CN106361701A - Liposome drug carrier and preparation method and application thereof - Google Patents
Liposome drug carrier and preparation method and application thereof Download PDFInfo
- Publication number
- CN106361701A CN106361701A CN201610792043.7A CN201610792043A CN106361701A CN 106361701 A CN106361701 A CN 106361701A CN 201610792043 A CN201610792043 A CN 201610792043A CN 106361701 A CN106361701 A CN 106361701A
- Authority
- CN
- China
- Prior art keywords
- preparation
- drug carrier
- liposome
- cholesterol
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
-
- 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
- A61K31/66—Phosphorus compounds
- A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
- A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
-
- 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
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- 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
- A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
Abstract
The invention belongs to the technical field of drug preparation development, in particular to a liposome drug carrier and a preparation method and application thereof. The liposome drug carrier includes the following ingredients: cetylic acid and cholesterol. By freeze drying or rotary drying the ingredients transform into membranes. Through the subsequent hydration and filming the final products are acquired. The liposome drug carrier can automatically form lipid microspheres which are quite stable. At the same time the drug carrier preserves the advantages of traditional liposome, namely, the drug carrier has a structure similarity to the bimolecular lamellar membrane structure of biological plasma membrane. The drug carrier has the advantages of excellent biological adaptability, stability, simple production and high encapsulation efficiency and the like. The liposome drug carrier can be used to carry biphosphonate drugs with a consistent and unified particle diameter, high encapsulation efficiency and high stability. The carrier has negative charges and can further enhance the ability that the drug enters cells. Compared with traditional lecithin liposome the carrier enhances the effects on macrophages.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, more particularly to a kind of lipidosome drug carrier and preparation method thereof and should
With.
Background technology
A kind of orientation medicine that liposome is the vesicle packaging medicine molecule being formed using phospholipid bilayer tunic and is formed
Thing carrier, is a kind of novel form of targeting drug delivery system.It is that the active substance in solution or solution is embedded in a diameter of receiving
In the microgranule of meter level.At present, the primary raw material of liposome is phospholipid, and phospholipid is the main chemical compositions constituting liposome, wherein
Most representational is lecithin.Cholesterol is also another important composition composition of liposome, and itself does not form film knot
Structure, but can be with certain mol ratio insertion immobilized artificial membrane.Add cholesterol can change the phase transition temperature of adipose membrane, thus shadow
Ring permeability and the mobility of film, increase the stability of immobilized artificial membrane.Multilamellar microcapsule can be formed when phospholipid is dispersed in water, and every layer
It is lipid bilayer, is separated by aqueous phase between each layer, this microcapsule is exactly liposome.Liposome can be divided into single chamber lipid
Body, multilamelar liposome, the liposome containing surfactant.
Phospholipid or phospholipid are mainly dissolved in wiring solution-forming in organic solvent with cholesterol, then by the method preparing liposome
Evaporating organic solvent, forms bilayer lipid membrane on wall, this thin film is collected and mixes with aqueous solution stirring
Form liposome.Liposome now is often the very big multilamelar liposome of diameter, makes fat with practical value through processing
Matter microsphere.The method preparing lipid microsphere is mainly ultrasound wave, ultra-filtration, high pressure pulverizing and mechanical disruption.
The application of liposome is quite varied.Various drug encapsulation can be become lipid microsphere (liposome) by phospholipid, and this lipid is micro-
Ball with contact human skin or enter human body in after, using its film feature similar to membrane structure, carry out with cell membrane
Merge or by endocytosises, medicine is carried in target cell.Medicine passes through lipid carrier, can pass through table
The horny layer of skin, is that the exploitation of various medicine for external use and personal care's articles for use provides effective method.And it is quick to oral cavity and stomach
The medicine of sense, after the parcel of liposome, can protect it from the destruction of oral cavity and gastric enzyme so as to the arrival intestinal of safety, using fat
Plasma membrane and intestinal wall cell membrane ground structure similarity, more effectively introduce a drug into intracellular, thus the treatment improving medicine refers to
Number, reduces the therapeutic dose of medicine and the toxicity reducing medicine.
Although liposome possesses many advantages and feature as the application of pharmaceutical carrier, just at present, yet deposit
In certain limitation: the stability of liposome is not good, liposome preservation process or run into temperature change and add not
With additive or adjuvant, liposome is likely to mutual adsorpting aggregation and merges, and leads to the unstable of liposome；Envelop rate is low,
Liposome with phospholipid as primary raw material is low to the envelop rate of medicine, especially for some water soluble drug envelop rates more at present
Low, and medicine seepage easily from liposome.Develop a kind of stable, easy production, that envelop rate is good novel lipide to overcome
The deficiency of existing liposome is very necessary at present.
