CN104689321B - Curcumin long-circulating nanoliposome carrier of enoxolone mediation and preparation method thereof - Google Patents
Curcumin long-circulating nanoliposome carrier of enoxolone mediation and preparation method thereof Download PDFInfo
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Abstract
The invention provides curcumin long-circulating nanoliposome carrier of a kind of enoxolone mediation and preparation method thereof, the nano-lipid carrier is prepared by curcumin, glycerin monostearate, caprylic/capric triglyceride, enoxolone phospholipid derivative, soybean lecithin for injection and the Strearate of Polyoxyethylene 40, first by lipid and curcumin heating water bath to 75 DEG C, adding absolute ethyl alcohol dissolves it, and rotary evaporation removes absolute ethyl alcohol and is used as oil phase；Water for injection is added in the Strearate of Polyoxyethylene 40, heating water bath is used as aqueous phase to 75 ± 2 DEG C after ultrasonic agitation is uniform；Aqueous phase is added dropwise in synthermal oil phase under magnetic agitation, colostrum is made in constant temperature stirring 5min, ultrasonic cell disruptor ultrasonic disperse 5min is used while hot, 0.22 μm of miillpore filter is crossed, room temperature ice-water bath cooling and solidifying obtains the curcumin long-circulating nanoliposome carrier dispersion liquid of enoxolone mediation.
Description
Technical field
The invention belongs to field of medicaments, a kind of curcumin long-circulating nanoliposome carrier of enoxolone mediation is especially provided
And preparation method thereof.
Background technology
Curcumin（Curcumin, Cur）It is extracted from plant turmeric（Curcuma longa L.）Dry rhizome, be a kind of
Polyphenol compound, is the main active in curcumin.It is widely used in food neck as colouring agent and colouring substance
Domain.In recent years, multiple studies have shown that, curcumin has various bioactivity and pharmacological action, can be used as anti-inflammatory agent, antioxygen
Agent, antineoplastic, hepatoprotective agent etc., its toxicity is low, with good clinical practice potentiality.But, curcumin solubility in water
It is small and stability is poor, cause poor bioavailability in vivo, medicine is inactivated in intestines and stomach intracellular metabolite quickly, and increased dosage amount and give
Medicine frequency can increase side effects of pharmaceutical drugs again, limit the clinical practice of curcumin.In order to improve the solubility of curcumin and steady
It is qualitative, its bioavilability is improved, at present correlative study in terms of curcumin microemulsion, polymer nanoparticle, liposome.Its
The research of middle liposome is relatively broad, the features such as liposome has good sustained release, targeting and biocompatibility in vivo, but
Liposome is unstable in vivo and in vitro, medicine is easily leaked during storage；Polymer nanoparticle can have residual in preparation process
The toxicants such as monomer, organic solvent, polymerization initiator, and biodegradable polymer used swallows in cell
Also cytotoxicity is often produced after degraded；Micro emulsion has that physical stability is poor, and medicine is limited in the low problem of solubility of oil phase
Its broad development and application.The novel form of curcumin is researched and developed, increases it water-soluble, it is to avoid inactivated, carry by gastrointestinal tract
High blood concentration, extends circulation time in vivo, improves bioavilability, preferably plays the pharmacological activity of curcumin, it is faced
Bed applies significant and vast potential for future development.
Nano-lipid carrier (nanostructured lipid carrier, NLC) is by solid lipid nano granule
A kind of novel partculate delivery system that (solid lipid nanoparticles, SLN) is developed.With the natural of solid-state or
The lipoid of synthesis such as lecithin, triacylglycerol etc. are carrier, and chemical differences very big fluid oil is added into Solid lipid, makes to receive
Medicine is wrapped up or is adsorbed in lipoid core the particle diameter through being made in 50- with crystal defect type or unformed presence by rice grain structure
Solid-state micelle drug delivery system between 1000nm.NLC is prepared by Solid lipid and liquid fatty substance mixing as matrix material,
Due to the addition of fluid oil, lattice structure has been upset, unformed structure carrier is formed, it is to avoid medicine is expelled from coming, and adds
Drugloading rate and envelop rate[23].Also have been reported that and think, NLC Solid lipids wrap small fluid oil formation nanometer room.When medicine is in liquid
When solubility is far above Solid lipid solubility in state lipid, medicine is dissolved in fluid oil, greatly improves drugloading rate, and
The Solid lipid wrapped also acts the effect of sustained release[24].And the addition of fluid oil does not influence the crystal structure of lipid,
Still it is present in solid form in dispersion liquid.NLC had both possessed the good physiological compatibility such as liposome, emulsion and had been easy to extensive
The advantage of industrialized production, but also with polymer nanoparticle stability height, the antileakaging feature of medicine, is optimized simultaneously
SLN（Solid lipid nano granule, Solid lipid nanoparticles）Envelop rate is low, the shortcomings of holding medicine is separated out, by
Increasing researcher's concern, as the medicine novel carriers with broad based growth future.
At present, conventional NLC preparation methods have solvent evaporation method, high-pressure stripping, micro emulsion method, film ultrasound etc..No
Same preparation method can all produce a certain degree of influence to the particle diameter, envelop rate and stability of nano-lipid carrier.
