CN110464708A - A kind of spirulina nanometer formulation and preparation method thereof - Google Patents

A kind of spirulina nanometer formulation and preparation method thereof Download PDF

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CN110464708A
CN110464708A CN201910663848.5A CN201910663848A CN110464708A CN 110464708 A CN110464708 A CN 110464708A CN 201910663848 A CN201910663848 A CN 201910663848A CN 110464708 A CN110464708 A CN 110464708A
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spirulina
taxol
water
parts
preparation
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王冰月
白莉
王�琦
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GUANGZHOU JIAYUAN PHARMACEUTICAL TECHNOLOGY Co Ltd
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GUANGZHOU JIAYUAN PHARMACEUTICAL TECHNOLOGY Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/748—Cyanobacteria, i.e. blue-green bacteria or blue-green algae, e.g. spirulina
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/113—Multiple emulsions, e.g. oil-in-water-in-oil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The present invention relates to contain spirulina-taxol composite nanostructure lipid vector preparation and preparation method thereof.The technical solution adopted is that: it is made by weight ratio including following raw material: 2-10 parts of spirulina, 2-10 parts of taxol, 30-150 parts of solid lipid, 10-50 parts of liquid fatty, 30-300 parts of fat-soluble emulsifier, 20-250 parts of water soluble emulsifier, 90-550 parts of surfactant.Two medicine preparations at composite nanostructure lipid carrier, are optimized and are investigated to its formulation and technology and physicochemical property by the present invention, prepare the nanostructure carrier of good properties, the multidrug resistance effect with reversing tumor cell.

Description

A kind of spirulina nanometer formulation and preparation method thereof
Technical field
The invention belongs to Chinese pharmacology fields, and in particular to a kind of composite nanostructure lipid for containing taxol and spirulina Carrier and preparation method thereof.
Background technique
Neighbouring tissue and organ can be invaded and be destroyed to the cell of malignant tumour, moreover, cancer cell can enter blood System and lymphatic system are transferred to other organs from primary position, form new tumour.With the increasing of chemotherapeutics usage amount Adding, cancer cell can automatically pump out chemotherapeutics, and cause the effective dose for reaching target cell to reduce, is unable to reach therapeutic dose, It may be said that tumor multi-medicine drug-resistant (MDR) is the main reason for causing chemotherapy of tumors to fail, therefore reverse multiple drug resistance of tumor drug And the project that those skilled in the art constantly studies.
Spirulina, also known as " arthrospira ".Cyanophyceae, Oscillariaceae.Frond is the not branch filamentous of single-row cell composition, glue Matter sheath without or only very thin sheath, and regular helical form, to form hormocystangium breeding.Without heterocyst and akinete.Nutrition Abundant, protein content is up to 60%-70%, has the toxicity for mitigating cancer radiation, chemotherapy, improves immune function, association Chemotherapeutics reversing multiple medicine resistance of tumor cells is helped to act on.Taxol is mainly derived from the drying of taxaceae plant yew Root, branches and leaves and bark are a kind of novel breast cancers, and the mechanism for embodying antitumor action is by promoting micro-pipe Albumen polymerize to inhibit depolymerization, then maintains the stabilization of tubulin, inhibits the mitosis of cell, inhibits the increasing of cancer cell It grows.Experiment in vitro proves that taxol has significant radiosensitizing effect, but the outlet effect of cancer cell is big, is also easy to produce Japanese yew Alcohol drug resistance.Therefore need to select a kind of suitable preparation formulation to reduce the exocytosis of taxol, enhanced sensitivity is antitumor Effect reduces the toxic side effect of taxol.
Nano structured lipid carrier (NLC) is that drug is wrapped in rouge using solid natural or synthetic lipid as carrier In core, solid-state micelle drug delivery system is made, this dosage form, which is compared, can embody preferably slow controlled release effect with other nano-formulations Fruit, while toxicity can be reduced, improve the drugloading rate of drug.
Chinese invention patent CN201710146446.9 discloses " a kind of spiral algae sheet and preparation method thereof " the invention piece Agent prescription is spirulina powder, glycerol, magnesium stearate, disintegrating agent, D-sorbite etc., and spirulina is prepared into ordinary tablet in the invention Agent；Chinese invention patent CN201710951391.9 discloses " a kind of Docetaxel pharmaceutical composition and preparation method thereof ", It is purple using-ten dihydroxystearic acid ester of new non-ionic surfactants polyethylene glycol, polyethylene glycol 400, citric acid and polyenoid China fir alcohol is prepared into a kind of Docetaxel pharmaceutical composition.Spirulina and paclitaxel composition are had no at present, have no spirulina It is prepared into nano structured lipid carrier with paclitaxel composition, has no spirulina and paclitaxel composition and its nanostructured lipid Carrier application report in reverse multiple drug resistance of tumor.