Content of the invention
The shortcoming of prior art in view of the above, it is an object of the invention to provide a kind of lipidosome drug carrier and its
Preparation method and application.Described lipidosome drug carrier includes following component: Palmic acid and cholesterol, by lyophilization or rotation
Turn hydration after being evaporated film forming, film excessively is obtained.Described lipidosome drug carrier can automatically form lipid microsphere and quite stable, simultaneously
The advantage remaining traditional liposomal, that is, similar to the basic bilayer membrane structure of organism plasma membrane, have biological simultaneous well
Capacitive, and have the advantages that stable, easily production and envelop rate is good.Described lipidosome drug carrier is used for loading diphosphonate medicine
Thing, uniform particle diameter, envelop rate are good, and stability is high, negatively charged, can improve the ability that medicine enters cell further, relatively
Traditional phospholipid lipoid plastid improves its effect to macrophage.
To achieve these goals and other related purposes, the present invention adopts the following technical scheme that
First aspect present invention provides a kind of lipidosome drug carrier, and described lipidosome drug carrier includes following component:
Palmic acid and cholesterol.
Described lipidosome drug carrier is stablized, easily produces, envelop rate is good and ph sensitivity.
Palmitic acid molecule formula is:The water-wet side of Palmic acid is carboxylic acid, and hydrophobic side is carbon
Chain, cholesterol and Palmic acid are formed stablizes tight lipid bilayer.The preferred Palmic acid of described lipid bilayer is in ph 6
～9 prepare gained, and Palmic acid is ionic state, and the carboxylic acid of Palmic acid hydrophilic section is non-protonated, and therefore gained liposome is negatively charged, electricity
Position is minus 60～70mv, has good stability.Described liposome hydrophilic section is the carboxylic acid of Palmic acid, in acid condition, carboxylic
Acid protonation, leads to the unstability of liposome bilayers, thus causing the leakage of medicine, so palmitic acid lipid plastid has
Ph sensitivity.
Preferably, Palmic acid and the mol ratio of cholesterol are 0.37～0.6:1.
Preferably, described lipidosome drug carrier is negatively charged, and current potential is -60～-95mv..
Second aspect present invention provides the preparation method of above-mentioned lipidosome drug carrier, appointing selected from following preparation method
One:
Preparation method one: Palmic acid and cholesterol are dissolved in organic solvent, lyophilization film forming, are then hydrated, crosses film and obtains final product
Lipidosome drug carrier；
Preparation method two: Palmic acid and cholesterol are dissolved in organic solvent, dries up film forming with nitrogen or rotation is evaporated film forming, so
After be hydrated, cross film obtain final product lipidosome drug carrier.
Preferably, also include one or more in following characteristics:
A) Palmic acid and the mol ratio of cholesterol are 0.37～0.6:1；
B) described organic solvent is selected from one or more of the tert-butyl alcohol, ethanol, methanol and chloroform；
C) Palmic acid and cholesterol are dissolved in organic solvent at 50～65 DEG C；
D), in preparation method one, the cryodesiccated time is 12～48h.
Third aspect present invention provides the purposes of above-mentioned lipidosome drug carrier, for loading biphosphonates.
Preferably, described biphosphonates are selected from disodium clodronate, Sodium Pamidronate, alendronate, etidronic acid
Sodium, Disodium tiludronate, olpadronic acid sodium, neridronic acid sodium, one of risedronate sodium, ibandronate and zoledronic acid or
Multiple.
Fourth aspect present invention provides a kind of pharmaceutical preparation, including the lipidosome drug carrier of any of the above-described, described fat
Liposome medicament carrier load has biphosphonates.
Preferably, biphosphonates and the mol ratio of described lipidosome drug carrier are 2～10:1.
Fifth aspect present invention provides the preparation method of said medicine preparation, arbitrary selected from following preparation method:
Preparation method one: Palmic acid and cholesterol are dissolved in organic solvent, lyophilization film forming, are subsequently adding diphosphonate medicine
The aqueous solution hydration of thing, crosses film and obtains final product pharmaceutical preparation；
Preparation method two: Palmic acid and cholesterol are dissolved in organic solvent, dries up film forming with nitrogen or rotation is evaporated film forming, so
Add the aqueous solution hydration of biphosphonates afterwards, cross film and obtain final product pharmaceutical preparation.
Preferably, also include one or more in following characteristics:
A) Palmic acid and the mol ratio of cholesterol are 0.37～0.6:1；
B) described organic solvent is selected from one or more of the tert-butyl alcohol, ethanol, methanol and chloroform；
C) Palmic acid and cholesterol are dissolved in organic solvent at 50～65 DEG C；
D), in preparation method one, the cryodesiccated time is 12～48h；
E) the aqueous solution ph value of biphosphonates is 6～9.
