
The killing of existing tumour cell.But that there are solubility is low for most of chemotherapeutics, stability is poor, and toxic side effect is strong, long-term administration
The defects of being easy to produce multidrug resistance.Consequently found that and being the new way for realizing treatment of cancer using a kind of new cell death way
Diameter.
2012, Stockwell et al. was by the lipid peroxide (lipid peroxidation, LPO) of iron dependence
Accumulation caused by apoptosis process be defined as iron apoptosis.Different from known apoptosis, necrosis, autophagy, iron withers
The accumulation for being mainly characterized by the LPO that a large amount of, iron ion relies on for dying generation, destroys redox equilibrium in cell, causes
Cell death.The lipid peroxidation that iron ion relies on is the major downstream feature of iron apoptosis, and LPO is to cause cell iron occurs
The major reason of apoptosis.As the key factor that iron apoptosis occurs, circulation iron is usually in conjunction with transferrins in body, with Fe3+
Form exist.Fe3+Enter cell by memebrane protein TfR 1 (transferrin receptor 1, TFR1),
And it is present in endosome (endosome), the Fe in endosome3+By iron oxidoreducing enzyme (iron oxide reductase,
Also termed STEAP3) it is reduced to Fe2+.Finally, Fe2+Transporter 1 (divalent metal transporter 1,
DMT1 Fe) is mediated2+From the labile iron library being discharged into cytoplasm in inner body, excessive iron rule is stored in ferritin
(ferritin) in.Contain a large amount of unsaturated lipids on cell membrane and organelle film, it can be by lipoxygenase
The oxidations such as (lipoxygenase, LOXs) and ROS form LPO.When the iron stable state in histocyte is destroyed, excessive iron
Pass through Fenton's reaction and H2O2Hydroxyl radical free radical is generated, intracellular unsaturated lipids is further caused and peroxidating occurs, is caused a large amount of
LPO be deposited in tumour cell, finally cause iron apoptosis.It is reported that LPO plays toxic effects by two kinds of approach: first because
Be responsible for maintaining the integrality of cell membrane for lipid, a large amount of lipid occur peroxidating can change the structure of lipid, composition, assembling and
The mobility of lipid.Furthermore high-activity compound, the catabolite of LPO such as malonaldehyde (malonaldehyde, MDA) are used as
With 4- hydroxyl nonenoic acid (4-hydroxynonenal, HNE) etc., epicyte protein can be made to crosslink, to make cell
The mobility and permeability of film change, and eventually lead to the change of eucaryotic cell structure and function, finally cause cell death.
Since iron apoptosis has biggish potentiality in oncotherapy, occur largely causing with Fenton's reaction in recent years
Load iron nanometer delivery system based on lipid peroxidation passes through the hydrogen peroxide in ferrous ion and tumour cell
(hydrogen peroxide, H2O2) Fenton's reaction generation hydroxyl radical free radical occurs, and then will be on cell membrane and organelle film
Unsaturated lipids are oxidized to lipid peroxide, final to cause iron apoptosis.
However, although tumor microenvironment pH be weak acid, be difficult optimum reaction condition needed for meeting Fenton's reaction (pH=
3-4)；And H in tumour cell2O2Content is limited, significantly limits the efficiency of Fenton's reaction, it usually needs is added a large amount of
Iron introduces exogenous H2O2Or stimulation of endogenous H2O2Generation for improving Fenton's reaction.The lipid mistake relied on based on iron ion
Oxide is the main executive of iron apoptosis, and general iron apoptosis delivery system cannot effectively realize the rouge that Fenton's reaction causes
Matter peroxidating, therefore, the limitation of Fenton's reaction low efficiency can be overcome and can produce a large amount of lipid peroxide by developing one kind
Iron apoptosis delivery system is of great significance for improving tumour iron apoptosis therapy.
Summary of the invention
Purpose: the present invention provides a kind of lipid peroxide generator and its preparation method and application, also by iron oxidation
Original occurs oxidation reaction to catalysis lipid and generates lipid peroxide, to overcome the limitation of Fenton's reaction low efficiency, improves tumour
Iron apoptosis therapy effect.
Technical solution: in order to solve the above technical problems, the technical solution adopted by the present invention are as follows:
A kind of lipid peroxide generator, the lipid peroxide generator includes lipid and source of iron, passes through liposome
Source of iron is wrapped in lipid and forms liposome by preparation method；
Contain one or more unsaturated bonds on the aliphatic chain of the lipid；The valence state of the source of iron is divalent, trivalent or divalent
It is existed simultaneously with trivalent；Lipid is catalyzed by iron redox couple, and oxidation reaction generation lipid peroxide occurs.
The lipid peroxide generator is catalyzed the principle of lipid peroxidation for unsaturated lipid by iron redox couple
Matter is oxidized to lipid peroxide.
The unsaturated lipids are selected from the lipid and are selected from phosphatidyl choline (phosphatidylcholine, PC), phosphorus
Acyl ethanol amine (phosphatidylethanolamine, PE), sphingomyelins (sphingomyelin, SM), phosphatidic acid
(phosphatidic acid, PA), phosphatidyl glycerol (phosphatidyl glycerol, PG), phosphatidylinositols
(phosphatidylinositol, PI), phosphatidylserine (phosphatidyl serine, PS), glycolipid, acyl are sweet
One of oil is a variety of, and each lipid has the aliphatic chain of different saturation.