Summary of the invention
It is an object of the invention to which spirulina and taxol are prepared into composite nanostructure lipid carrier, to its prescription work Skill and physicochemical property are optimized and are investigated, and prepare the nanostructure carrier of good properties, for further research reversing tumor The multidrug resistance effect of cell provides basis.
The technical solution adopted by the present invention is that: one kind containing spirulina-taxol composite nanostructure lipid vector preparation, It is made by weight ratio including following raw material:
The solid lipid is glycerin monostearate, isopropyl palmitate (different monooctyl ester), one in Compritol 888 ATO Kind or two kinds or more of combination.
The liquid fatty is one of monoglyceride, isopropyl myristate, isopropyl laurate or oleic acid or two kinds Above mixing.
The fat-soluble emulsifier is Emulsifier EL-60 EL.
The water soluble emulsifier is sodium glycocholate, natrii tauroglycocholas, one kind of poloxamer or NaTDC or two kinds Above mixing.
The poloxamer is poloxamer188, Pluronic/Lutrol F 108 or PLURONICS F87；It is preferred that Pluronic/Lutrol F 108.
The surfactant is Tween 80.
It is above-mentioned contain spirulina-taxol composite nanostructure lipid vector preparation the preparation method is as follows:
1) taxol, solid lipid, liquid fatty and fat-soluble emulsifier are sequentially added in appropriate organic solvent, 65- It is dissolved by heating in 75 DEG C of water-baths, ultrasonic (200~400W, 2~5min) dispersion mixes, as organic phase；
The organic solvent is dehydrated alcohol；
2) spirulina and water soluble emulsifier are dissolved in appropriate water for injection, are dissolved by heating in 65-75 DEG C of water-bath, stirred Uniformly, as inner aqueous phase；
3) surfactant is dissolved in appropriate water for injection, dissolves by heating, stirs evenly in 65-75 DEG C of water-bath, as Outer aqueous phase；
4) under 500-1200r/min stirring, inner aqueous phase is slowly injected into organic phase, stirring 15-20min is formed just Cream；
5) under 500-1200r/min stirring, colostrum is slowly injected into outer aqueous phase, emulsification is sufficiently stirred and forms emulsion；
6) it is poured into rapidly after emulsion being concentrated in ice water (2-4 DEG C), low-temperature setting 1-2h, after filtering with microporous membrane Liquid phase contains spirulina-taxol composite nanostructure lipid vector preparation；
Preferably, the micro porous filtration is to use aperture for 0.22 μm or 0.45 μm of water system membrane filtration；
7) to liquid phase contain in spirulina-taxol composite nanostructure lipid vector preparation be added freeze drying protectant it is molten Liquid, freeze-drying, obtains solid phase and contains spirulina-taxol composite nanostructure lipid vector preparation.
Preferably, the freeze drying protectant is the mannitol solution that mass percent is 10%.
The beneficial effects of the present invention are: the present invention carries taxol and both drugs of spirulina using nanostructured lipid Body (NLC) is wrapped up, and a kind of novel nano drug delivery system is made, and the liquid fatty incompatible with solid lipid is added to solid Incomplete crystal form is formed in body fat matter, effectively prevent the leakage of drug.It is prepared by the method for the present invention to contain spirulina- The composite nanostructure lipid vector preparation (spirulina-taxol-NLC) of taxol is round or similar round entity ball, partial size For (92.7 ± 1.2) nm, polydispersity coefficient is 0.18 ± 0.08, and Zeta potential is (- 18.06 ± 0.24) mV, taxol encapsulating Rate is up to 85% or more, and spirulina encapsulation rate is up to 72% or more, the spirulina-taxol-NLC extracorporeal releasing experiment result Show that it meets Bidirectional power model, taxol and the substantially all release in 20h of spirulina drug solution are complete, spiral There is phenomenon of burst release in preceding 5h in spirulina in algae-taxol-NLC nano-solution, and then slow controlled release is preferable for taxol, therefore this hair Bright contains spirulina-taxol composite nanostructure lipid vector preparation with very good slow controlled release properties.The present invention, By the way that taxol and spirulina are prepared into NLC, bioavilability not only can be improved, but also to the multidrug resistance of tumour cell Property has good reversing effect.