Preferably, described biphosphonates are selected from disodium clodronate, Sodium Pamidronate, alendronate, etidronic acid
Sodium, Disodium tiludronate, olpadronic acid sodium, neridronic acid sodium, one of risedronate sodium, ibandronate and zoledronic acid or
Multiple.
Sixth aspect present invention provides a kind of medical composition, including the said medicine preparation of therapeutically effective amount.
Traditional liposomal is a kind of vesicle being formed by phosphoric acid class bi-layer membrane, and typical liposome is microspheroidal, micro-
The surfaces externally and internally of ball is respectively polar hydrophilic phosphate ester bimolecular top layer, and the microsphere inner chamber of polar hydrophilic can the hydrophilic medicine of encapsulation
Molecule.Traditional phosphate lipid plastid encapsulating water solublity bisphosphonates envelop rate is low, and stability is poor.Unstability: performance
In the easily oxidized property of phosphoric acid ester molecule and facile hydrolysiss；Another kind is then that liposome is mutually assembled, and mutually merges and forms super large fat
Plastid, leads to contain the leakage of medicine, reduces embedding rate.Therefore screen a kind of more stable, there is self vesicle form function
Lipid is to solve the not enough primary factor of traditional liposomal as raw material.The molecule forming liposome must be not only hydrophilic but also close
Parents' molecule of fat, the structure of the hydrophilic group carboxylic acid (head) of Palmic acid and the carbochain (afterbody) of lipophilic meets formation liposome
Requirement it is preferable that the liposome that Palmic acid and cholesterol are prepared with 0.37～0.6:1 mol ratio improves diphosphonate lipoid
The envelop rate of plastid, stability, thus increased its practicality.
In sum, compared with prior art, the invention has the following beneficial effects:
The present invention includes following component: Petiolus Trachycarpi by extensively in-depth study discovery, a kind of new Lipid pharmaceutical carrier
Acid and cholesterol, described lipidosome drug carrier can automatically form lipid microsphere and quite stable, remain traditional liposomal simultaneously
The advantage of body, that is, similar to the basic bilayer membrane structure of organism plasma membrane, there is good bio-compatibility, and have steady
The advantages of fixed, easy production and envelop rate are good.Described lipidosome drug carrier is used for loading biphosphonates, uniform particle diameter, bag
Envelope rate is good, and stability is high, negatively charged, can improve the ability that medicine enters cell, relatively conventional phospholipid lipoids further
Body improves its effect to macrophage.
Brief description
Fig. 1 is the phenogram of disodium clodronate Liposomal formulation shelf-stability.
Wherein, 1a is epc1, particle diameter distribution in 40 days for hspc1, the pa2 liposome and comparing.
1b is epc1, particle diameter distribution coefficient in 40 days for hspc1, the pa2 liposome and comparing.
1c is epc1, and slip in 40 days for hspc1, the pa2 liposome is distributed and compares.
Fig. 2 is the phenogram of disodium clodronate Liposomal formulation ph stability.
Wherein, 2a is hspc1, particle diameter distribution under different ph for the pa2 liposome and comparing.
2b is hspc1, particle diameter distribution coefficient under different ph for the pa2 liposome and comparing.
2c is hspc1, Potential Distributing under different ph for the pa2 liposome and comparing.
2d is hspc1, and percolation ratio under different ph for the pa2 liposome compares.
Fig. 3 is the interpretation of result of disodium clodronate Liposomal formulation macrophage toxicity.
Wherein, 3a is the cell survival rate distribution after phospholipid and Petiolus Trachycarpi acids blank liposome effect macrophage 24h
And compare.
3b is that the cell survival rate after hspc1 and pa2 acts on macrophage 6h is distributed and compares.
3c is that the cell survival rate after hspc1 and pa2 acts on macrophage 12h is distributed and compares.
3d is that the cell survival rate after hspc1 and pa2 acts on macrophage 24h is distributed and compares.
3e is pa2, and pa4, pa5 liposome acts on the cell survival rate distribution in macrophage 6h and compares.
3f is pa2, and pa4, pa5 liposome acts on the cell survival rate distribution in macrophage 12h and compares.
3g is pa2, and pa4, pa5 liposome acts on the cell survival rate distribution in macrophage 24h and compares.
Fig. 4 is the grain size distribution of disodium clodronate Liposomal formulation.
Wherein, 4a is the grain size distribution of pa3.
4b is the grain size distribution of hspc1.
4c is the grain size distribution of pa2.
4d is the grain size distribution of epc1.
4d is the grain size distribution of pa1.
Fig. 5 is concentration standard curve.The concentration standard curve (ultraviolet detection) of 5a disodium clodronate.
The concentration standard curve (ultraviolet detection) of 5b Sodium Pamidronate.
The concentration standard curve (ultraviolet detection) of 5c alendronate.