The content of the invention
It is an object of the invention to provide a kind of curcumin of the enoxolone mediation with high encapsulation rate and drugloading rate is long
Circulating nanoliposome carrier and preparation method thereof.
The present invention specifically provides a kind of curcumin long-circulating nanoliposome carrier of enoxolone mediation, and its feature exists
In：The curcumin long-circulating nanoliposome carrier of enoxolone mediation be by curcumin, glycerin monostearate, octanoic acid/
Triglyceride DDD, enoxolone-phospholipid derivative, soybean lecithin for injection and Polyoxyethylene 40
What Strearate was prepared, wherein, the synthetic method of enoxolone-phospholipid derivative is：
GA, DCC, NHS three is taken to be dissolved in water removal dichloromethane, 3~4h of reaction activation under normal temperature；Separately take DSPE-
PEG2000-NH2Dichloromethane solution is dissolved in, is added dropwise in above-mentioned activator, 48~50h of the lower reaction of room temperature under nitrogen protection；Filtering
Except the accessory substance of dereaction, the extraction of ice ether is added, unreacted GA in supernatant is removed, deposit is target product,
DMF is fitted into bag filter the 72h that dialyses after redissolving, freeze-drying obtains enoxolone-phospholipid derivative.
The preparation method of the curcumin long-circulating nanoliposome carrier mediated present invention also offers the enoxolone, its
It is characterised by, preparation process is as follows：
By lipid（Glycerin monostearate and caprylic/capric triglyceride）, enoxolone-phospholipid derivative and curcumin
Heating water bath is to 75 DEG C, and adding absolute ethyl alcohol dissolves it, and rotary evaporation removes absolute ethyl alcohol and is used as oil phase；
Water for injection is added in the Strearate of Polyoxyethylene 40, heating water bath is made to 75 ± 2 DEG C after ultrasonic agitation is uniform
For aqueous phase；Aqueous phase is added dropwise in synthermal oil phase under magnetic agitation, constant temperature stirring 5min is made colostrum, ultrasonic wave is used while hot
Cell disruptor ultrasonic disperse 5min, crosses 0.22 μm of miillpore filter, and room temperature ice-water bath cooling and solidifying obtains the ginger of enoxolone mediation
Flavine long-circulating nanoliposome carrier dispersion liquid.
The present invention makees compound emulsifying agent using soybean lecithin for injection and the poly- ester of hydrogen-oxygen 40 of stearic acid, due to curcumin fat
Dissolubility is general, is directly added into solubility in oil phase poor.And add soybean lecithin for injection in oil phase, considerably increase
Solubility in oil phase.The easy oxidation deterioration but phosphatide is heated for a long time, so as to lose emulsifying capacity and its catabolite
The adverse reactions such as haemolysis are easily caused, so should try one's best the shortening heat time.And the poly- ester of hydrogen-oxygen 40 of stearic acid is water soluble emulsifier,
Add it to stirring, ultrasound in aqueous phase and be allowed to scattered.
During preparing nano-lipid carrier, control operation temperature must be more than the fusing point of lipid, oil phase and water
It must mutually be heated to mix after 75 DEG C.Oil phase is added dropwise or when pouring into aqueous phase at room temperature, because lipid viscosity is larger, now temperature
Less than lipid fusing point, oil phase can solidify quickly, operate more difficult.And select under magnetic agitation by heat aqueous phase be added dropwise to oil phase,
Rapid dispersion is into colostrum again, and dispersion effect is evenly, simple to operate.
The preparation method of the curcumin long-circulating nanoliposome carrier of enoxolone mediation of the present invention, its feature exists
In：In the 5min with ultrasonic cell disruptor ultrasonic disperse, ultrasonic 1s, interval 1s.
The preparation method of the curcumin long-circulating nanoliposome carrier of enoxolone mediation of the present invention, its feature exists
In：When aqueous phase is added dropwise in synthermal oil phase, rate of addition is 10mLmin-1.When rate of addition is too fast, lipid phase and water
Mutually it is layered, it is difficult to obtain uniform dispersion.
When aqueous phase is instilled into oil phase, high-speed stirred must be carried out.The higher gained colostrum particle diameter of mixing speed is smaller, and system is more steady
It is fixed；But the too high loss that oil phase can be made to splash in bottle wall, cause material and medicine of mixing speed.Mixing speed can cause two slowly excessively
Mix irregular, oil phase floats over aqueous top layer, it is impossible to form stable colostrum, influence the preparation of next step nano-lipid carrier.
Through testing repeatedly, select with 3000rmin-1It is used as the mixing speed for preparing colostrum.
In 75 DEG C, 3000rmin-1Prepare after colostrum, while hot carry out colostrum at Probe Ultrasonic Searching under the conditions of stirring 5min
Reason, using particle diameter, envelop rate as index, investigates the influence of ultrasonic power and time to NLC.Experiment finds that ultrasonic power is bigger, carries
The damage capability of confession is stronger, and the particle diameter of nano-lipid carrier is smaller.But power, which crosses conference, causes the size distribution of nanoparticle uneven
Even, system surface free energy is excessive and unstable.And ultrasonic power it is too small there is provided damage capability be not enough to destroy micro emulsion, obtain
To sufficiently small particle.Therefore, selection ultrasonic power is 400W.