For the present invention by the way that taxol and spirulina are prepared into composite nanostructure lipid carrier, it is resistance to that spirulina plays reverse The auxiliary therapeutic action of medicine, fat-soluble taxol play main antineoplastic treatment function, and being used in combination for the two can be effective The effective accumulation of drug in the cell is improved, therapeutic effect is promoted, simultaneously because the advantage of its dosage form, can slow down drug Excretion, reaches slow-releasing and controlled-releasing action.The two is carried out compound preparation using simple preparation process by the present invention, and obtain 70% with On encapsulation rate, partial size is convenient for industrialized production in 98nm or so, simple process.
Detailed description of the invention
Fig. 1 is liquid chromatogram, in which: Figure 1A is the chromatogram of blank NLC in embodiment 1；Figure 1B is spiral shell in embodiment 1 Revolve the chromatogram of algae solution；Fig. 1 C is the chromatogram of paclitaxel solution in embodiment 1；Fig. 1 D is rotation algae-taxol in embodiment 1 The chromatogram of mixed solution；Fig. 1 E is spirulina in embodiment 1-taxol-NLC solution chromatogram.
Fig. 2 is spirulina-taxol-NLC transmission electron microscope picture prepared by embodiment 1.
Fig. 3 is spirulina-taxol-NLC grain size distribution prepared by embodiment 1.
Fig. 4 is spirulina-taxol-NLC Zeta potential distribution map prepared by embodiment 1.
Fig. 5 is spirulina in embodiment 1, taxol and spirulina-taxol-NLC drug release patterns in vitro.
Specific embodiment
Embodiment 1 contains spirulina-taxol composite nanostructure lipid vector preparation (spirulina-taxol-NLC)
(1) the preparation method is as follows:
1) by 5mg taxol, 30mg solid lipid (glycerin monostearate), 25mg liquid fatty (monoglyceride) and 150mg fat-soluble emulsifier (Emulsifier EL-60 EL) sequentially adds in 20ml dehydrated alcohol, dissolves by heating in 70 DEG C of water-baths, Ultrasonic (200~400W, 2~5min) dispersion mixes, as organic phase；
2) 5mg spirulina, 150mg water soluble emulsifier (PLURONICS F87) are dissolved in 5ml water for injection, 70 DEG C of water It dissolves by heating, stirs evenly in bath, as inner aqueous phase；
3) 200mg surfactant (Tween 80) is dissolved in 15ml water for injection, is dissolved by heating in 70 DEG C of water-baths, stirred Uniformly, as outer aqueous phase；
4) under 1200r/min stirring, 5ml inner aqueous phase is slowly injected into 20ml organic phase, stirring 20min is formed just Cream；
5) under 1200r/min stirring, above-mentioned colostrum is slowly injected into 15ml outer aqueous phase, stirring and emulsifying forms emulsion；
6) when emulsion is concentrated by evaporation to about 10ml, emulsion is poured into rapidly in ice water (2-4 DEG C), low-temperature setting 1.5h, The water system membrane filtration of 0.45um obtains liquid phase spirulina-taxol-NLC；
7) 10% mannitol solution 2ml is added into 5ml liquid phase spirulina-taxol-NLC, is freeze-dried, obtains solid phase spiral shell Revolve algae-taxol-NLC.
(2) testing result
1. specificity is tested
Chromatographic column: YMC 2 pack CN 150mm × 6mm i dl, 5 μm；Mobile phase: methanol-water (45: 55)；Flow velocity: 0.8mL/min；Ultraviolet detection wavelength: 227nm；Column temperature: 25 DEG C；Sample volume: 10 μ L.
Method: it is blank NLC (taxol and spirulina is not added) simultaneously by above-mentioned steps, takes blank NLC and spiral respectively Algae-taxol-NLC is placed in right amount in 10ml volumetric flask, and 5ml mobile phase ultrasound is added and is demulsified within 15 minutes, uses after being placed in room temperature It is spare to cross 0.45 μm of organic filter membrane for mobile phase constant volume.It weighs a certain amount of taxol control product and spirulina reference substance is each appropriate, It is respectively placed in 10ml volumetric flask, adds ultrasound after 5ml mobile phase to make sufficiently to dissolve for about 10 minutes, be placed in room temperature, constant volume, mistake 0.45 μm of organic filter membrane saves backup.Precision weighs 5mg taxol control product and 5mg spirulina reference substance respectively, same to be placed in It in 100ml volumetric flask, is added 20mml mobile phase ultrasound about 10 minutes, places after dissolution to room temperature and constant volume, be configured to 50 μ g/ The spirulina of ml-taxol mixed reference substance solution.Sample introduction, which is distinguished, by five solution that above-mentioned condition configures observes each ingredient Disturbed condition.