Specific embodiment
Below by way of specific instantiation, embodiments of the present invention are described, those skilled in the art can be by this specification
Disclosed content understands other advantages and effect of the present invention easily.The present invention can also be by addition different concrete realities
The mode of applying is carried out or applies, and the every details in this specification can also be based on different viewpoints and application, without departing from
Carry out various modifications and changes under the spirit of the present invention.
It should be clear that the process equipment specifically not indicated in the following example or device all using conventional equipment in the art or
Device.
In addition, it is to be understood that the one or more method and steps mentioned in the present invention do not repel before and after described combination step
Can also exist and can also insert additive method step between additive method step or the step that specifically mentions at these, unless separately
It is described；It should also be understood that the combination annexation between the one or more equipment/devices mentioned in the present invention is not repelled
Can also exist before and after described unit equipment/device other equipment/device or two equipment/devices specifically mentioning at these it
Between can also insert other equipment/device, unless otherwise stated.And, unless otherwise stated, the numbering of various method steps is only
Differentiate the convenient tool of various method steps, rather than for limiting the ordering of various method steps or limiting the enforceable model of the present invention
Enclose, being altered or modified of its relativeness, in the case of no essence change technology contents, enforceable when being also considered as the present invention
Category.
In the present invention, dummy suffix notation is expressed as follows:
Abbreviation title Chinese full name
Pdi particle diameter distribution coefficient
Zeta potential electrokinetic potential or eletrokinetic potential
Chol cholesterol
Pa Palmic acid (hexadecanoic acid)
Hspc hydrogenated soy phosphatidyl choline
Epc Egg Yolk Lecithin (PC-98T)
Uv ultraviolet detection
Clodrolip disodium clodronate liposome
Pamidrolip pamidronic acid sodium lipidosome
Alendrolip Alendronic acid sodium lipidosome
Embodiment 1 phospholipid lipoid plastid loads the preparation of bisphosphonates
1.1 reagent and the preparation of storing solution
Chloroform (AG)；
Phosphate buffer: the nacl taking 12.2mm disodium hydrogen phosphate and 8.2g/l, in ultra-pure water, adjusts ph to 7.40；
The epc/hspc of storing solution: 100mg/ml, takes 100mgepc/hspc to be dissolved in chloroform；
The chol of 20mg/ml, takes 20mg chol to be dissolved in chloroform and obtains；
Bisphosphonates solution, takes and is dissolved in ultra-pure water in right amount, is adjusted to ph 7.4 with 5n naoh.
1.2 preparation methoies-film dispersion method
A) take storing solution Egg Yolk Lecithin (PC-98T) epc or hydrogenated soy phosphatidyl choline hspc and cholesterol by the mol ratio of 2:1 in eggplant
In type bottle；
B) nitrogen slowly dries up or the rotation of water-bath low pressure volatilizes chloroform, forms thin film in eggplant type bottle；
C) storing solution bisphosphonates or phosphate buffered solution hydration are added, room temperature preservation 2h or 4 DEG C are overnight protected
Deposit, make liposome fully swelling；
D) extruder crosses 200nm, each 8-10 time of 100nm, 80nm film, obtains liposome turbid liquor.
1.3 Liposomal formulations separate-ultrafiltration centrifuging with free drug
A) take Liposomal formulation in 15ml, 100kd super filter tube, 15000 × g in centrifuge, 10 DEG C of centrifugation 30-60h,
Concentrating and separating free drug；
B), after removing free drug, add phosphate buffer in Liposomal formulation super filter tube to concentrating, resuspended liposome,
15000 × g in centrifuge, 10 DEG C of centrifugation 30-60h；
C) so resuspended-washing liposome 3 times, final Liposomal formulation is resuspended in phosphate buffer, stand-by.
Embodiment 2 Palmic acid and the preparation of cholesterol liposomes loading bisphosphonates
2.1 reagent and the preparation of storing solution
Chloroform (AG)；
The tert-butyl alcohol (AG)；
Phosphate-buffered night: the nacl taking 12.2mm disodium hydrogen phosphate and 8.2g/l, in ultra-pure water, adjusts ph to 7.40；
The pa of storing solution: 100mg/ml, takes 100mgpa to be dissolved in chloroform or the tert-butyl alcohol；
The chol of 20mg/ml, takes 20mg chol to be dissolved in chloroform and obtains；
Bisphosphonates solution, takes and is dissolved in ultra-pure water in right amount, is adjusted to ph for 7.60 with 5n naoh.
2.2 preparation method one-film dispersion methods
A) take storing solution Palmic acid pa and cholesterol by 3:5,3:7,3:8 mol ratio in eggplant type bottle；
B) nitrogen slowly dries up or the rotation of water-bath low pressure volatilizes chloroform, forms thin film in eggplant type bottle；
C) storing solution bisphosphonates or phosphate solution hydration are added, room temperature preservation 2h or 4 DEG C overnight preserve, and make
Liposome is fully swelling；
D) extruder crosses 200nm, each 8-10 time of 100nm, 80nm film, obtains liposome turbid liquor.