The molysite is selected from ferric citrate, ferric nitrate, iron-dextrin, Iron Sorbitex, iron sucrose, ferric phosphate, burnt phosphorus
Sour iron, ferric carbonate, iron chloride, frerrous chloride, ironic citrate, ferrous citrate, ferric sulfate, ferrous sulfate, tartaric acid iron, winestone
Sour ferrous iron, ferrous fumarate, ferrous fumarate, ferric succinate, ferrous succinate, iron edta sodium salt, ferric ferrocyanide,
Ferrocene, ferrous acetate.
The ferri nano particle is selected from prussian blue nano particle, hollow mesoporous prussian blue nano particle, three oxidations
Two iron nano-particles, ferroferric oxide nano granules, manganese iron oxygen nano particle, iron sulphur copper nano particles, nanometer Fe-Pt particle.
The preparation method of the lipid peroxide generator, comprising:
(1) source of iron is dissolved in water or aqueous solution, obtains water phase；
(2) lipid is dissolved in organic solvent, obtains organic phase；
(3) by the preparation method of liposome, water phase and organic phase are combined；
(4) saturating in water, phosphate buffered saline solution PBS or physiological saline using the bag filter that molecular cut off is 0.5-14 KDa
Analysis obtains liposome after removing organic solvent and the source of iron that do not wrap up.
According to another aspect of the present invention, the compound of a kind of lipid peroxide generator and drug, including rouge are provided
Source of iron and drug are wrapped in lipid by the preparation method of liposome and form liposome by matter, source of iron and drug；
Contain one or more unsaturated bonds on the aliphatic chain of the lipid；The valence state of the source of iron is divalent, trivalent or divalent
It is existed simultaneously with trivalent；Lipid is catalyzed by iron redox couple, and oxidation reaction generation lipid peroxide occurs；
The drug is one or more of water soluble drug, hydrophobic drug, is selected from iron inducer of apoptosis, immunization therapy medicine
Object, chemotherapeutic agent, radiotherapeutic agent, optical dynamic therapy medicine, photo-thermal therapy drug.
The iron inducer of apoptosis be selected from l-Butionine sulfoximine, salicylazosulfapyridine, glutamic acid, Sorafenib,
One or more of Erastin, RSL-3, RSL-5.
The immunotherapy medicaments are selected from one or more of anti-PD-1, anti-PD-L1, anti-CTLA-4 antibody.
The chemotherapeutic agent is selected from adriamycin, mitomycin, daunorubicin, cis-platinum, carboplatin, cyclophosphamide, first ammonia
One or more of pterin, fluorouracil, gemcitabine, taxol, vincristine.
The radiotherapeutic agent in 32P, 89Sr, 90Y, 131I, 153Sm, 188Re, 117mSn, 117Lu one
Kind is several.
The optical dynamic therapy medicine is selected from chlorin e 6, hematoporphyrin derivative, ferroheme, indocyanine green, methylene
Blue, phytochrome II, benzoporphyrin derivative mono-acid ring A, Verteporfin, 5-ALA, in Porfimer Sodium, metal phthalocyanine class
One or more.
The photo-thermal therapy drug is selected from one of gold nano grain, prussian blue nano particle, poly-dopamine or several
Kind.
The preparation method of the compound of the lipid peroxide generator and drug, comprising the following steps:
(1) source of iron or source of iron are mixed with water soluble drug addition water or aqueous solution, obtains water phase；
(2) organic solvent is added with hydrophobic drug in lipid or lipid to mix, obtains organic phase；
(3) by the preparation method of liposome, water phase and organic phase are combined；
(4) saturating in water, phosphate buffered saline solution PBS or physiological saline using the bag filter that molecular cut off is 0.5-14 KDa
Analysis obtains the compound of lipid peroxide generator and drug after removing organic solvent and the source of iron that do not wrap up, drug.
The preparation method of liposome includes: film dispersion method, crosses film extrusion, French extrusion, reverse phase evaporation, change
Learn gradient method etc..
According to another aspect of the present invention, above-mentioned lipid peroxide generator, above-mentioned lipid peroxidation are also provided
Application of the compound of object generator and drug in the drug of preparation prevention and/or treatment and tumor-related illness.
Preferably a kind of preparation method of lipid peroxide generator of the present invention, comprising the following steps: film was used to squeeze
Method is used by the normal saline solution for being added dropwise to source of iron dissolved with the ethanol solution of unsaturated lipids under vorticity
The bag filter that molecular cut off is 0.5-14 KDa dialysed overnight in physiological saline aqueous solution, removes organic solvent and does not wrap up
Source of iron；It is squeezed out using liposome extruder, the smaller and uniform lipid peroxide generator of partial size is prepared.
The preparation method of the compound of preferably another lipid peroxide generator of the present invention and drug, film dispersion
Method, comprising the following steps: lipid is mixed with hydrophobic drug first, obtains organic phase, revolving removes organic solvent, obtains
To lipid membrane；Then source of iron is mixed with water soluble drug, obtains water phase, be added in above-mentioned lipid membrane and carry out water
Change；It is dialysed in water (PBS or physiological saline) after removing free drug using the bag filter that molecular cut off is 0.5-14 KDa,
The compound of lipid peroxide generator and drug is prepared.
The present invention designs a kind of lipid peroxide generator by treatment means of iron apoptosis, is touched based on iron redox couple
Sending out peroxidatic reaction of lipid is a kind of lipid peroxidation mode caused independent of Fenton's reaction.