Nano-lipid dispersion liquid after ultrasound needs to cool immediately and maintains sufficiently long cool time just to make nanometer
Grain is solidified enough, stable.Cooling rate is too slow or cool time it is too short can cause particle buildup, particle diameter increase, system
Stability is reduced, and causes the failure of an experiment.The cooling of selection room temperature ice-water bath and water-bath cooling two ways are compared the present invention respectively
Compared with.As a result show, it is slow to cool and solidify speed under room-temperature water bath, particle diameter is larger and light under clarity it is poor.Therefore preferably 0~
2 DEG C of ice-water baths are cooled and solidified rapidly.
The preparation method of the curcumin long-circulating nanoliposome carrier of enoxolone mediation of the present invention, its feature exists
In raw materials used mass ratio is curcumin：Glycerin monostearate：Caprylic/capric triglyceride：Enoxolone-phosphatide spreads out
It is biological：Soybean lecithin for injection：Polyoxyethylene 40 Strearate=1：28~30：12~13：6~7：9~
10：31~32.Most preferably curcumin：Glycerin monostearate：Caprylic/capric triglyceride：Enoxolone-phosphatide derives
Thing：Soybean lecithin for injection：Polyoxyethylene 40 Strearate=1：29：12.5：6.2：9.1：32.
The preparation method of the curcumin long-circulating nanoliposome carrier of enoxolone mediation of the present invention, its feature exists
In：In the synthetic method of the enoxolone-phospholipid derivative, the mol ratio of GA, DCC, NHS three are 1.5:1.2:1.2.
The preparation method of the curcumin long-circulating nanoliposome carrier of enoxolone mediation of the present invention, its feature exists
In：In the synthetic method of the enoxolone-phospholipid derivative, DSPE-PEG2000-NH2, GA, DCC, NHS mol ratio be 1：
1.5:1.2:1.2。
The preparation method of the curcumin long-circulating nanoliposome carrier of enoxolone mediation of the present invention, its feature exists
In：The addition of enoxolone-phospholipid derivative is 5~15% (W/W) of total lipid consumption, wherein most preferably 10% (W/W),
By experimental results demonstrate, when enoxolone-phospholipid derivative addition be total lipid consumption 10% when, gained nanometer fat
Matter carrier has most strong tumor cytotoxicity effect.
The preparation method of the curcumin long-circulating nanoliposome carrier of enoxolone mediation of the present invention, its feature exists
In the optimal preparation technology of the curcumin long-circulating nanoliposome carrier of the enoxolone mediation is as follows：
GA, DCC, NHS three is taken to be dissolved in 10mL water removal dichloromethane, reaction activation 3h under normal temperature；Separately take 1mmol
DSPE-PEG2000-NH25mL dichloromethane solution is dissolved in, is added dropwise in above-mentioned activator, room temperature under nitrogen protection is lower to react
48h；The accessory substance of reaction is filtered to remove, the extraction of ice ether is added, removes unreacted GA in supernatant, deposit is mesh
Product is marked, DMF is fitted into bag filter the 72h that dialyses after redissolving, freeze-drying obtains enoxolone-phospholipid derivative；
Weigh the glycerin monostearate of recipe quantity, caprylic/capric triglyceride, enoxolone-phospholipid derivative, injection
With soybean lecithin and curcumin heating water bath to 75 DEG C, adding 5ml absolute ethyl alcohols dissolves it, and rotary evaporation removes anhydrous second
Alcohol obtains well mixed melting behaviors as oil phase, and the wherein addition of enoxolone-phospholipid derivative is total lipid consumption
10%(W/W)；The Strearate of Polyoxyethylene 40 of recipe quantity are weighed, water for injection are added, after ultrasonic agitation is uniform
Heating water bath is used as aqueous phase to 75 DEG C；In 3000rmin-1Aqueous phase is added dropwise in synthermal oil phase under magnetic agitation, is added dropwise
Speed control is in 10mLmin-1；Constant temperature stirs 5min, and colostrum is made, while hot divides colostrum ultrasonic cell disruptor again
Dissipate, ultrasonic power 400W, ultrasonic 1s, interval 1s, time 5min, after 0.22 μm of filtering with microporous membrane, moisturizing to 10mL, frozen water
The rapid cooling and solidifying of bath, produces the curcumin long-circulating nanoliposome carrier of mediation.
The present invention adds long-circulation fat material and is prepared for curcumin on the basis of curcumin nano-lipid carrier prescription
Long-circulating nanoliposome carrier, the hydrophily on nanoparticle surface is added using the PEG hydrated sheaths formed, internal phagocytosis is reduced
Cell is to its phagocytosis, so as to extend internal circulation time, makes it have long circulating performance.