As a result: as in figs. 1 a-e.By the visible taxol of Figure 1A-Fig. 1 E, spirulina, blank NLC, spirulina-Japanese yew It is noiseless between alcohol-NLC and spirulina-taxol each sample, separating degree R > 1.5 between adjacent peak.Illustrate detection of the invention Method is feasible.
2. linear relationship is investigated
Method: precision measures taxol control product 1mg and spirulina reference substance 1mg, with being placed in 10ml volumetric flask, adds stream Constant volume after dynamic phase ultrasonic dissolution, is configured to the mother liquor of 100 μ g/ml of concentration, then measures mother liquor respectively in right amount with flowing phase dilution The array of linear strength solution for being 2.0,4.0,8.0,16.0,32.0,50 μ g/ml at concentration surveys each line with above-mentioned HPLC condition Property solution peak area, using peak area as ordinate (Y), reference substance concentration be abscissa (X), carry out linear regression.
As a result: the calibration curve equation Y=549.626X-152.54 (r of taxol2=0.9997)；The standard of spirulina Curvilinear equation Y=5187.1X+40.52 (r2=0.9995), show that taxol and spirulina are linear within the scope of 2~50 μ g/mL Relationship is good.
3. Precision Experiment
Method: taking basic, normal, high three strength solutions that concentration is 4,16,32 μ g/ml in above-mentioned linear solvent, and use is exclusive Property in the measurement of HPLC condition, respectively at being measured in parallel 5 times in 1 day, METHOD FOR CONTINUOUS DETERMINATION 5 days, calculating was in a few days and day to day precision RSD。
It the results are shown in Table 1.Show that precision is good by table 1.
In table 1 day and day to day precision result (n=5)
4. the rate of recovery is tested
Method: taking 5ml blank NLC, is separately added into taxol control product and spirulina reference substance solution 0.8,3.2,6.4ml The sample of basic, normal, high three concentration, 0.45 μm of organic filter membrane are made into mobile phase constant volume after 10ml volumetric flask, ultrasonic emulsion breaking Filtering, 10 μ L sample introductions calculate the rate of recovery.
As a result: result such as table 2.As the result is shown three strength solution rate of recovery of taxol and spirulina 94%~ Between 106%, RSD < 2% shows that this method rate of recovery is good.
2 rate of recovery experimental result (n=3) of table
5. stability experiment
Method: taking concentration in above-mentioned linear solvent is spirulina-paclitaxel solution of 8 μ g/ml, respectively at 0,2,4,6, 12, for 24 hours in sample introduction, calculate RSD.
The results show that peak area RSD of the mixed solution in for 24 hours is respectively 1.82% and 1.29%, respectively less than 2%, Show that mixed solution is good in internal stability for 24 hours.
6. entrapment efficiency determination method
Method: experiment uses its encapsulation rate of ultrafiltration centrifugal determination.Take 4ml spirulina-taxol-NLC solution in ultrafiltration In centrifuge tube, 12000r/min is centrifuged 20min, takes bottom filtrate 10ul in ultra-filtration centrifuge tube to inject high performance liquid chromatograph, obtains To peak area calculated according to linear formula, obtained result is as free drug amount (WF)；Separately take 4.0ml spirulina-Japanese yew Alcohol-NLC solution adds methanol 5ml in 10ml volumetric flask, is demulsified within ultrasound 30 minutes, flowing constant volume is used after placing to room temperature, The organic membrane filtration of 0.45um, takes filtrate 10ul to inject high performance liquid chromatograph, and 10 times of measurement result are used as total dose (WT)。 Then encapsulation rate=(WT-WF)/WT× 100%.
As a result: taxol encapsulation rate is 86.56%, and spirulina encapsulation rate is 79.48%.
7. morphology is investigated
Method: spirulina-taxol-NLC is taken on the copper mesh with carbon film, to be dyed with 2% phosphotungstic acid, dried in the air naturally in right amount Dry be placed under transmission electron microscope is observed.
As a result well dispersed as shown in Fig. 2, from Figure 2 it can be seen that nanoparticle is round or similar round entity bead, substantially without It is adhered.
8. partial size and Zeta potential measurement
Method: taking spirulina-taxol-NLC to be diluted in right amount, is carried out using Zetasizer nanometer laser particle size analyzer The measurement of partial size and current potential.
As a result as shown in Figure 3 and Figure 4, by Fig. 3 and Fig. 4 as it can be seen that the partial size of nanoparticle is 92.97nm, PDI is 0.138 (n =3), current potential is -18.3mV, it is seen that the nano-carrier partial size of preparation is small, is uniformly dispersed, and has preferable stability.