One is preferred, selects the soybean lecithin for injection containing unsaturated lipids and small molecule source of iron ferric citrate
(FAC) preparation carries iron liposome (Lip@FAC), and FAC is reduced to Fe in tumour cell under the action of higher GSH2+, generation
Iron redox couple can be catalyzed the unsaturated lipids in lecithin and lipid peroxidation occurs, and cause a large amount of lipid peroxide
It is deposited in tumour cell, realizes tumour iron apoptosis therapy.
It is another preferred, select the soybean lecithin for injection containing unsaturated lipids and small molecule source of iron ferrous sulfate
(FeSO4) preparation load iron liposome (Lip@FeSO4), FeSO4Metal oxidizing ferment and higher H in tumour cell2O2Work
Fe is oxidized under3+, the iron redox couple of generation can be catalyzed the unsaturated lipids in lecithin occur lipid peroxidation, make
It is deposited in tumour cell at a large amount of lipid peroxide, realizes tumour iron apoptosis therapy.
The utility model has the advantages that lipid peroxide generator provided by the invention is compared with prior art, have the advantage that
The preparation method of the invention is simple, innovatively applies the principle system of iron redox couple triggering peroxidatic reaction of lipid
For the lipid peroxide generator tumour iron apoptosis therapy non-dependent for Fenton's reaction；The present invention uses small molecule FAC
As source of iron, it rapid, high volume can be discharged in acid lysosome environment (pH 4.5-5.5), lead to intracellular iron ion sharply
It increases, can effectively improve the efficiency of iron redox couple triggering peroxidatic reaction of lipid；The load iron liposome has safety
Advantage high, stability is strong, pH responsiveness discharges, and overcome the defect of Fenton's reaction low efficiency.The iron apoptosis delivery system
It is greatly expanded function and application range of the liposome as nano-carrier, for safe and stable, the high-efficiency delivery for realizing tumour
Practical platform is built.
Detailed description of the invention
Fig. 1 is the GSH that various concentration is added in embodiment 1 into FAC, and ferrous yield, result verification GSH can
Using by ferric iron back as ferrous iron.
Fig. 2 be in embodiment 2 by Lip and Lip@FAC respectively with (1) GSH, (2) Fe2+It mixes, the rouge under different condition
Plastid appearance.
Fig. 3 be in embodiment 2 by Lip and Lip@FAC respectively with (1) GSH, (2) Fe2+It mixes, product under different condition
Mass spectrogram.
Fig. 4 is the transmission electron microscope picture of Lip@FAC liposome in embodiment 4.
The release behavior that Fig. 5 is the Lip@FAC in the PBS solution of different pH value in embodiment 5.
Fig. 6 is to measure intake of the Lip@C6 on 4T1 cell using flow cytometer in embodiment 6.
Fig. 7 be in embodiment 7 Lip@FAC to tumor cell line MCF-7/ADR, MCF-7,4T1 and normal cell strain L02
Cytotoxicity, the reduction of cell survival rate demonstrate the good antitumous effect of the invention.
Fig. 8 is that embodiment 8 is separately added into Fer-1, DFO, APO, Nec-1, Aut, VE, VC, waits the Lip of processing
Cytotoxicity of the FAC to 4T1 cell.
Fig. 9 is the Mitochondrial Shape for observing Lip@FAC treated cell in embodiment 9 using biological electron microscope.
Figure 10 is the horizontal characterization of reactive oxygen species in embodiment 10.
Figure 11 is to measure lipid within endothelial cells peroxide result figure using BODIPY-C11 in embodiment 11.
Figure 12 is the fluorescence intensity for observing each experimental group lipid peroxide in embodiment 12 under laser co-focusing.
Figure 13 is the amount that intracellular malonaldehyde is measured in embodiment 13.
Figure 14 is permeability of the Lip@C6 in tumour ball in embodiment 14.
Figure 15 is that Lip@FAC evaluates the antitumous effect of tumor-bearing mice in embodiment 15.
Specific embodiment
The present invention will be further explained in the following with reference to the drawings and specific embodiments.
Embodiment 1
Fe is detected using Phen reagent2+, GSH and the Phen detection of a series of concentration is added into FAC solution first
Reagent is vortexed after shaking up 37oC constant-temperature table reacts 24 h, detects the light absorption value at 510 nm using microplate reader.Fig. 1 is this
The GSH of various concentration is added in embodiment into FAC, ferrous yield, result verification GSH can by ferric iron back
For ferrous iron.As the result is shown: as GSH concentration increases, light absorption value of the mixture at 510 nm is gradually increased, and works as GSH
When greater than 1 mM, mixture absorption value and positive controls FeSO4It is identical, thus illustrate FAC in the tumour with high concentration GSH
Fe can be effectively reduced into the cell2+, and then the iron ion of two kinds of valence states exists simultaneously to form iron redox couple.