2.3 preparation method two-lyophilizing hydration methods
A) take storing solution Palmic acid pa and cholesterol by 3:5,3:7,3:8 mol ratio in lyophilizing bottle；
B) lyophilizing 24h in freeze dryer, the tert-butyl alcohol volatilizes completely；
E) storing solution bisphosphonates or phosphate solution hydration are added；
F) extruder crosses 200nm, each 8-10 time of 100nm, 80nm film, obtains liposome turbid liquor.
2.4 Liposomal formulations separate-ultrafiltration centrifuging with free drug
A) take Liposomal formulation in 15ml, 100kd super filter tube, 15000 × g in centrifuge, 10 DEG C of centrifugation 30-60h,
Concentrating and separating free drug；
B), after removing free drug, add phosphate buffer in Liposomal formulation super filter tube to concentrating, resuspended liposome,
15000 × g in centrifuge, 10 DEG C of centrifugation 30-60h；
C) so resuspended-washing liposome 3 times, final Liposomal formulation is resuspended in phosphate buffer, stand-by.
The above-mentioned liposome of embodiment 3 load bisphosphonates evaluation with compare
3.1 particle diameters, pdi, potential measurement
Instrument: dynamic light scattering laser particle analyzer and zeta potentiometric analyzer (zetasizer3000)；
Condition: set sample refractive index as the refractive index (1.59) of silicon dioxide, dispersion phase is that water (glue by refractive index 1.36
Degree 0.8cp), 25 degree of detection temperature, balance 60 seconds in detection cavity temperature before sample detection.Optical maser wavelength is 633nm, refraction angle
For 90 °；
Particle diameter and pdi measure: take sample 50 μ l, pure water dilutes 10 times, is transferred to particle diameter pond, each sample parallel assay three
Secondary.
Potential measurement: take sample 50 μ l, 10%hepes solution dilutes 10 times, is transferred to current potential pond, the parallel survey of each sample
Fixed three times.
3.2 entrapment efficiency determination
3.2.1 the preparation of reagent and storing solution
Ultra-pure water；
Chloroform (AG)；
The tert-butyl alcohol (AG)；
Phosphate-buffered night: the nacl taking 12.2mm disodium hydrogen phosphate and 8.2g/l, in ultra-pure water, adjusts ph to 7.40；
Standard bisphosphonates solution: bisphosphonates are dissolved in ultra-pure water, configures 10mg/ml, and 5n naoh is adjusted to
ph7.40；
90% phenol solution；
4mm cuso4Solution: by 0.64g cuso4It is dissolved in 1l ultra-pure water；
hno3: 65% hno3Dilute 100 times with ultra-pure water；
3.2.2 liposome breakdown of emulsion
A) 1ml diphosphonate liposome, 1ml standard bisphosphonate solutions 10mg/ml are taken, 1ml phosphate-buffered night is in ep
Guan Zhong；
B) add 8ml phenol/chloroform solution (volume ratio 1:2) in every pipe；
C) strenuous vibration vortex ep pipe, stands more than 15min under room temperature；
D) centrifugation 1125 × g, 10 DEG C, 10min；
E) stand more than 10min under room temperature, until water-oil phase is substantially layered；
F) pipette upper strata aqueous phase in clean ep pipe, add 6ml chloroform, be vortexed and extract again；
G) stand more than 5min under room temperature, be centrifuged (1125 × g, 10 DEG C, 10min)；
H) pipette supernatant aqueous phase, stand-by.
3.2.3 prepared by bisphosphonates standard curve
A) the standard bisphosphonates medicine 0,10,20,40,50,70,80ul taking above-mentioned extraction respectively in volumetric flask, plus
Entering phosphoric acid buffer night is diluted to 1ml.
B) add 2.25ml 4mm cuso in every bottle4Solution, 0.05ml hno3Solution；
C) acutely shake, after vortex, survey ultraviolet absorptivity at 240nm.
3.2.4 Liposomal formulation entrapment efficiency determination
Take prepared bisphosphonates Liposomal formulation respectively, with phenol/chloroform breakdown of emulsion, add developer, by 3.2.3
Operation, in dilute sample to standard curve range, acutely shakes, and after vortex, surveys ultraviolet absorptivity at 240nm.
The short-term shelf-stability of 3.3 Liposomal formulations is investigated
The Liposomal formulation of preparation is placed January at 4 DEG C, 15000 × g, 4 DEG C, 30min, 2 removal free drugs, make
Measure particle diameter, particle diameter distribution coefficient and current potential with Malvern laser particle analyzer；Take appropriate preparation with phenol/chloroform breakdown of emulsion, ultraviolet
Spectrophotometric determination envelop rate.Assess its short-term and place and stablize stability.