9. the foundation of release profiles
Method: 5ml spirulina-taxol-NLC is taken to be placed in bag filter (molecular weight 10KDa), while phase is respectively configured Bag filter is placed in 200ml and contains 10% Tween 80 by taxol and spirulina solution 5ml with concentration in identical bag filter Buffer salt dissolution medium (pH 7.4) in, revolving speed 80r/min, (37 ± 0.5) DEG C dynamic dialysis, respectively at 0.5,1,2,3,4, 5,6,8,12,24,36h point in time sampling 1m, spare sample introduction after filtering, while adding the dissolution medium of the same race at identical temperature 1ml.Dissolution medium drug concentration is measured using HPLC method, and calculates preparation.
As a result as shown in figure 5, as seen from the figure, carrying out models fitting to it, meeting Bidirectional power equation.Taxol and spiral shell Revolving algae drug solution, completely, spirulina is in preceding 5h memory in spirulina-taxol-NLC nano-solution for release substantially in 20h In burst effect, it is preferable that spirulina then delays controlled release, therefore the nano-carrier has good slow controlled release properties.
10. the research of reverse multidrug cells of resistant tumors strain drug resistance
Hepatoma H22 cells/S is sensitive strain, and HepG2/MDR is multidrug resistance strain, both cell strains are cultivated In the RPMI-1640 culture solution containing 10% fetal calf serum.
Contained Serum preparation: for KM mouse adaptive feeding after 7 days, weighing is randomly divided into high dose according to random numbers table Totally 4 groups of group, middle dose group, low dose group, blank control group, every group 15.
Medication and dosage: spirulina-taxol composite nanostructure lipid high dose group presses 16g/kg gastric infusion, Middle dose group presses 8g/kg gastric infusion, and low dose group presses 4g/kg gastric infusion, and blank control group is with physiological saline stomach-filling.
Above-mentioned 4 groups of mouse take blood after 2.5h is administered, and 3000g, 4 DEG C are centrifuged 10 minutes, separate serum, 56 DEG C of inactivations 35min prepares the serum free culture system liquid of various concentration with RPMI1640.
The HepG2/MDR cell of logarithmic growth phase is with 1 × 105/ ml, HepG2/S is with 0.5 × 105/ ml is inoculated in respectively In 96 well culture plates, 150 holes μ l/.In 37 DEG C, 5%CO2Under the conditions of after overnight incubation, respectively plus the tested drug containing of various concentration Each 100 μ l of serum, zeroing group and control group add the culture solution of respective volume, and every group sets 4 parallel holes.After culture for 24 hours, every hole adds The 10 μ l of MTT solution (except zeroing group) of 5mg/ml, is further cultured for 2h, discards culture solution, and 150 hole μ l/ DMSO is added, is placed in vibration Swing and shaken 10 minutes on device, it is to be crystallized be completely dissolved after, read absorbance after the zeroing of zeroing group at the wavelength 570nm with microplate reader (A) value.
Take the mean of 4 hole A values that cell inhibitory rate, cell inhibitory rate (IR)=[1- (test hole A mean value/right is calculated by formula According to hole A mean value)] × 100%；It calculates IR and finds out half-inhibitory concentration (IC50), the above experiment is repeated 3 times；Reversal index=sky White control group IC50/ medication group IC50, the results are shown in Table 3, and different dosing concentration is 1.35- to HepG2/MDR reversal index 6.18, and be positively correlated in concentration, prompt spirulina-taxol-NLC in the present invention that there is the drug resistance of HepG2/MDR cell Reverse effect.
3 reversal of drug resistance effect of table
Embodiment 2 contains spirulina-taxol composite nanostructure lipid vector preparation (spirulina-taxol-NLC)
The preparation method is as follows:
1) by 5mg taxol, 100mg solid lipid (isopropyl palmitate), 40mg liquid fatty (myristic acid isopropyl Ester) and 100mg fat-soluble emulsifier (Emulsifier EL-60 EL) sequentially add in 20ml dehydrated alcohol, heated in 75 DEG C of water-baths Dissolution, ultrasonic (200~400W, 2~5min) dispersion mixes, as organic phase；
2) 5mg spirulina, 100mg water soluble emulsifier (PLURONICS F87 90mg+ NaTDC 10mg) are dissolved in In 5ml water for injection, dissolves by heating, stir evenly, as inner aqueous phase in 75 DEG C of water-baths；
3) 100mg surfactant (Tween 80) is dissolved in 15ml water for injection, is dissolved by heating in 75 DEG C of water-baths, stirred Uniformly, as outer aqueous phase；
4) under 1000r/min stirring, 5ml inner aqueous phase is slowly injected into 20ml organic phase, stirring 15min is formed just Cream；
5) under 1000r/min stirring, above-mentioned colostrum is slowly injected into 15ml outer aqueous phase, stirring and emulsifying forms emulsion；
6) when emulsion is concentrated by evaporation to about 10ml, emulsion is poured into rapidly in ice water (2-4 DEG C), low-temperature setting 1.5h, The water system membrane filtration of 0.45um obtains liquid phase spirulina-taxol-NLC；
7) 10% mannitol solution 2ml is added into 5ml liquid phase spirulina-taxol-NLC, is freeze-dried, obtains solid phase spiral shell Revolve algae-taxol-NLC.