Brief description of the drawings
The process route chart of Fig. 1 enoxolones (GA)-phospholipid derivative synthesis；
Fig. 2 GA infrared spectrums；
Fig. 3 DSPE-PEG2000-NH2Infrared spectrum；
Fig. 4 DSPE-PEG2000- GA infrared spectrums；
Fig. 5 GA nuclear magnetic resonance maps；
Fig. 6 DSPE-PEG2000-NH2Nuclear magnetic resonance map；
Fig. 7 DSPE-PEG2000- GA nuclear magnetic resonance maps；
Fig. 8 nano-lipid carrier configuration of surface figures（Wherein a：Cur-NLC；b：Cur-PEG-NLC；c：Cur-GA 10%-
PEG-NLC）；
Fig. 9 nano-lipid carrier Zeta potential measurement results（A：Cur-NLC；B：Cur-PEG-NLC；C：Cur-GA10%-
PEG-NLC）；
Figure 10 difference target head amount nano-lipid carrier intake comparison diagrams；
Intake of Figure 11 HepG2 cells to the free medicine of curcumin and each preparation；
Figure 12 cell inhibitory rate experimental result pictures.
Embodiment
Embodiment 1
Enoxolone-phospholipid derivative（GA-PEG2000-DSPE）Synthesis：
GA, DCC, NHS three is taken to be dissolved in 10mL water removal dichloromethane, wherein GA is 1.5mmol, three's mol ratio is
1.5:1.2:1.2, reaction activation 3h under normal temperature.Separately take 1mmolDSPE-PEG2000-NH25mL dichloromethane solution is dissolved in, by
It is added dropwise in above-mentioned activator, the lower reaction 48h of room temperature under nitrogen protection.The accessory substance of reaction is filtered to remove, the extraction of ice ether is added,
Unreacted GA in supernatant is removed, deposit is target product, and DMF is fitted into bag filter the 72h that dialyses after redissolving, and freezes
It is dried to obtain product.Process route is shown in Fig. 1.
FTIR is characterized：Reactant GA, DSPE-PEG are determined respectively with the type infrared spectrometers of Bruker vector 222000-
NH2With polymerizate DSPE-PEG2000- GA infrared spectrum, method is solid sample and potassium bromide powder mixed grinding, progress
Tabletting, is then directly tested, and scanning times are 256 times, and resolution ratio is 2cm-1.Experimental result is shown in Fig. 2,3,4.
In GA collection of illustrative plates ,-OH ,-CH2,-CH3、O-CH2, C=C feature peak position be respectively 3438cm-1、2926cm-1、
1384cm-1、1177cm-1、1663cm-1。DSPE-PEG2000In-NH2 collection of illustrative plates, N-C=O ,-CH2、-CH3、O-CH2, C=C spy
Levy peak position respectively 1739cm-1、2917cm-1、1359cm-1、1113cm-1、1669cm-1.The characteristic absorption peak figure of amido link is
The identification mark peak of product infrared absorpting light spectra in building-up process.End-product GA-PEG2000In-DSPE collection of illustrative plates, 1741cm-1、1645cm-1、1354cm-1Appearance show N-C=O enhancing.
H-NMR is characterized：Using Bruker ARX-300 nuclear magnetic resonance chemical analysers to reactant GA, DSPE-PEG2000-NH2With
Polymerizate DSPE-PEG2000- GA structure and composition is carried out1H-NMR is tested.Test frequency is 300M and 600M, test solvent
For CDCl3.Experimental result is shown in Fig. 5,6,7.
In GA collection of illustrative plates, δ 0.75-1.5ppm are the chemical shift of methyl proton in GA, the characteristic absorption peak position for being GA.
DSPE-PEG2000-NH2Collection of illustrative plates in, at δ 1.253ppm be-NH2It is-CH in PEG at-characteristic absorption peak, δ 3.64ppm2-
Characteristic peak.Synthetic product DSPE-PEG2000In-GA collection of illustrative plates, the characteristic absorption peak containing GA at δ 0.75-1.5ppm, in δ
- NH- characteristic absorption peak is remained at 1.254ppm, while remaining at δ 3.64ppm-CH in PEG2- characteristic absorption peak.
Embodiment 2
（One）, curcumin nano-lipid carrier（Cur-NLC）Preparation：
Raw material：
Weigh glycerin monostearate, caprylic/capric triglyceride, soybean lecithin for injection and the curcumin of recipe quantity
Heating water bath is to 75 DEG C, and adding 5ml absolute ethyl alcohols dissolves it, and rotary evaporation removes absolute ethyl alcohol and obtains well mixed melting
It is used as oil phase；The Strearate of Polyoxyethylene 40 of recipe quantity are weighed, water for injection is added, ultrasonic agitation is uniform
Heating water bath is used as aqueous phase to 75 DEG C afterwards；In 3000rmin-1Aqueous phase is added dropwise in synthermal oil phase under magnetic agitation, dripped
Acceleration Control is in 10mLmin-1；Constant temperature stirs 5min, and colostrum is made, while hot divides colostrum ultrasonic cell disruptor again
Dissipate, ultrasonic power 400W, ultrasonic 1s, interval 1s, time 5min, after 0.22 μm of filtering with microporous membrane, moisturizing to 10mL, frozen water
The rapid cooling and solidifying of bath, produces Cur-NLC dispersion liquids.