Embodiment 2
Fe is added into Lip and Lip@FAC first2+, 37oReaction is stayed overnight in C constant-temperature table, blank liposome Lip solution
Fe is being added2+The equal clear in front and back, appearance do not change significantly, while appearance is not yet after Lip FAC solutions overnight
Apparent variation.When Fe is added into Lip@FAC solution2+, there is yellow mercury oxide in solution after reaction overnight.Then to Lip and
GSH is separately added into Lip@FAC, Lip solution is rear before addition, and there is no the apparent variations of generation, and Lip@FAC is in addition GSH
There is a large amount of yellow mercury oxide in front and back；As shown in Figure 2.The above substance is analyzed by mass spectrometry, as shown in figure 3, as the result is shown
Fe is being added in Lip2+, before and after GSH and Lip@FAC itself, there is no significant changes for molecular weight, occur 804 molecule from
Sub- peak, but there is 836 molecular ion peak in the group for appearing above yellow mercury oxide, and molecular weight increases by 32, this prompt yellow is heavy
Oxidation product in shallow lake contains lipid peroxide.It further relates to, FAC can be reduced to Fe by GSH2+, and then in FAC/Fe2+
The unsaturated lipids in soybean lecithin are aoxidized under conditions of existing simultaneously.
Embodiment 3
This experiment used film extrusion, and the ethanol solution of soybean lecithin is added dropwise to FAC physiology under vorticity
In saline solution, using the bag filter that molecular cut off is 14 KDa in physiological saline dialysed overnight, remove and ethyl alcohol and do not wrap
The FAC wrapped up in；Liposome extruder is further used on this basis, and the smaller and uniform liposome of partial size is prepared.
Embodiment 4
Use the physical aspect of transmission electron microscope observation Lip@FAC.It takes Lip@FAC solution a little, drops to Electronic Speculum copper mesh
On, it after natural drying, is placed in transmission electron microscope and is observed and taken pictures, Fig. 4 is the transmission of Lip@FAC liposome in the present embodiment
Electron microscope, the liposome has well-regulated spherical structure as the result is shown, is equally distributed monodisperse body.
Embodiment 5
Use the release behavior of Bag filter method measurement Lip@FAC.By the Lip@FAC of known content be dissolved in different pH value (7.4,
6.5,5.5) in the acetate buffer of phosphate buffer and pH 4.5, then adding to molecular cut off is the saturating of 14 KDa
Analyse both ends ligation in bag.Under the premise of meeting sink conditions, the bag filter equipped with Lip@FAC solution is placed into corresponding pH value
PBS dissolution medium in, take out 3 mL dissolution mediums in 1,2,4,6,8,12,24 h respectively, and add isometric release liquid,
Finally calculate accumulative releasing degree of the FAC under condition of different pH.Fig. 5 be the present embodiment in the PBS solution of different pH value
The release behavior of Lip@FAC, FAC discharges in the neutral environment of pH 7.4 and the microenvironment of tumour weak acid pH 6.8 as the result is shown
Slowly；And with the increase of dissolution medium acidity, the quick release of pH responsiveness is presented in FAC: simulating intracellular lysosome
In the environment of pH 4.5 and intracellular endosome pH 5.5, the accumulative releasing degree of drug can respectively reach 49.05 % in 24 h
With 31.17 %, with pH responsiveness release property, these results suggest that Lip@FAC in the acidic environment of tumour cell more
It is easy to release FAC, free FAC is conducive to lipid peroxidation.
Embodiment 6
Preparation carries the liposome of C6, and using intake situation of the flow cytometer measurement Lip C6 on 4T1 tumour cell, can be with
Reflect intake behavior of the Lip@FAC on tumour cell.By the 4T1 cell inoculation of logarithmic growth phase into 24 orifice plates, every hole paving
Plate density is 1.0 × 105/ 1 mL, the overnight incubation in cell incubator.80 % of hole plate suqare are grown to cell, point
The free C6 and Lip@C6 of 0.1 μM, 0.2 μM is not given, and cultivates 1 h, 2 h, 4 h in administration respectively, is used after 6 h cold
Then plus 200 μ L pancreatin appropriateness vitellophags PBS is cleaned three times, immediately siphons away pancreatin and 500 μ L are added and not exclusively train
It supports base and collects cell.After filtering out the large granular impurity in cell liquid with nylon gauze, free C6 is measured using flow cytometer
With intake of the Lip@C6 on 4T1 cell, as shown in fig. 6, C6 and Lip@C6 intake with the incubation of two kinds of substances when
Between and dosage increase and increase, i.e., the intake of two kinds substances is presented time and dose dependent, and Lip@C6 takes the photograph
Taken amount is higher than free C6.This prompt, which carries FAC liposome, has higher intake relative to free FAC, higher to improve FAC
Bioavilability, for play iron apoptosis strong condition is provided.(channel BD, FL1)
Embodiment 7
Using mtt assay measurement Lip@FAC to the inhibiting rate of different types of cell Proliferation.Select three plants of tumour cells and one plant just
Normal cell: MCF-7/ADR, MCF-7,4T1, L02, by logarithmic growth phase cell inoculation into 96 orifice plates, the cell density in every hole
It is 1.0 × 104/ 200 μ L, in cell incubator overnight incubation.80 % for growing to hole plate suqare to cell start to be administered,
Experimental setup control group, FAC group, Lip group, Lip@FAC group, in each group corresponding FAC administration concentration be 10,15,20,25,
30,35 μM, corresponding Lip administration concentration is 20,30,40,50,60,70 μM, after 48 h are administered, is added under the conditions of being protected from light
The MTT of 5 mg/mL of 20 μ L continues to cultivate 4 h in cell incubator.The solution in 96 orifice plates is siphoned away with 1 mL syringe,
150 μ L DMSO are added, shake 15 min in 37 DEG C of shaking tables, detect the light absorption value in each hole at 570 nm using microplate reader
(A), cells survival rate is calculated.As shown in fig. 7, for Lip@FAC in the present embodiment to tumor cell line MCF-7/ADR, MCF-7,
The cytotoxicity of 4T1 and normal cell strain L02, the reduction of cell survival rate demonstrate the good antitumous effect of Lip@FAC.