Spirulina-taxol-NLC is round, similar round entity ball, and taxol encapsulation rate is up to 85.8%, spirulina packet Envelope rate is up to 74.0%.
Embodiment 3 contains spirulina-taxol composite nanostructure lipid vector preparation (spirulina-taxol-NLC)
The preparation method is as follows:
1) by 5mg taxol, 150mg solid lipid (Compritol 888 ATO), 50mg liquid fatty (isopropyl laurate) It sequentially adds in 20ml dehydrated alcohol, is heated in 65 DEG C of water-baths molten with 200mg fat-soluble emulsifier (Emulsifier EL-60 EL) Solution, ultrasonic (200~400W, 2~5min) dispersion mixes, as organic phase；
2) 5mg spirulina, 200mg water soluble emulsifier (Pluronic/Lutrol F 108 100mg+ natrii tauroglycocholas 100mg) are dissolved in In 5ml water for injection, dissolves by heating, stir evenly, as inner aqueous phase in 65 DEG C of water-baths；
3) 300mg surfactant (Tween 80) is dissolved in 15ml water for injection, is dissolved by heating in 65 DEG C of water-baths, stirred Uniformly, as outer aqueous phase；
4) under 500r/min stirring, 5ml inner aqueous phase is slowly injected into 20ml organic phase, stirring 20min forms colostrum；
5) under 500r/min stirring, above-mentioned colostrum is slowly injected into 15ml outer aqueous phase, stirring and emulsifying forms emulsion；
6) when emulsion is concentrated by evaporation to about 5ml, emulsion is poured into rapidly in ice water (2-4 DEG C), low-temperature setting 2h, 0.22um Water system membrane filtration, obtain liquid phase spirulina-taxol-NLC；
7) 10% mannitol solution 2ml is added into 2ml liquid phase spirulina-taxol-NLC, is freeze-dried, obtains solid phase spiral shell Revolve algae-taxol-NLC.
Spirulina-taxol-NLC is round, similar round entity ball, and taxol encapsulation rate is up to 85.4%, spirulina packet Envelope rate is up to 72.3%.
Embodiment 4 contains spirulina-taxol composite nanostructure lipid vector preparation (spirulina-taxol-NLC)
The preparation method is as follows:
1) by 5mg taxol, 50mg solid lipid (glycerin monostearate 25mg+ isopropyl palmitate 25mg), 10mg Liquid fatty (monoglyceride 8mg+ oleic acid 2mg) and 30mg fat-soluble emulsifier (Emulsifier EL-60 EL) sequentially add 20ml without It in water-ethanol, is dissolved by heating in 70 DEG C of water-baths, ultrasonic (200~400W, 2~5min) dispersion mixes, as organic phase；
2) 5mg spirulina, 50mg water soluble emulsifier (poloxamer188) are dissolved in 5ml water for injection, 70 DEG C of water-baths Middle heating for dissolving, stirs evenly, as inner aqueous phase；
3) 150mg surfactant (Tween 80) is dissolved in 15ml water for injection, is dissolved by heating in 70 DEG C of water-baths, stirred Uniformly, as outer aqueous phase；
4) under 1200r/min stirring, 5ml inner aqueous phase is slowly injected into 20ml organic phase, stirring 20min is formed just Cream；
5) under 1200r/min stirring, above-mentioned colostrum is slowly injected into 15ml outer aqueous phase, stirring and emulsifying forms emulsion；
6) when emulsion is concentrated by evaporation to about 8ml, emulsion is poured into rapidly in ice water (2-4 DEG C), low-temperature setting 2h, 0.45um Water system membrane filtration, obtain liquid phase spirulina-taxol-NLC；
7) 10% mannitol solution 2ml is added into 5ml liquid phase spirulina-taxol-NLC, is freeze-dried, obtains solid phase spiral shell Revolve algae-taxol-NLC.