（Two）, PEG modification curcumin nano-lipid carrier（Cur-PEG-NLC）Preparation：
In the case where curcumin nano-lipid carrier technique and other prescriptions are constant, the another total lipid that adds is used in oil phase
Measure 15% (W/W) DSPE-PEG2000, finally prepare Cur-PEG-NLC.
（Three）, GA mediation curcumin long-circulating nanoliposome carrier preparation：
In the case where curcumin nano-lipid carrier technique and other prescriptions are constant, the another total lipid that adds is used in oil phase
5%, 10%, 15% (W/W) of amount enoxolone-phospholipid derivative（It is made by embodiment 1）, finally prepare Cur-GA5%-PEG-
The curcumin long circulating of tri- kinds of mediations containing different target head amount GA of NLC, Cur-GA10%-PEG-NLC, Cur-GA15%-PEG-NLC
Nano-lipid carrier.
（1）, preparation outward appearance：
Nano-lipid carrier dispersion liquid Cur-NLC, Cur-PEG-NLC, Cur-GA5%-PEG-NLC, Cur-GA10%-PEG-
NLC, Cur-GA15%-PEG-NLC outward appearance are yellow clear solution, and have obvious opalescence.Visually observe have no it is insoluble into
Divide or block aggregate, turmeric cellulose crystal is not observed under ordinary optical microscope.
（2）, morphologic observation：
Using the configuration of surface of transmission electron microscope observing nano-lipid carrier.Nano-lipid carrier dispersion liquid is taken, distilled water is dilute
Release to suitable concentration, take a small amount of dropwise addition on the copper mesh of covering carbon film, then 2% phosphotungstic acid aqueous solution progress negative staining is added dropwise, it is natural
After drying, its form is observed under transmission electron microscope, Fig. 8 is as a result seen.Because the curcumin that the enoxolone of different target head amounts is modified is received
Mizhi matter diameter of carrier is more or less the same, and only provides Cur-GA10%-PEG-NLC configuration of surface figure.
（3）, particle size determination：
Five kinds of nano-lipid carriers are determined using Malvern Zetasizer Nano 2S-90 laser particles analyzers respectively
Particle size and distribution.Experimental result is shown in Table 1.
Each nano-lipid carrier Average Particle Diameters of table 1
The particle diameter of the curcumin nano-lipid carrier of PEG modifications is significantly greater than curcumin nano-lipid carrier, and with conjunction
Into the addition of phospholipid derivative part, particle diameter slightly has increase, substantially in the range of 123~132nm, changes unobvious.
（4）, Zeta potential determine：
Five kinds of nano-lipid carriers are determined using Malvern Zetasizer Nano 2S-90 laser particles analyzers respectively
Zeta potential（Fig. 9）, measurement result is shown in Table 2.
The average Zeta values of each nano-lipid carrier of table 2
The curcumin long-circulating nanoliposome carrier that the curcumin nano-lipid carrier and enoxolone of PEG modifications are mediated
Zeta potential absolute value is slightly below common curcumin nano-lipid carrier, but changes unobvious.The nano-lipid of different target head amounts
Carrier Zeta potential is substantially unchanged.
（5）, envelop rate and drugloading rate：
Three crowdes of Cur-NLC, Cur-PEG-NLC, Cur-GA5%-PEG-NLC, Cur- are made by the present embodiment preparation technology
GA10%-PEG-NLC, Cur-GA15%-PEG-NLC, determine its average envelop rate be respectively 93.48%, 97.12%, 95.31%,
93.11%th, 90.06%, drugloading rate is 2.25%, 2.34%, 2.30%, 2.24%, 2.17%.
（6）, stability
Five kinds of nano-lipid carriers by best prescription and technique preparation are kept in dark place in 4 DEG C of refrigerators, the 1st, 5,
10th, 15 days when take out respectively, using the envelop rate of micro-column centrifugal determination nano-lipid carrier, carried while investigating nano-lipid
The size distribution situation of body, observation size distribution changes with time trend.As a result show, Cur-NLC envelop rates in 15 days
With size distribution all without significant changes, preferably, its stability of other four kinds of carriers is in a slight decrease for stability, 4 DEG C of lucifuge storages 10
It is more stable.
Embodiment 3
The cell in vitro of the curcumin long-circulating nanoliposome carrier of enoxolone mediation is evaluated：
Reagent and material：
Nutrient solution is prepared：
Phosphate buffer (PBS)：Weigh respectively disodium hydrogen phosphate 1.44g, potassium dihydrogen phosphate 0.24g, sodium chloride 8.0g,
Potassium chloride 0.2g, is dissolved in 700mL redistilled waters, is mixed and stirred for uniform.Use 1molL-1HCl adjust PH to 7.4, redistilled water
1000mL is settled to, concentration is made into for 0.01molL-1Phosphate buffer (pH7.4).Autoclaving 30min, packing, 4
DEG C storage is standby.