Embodiment 8
Using the variation of cytotoxicity of the cell of mtt assay measurement Lip@FAC processing after different inhibitor are added.Logarithm is raw
For long-term 4T1 cell inoculation into 96 orifice plates, the cell density in every hole is 1.0 × 104/ 200 μ L, in cell incubator culture
Overnight.80 % that hole plate suqare is grown to cell, final concentration of 20 μM for giving final preparation Lip FAC(FAC first),
It is then respectively adding inhibitor, this experiment selection iron inhibitors of apoptosis: Ferrostatin-1(Fer-1,2 μM), Deferoxamine
(DFO, 100 μM), inhibitors of apoptosis: Z-VAD-FMK(Apo, 160 μM)；Downright bad inhibitor: Necrostatin-1(Nec-1,
1 μM)；Autophagy inhibitor, trimethyl adenine (Atu, 60 μM)；Water-soluble active oxygen killer: vitamin C (VC, 200 μ
M)；Lipid activity oxygen killer: vitamin E (VE, 200 μM).For the importance for investigating iron redox couple, this experiment preparation
Mixture (the Zn of PC and zinc chloride2+, 20 μM), for the importance for investigating unsaturated lipids, it is prepared for two myristoyl phosphatide
The mixture of phatidylcholine (DMPC, 40 μM) and FAC.After cultivating 48 h, it is added 5 mg/mL's of 20 μ L under the conditions of being protected from light
MTT continues to cultivate 4 h in cell incubator.The solution in 96 orifice plates is siphoned away with 1 mL syringe, 150 μ L DMSO are added,
15 min are shaken in 37 DEG C of shaking tables, detect the light absorption value (A) in each hole at 570 nm using microplate reader, calculate cells survival
Rate, as a result as shown in figure 8, Lip@FAC enter after cell can be by the redox mode that iron ion relies on by the insatiable hunger in Lip
It is LPO with lipid oxidation, and then causes iron apoptosis, wherein iron and unsaturated lipids is indispensable key factor.
Embodiment 9
By logarithmic growth phase cell inoculation into 6 orifice plates, the cell density in every hole is 2.5 × 105/ 2 mL, in cell culture
Case overnight incubation.80 % that hole plate suqare is grown to cell give final concentration of 20 μM of final preparation Lip FAC(FAC),
Culture solution is discarded after 48 h are administered, is just rapidly added Electronic Speculum fixer (2.5 % glutaraldehyde solutions, 1 mL) to cell without rinsing
It is fixed, gently scrapes cell with clean cell scraper and is collected into centrifuge tube and (trypsin digestion cell cannot be used), 1500
Rpm is centrifuged 3 min and collects a certain amount of cell (naked eyes visible cell is wanted to precipitate sesame to mung bean size), adds after discarding fixer
Enter new Electronic Speculum fixer liquid-transfering gun piping and druming to be resuspended, room temperature is transferred to 4 °C of refrigerators preservations after fixing 2 h.Later with four oxidations
Osmium is fixed after carrying out, and then uranium acetate carries out block dye, with the ethyl alcohol (30 ~ 100 %) of continuous concentration by low concentration to highly concentrated
Degree dehydration.Next it is impregnated with propylene oxide and Spurr ' s resin (embedding medium), embedding medium is made gradually to infiltrate through histocyte
Interior, the embedding medium being finally embedded in is 70oUnder the conditions of C overnight, solidify resin polymerization.Then by sample sections, it is placed in copper mesh
On.Uranium acetate and lead citrate dyeing are finally carried out, finally using the state of biological electron microscope observation mitochondria, as a result as schemed
Shown in 9, the Mitochondrial Shape of Control group is uniform, and mitochondrial membrane is high-visible with cristal membrane.Such as black arrow institute in figure
Refer to, increase by Lip@FAC treated mitochondrial membrane density, cristal membrane thickens, and profile is unintelligible, and mitochondria becomes smaller simultaneously
There is the phenomenon that rupture.The variation of the above mitochondria meets cellular change caused by iron apoptosis, therefore can further verify and give
Iron apoptosis has occurred in the 4T1 cell of Lip@FAC.