Spirulina-taxol-NLC is round, similar round entity ball, and taxol encapsulation rate is up to 90.2%, spirulina packet Envelope rate is up to 87.6%.
Embodiment 5 contains spirulina-taxol composite nanostructure lipid vector preparation (spirulina-taxol-NLC)
The preparation method is as follows:
1) by 5mg taxol, 80mg solid lipid (isopropyl palmitate 40mg+ Compritol 888 ATO 40mg), 20mg liquid Body fat matter (isopropyl myristate 8mg+ oleic acid 12mg) and 50mg fat-soluble emulsifier (Emulsifier EL-60 EL) successively add Enter in 20ml dehydrated alcohol, dissolved by heating in 70 DEG C of water-baths, ultrasonic (200~400W, 2~5min) dispersion mixes, as organic Phase；
2) 5mg spirulina, 30mg water soluble emulsifier (Pluronic/Lutrol F 108) are dissolved in 5ml water for injection, 70 DEG C of water-baths Middle heating for dissolving, stirs evenly, as inner aqueous phase；
3) 90mg surfactant (Tween 80) is dissolved in 15ml water for injection, is dissolved by heating in 70 DEG C of water-baths, stirred Uniformly, as outer aqueous phase；
4) under 1200r/min stirring, 5ml inner aqueous phase is slowly injected into 20ml organic phase, stirring 15min is formed just Cream；
5) under 1200r/min stirring, above-mentioned colostrum is slowly injected into 15ml outer aqueous phase, stirring and emulsifying forms emulsion；
6) when emulsion is concentrated by evaporation to about 5ml, emulsion is poured into rapidly in ice water (2-4 DEG C), low-temperature setting 1h, 0.45um Water system membrane filtration, obtain liquid phase spirulina-taxol-NLC；
7) 10% mannitol solution 2ml is added into 2ml liquid phase spirulina-taxol-NLC, is freeze-dried, obtains solid phase spiral shell Revolve algae-taxol-NLC.
Spirulina-taxol-NLC is round, similar round entity ball, and taxol encapsulation rate is up to 87.5%, spirulina packet Envelope rate is up to 79.0%.
Embodiment 6 contains spirulina-taxol composite nanostructure lipid vector preparation (spirulina-taxol-NLC)
Preparation method:
1) by 5mg taxol, 90mg solid lipid (glycerin monostearate 20mg+ Compritol 888 ATO 70mg), 30mg Liquid fatty (monoglyceride 5mg+ isopropyl myristate 25mg) and 250mg fat-soluble emulsifier (Emulsifier EL-60 EL) according to In secondary addition 20ml dehydrated alcohol, being dissolved by heating in 70 DEG C of water-baths, ultrasonic (200~400W, 2~5min) dispersion mixes, as Organic phase；
2) 5mg spirulina, 40mg water soluble emulsifier (PLURONICS F87 20mg+ sodium glycocholate 20mg) are dissolved in 5ml note It penetrates in water, dissolves by heating, stir evenly, as inner aqueous phase in 70 DEG C of water-baths；
3) 400mg surfactant (Tween 80) is dissolved in 15ml water for injection, is dissolved by heating in 70 DEG C of water-baths, stirred Uniformly, as outer aqueous phase；
4) under 1200r/min stirring, 5ml inner aqueous phase is slowly injected into 20ml organic phase, stirring 20min is formed just Cream；
5) under 1200r/min stirring, above-mentioned colostrum is slowly injected into 15ml outer aqueous phase, stirring and emulsifying forms emulsion；
6) when emulsion is concentrated by evaporation to about 10ml, emulsion is poured into rapidly in ice water (2-4 DEG C), low-temperature setting 2h, The water system membrane filtration of 0.22um obtains liquid phase spirulina-taxol-NLC；
7) 10% mannitol solution 2ml is added into 5ml liquid phase spirulina-taxol-NLC, is freeze-dried, obtains solid phase spiral shell Revolve algae-taxol-NLC.
Spirulina-taxol-NLC is round, similar round entity ball, and taxol encapsulation rate is up to 86.9%, spirulina packet Envelope rate is up to 77.4%.