The ph study on the stability of 3.4 Liposomal formulations
Phospholipid and Palmic acid cholesterol liposomes preparation 200ul is taken to add the ph 2.51,3.51 of 600ul respectively,
In 4.51,5.51,6.51,7.51,8.51,9.51 phosphate buffer, after 37 DEG C of shaking tables shake 48h, take out centrifugation
(15000 × g, 4 DEG C, 30min, 2 times) remove free drug, measure particle diameter, particle diameter distribution system using Malvern laser particle analyzer
Number and current potential；Take appropriate preparation with phenol/chloroform breakdown of emulsion, ultraviolet spectrophotometer measures envelop rate.
3.5 interpretation of result
3.5.1 bisphosphonates Liposomal formulation is characterized as below:
As table, the bisphosphonates Liposomal formulation of preparation, particle diameter distribution more uniform (epc1, hspc1, pa1, pa2 and
Pa3 grain size distribution is shown in Fig. 5), dispersibility is preferable；Phosphoric acid cholesterol liposomes relatively, Palmic acid and cholesterol liposomes have
Preferably envelop rate and stability.
3.5.2 shelf-stability:
Above-mentioned 4 DEG C of placements of preparation 40 days, ultrafiltration removed free drug, and preparation characterizes sees Fig. 1.
After 4 DEG C of disodium clodronate Liposomal formulation is placed 40 days, the particle diameter of the clodrolip of pa-chol preparation, particle diameter divides
Cloth coefficient is almost unchanged, and envelop rate is slightly decreased, and possible ultrafiltration centrifugation free drug is relevant, but all in tolerance interval
Interior, that is, liposome is placed in 4 DEG C of short-terms is stable.
3.5.3 diphosphonate lipoid plastid ph stability:
See Fig. 2.It can be seen that, disodium clodronate Liposomal formulation after different ph act on, pa-chol preparation clodrolip
Particle diameter distribution coefficient with respect to the clodrolip of hspc-chol preparation and potential change reduce with ph and reduce, and slip is with ph
Reduce and decline, thus, the clodrolip of pa-chol preparation has ph sensitivity.
Embodiment 4 disodium clodronate liposome to the effect of macrophage with compare
4.1 cytotoxicity-cck8
A) prepare cell (raw264.7) suspension: cell counting；
B) it is inoculated in 96 orifice plates: according to suitable bed board cell number, every hole 200ul cell suspension, same sample can
Do 3 repetitions；
C) cultivate 24h overnight in 37 DEG C of incubators；
D) add the different pharmaceutical concentration being obtained by disodium clodronate liposome/blank liposome with culture medium；
E) cultivate 6h in 37 DEG C of incubators, after 12h, 24h, remove pharmaceutical culture medium, add fresh culture 100ul；
F) add 10ul cck8, gently tap culture plate after adding reagent to help mix.
G) cultivate 1-4 hour: cell category is different, the amount of the formazan of formation is also different.If colour developing is inadequate
Words, can continue to cultivate, to confirm optimum condition.
H) measure 450nm absorbance.
4.2 macrophage toxicity interpretations of result
As shown in figure 3, the toxicity after Palmic acid and cholesterol lipid carrier load disodium clodronate medicine, to macrophage
Effect is remarkably reinforced, and possible cause is nagative potential for Palmic acid cholesterol liposomes, and phospholipid lipoid plastid is more easy to enter carefully relatively
Born of the same parents, show more preferable toxic action, and second filial generation bisphosphonates Sodium Pamidronate and third generation Alendronic acid sodium toxicity are made
With higher.
Above-described embodiment only principle of the illustrative present invention and its effect, not for the restriction present invention.Any ripe
The personage knowing this technology all can carry out modifications and changes without prejudice under the spirit and the scope of the present invention to above-described embodiment.Cause
This, those of ordinary skill in the art is complete with institute under technological thought without departing from disclosed spirit such as
All equivalent modifications becoming or change, must be covered by the claim of the present invention.
Claims (12)
1. a kind of lipidosome drug carrier is it is characterised in that described lipidosome drug carrier includes following component: Palmic acid and gallbladder
Sterin.
2. lipidosome drug carrier as claimed in claim 1 is it is characterised in that Palmic acid is 0.37 with the mol ratio of cholesterol
～0.6:1.
3. lipidosome drug carrier as claimed in claim 1 is it is characterised in that described lipidosome drug carrier is negatively charged, electric
Position is -60～-95mv.