Embodiment 10
Intracellular active oxygen can aoxidize non-blooming DCFH and generate the DCF for having fluorescence, this experiment is detected thin using DCFH-DA
The level of intracellular reactive oxygen species generation.By logarithmic growth phase cell inoculation into 24 orifice plates, the cell density in every hole is 1.0 × 105 / 1
ML, in cell incubator overnight incubation.80 % for growing to hole plate suqare to cell start to be administered, experimental setup control group,
FAC group, Lip group, Lip@FAC group, corresponding FAC administration concentration is 20 μM in each group, and the administration concentration of corresponding Lip is 40
μM.Medical fluid is discarded after 24 h are administered, DCFH-DA is diluted to 5 μM using incomplete culture medium, every hole is added 500 μ L and places
30 min are dyed in cell incubator.Then plus 200 μ L pancreatin dyestuff is discarded after dyeing, is cleaned three times with cold PBS,
Appropriate vitellophag immediately siphons away pancreatin and 500 μ L incomplete culture mediums is added and collects cell.It is filtered with nylon gauze
After falling the large granular impurity in cell liquid, the content of ROS in each experimental group cell is measured using flow cytometer, as a result such as Figure 10
Shown, there was no significant difference compared with Control group with the ROS level of Lip group for FAC group, and Lip@FAC group is relative to control group
It is horizontal with up to 16 times of ROS.Thus illustrate that Lip FAC can be such that intracellular ROS level significantly improves, and then destroy
Intracellular redox equilibrium, finally causes cell death.(channel BD, FL1)
Embodiment 11
By logarithmic growth phase cell inoculation into 24 orifice plates, the cell density in every hole is 1.0 × 105/ 1 mL, in cell culture
Case overnight incubation.80 % for growing to hole plate suqare to cell start to be administered, experimental setup control group, FAC group, Lip group,
Lip@FAC group, corresponding FAC administration concentration is 20 μM in each group, and the administration concentration of corresponding Lip is 40 μM.24 h are administered
After discard medical fluid, BODIPY-C11 is diluted to 5 μM using incomplete culture medium[8], every hole is added 500 μ L and is placed in cell
30 min are dyed in incubator.Dyestuff is discarded after dyeing, is cleaned three times with cold PBS, and then plus 200 μ L pancreatin moderately disappear
Change cell, immediately siphons away pancreatin and 500 μ L incomplete culture mediums are added and collect cell.Cell is filtered out with nylon gauze
After large granular impurity in liquid, the content of lipid ROS in each experimental group cell is measured using flow cytometer, as a result such as Figure 11 institute
Show, there was no significant difference with Control group for the fluorescence intensity of FAC group and Lip group, and Lip@FAC group fluorescence intensity is stronger, about
2 times of control group.It is possible thereby to illustrate that individually giving FAC or Lip can not be such that intracellular lipid ROS increases, and Lip FAC
Into after cell, FAC therein can be reduced to Fe by the GSH of higher concentration2+, the iron redox couple of formation is by unsaturated lipid
Matter is oxidized to lipid peroxide, therefore lipid ROS is increased in cell.The present embodiment further proves that Lip@FAC can cause
Iron apoptosis occurs for cell.(channel BD, FL2)
Embodiment 12
By in the laser co-focusing ware of logarithmic growth phase cell inoculation to 3.5 cm, the cell density in every hole is 3.0 × 105 / 2
ML, in cell incubator overnight incubation.80 % for growing to laser co-focusing ware area to cell start to be administered, experimental setup
Control group, FAC group, Lip group, Lip@FAC group, corresponding FAC administration concentration is 20 μM in each group, and corresponding Lip's gives
Concentration is 40 μM.Medical fluid is discarded after 24 h are administered, BODIPY-C11 is diluted to 5 μM using incomplete culture medium, every hole
It 1 mL is added is placed in cell incubator and dye 30 min.It discards culture medium and is cleaned three times using cold PBS, then addition is
Continued to cultivate 15-20 min in cell incubator with type Hoechst dyeing liquor (50 μ L are diluted to 10 mL), PBS cleaning three
It after secondary, infiltrate in incomplete culture medium, the fluorescence intensity of each experimental group lipid peroxide is observed under laser co-focusing, tie
Fruit is as shown in figure 12, Control group, and the fluorescence intensity of FAC group and Lip group lipid ROS are smaller, and after giving Lip@FAC, cell
Interior fluorescence intensity significantly increases, and lipid ROS is distributed in cytoplasm.The present embodiment further illustrates Lip@FAC entrance
After cell can significantly raised lipid ROS it is horizontal.
Embodiment 13
Unsaturated lipids generate lipid peroxide after being oxidized, be then gradually decomposed into the compound of a series of complex, wherein
Including MDA.This experiment measures the amount of intracellular MDA, the level of indirect determination lipid peroxide using MDA detection kit.
By logarithmic growth phase cell inoculation into 6 orifice plates, the cell density in every hole is 3.0 × 105/ 2 mL are trained in cell incubator
It supports overnight.80 % for growing to hole plate suqare to cell start to be administered, experimental setup control group, FAC group, Lip group, Lip
FAC group, corresponding FAC administration concentration is 20 μM in each group, and the administration concentration of corresponding Lip is 40 μM.It is abandoned after 24 h are administered
Medical fluid is removed, incomplete culture medium is added after pancreatin digestion and neutralizes, cell is collected by centrifugation, cell pyrolysis liquid is added in ice after removing supernatant
Upper cracking makes cracking uniformly at interval of 5 min vortex, and rear 12000 rpm are centrifuged 5 min in triplicate, collect supernatant, then press
It is detected according to MDA kit specification, as a result as shown in figure 13, MDA kit detects in the cell of Lip@FAC processing
The amount of LPO is twice of Control, and the MDA content of FAC group and Lip group does not change significantly.Thus illustrate Lip@
FAC can cause lipid within endothelial cells peroxide content to increase, and then MDA is also increased.
Embodiment 14
This experiment uses tumour ball made above, sucks culture medium in 96 orifice plates, gives free C6 and Lip@C6 respectively, is incubated for
After 24 h, culture solution is discarded, and cannots be used up full culture medium repeated washing three times, then observes C6 under laser confocal microscope
In the osmosis of tumour ball, as a result as shown in figure 14, fluorescence intensity of the C6 in tumour ball of dissociating is weaker, and Lip@C6 is swollen
Fluorescence intensity in tumor ball is stronger, and tomoscan is shown, fluorescence can penetrate into inside tumour ball.It follows that Lip@
C6 has preferable intake and permeability on 4T1 cell tumour ball, is conducive to drug and is sufficiently distributed in entire tumor tissues, thorough
Bottom kills tumour.
Embodiment 15
Inhibiting tumor assay final choice gross tumor volume is 50-100 mm3Lotus knurl Balb/c mouse model as research object, at random
It is divided into 4 groups, every group 6.4 groups of experiment mices give the physiological saline of 100 μ L, FAC (1 mM/100 μ L), Lip (2 respectively
MM/100 μ L) and Lip@FAC (1 mM FAC-2 mM Lip).Be calculated as the 0th day with first time administration time, then respectively at
It is administered within 0th, 3,6,9,12,15,18 day, and observes the survival condition of mouse before each administration, measure the weight of mouse
And gross tumor volume.After experiment, the tumor tissue section of each group experiment mice is taken out, is investigated using BODIPY-C11 dyeing swollen
Then the production quantity of the lipid peroxide at tumor position carries out hematoxylin-eosin (H&E) dyeing for investigating the dead of tumour cell
Situation is died, as a result as shown in figure 15, in tumor-bearing mice body after successive administration 7 times, FAC group is compared with physiological saline group without obvious
Difference illustrates the iron apoptosis that cannot individually cause tumor tissues to iron；Lip group is compared with physiological saline group also without conspicuousness
Difference illustrates that individually iron apoptosis can not be induced by giving blank liposome；And final preparation Lip@FAC group gross tumor volume obviously becomes
It is small, illustrate that tumor locus can be accumulated in by EPR effect and penetrate into tumor tissues deep by carrying iron liposome, when load iron liposome
Entered in tumour cell by endocytosis, ferric ion can be to be reduced to ferrous iron under the action of GSH in the cell, then
The iron redox couple of formation can be catalyzed liposome and form lipid peroxide, kill tumour eventually by iron apoptosis.
BODIPY-C11 dyeing display, compared with physiological saline group, the fluorescence intensity of FAC group and Lip group does not have significant difference, and
Then there is stronger fluorescence intensity in Lip@FAC group, illustrates that the Lip@FAC that tail vein is given can be generated largely in tumor locus
LPO, and then can trigger iron apoptosis.There is more Dead tumor cell and with more in H&E dyeing display Lip@FAC group
Thus vacuole illustrates that Lip@FAC can effectively cause tumor tissues that iron apoptosis occurs, generates preferable tumor killing effect.
The above is only a preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of lipid peroxide generator, which is characterized in that the lipid peroxide generator includes lipid and source of iron,
Source of iron is wrapped in lipid by the preparation method of liposome and forms liposome；
Contain one or more unsaturated bonds on the aliphatic chain of the lipid；The valence state of the source of iron is divalent, trivalent or divalent
It is existed simultaneously with trivalent；Lipid is catalyzed by iron redox couple, and oxidation reaction generation lipid peroxide occurs.
2. lipid peroxide generator according to claim 1, which is characterized in that the lipid is selected from phosphatidyl gallbladder
Alkali, phosphatidyl-ethanolamine, sphingomyelins, phosphatidic acid, phosphatidyl glycerol, phosphatidylinositols, phosphatidylserine, glycolipid, acyl are sweet
One of oil is a variety of.
3. lipid peroxide generator according to claim 1, which is characterized in that the source of iron is molysite or iron-containing
Nano particle；
The molysite be selected from ferric citrate, ferric nitrate, iron-dextrin, Iron Sorbitex, iron sucrose, ferric phosphate, ferric pyrophosphate,
Ferric carbonate, iron chloride, frerrous chloride, ironic citrate, ferrous citrate, ferric sulfate, ferrous sulfate, tartaric acid iron, tartaric acid are sub-
Iron, ferrous fumarate, ferrous fumarate, ferric succinate, ferrous succinate, iron edta sodium salt, ferric ferrocyanide, two cyclopentadienyls
Iron, ferrous acetate；
The ferri nano particle is selected from prussian blue nano particle, hollow mesoporous prussian blue nano particle, di-iron trioxide
Nano particle, ferroferric oxide nano granules, manganese iron oxygen nano particle, iron sulphur copper nano particles, nanometer Fe-Pt particle.
4. lipid peroxide generator according to claim 1, which is characterized in that the preparation method packet of the liposome
It includes film dispersion method, cross film extrusion, French extrusion, reverse phase evaporation, chemical gradient method.
5. lipid peroxide generator according to claim 1, which is characterized in that the lipid peroxide generator
Preparation method, comprising:
(1) source of iron is dissolved in water or aqueous solution, obtains water phase；
(2) lipid is dissolved in organic solvent, obtains organic phase；
(3) by the preparation method of liposome, water phase and organic phase are combined；
(4) saturating in water, phosphate buffered saline solution PBS or physiological saline using the bag filter that molecular cut off is 0.5-14 KDa
Analysis obtains liposome after removing organic solvent and the source of iron that do not wrap up.
6. a kind of compound of lipid peroxide generator and drug, which is characterized in that including lipid, source of iron and drug, lead to
Source of iron and drug are wrapped in lipid by the preparation method for crossing liposome forms liposome；
Contain one or more unsaturated bonds on the aliphatic chain of the lipid；The valence state of the source of iron is divalent, trivalent or divalent
It is existed simultaneously with trivalent；Lipid is catalyzed by iron redox couple, and oxidation reaction generation lipid peroxide occurs；
The drug is one or more of water soluble drug, hydrophobic drug, is selected from iron inducer of apoptosis, immunization therapy medicine
Object, chemotherapeutic agent, radiotherapeutic agent, optical dynamic therapy medicine, photo-thermal therapy drug.
7. the compound of lipid peroxide generator and drug according to claim 6, which is characterized in that
The iron inducer of apoptosis be l-Butionine sulfoximine, salicylazosulfapyridine, glutamic acid, Sorafenib, Erastin,
RSL-3,RSL-5；
The immunotherapy medicaments are anti-PD-1, anti-PD-L1, anti-CTLA-4 antibody；
The chemotherapeutic agent be selected from adriamycin, mitomycin, daunorubicin, cis-platinum, carboplatin, cyclophosphamide, methotrexate (MTX),
Fluorouracil, gemcitabine, taxol, vincristine；
The radiotherapeutic agent is selected from 32P, 89Sr, 90Y, 131I, 153Sm, 188Re, 117mSn, 117Lu；
The optical dynamic therapy medicine is selected from chlorin e 6, hematoporphyrin derivative, ferroheme, indocyanine green, methylene blue, light
Quick element II, benzoporphyrin derivative mono-acid ring A, Verteporfin, 5-ALA, Porfimer Sodium, metal phthalocyanine class；
The photo-thermal therapy drug is selected from gold nano grain, prussian blue nano particle, poly-dopamine.
8. the compound of lipid peroxide generator and drug according to claim 6 or 7, which is characterized in that lipid
The preparation method of body includes: film dispersion method, crosses film extrusion, French extrusion, reverse phase evaporation, chemical gradient method.
9. the preparation method of the compound of lipid peroxide generator according to claim 6 or 7 and drug, including with
Lower step:
(1) source of iron or source of iron are mixed with water soluble drug addition water or aqueous solution, obtains water phase；
(2) organic solvent is added with hydrophobic drug in lipid or lipid to mix, obtains organic phase；
(3) by the preparation method of liposome, water phase and organic phase are combined；
(4) saturating in water, phosphate buffered saline solution PBS or physiological saline using the bag filter that molecular cut off is 0.5-14 KDa
Analysis obtains the compound of lipid peroxide generator and drug after removing organic solvent and the source of iron that do not wrap up, drug.
10. lipid peroxidation described in the described in any item lipid peroxide generators of claim 1-5, claim 6 or 7
Application of the compound of object generator and drug in the drug of preparation prevention and/or treatment and tumor-related illness.
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| CN111671769A (en) * | 2020-05-26 | 2020-09-18 | 赣南医学院 | Application of copper-containing ferrite nano-particles in preparation of antitumor drugs |
| CN111803446A (en) * | 2020-07-14 | 2020-10-23 | 南方医科大学 | Photosensitive liposome and application thereof |
| CN112274495A (en) * | 2020-10-31 | 2021-01-29 | 郑州大学 | H2O2Preparation method and application of self-supply type calcium peroxide loaded curcumin nanoparticles |
| CN112402453A (en) * | 2020-11-18 | 2021-02-26 | 中国药科大学 | Enzyme and insoluble drug co-carried liposome and preparation method and application thereof |
| CN115888773A (en) * | 2022-11-08 | 2023-04-04 | 青岛农业大学 | Multifunctional cascade nanoenzyme, preparation method and application thereof |
| CN116808172A (en) * | 2023-01-07 | 2023-09-29 | 王晓娟 | Sunflower disc liposome and application thereof in preparation of products for reducing uric acid and dissolving tophus |
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| CN111671769A (en) * | 2020-05-26 | 2020-09-18 | 赣南医学院 | Application of copper-containing ferrite nano-particles in preparation of antitumor drugs |
| CN111803446A (en) * | 2020-07-14 | 2020-10-23 | 南方医科大学 | Photosensitive liposome and application thereof |
| CN111803446B (en) * | 2020-07-14 | 2021-10-01 | 南方医科大学 | Photosensitive liposome and application thereof |
| CN112274495A (en) * | 2020-10-31 | 2021-01-29 | 郑州大学 | H2O2Preparation method and application of self-supply type calcium peroxide loaded curcumin nanoparticles |
| CN112274495B (en) * | 2020-10-31 | 2022-05-03 | 郑州大学 | H2O2Preparation method and application of self-supply type calcium peroxide loaded curcumin nanoparticles |
| CN112402453A (en) * | 2020-11-18 | 2021-02-26 | 中国药科大学 | Enzyme and insoluble drug co-carried liposome and preparation method and application thereof |
| CN115888773A (en) * | 2022-11-08 | 2023-04-04 | 青岛农业大学 | Multifunctional cascade nanoenzyme, preparation method and application thereof |
| CN115888773B (en) * | 2022-11-08 | 2023-08-11 | 青岛农业大学 | Multifunctional cascade nanoenzyme, preparation method and application thereof |
| CN116808172A (en) * | 2023-01-07 | 2023-09-29 | 王晓娟 | Sunflower disc liposome and application thereof in preparation of products for reducing uric acid and dissolving tophus |
| CN116808172B (en) * | 2023-01-07 | 2024-02-13 | 龚曙初 | Sunflower disc peptide, composite liposome and application thereof in preparation of products for reducing uric acid and dissolving tophus |
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