Claims (4)

1. a kind of contain spirulina-taxol composite nanostructure lipid vector preparation, which is characterized in that described to contain spiral Algae-taxol composite nanostructure lipid vector preparation belongs to sustained-release preparation, the preparation method is as follows:
1) taxol, solid lipid, liquid fatty and fat-soluble emulsifier are sequentially added in organic solvent, 65-75 DEG C of water-bath Middle heating for dissolving, ultrasound mixes dispersion, as organic phase；
2) spirulina and water soluble emulsifier are dissolved in appropriate water for injection, are dissolved by heating in 65-75 DEG C of water-bath, stirring is equal It is even, as inner aqueous phase；
3) surfactant is dissolved in appropriate water for injection, dissolves by heating, stirs evenly in 65-75 DEG C of water-bath, as outer water Phase；
4) under stiring, inner aqueous phase is slowly injected into organic phase, stirs 15-20min, forms colostrum；
5) under stiring, colostrum is slowly injected into outer aqueous phase, emulsification is sufficiently stirred and forms emulsion；
6) it is poured into rapidly after being concentrated by evaporation emulsion in ice water (2-4 DEG C), 1~2h of low-temperature setting obtains liquid phase after micro porous filtration Contain spirulina-taxol composite nanostructure lipid vector preparation；
Wherein, following raw material is as follows by weight ratio: 2-10 parts of spirulina, 2-10 parts of taxol, solid lipid 30-150 Part, 10-50 parts of liquid fatty, 30-300 parts of fat-soluble emulsifier, 20-250 parts of water soluble emulsifier, surfactant 90-550 Part；
The solid lipid be one of glycerin monostearate, isopropyl palmitate (different monooctyl ester), Compritol 888 ATO or Two kinds or more of combination；
The liquid fatty is one of monoglyceride, isopropyl myristate, isopropyl laurate or oleic acid or two kinds or more Mixing；
The fat-soluble emulsifier is Emulsifier EL-60 EL；
The water soluble emulsifier is the mixed of one of sodium glycocholate, natrii tauroglycocholas, poloxamer, NaTDC or two kinds It closes；
The surfactant is Tween 80.
2. a kind of contain spirulina-taxol composite nanostructure lipid vector preparation, which is characterized in that described to contain spiral Algae-taxol composite nanostructure lipid vector preparation is sustained-release preparation, the preparation method is as follows:
1) taxol, solid lipid, liquid fatty and fat-soluble emulsifier are sequentially added in organic solvent, 65-75 DEG C of water-bath Middle heating for dissolving, ultrasound mixes dispersion, as organic phase；
2) spirulina and water soluble emulsifier are dissolved in appropriate water for injection, are dissolved by heating in 65-75 DEG C of water-bath, stirring is equal It is even, as inner aqueous phase；
3) surfactant is dissolved in appropriate water for injection, dissolves by heating, stirs evenly in 65-75 DEG C of water-bath, as outer water Phase；
4) under stiring, inner aqueous phase is slowly injected into organic phase, stirs 15-20min, forms colostrum；
5) under stiring, colostrum is slowly injected into outer aqueous phase, emulsification is sufficiently stirred and forms emulsion；
6) it is poured into rapidly after being concentrated by evaporation emulsion in ice water (2-4 DEG C), 1~2h of low-temperature setting obtains liquid phase after micro porous filtration Contain spirulina-taxol composite nanostructure lipid vector preparation；
7) addition frozen-dried protective agent solution in spirulina-taxol composite nanostructure lipid vector preparation is contained to liquid phase, Freeze-drying, obtains solid phase and contains spirulina-taxol composite nanostructure lipid vector preparation；
Wherein, following raw material is as follows by weight ratio: 2-10 parts of spirulina, 2-10 parts of taxol, solid lipid 30-150 Part, 10-50 parts of liquid fatty, 30-300 parts of fat-soluble emulsifier, 20-250 parts of water soluble emulsifier, surfactant 90-550 Part；
The solid lipid be one of glycerin monostearate, isopropyl palmitate (different monooctyl ester), Compritol 888 ATO or Two kinds or more of combination；
The liquid fatty is one of monoglyceride, isopropyl myristate, isopropyl laurate or oleic acid or two kinds or more Mixing；
The fat-soluble emulsifier is Emulsifier EL-60 EL；
The water soluble emulsifier is the mixed of one of sodium glycocholate, natrii tauroglycocholas, poloxamer, NaTDC or two kinds It closes；
The surfactant is Tween 80.
3. containing spirulina-taxol composite nanostructure lipid vector preparation, feature as claimed in claim 1 or 2 Be: micro porous filtration described in step 6) is to use aperture for 0.22 μm or 0.45 μm of water system membrane filtration.
4. spirulina-taxol composite nanostructure lipid vector preparation of any of claims 1 or 2 that contains is reversed in preparation Application in tumor multi-medicine drug-resistant drug.
CN201910663848.5A 2019-07-12 2019-07-12 A kind of spirulina nanometer formulation and preparation method thereof Withdrawn CN110464708A (en)

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