4. lipidosome drug carrier as claimed in claim 1 preparation method it is characterised in that selected from following preparation method it
Arbitrary:
Preparation method one: Palmic acid and cholesterol are dissolved in organic solvent, lyophilization film forming, are then hydrated, crosses film and obtains final product lipid
Body pharmaceutical carrier；
Preparation method two: Palmic acid and cholesterol are dissolved in organic solvent, dries up film forming with nitrogen or rotation is evaporated film forming, Ran Houshui
Close, cross film and obtain final product lipidosome drug carrier.
5. preparation method as claimed in claim 4 is it is characterised in that also include one or more in following characteristics:
A) Palmic acid and the mol ratio of cholesterol are 0.37～0.6:1；
B) described organic solvent is selected from one or more of the tert-butyl alcohol, ethanol, methanol and chloroform；
C) Palmic acid and cholesterol are dissolved in organic solvent at 50～65 DEG C；
D), in preparation method one, the cryodesiccated time is 12～48h.
6. lipidosome drug carrier as claimed any one in claims 1 to 3 is used for loading biphosphonates.
7. purposes as claimed in claim 6 is it is characterised in that described biphosphonates are selected from disodium clodronate, handkerchief rice phosphine
Sour sodium, alendronate, etidronate, Disodium tiludronate, olpadronic acid sodium, neridronic acid sodium, risedronate sodium, her class's phosphine
One or more of sour sodium and zoledronic acid.
8. a kind of pharmaceutical preparation it is characterised in that include lipidosome drug carrier as claimed any one in claims 1 to 3,
Described lipidosome drug carrier is mounted with biphosphonates.
9. the preparation method of pharmaceutical preparation as claimed in claim 8 is it is characterised in that be selected from the arbitrary of following preparation method:
Preparation method one: Palmic acid and cholesterol are dissolved in organic solvent, lyophilization film forming, are subsequently adding biphosphonates
Aqueous solution is hydrated, and crosses film and obtains final product pharmaceutical preparation；
Preparation method two: Palmic acid and cholesterol are dissolved in organic solvent, dries up film forming with nitrogen or rotation is evaporated film forming, Ran Houjia
Enter the aqueous solution hydration of biphosphonates, cross film and obtain final product pharmaceutical preparation.
10. the preparation method of pharmaceutical preparation as claimed in claim 9 is it is characterised in that also include one in following characteristics
Or multinomial:
A) Palmic acid and the mol ratio of cholesterol are 0.37～0.6:1；
B) described organic solvent is selected from one or more of the tert-butyl alcohol, ethanol, methanol and chloroform；
C) Palmic acid and cholesterol are dissolved in organic solvent at 50～65 DEG C；
D), in preparation method one, the cryodesiccated time is 12～48h；
E) the aqueous solution ph value of biphosphonates is 6～9.
The preparation method of 11. pharmaceutical preparatioies as claimed in claim 9 is it is characterised in that described biphosphonates are selected from chlorine
Phosphonic acids disodium, Sodium Pamidronate, alendronate, etidronate, Disodium tiludronate, olpadronic acid sodium, neridronic acid sodium, profit
One or more of plug Alendronate, ibandronate and zoledronic acid.
A kind of 12. medical compositions are it is characterised in that include the pharmaceutical preparation as claimed in claim 8 of therapeutically effective amount.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610792043.7A CN106361701B (en) | 2016-08-31 | 2016-08-31 | A kind of lipidosome drug carrier and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610792043.7A CN106361701B (en) | 2016-08-31 | 2016-08-31 | A kind of lipidosome drug carrier and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106361701A true CN106361701A (en) | 2017-02-01 |
| CN106361701B CN106361701B (en) | 2019-08-27 |
Family
ID=57899687
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610792043.7A Active CN106361701B (en) | 2016-08-31 | 2016-08-31 | A kind of lipidosome drug carrier and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106361701B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107693489A (en) * | 2017-09-12 | 2018-02-16 | 甘肃博睿生物科技有限公司 | Chlorine disodium hydrogen phosphate liposome enema and preparation method and its application in preventing and treating Colon and rectum inflammation and colorectal cancer |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000003677A2 (en) * | 1998-07-14 | 2000-01-27 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Use of bisphosphonates in the treatment of vascular restenosis |
| CN1261892A (en) * | 1997-07-09 | 2000-08-02 | 杰安杰有限公司 | Diphosphonic acid salts for treatment of osteoporsis |