DMEM nutrient solutions：DMEM dry powder 10.4g are taken, are dissolved with a small amount of double distilled water, sodium acid carbonate 3.7g is added,
HEPES2.0g, 100units/mL penicillin, 100units/mL streptomysins, which are mixed, dissolves it, and redistilled water is settled to
1000mL.Use 1molL-1HCl adjust pH to 7.0, packing degerming through 0.22 μm of membrane filter, 4 DEG C of storages are standby.Use
It is 10% that preceding addition hyclone, which is adjusted to concentration,.
Digestive juice：EDTA 0.02g, trypsase 0.25g are taken respectively, are dissolved in 100mL PBS (pH7.4), super-clean bench
Lower degerming through 0.22 μm of membrane filtration, packing, 4 DEG C of storages are standby.
Cell culture：
Cell recovery and culture：The HepG2 cells frozen are taken out from liquid nitrogen, are put in rapidly in 37 DEG C of water-baths, quickly
Melt and thaw.Cell suspension is transferred in centrifuge tube under super-clean bench, cell culture fluid is added to 10mL, slight piping and druming makes thin
1000rmin after born of the same parents are scattered-15min is centrifuged, supernatant is abandoned, nutrient solution is added in right amount, it is single equal that piping and druming cell is dispersed into it
Even cell liquid, is inoculated in blake bottle, in relative humidity 90%, 37 DEG C, 5%CO2Cultivated in incubator, training is changed every other day
Nutrient solution, it is good and keep exponential phase to cell state, it is standby.
Passage：When in blake bottle cell growth area to blake bottle culture face 75%~80% when, discard nutrient solution,
3mL PBS are added, are cleaned 2 times.400 μ L pancreatin digestive juices are added into blake bottle, is allowed to outwell after fully soaking bottle wall, only uses
Remaining pancreatin is digested.It is placed in incubator after 2~3min, takes out and be inverted in micro- Microscopic observation, work as space between cells
Increase, cellular contraction becomes bowlder, add the DMEM nutrient solutions containing 10% hyclone, it is slight to blow and beat bottle wall for several times, to attached cell
Nutrient solution is fully entered, continues slight blow and beat to it and is separated into individual cells.Cell suspension is equally divided into 2~3 bottles of cultures, mended
Sufficient fresh medium is to 6mL.
（1）, intake of the HepG2 cells to the curcumin preparation of different target head amounts
After exponential phase HepG2 cell lines pancreatin is digested, with the DMEM cell culture fluid systems added with 10% hyclone
Into cell suspension.After counting, with every hole 106Individual cell concentration is inoculated in 6 orifice plates, and cell is placed in into 5%CO2, 37 DEG C, saturation it is wet
24h is cultivated in incubator under the conditions of degree.Culture plate is taken out, nutrient solution in hole is carefully siphoned away, then be separately added into containing Cur-
GA5%-PEG-NLC, Cur-GA10%-PEG-NLC, Cur-GA15%-PEG-NLC cell culture fluid, every kind of 3 multiple holes of preparation.
It is placed in constant temperature CO2In incubator 37 DEG C be incubated 0.5 respectively, 1,2,4, after 6 hours, under the conditions of lucifuge, suction out liquid in hole, will train
The cell supported in plate is cleaned 2 times with the pH7.4 phosphate buffers (PBS) of 4 DEG C of precoolings, adds trypsin digestion cell.Use 2.5mL
Absolute ethyl alcohol is collected cell and diluted, and is transferred in 4mL EP pipes, Probe Ultrasonic Searching under ice-water bath（Work 1s, interval 1s, power
40W, ultrasonic time 5min）.It is put in again in supercentrifuge, 15000rmin-1, 10min is centrifuged, supernatant is taken in fluorescence point
Detected under light photometer and record fluorescent value, and bring standard curve into and calculate content.Experimental result is shown in Figure 10.
As seen from Figure 10, the curcumin long-circulating nanoliposome carrier intake of enoxolone mediation prepared by three kinds of target head amounts
Amount is more, when addition is 10%, and at most, selection Cur-GA10%-PEG-NLC preparation progress is next for cancer cell intake
Step is investigated.
（2）, HepG2 cells dissociate the intake of medicine and each preparation to curcumin：
The above method is operated, and investigates HepG2 cells to Cur solution, Cur-NLC, Cur-PEG-NLC, Cur-GA10%-
PEG-NLC intake situation.Experimental result is shown in Figure 11.
Cellular uptake experiment shows that HepG2 cells are equal to the intake of free drug group under same concentrations and each preparation group
With time dependence, with the extension of time, intake increase, intake increase is slow after 6 hours, illustrates cell to preparation
Intake there is saturability, belong to active absorption process.The HepG2 cellular uptake trend for being incubated 6h altogether is descending for Cur-
GA10%-PEG-NLC>Cur-NLC>Cur-PEG-NLC>Cur solution.Preparation group Cur is contained in nano-lipid carrier by interior
The effect of gulping down enters inside tumor cells, and cellular uptake amount is above Cur solution groups, increased with incubation time, and preparation group and Cur are molten
The difference of liquid group cellular uptake amount also becomes big therewith.In three groups of preparations, Cur-GA10%-PEG-NLC preparation group cellular uptakes amount is most
Height, and Cur-PEG-NLC preparation group cellular uptake amounts are relatively relatively low.Cur-GA10%-PEG-NLC is suitably close due to surface modification
GA and HepG2 the cell surface receptor interaction of degree, cause cellular uptake amount to dramatically increase.In addition, different target head volume preparations
The cellular uptake of group all has time dependence, with the increase of time lengthening intake.At 0.5h, 1h time point, three kinds of preparation groups
Intake quite, is incubated after 2h, Cur-GA10%-PEG-NLC preparation group cellular uptake amounts are higher than other two preparation groups.
From experimental result, not the higher the better for nano-lipid carrier surface modification GA amounts, and the receptor number of cell surface is limited, mistake
It is many ligand modified not but not to improve the efficiency that part is combined with acceptor, it is also possible to because of its relatively large steric interference
To the combination of part and acceptor, further raising of the tumour cell to nano-lipid carrier intake is unprofitable to.
（3）, cellulotoxic experiment：
MTT powder is taken to be dissolved in PBS (pH7.4), compound concentration is 5mg/mL MTT solution, through 0.22 μm of filter membrane mistake
Bacterium is filtered out, is dispensed, -20 DEG C of stored protected from light, 2~8 DEG C of refrigerators thaw standby in advance.
When taking HepG2 cell lines in exponential phase, digested with pancreatin, add the DMEM containing 10% hyclone thin
Cell suspension is made in born of the same parents' nutrient solution.After being counted under microscope, 96 orifice plates are inoculated in every 8000 cell concentrations in hole, cell is put
In 5%CO2, 37 DEG C, cultivate 24h in the incubator under the conditions of saturated humidity.Culture plate is taken out, nutrient solution in hole is carefully removed, then
Concentration containing curcumin is separately added into for 0.2,1,5,10,25,50,100 μ gmL-1Cur solution, Cur-NLC, Cur-PEG-
NLC, Cur-GA10%-PEG-NLC cell culture fluid, 200 μ L, each 4 multiple holes of concentration are added per hole.Wherein, control group is every
Hole adds 200 μ L culture medium solution, and blank well is not added with cell and only adds nutrient solution.Respectively after effect 24h, 48h, 72h, culture is taken out
MTT solution (5 μ gmL are added under the conditions of plate, lucifuge per hole-1, 20 μ l), continue to be placed in and taken out after incubator culture 4h, remove hole
Interior liquid, the μ L of DMSO 150 are added per hole.On constant-temperature table, lucifuge shaking 10min makes sediment fully dissolve.It is engraved in
ELIASA wavelength (λ) is that absorbance (A) is determined at 490nm, records result, according to the following formula cell inhibitory rate.Experiment knot
Fruit sees Figure 12.
Cell inhibitory rate=[(AControl group- AAdministration group)/(AControl group- ABlank group)]×100%
Use IC50Software for calculation processing, obtain respectively free drug and each preparation to HepG2 cytosiies 24h, 48h,
72h IC50Value, the results are shown in Table 3.
The cell inhibitory rate of table 3
Cellulotoxic experiment shows that free drug group and each preparation group are respectively provided with concentration to the inhibiting rate that HepG2 cells are bred
With the dependence of time.Same agents extension over time and the increase of concentration, inhibiting rate and toxicity enhancing to tumour.
The cytotoxicity of 48h and 72h preparation groups is significantly higher than free drug group, and its reason is probably drug encapsulation in nano-lipid carrier
Core in, make insoluble drug release slow, but increase over time, gradually discharge.In preparation group there is enoxolone to modify
Preparation cytotoxicity it is most notable.It can be seen that enoxolone utilizes ligand-receptor binding mechanism, target site is efficiently identified, makes ginger
Flavine is in diseased region concentration, with stronger lethal effect.
The above embodiments merely illustrate the technical concept and features of the present invention, and its object is to allow person skilled in the art
Scholar can understand present disclosure and implement according to this, and it is not intended to limit the scope of the present invention.It is all according to the present invention
The equivalent change or modification that Spirit Essence is made, should all be included within the scope of the present invention.
Claims (8)
1. a kind of curcumin long-circulating nanoliposome carrier of enoxolone mediation, it is characterised in that：The enoxolone mediation
Curcumin long-circulating nanoliposome carrier be by curcumin, glycerin monostearate, caprylic/capric triglyceride, radix glycyrrhizae time
What acid-phospholipid derivative, soybean lecithin for injection and Polyoxyethylene 40Strearate were prepared, wherein,
Raw materials used mass ratio is curcumin：Glycerin monostearate：Caprylic/capric triglyceride：Enoxolone-phosphatide derives
Thing：Soybean lecithin for injection：Polyoxyethylene 40Strearate=1：28~30：12~13：6~7：9~10：
31~32；The addition of enoxolone-phospholipid derivative is 5~15% (W/W) of total lipid consumption, and enoxolone-phosphatide spreads out
Biological synthetic method is：
GA, DCC, NHS three is taken to be dissolved in water removal dichloromethane, 3~4h of reaction activation under normal temperature；Separately take DSPE-PEG2000-NH2
Dichloromethane solution is dissolved in, is added dropwise in above-mentioned activator, 48~50h of the lower reaction of room temperature under nitrogen protection；It is filtered to remove reaction
Accessory substance, add the extraction of ice ether, remove unreacted GA in supernatant, deposit is target product, after DMF redissolves
It is fitted into bag filter the 72h that dialyses, freeze-drying obtains enoxolone-phospholipid derivative.
2. the preparation method for the curcumin long-circulating nanoliposome carrier that enoxolone described in a kind of claim 1 is mediated, it is special
Levy and be, preparation process is as follows：
By lipid, enoxolone-phospholipid derivative and curcumin heating water bath to 75 DEG C, adding absolute ethyl alcohol dissolves it, revolves
Turn evaporation removing absolute ethyl alcohol and be used as oil phase；Water for injection is added in the Strearate of Polyoxyethylene 40, ultrasound
Heating water bath is used as aqueous phase to 75 ± 2 DEG C after stirring；Aqueous phase is added dropwise in synthermal oil phase under magnetic agitation, constant temperature
5min is stirred, colostrum is made, while hot with ultrasonic cell disruptor ultrasonic disperse 5min, 0.22 μm of miillpore filter, room temperature ice is crossed
Water-bath is cooled and solidified, and obtains the curcumin long-circulating nanoliposome carrier dispersion liquid of enoxolone mediation.
3. the preparation method of the curcumin long-circulating nanoliposome carrier mediated according to enoxolone described in claim 2, it is special
Levy and be：In the 5min with ultrasonic cell disruptor ultrasonic disperse, ultrasonic 1s, interval 1s.
4. the preparation method of the curcumin long-circulating nanoliposome carrier mediated according to enoxolone described in claim 2, it is special
Levy and be：When aqueous phase is added dropwise in synthermal oil phase, rate of addition is 10mLmin-1。
5. the preparation method of the curcumin long-circulating nanoliposome carrier mediated according to enoxolone described in claim 2, it is special
Levy and be：Raw materials used mass ratio is curcumin：Glycerin monostearate：Caprylic/capric triglyceride：Enoxolone-phosphorus
Fat derivative：Soybean lecithin for injection：Polyoxyethylene 40Strearate=1：29：12.5：6.2：9.1：32.
6. the preparation method of the curcumin long-circulating nanoliposome carrier mediated according to enoxolone described in claim 2, it is special
Levy and be：In the synthetic method of the enoxolone-phospholipid derivative, the mol ratio of GA, DCC, NHS three are 1.5:1.2:
1.2。
7. the preparation method of the curcumin long-circulating nanoliposome carrier mediated according to enoxolone described in claim 2, it is special
Levy and be：In the synthetic method of the enoxolone-phospholipid derivative, DSPE-PEG2000-NH2, GA, DCC, NHS mol ratio
For 1：1.5:1.2:1.2.
8. the preparation method of the curcumin long-circulating nanoliposome carrier mediated according to enoxolone described in claim 2, it is special
Levy and be, the specific preparation process of the curcumin long-circulating nanoliposome carrier of the enoxolone mediation is as follows：
GA, DCC, NHS three is taken to be dissolved in 10mL water removal dichloromethane, reaction activation 3h under normal temperature；Separately take 1mmol DSPE-
PEG2000-NH25mL dichloromethane solution is dissolved in, is added dropwise in above-mentioned activator, the lower reaction 48h of room temperature under nitrogen protection；Cross
The accessory substance of dereaction is filtered out, the extraction of ice ether is added, unreacted GA in supernatant is removed, deposit is target production
Thing, DMF is fitted into bag filter the 72h that dialyses after redissolving, freeze-drying obtains enoxolone-phospholipid derivative；
Weigh the glycerin monostearate of recipe quantity, caprylic/capric triglyceride, enoxolone-phospholipid derivative, injection big
Beans lecithin and curcumin heating water bath are to 75 DEG C, and adding 5ml absolute ethyl alcohols dissolves it, and rotary evaporation removes absolute ethyl alcohol and obtained
To well mixed melting behaviors as oil phase, the wherein addition of enoxolone-phospholipid derivative is the 10% of total lipid consumption
(W/W)；The Polyoxyethylene 40Strearate of recipe quantity are weighed, water for injection, water-bath after ultrasonic agitation is uniform is added
75 DEG C are heated to, aqueous phase is used as；In 3000rmin-1Aqueous phase is added dropwise in synthermal oil phase under magnetic agitation, rate of addition
Control is in 10mLmin-1；Constant temperature stirs 5min, and colostrum is made, while hot by colostrum ultrasonic cell disruptor redisperse, surpasses
Acoustical power 400W, ultrasonic 1s, interval 1s, time 5min, after 0.22 μm of filtering with microporous membrane, moisturizing to 10mL, ice-water bath is fast
Speed cooling and solidifying, produces the curcumin long-circulating nanoliposome carrier of mediation.
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