| CN101977609A (en) * | 2008-03-18 | 2011-02-16 | A·V·迪科夫斯基 | Pharmaceutical composition for preventing and treating bone resorption of different etiology |
| CN103040863A (en) * | 2012-12-17 | 2013-04-17 | 海南百思特医药科技有限公司 | Disodium clodronate liposome injection |
| CN103505419A (en) * | 2013-09-24 | 2014-01-15 | 上海纳米技术及应用国家工程研究中心有限公司 | Oxygen-carrying lipidosome with low surface tension and preparation method thereof |
-
2016
- 2016-08-31 CN CN201610792043.7A patent/CN106361701B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1261892A (en) * | 1997-07-09 | 2000-08-02 | 杰安杰有限公司 | Diphosphonic acid salts for treatment of osteoporsis |
| WO2000003677A2 (en) * | 1998-07-14 | 2000-01-27 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Use of bisphosphonates in the treatment of vascular restenosis |
| CN101977609A (en) * | 2008-03-18 | 2011-02-16 | A·V·迪科夫斯基 | Pharmaceutical composition for preventing and treating bone resorption of different etiology |
| CN103040863A (en) * | 2012-12-17 | 2013-04-17 | 海南百思特医药科技有限公司 | Disodium clodronate liposome injection |
| CN103505419A (en) * | 2013-09-24 | 2014-01-15 | 上海纳米技术及应用国家工程研究中心有限公司 | Oxygen-carrying lipidosome with low surface tension and preparation method thereof |
Non-Patent Citations (7)
| Title |
|---|
| CARBAJAL GUSTAVO，等: "Non-phospholipid liposomes with high sterol content display a very limited permeability", 《SCIENCE CHINA-CHEMISTRY》 * |
| GUILLAUME BASTIAT，等: "Development of Non-Phospholipid Liposomes Containing a High Cholesterol Concentration", 《LANGMUIR》 * |
| NICOLAS COTTENYE，等: "Formation, stability, and pH sensitivity of freefloating,giant unilamellar vesicles using palmitic acid-cholesterol mixtures", 《SOFT MATTER》 * |
| WILLIAM G.LOVE,等: "Efficient Clodronate Entrapment Within Multilamellar and Unilamellar Liposomes", 《JOURNAL OF PHARMACOLOGICAL AND TOXICOLOGICAL METHODS》 * |
| 崔福德，等: "《药剂学》", 31 August 2011, 人民卫生出版社 * |
| 梁彬，等: "《临床肿瘤学相关进展》", 31 October 2012, 辽宁科学技术出版社 * |
| 高建川，等: "脂质体包裹Clodronate对单核/巨噬细胞选择性清除作用的研究", 《解放军医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107693489A (en) * | 2017-09-12 | 2018-02-16 | 甘肃博睿生物科技有限公司 | Chlorine disodium hydrogen phosphate liposome enema and preparation method and its application in preventing and treating Colon and rectum inflammation and colorectal cancer |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106361701B (en) | 2019-08-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tai et al. | Effect of β-sitosterol on the curcumin-loaded liposomes: Vesicle characteristics, physicochemical stability, in vitro release and bioavailability | |
| El-Samaligy et al. | Increasing bioavailability of silymarin using a buccal liposomal delivery system: preparation and experimental design investigation | |
| US11964019B2 (en) | Cochleates made with soy phosphatidylserine | |
| Elnaggar et al. | Anionic versus cationic bilosomes as oral nanocarriers for enhanced delivery of the hydrophilic drug risedronate | |
| Liang et al. | Encapsulation of ATP into liposomes by different methods: optimization of the procedure | |
| WO2011129588A2 (en) | Polymer/liposome nanocomposite composition for percutaneous absorption, and method for preparing same | |
| Vural et al. | Chitosan coated furosemide liposomes for improved bioavailability | |
| CN103040748A (en) | Pemetrexed disodium liposome injection | |
| Zhong et al. | Azithromycin cationic non-lecithoid nano/microparticles improve bioavailability and targeting efficiency | |
| Dubey et al. | Effects of linkers on the development of liposomal formulation of cholesterol conjugated cobalt bis (dicarbollides) | |
| CN106361701A (en) | Liposome drug carrier and preparation method and application thereof | |
| Pawar | Nanocochleate: a novel drug delivery system | |
| CN102716089B (en) | Gemcitabine hydrochloride liposome injection | |
| CN102247324B (en) | Flumazenil liposome injection | |
| Wasankar et al. | Liposome as a drug delivery system-a review | |
| Gupta et al. | Novel clindamycin loaded transfersomes formulation for effective management of acne | |
| CN107530235A (en) | For producing the one-step method of microminiature lipid conformation | |
| CN104546718B (en) | A kind of long circulating Rabeprazole liposome composition and its preparation method and application | |
| CN103040723B (en) | Xiyanping lipidosome injection | |
| Amjad et al. | Development and characterization of liposome-enriched ketoprofen liposomal hydrogels | |
| CN105395485B (en) | A kind of bicyclic alcohols liposome and preparation method thereof | |
| CN103040764A (en) | Bleomycin hydrocloride lipidosome injection | |
| CN103040753B (en) | Ginkgolide lipidosome injection | |
| Nadaf et al. | Novel Liposome Derived Nanoparticulate Drug Delivery System: Fabrication and Prospects | |
| CN103040742B (en) | Ginkgolide B lipid microsphere injection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |