CN107028885A - Bone targeting liposome for treating osteoporosis and preparation method thereof - Google Patents
Bone targeting liposome for treating osteoporosis and preparation method thereof Download PDFInfo
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
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- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
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- 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
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Abstract
The invention discloses a kind of Bone targeting liposome for being used to treat osteoporosis and preparation method thereof, the liposome is made up of the raw material of following parts by weight：4 ~ 6 parts of lecithin, 4 ~ 6 parts of cholesterol, 0.2 ~ 0.4 part of Isopsoralen, 0.4 ~ 0.6 part of vitamin E, 4 ~ 6 parts of cholesterol polyethylene glycol two banks, 0.08 ~ 0.12 part of glucose.Preparation method uses film dispersion method, and the liposome prepared is generally the spheroidal particle of class, and particle diameter is about 200~300nm, and average grain diameter is 224.3nm, and the stability of the particle size range in vivo is higher；Envelop rate is higher, and vitro release is more stable.
Description
Technical field
The present invention relates to pharmaceutical technology field, and in particular to it is a kind of be used for treat osteoporosis Bone targeting liposome and
Its preparation method.
Background technology
Liposome (liposomes) is a kind of small vesica of the bilayer similar to biomembrane tissue, can be used as one
Targeted drug carrier is planted, belongs to a kind of targeting drug delivery system (targeting drug delivery system) new technology.
It can not only make pharmaceutical preparation that there is the targeting of height by the use of liposome as pharmaceutical carrier, and the dissolving of medicine can be increased
Property, the bioavilability of medicine is improved, the therapeutic dose of medicine is reduced.Traditional Chinese medicine liposome, which is meant, encapsulates middle the effective elements of the medicine
The spherical drug carriers formulations of a kind of superminiature being made in lipoids bilayer.As a kind of novel dosage forms of Chinese medicine, it
Prepare simply, can multipurpose administration using convenience.It has its unique advantage as pharmaceutical carrier, including medicine can be protected to exempt from
Degraded, reach targeting moiety and reduce toxic side effect.Its action principle be between liposome and target cell can by endocytosis, melt
The modes such as conjunction, contact release, absorption, lipid exchange work and change the distribution of medicine in vivo, reach targeting drug release.And
Liposome has the characteristic of encapsulating fat-soluble medicine or water soluble drug, and fat-soluble medicine is presented in vivo by after liposomal encapsulated
The characteristics of going out different in itself from medicine.When drug encapsulation in liposome due to liposome package action or phosphatide and medicine it
Between the effect such as hydrogen bond, slowly release medicine, so as to extend the action time of medicine, show certain sustained release
Property；, can be for a long time to normal cell and the harmless and inhibitory action of tissue because liposome is analogous to the vesica of biofilm structure
It is adsorbed in around target cell, medicine is fully permeated to target cell target tissue, show cellular affinity and histocompatbility.
In recent years, liposome turns into one of focus as the research of pharmaceutical carrier.In addition its be adapted in vivo degraded, it is nontoxic
Property and non-immunogenicity, particularly it as pharmaceutical carrier can improve drug therapeutic indices, reduction drug toxicity and reduce medicine
The advantages of toxic side effect, it is set more and more to be taken seriously as the research of pharmaceutical carrier.If Components In Scutellaria Baicalensis baicalein is that have
Antibacterial, the antiviral drugs of effect, but they are oxidizable, by thermally labile, and water insoluble, limits them in practice
Using, and Liposomal formulation just changes this shortcoming, there is its unique advantage again.Therefore this kind of medicine is developed only not heavy
The clinical meaning wanted, it may have wide market prospects.But the physical chemistry of the liposome of osteoporosis is treated at present
Matter, such as particle diameter, envelop rate and drugloading rate, are not highly desirable, seriously reduce the curative effect of liposome medicament.For the technology
Problem, still lacks effective solution.
The content of the invention
For prior art, it is an object of the invention to provide a kind of liposome and its preparation for being used to treat osteoporosis
Method, is made a kind of liposome Bone targeting preparation containing Isopsoralen, can improve the therapeutic effect of traditional Chinese medicine, improve it
The utilization rate and bioavilability of active ingredient, reduce dosage, save limited natural resources of Chinese medicinal materials.
To realize object above, the technical solution adopted by the present invention is as follows：
First purpose of the present invention is to provide a kind of Isopsoralen liposome of Bone targeting modification, is by following weight
What the raw material of part was made：
4~6 parts of lecithin, 4~6 parts of cholesterol, 0.2~0.4 part of Isopsoralen, 0.4~0.6 part of vitamin E, courage are solid
Alcohol -4~6 parts of polyethylene glycol-two banks, 0.08~0.12 part of glucose.
In the present invention Bone targeting modification Isopsoralen liposome, the present invention from the particle diameter of liposome, envelop rate and
The factors such as drugloading rate are set out, and have obtained one group of preferably liposome formula by screening and optimizing experiment, each raw material components are one
Organic whole, it is indispensable.Inventor has found in research process, changes the proportional quantity in above-mentioned formula, then liposome is whole
Body action effect is significantly reduced.
Most preferably, for the particle diameter, envelop rate and drugloading rate of liposome, the Isopsoralen of the Bone targeting modification
Liposome, is made up of following mass concentration raw material：
Lecithin 5mgmL-1, cholesterol 5mgmL-1, Isopsoralen 0.3mgmL-1, vitamin E 0.5mg
mL-1, CPEG-two banks 5mgmL-1, glucose 0.1mgmL-1。
Wherein, lecithin 5mgmL-1Refer to use 5mg lecithin in every milliliter of liposome turbid liquor, other raw materials have
With this identical implication.
Second object of the present invention is to provide the preparation method of the Isopsoralen liposome of the Bone targeting modification, bag
Include following steps：
(1) weigh lecithin, cholesterol, Isopsoralen and vitamin E according to setting ratio to be dissolved in chloroformic solution, so
Uniform film is obtained using film dispersion method afterwards；
(2) PBS and CPEG-two banks are added into film again, it is mixed in 20~30 DEG C of ultrasounds
Even, filter membrane obtains liposome turbid liquor, adds the osmotic pressure that glucose adjusts the liposome turbid liquor, obtains bone
The Isopsoralen liposome of targeting modification.
The present invention has attempted a variety of preparation methods, the newborn method of such as high pressure, film dispersion method and reverse phase evaporation, from what is obtained
Particle diameter, envelop rate and the drugloading rate of liposome consider that screening and optimizing of the present invention obtains using film-ultrasonic wave dispersion technique, and it is prepared
The particle diameter of the Isopsoralen liposome of Bone targeting modification meets the requirements, and envelop rate and drugloading rate are higher.
In step (1), for enable lecithin, cholesterol, Isopsoralen and vitamin E excellent dissolution so as to
Uniform film is preferably formed, by verification experimental verification, present invention selection chloroformic solution.
The rotary temperature of the film dispersion method is 30~45 DEG C, and the present invention considers to obtain the thermal sensitivity and lecithin of medicine
Phase transition temperature, preferable temperature is 30 DEG C.During less than 30 DEG C, liposomal encapsulated Isopsoralen is easily revealed；Higher than 30 DEG C
When, the physical stability of liposome is bad, it is not easy to form spherical liposome.
In step (2), for the effect of stable homogeneous for improving influence liposome, by verification experimental verification, the present invention
Ultrasonic temperature select at 20~30 DEG C.
The stability of liposome of the consumption of the PBS of the present invention with ultimately forming is relevant, by verification experimental verification, sheet
Invention selection usage ratio is PBS and lecithin=5mL：4~6mg, preferably 1mL：1mg.
The ultrasonic power and time of the present invention has important shadow to the form of the liposome of formation, particle diameter and envelop rate
Ring, by verification experimental verification, ultrasonic power of the invention is 100~200W (preferably 150W), and ultrasonic time is selected in 15-20min,
To cause the uniform particle sizes of liposome, particle size is met for 200~300nm, and make its envelop rate higher.If ultrasonic work(
Rate is excessive, and ultrasonic time is long, the oxidation of liposome can be caused to be damaged, the Isopsoralen seepage of encapsulating.Ultrasonic power is small, surpasses
The sound time is long, and the stability of liposome is not good enough, and the particle diameter of the liposome of formation is undesirable, and particle diameter is smaller, in vivo easily
Disposed by lymphatic system, lose curative effect.
Heretofore described CPEG-two banks (BP-PEG-CHOL) are that can routinely make in the prior art
Standby obtained material, for example：Synthetic intermediate cholesterol methanesulfonate ester, synthetic intermediate CPEG first
(CHOL-PEG), the derivative (CHOL-PEG-NH2) of synthesizing polyethylene glycol terminal amino group, final synthetic cholesterol-poly- second two
Alcohol-two banks (CHOL-PEG-BP).Specifically, the present invention is prepared using following methods, cholesterol is dissolved in dichloromethane
In, plus triethylamine does acid binding agent, is cooled to -10 DEG C, methylsufonyl chloride stirring reaction is slowly added dropwise, after completion of the reaction processing water
Wash and filter to obtain white powdery solids.After PEG2000 fully dryings and vacuumizing, under argon gas protection, dry two are added
Methyl sulfoxide (DMSO) and tetrahydrofuran (THF), are then rapidly added sodium hydride, after room temperature activation, the courage for being dissolved in THF are consolidated
Alcohol methanesulfonate ester, which is added dropwise in above-mentioned solution, to react, and reaction solution is inclined into people into frozen water, dichloromethane is extracted, and is spin-dried for, through fast
Fast column chromatography for separation obtains CHOL-PEG.Above-mentioned product and methylsufonyl chloride reaction are prepared to the intermediate of methanesulfonate ester, are dissolved in
In dimethylformamide (DMF), the Sodium azide of addition is in 50-80 DEG C of reaction reduction generation primary amine.Bis phosphoric acid derivative is dissolved in
In anhydrous THF, stirring is lower to add CHOL-PEG-NH2, dicyclohexylcarbodiimide (DCC) is added, is filled into after room temperature reaction
In THF suspensions, filtering, washing, anhydrous sodium sulfate drying, concentrate are through silica gel rapid column chromatography, with methylene chloride/methanol ladder
Degree elutes to obtain Bone targeting binding element BP-PEG-CHOL.
In step (2), the detailed process of filter membrane is：0.8 and 0.45um filter membrane is crossed successively.
The Isopsoralen liposome that third object of the present invention is to provide above-mentioned Bone targeting modification is preparing treatment bone
Application in the loose disease drug of matter.
Above-mentioned technical proposal has the advantages that：
(1) present invention is prepared from suitable raw material turns into the Isopsoralen liposome that Bone targeting is modified, due to using
Suitable raw material and amount of preparation and preparation method, the liposome prepared is generally the spheroidal particle of class, particle diameter is about
200~300nm, average grain diameter is 224.3nm, and the stability of the particle size range in vivo is higher；Envelop rate and drugloading rate are higher,
Vitro release is more stable.
(2) preparing Isopsoralen turns into the Isopsoralen liposome that Bone targeting is modified, and has to osteoporosis
Significant therapeutic effect, has significant treatment prospect to senile bone metabolic disease, be intractable bone metabolic disease patient with
Carry out Gospel.
(3) liposome of the invention can effectively protect Isopsoralen as pharmaceutical carrier, improve Isopsoralen
Bioavilability, makes Isopsoralen have targeting, so as to strengthen the therapeutic effect of osteoporosis.
Brief description of the drawings
Fig. 1 is the Isopsoralen liposome structure schematic diagram of Bone targeting modification.
Fig. 2 is the Isopsoralen liposome transmission electron microscope (X20000) of Bone targeting modification.
Fig. 3 is the Isopsoralen liposomal particle size distribution map of Bone targeting modification.
Fig. 4 is the Isopsoralen liposome encapsulation and drugloading rate bioassay standard curve map of Bone targeting modification.
Fig. 5：The Isopsoralen liposome of Bone targeting modification to removal ovary C57/BL6 mouse femur hypomere RUNX2 and
PPAR- γ expression influences.
Fig. 6：Shadow of the Isopsoralen liposome of Bone targeting modification to removal ovary C57/BL6 mouse femur hypomere bone amount
Ring.
Embodiment
It is noted that described further below is all exemplary, it is intended to provide further instruction to the present invention.Unless another
Indicate, all technologies used herein and scientific terminology are with usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe embodiment, and be not intended to restricted root
According to the illustrative embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in this manual using term "comprising" and/or " bag
Include " when, it indicates existing characteristics, step, operation and/or combinations thereof.
Agents useful for same and material are that those skilled in the art can routinely obtain in the present invention.
In order that technical scheme can clearly be understood by obtaining those skilled in the art, below with reference to tool
The embodiment of body describes technical scheme in detail with comparative example.
Embodiment 1
A kind of preparation method of the Isopsoralen liposome of Bone targeting modification, comprises the following steps：
Lecithin, cholesterol, Isopsoralen, vitamin E, chloroformic solution are weighed respectively, it is standby after filtering.By embodiment
The arrangement of orthogonal test table in 2, it is accurate successively to draw above-mentioned filtrate and be placed in right amount in 25mL round-bottomed flask, in suitable temperature
Rotation is evaporated in bottle wall and forms uniform film under water bath condition, continues to be pumped to chloroform and fully volatilizees, adds appropriate PBS and delay
Fliud flushing (being a kind of conventional phosphate buffered saline solution) and CPEG-two banks, under 20-30 DEG C of water bath condition
Ultrasound, forms the liposome turbid liquor that near-transparent shows slightly opalescence；0.8 and 0.45um filter membrane is crossed successively, adds glucose tune
The osmotic pressure of the liposome turbid liquor is saved, the Isopsoralen liposome of Bone targeting modification is obtained.
The Isopsoralen liposome of Bone targeting modification is obtained, is made up of each raw material of following parts by weight：Lecithin
5mg·mL-1, cholesterol 5mgmL-1, Isopsoralen 0.3mgmL-1, vitamin E 0.5mgmL-1, cholesterol-poly- second
Glycol-two banks 5mgmL-1, glucose 0.1mgmL-1。
So that the optimal condition of Isopsoralen liposome effect is：Rotating evaporation temperature is 30 DEG C, buffer solution consumption
5ml, ultrasonic power 150W and ultrasonic time 15 minutes.
Comparative example 1
The Isopsoralen liposome of Bone targeting modification is made up of each raw material of following parts by weight：Lecithin 10mg
mL-1, cholesterol 10mgmL-1, Isopsoralen 0.3mgmL-1, vitamin E 0.8mgmL-1, CPEG-
Two banks 5mgmL-1, glucose 0.1mgmL-1.Experimental procedure is same as Example 1, and condition is：Rotating evaporation temperature is
30 DEG C, (adding proportion of PBS and lecithin is 1mL to buffer solution consumption 5ml：1mg), ultrasonic power 150W and ultrasound when
Between 15 minutes, difference is in the liposome turbid liquor to be formed, and the content of each raw material is different, and the mass action effect of liposome shows
Write reduction.
Embodiment 2
A kind of research of the preparation technology of the Isopsoralen liposome of Bone targeting modification, comprises the following steps：
Focused on to have investigated the consumption of rotating evaporation temperature (a), buffer solution according to the preparation process of film-ultrasonic wave dispersion technique
(b), the influence of ultrasonic power (c), the factor of ultrasonic time (d) four to liposome encapsulation, has worked out L9(34) experimental factor level
Table (is shown in Table 1).
The preparation technology orthogonal test table of table 1
As a result it is that A1B3C2D3, i.e. rotating evaporation temperature are 30 DEG C to show optimum preparating condition, buffer solution consumption 5ml, is surpassed
Acoustical power 150W and ultrasonic time 15 minutes.
Embodiment 3
The quality investigation of the Isopsoralen liposome for the Bone targeting modification that effect is optimal in embodiment 1, including following step
Suddenly：
The size of liposomal particle size directly affects its stability in vivo, liposome in vivo when due to high density fat
Protein cholesterol (HDL) combination can cause the change of particle diameter.The liposome that particle diameter is more than 300nm lacks vasopermeability, easily quilt
Reticuloendothelial system phagocytic, it is difficult to leave the circulatory system, and the liposome less than or equal to l00nm is easily disposed by lymphatic system.
Small unilamellar vesicle (20nm-50nm) adds aggregation and the extension half-life period in blood of internal target site, but multicell
Liposome medicament infiltration is difficult.This experiment prepares the transmission electron microscope results (see Fig. 2, Fig. 3) of liposome, and the present invention is each by control
The proportional quantity of raw material, prepared liposome is generally the spheroidal particle of class, and particle diameter is about 200~300nm, and average grain diameter is
224.3nm, the Isopsoralen liposome stability in vivo of the Bone targeting modification of the particle size range is higher.
By determining, the particle size range of the liposome of comparative example 1 is in more than 300nm, and this can influence liposome in vivo steady
It is qualitative.The consumption that this explanation changes liposome each component influences larger, inappropriate proportion relation to the particle size of liposome
The obtained liposome of each raw material be unsatisfactory for requiring.
The envelop rate and load medicine of the Isopsoralen liposome for the Bone targeting modification that effect is optimal in the embodiment 1 of embodiment 4
The measure of amount, comprises the following steps：Precision draw above-mentioned obtained liposome liquid 1.0mL low temperature ultracentrifugation (4 DEG C,
16000r.rain-1) 30min, take supernatant to be diluted to l0mL with PBS, with filtering with microporous membrane, take filtrate 10uL, sample introduction is determined
Peak area, substitutes into standard curve and obtains Isopsoralen biological concentration, so as to obtain liposome middle reaches divorced psoralen derivative
The content of thing, by following equation computational envelope rate (Entrapment Efficiency, EE) and drugloading rate (Drug Loading,
DL).Calculation formula：EE=(total-W trips of W)/W total x100%, DL=(total-W trips of W)/W lipoid xl00%, wherein W is always dispensing
Amount, W trips are the free dose for not wrapping into liposome, and W lipoids are lipoid total amount in prescription (see Fig. 4).
It can be obtained by measure, the envelop rate EE of the liposome in embodiment 1 is 91.2%, and drugloading rate DL is 38.3%.It is right
The envelop rate EE of the liposome of ratio 1 is 80.3%, and drugloading rate DL is 29.7%, is had compared with the effect in embodiment 1 notable
Difference, the consumption that this explanation changes liposome each component is larger on the influence of the envelop rate and drugloading rate of liposome, inappropriate
The envelop rate and drugloading rate for the liposome that each raw material of proportion relation is obtained are relatively low, and both effects have significant difference.
The experiment effect of embodiment 5
1.1 drug candidate
Isopsoralen (purity ＞ 99%, molecular weight 186.1635, Sigma, USA)；
The Isopsoralen liposome for the Bone targeting modification that effect is optimal in embodiment 1.
1.2 animal packets are set up with model
24 C57/BL6 female mice stochastic averaginas be divided into sham-operation group (Shame), ovariectomized group (OVX) and removal ovary add it is different
Psoralen group (OVX+ISO1), removal ovary add the Isopsoralen liposome (OVX+ISO2) that Bone targeting is modified, each group quantity
6.Muscle at OVX groups, OVX+ISO1 the and OVX+ISO2 groups separation nearly ilium ridge in back, takes out bilateral ovaries, ligatures the defeated ovum in top
Pipe, cuts off bilateral salpingo.The partial fat tissue around parcel ovary is cut off after the identical approach of Shame groups.OVX+ISO1 and
OVX+ISO2 groups start the Isopsoralen liposome of gavage Isopsoralen respectively and Bone targeting modification for 5 days before removal ovary,
Given low is 20mg/ (kgd), and 2 months gavage duration, Shame groups group is with grade dosage physiological saline gavage.
1.3 observation index and method
1.3.1 bone specimen is collected：Postoperative to feed 3 months, the neck that breaks puts to death mouse, takes both sides femur, shin bone, carefully rejects week
The soft tissues such as the muscle of attachment are enclosed, while left femur is put into the physiological saline containing 0.1% Sodium azide, standby bone tissue is micro-
Structure determination；Right side femur is used for immunohistochemistry, Immunofluorescence test RUNX2 and peroxisome proliferator-activated receptor
Body γ (PPAR- γ) protein expressions and histomorphometric analysis.
1.3.2 femoral inferior segment HE dyeing and fat cell quantitative analysis：Take the neutral paraformaldehyde normal temperature of experimental mouse femur 4%
It is fixed, carry out decalcification after rinsing, 4 DEG C of decalcifications 21 days change 1 decalcifying Fluid in every 3 days, decalcification process was observed every 3 days, femur is curved
Song thinks that decalcification can be terminated completely to 90 °.To complete the sample rinsing of decalcification, tissue dewatering, FFPE, section,
Roasting piece, carries out HE dyeing.Ordinary optical microscope is taken pictures to femoral inferior segment metaphysis, quantitative fat cell, analytical parameters
For:Adipocyte number (AD#, per mm2), total fat cell area accounts for ossis ratio (AV/TV).All sections are by 3
Individual different people is unified to shoot the same area, and shooting order is to the right averagely split picture in 3 areas by picture lower left corner metaphysis
Domain is shot, and is then to the right shot picture segmentation into 3 regions out of the picture upper left corner cortex bone, carries out fat thin
Born of the same parents do not include disrupted fat cell when counting.To avoid producing bias to final result during analysis, all figures are unknown
Each self-grouping (Sham, OVX, OVX+ISO1 group and OVX+ISO2) of sample.
1.3.3 femoral inferior segment Micro-CT is scanned：Distal femur carries out Micro-CT scanning (Scanco Medical, μ CT 80) point
Analysis:Mouse CO2Anesthesia, distal part of femur bone trabecula more substantially passes through Micro-CT scanning (μ CT) with the region concentrated and analyzed and Three-dimensional Gravity
Build, arrange parameter is scanning voltage 70KV, power 30W, the μ A of sweep current 429, and 5 μm of thickness is scanned every time.Analyze data includes:
Bone trabecula thickness (Tb.Th), TBV (Tb.Sp), bone volume (BV)/cumulative volume (TV) and bone trabecula quantity (Tb.N).
1.3.4 femoral inferior segment RUNX2, PPAR- γ immunofluorescences are expressed:Remove ovary and gavage Isopsoralen C57/
BL6 mouse femurs, retain knee joint and mid distal femur, and 4% neutral paraformaldehyde room temperature is fixed, and are rinsed, decalcification, floated again
Wash, tissue dewatering, FFPE, the roasting piece of section, roasting wax, dewaxing, antigen retrieval, be incubated antibody etc., it is rear to use mountant containing DIPI
Mounting, is kept in dark place after 3 minutes confocal microscope observation and takes pictures, and 5 orientation take 5 non-heavy at random from up and down
The folded visual field, average optical density value is calculated using Image-Pro Plus (IPP) software.
1.4 interpretation of result
1.4.1 drug candidate is to removal ovary C57/BL6 mouse femur hypomere RUNX2 and PPAR- γ expressions of results：
The core associated proteins factor 2 (RUNX2) is the key regulator of osteoblast differentiation, for measure of cell skeletonization
Or into the ability of fat.PPAR- γ are required cell transcription factors during adipocyte maturation, and its expression is strong and weak and active
Height can determine mouse BMSCs to Gegenbaur's cell or Adipocyte Differentiation.
As a result：The prompting of RUNX2 and PPAR- γ immunofluorescences, Bone targeting are carried out to removal ovary C57/BL6 mouse femurs hypomere
The Isopsoralen liposome therapeutic of modification can increase femoral inferior segment due to RUNX2 expression reduction caused by removal ovary, while energy
Suppress PPAR- γ expression increases, i.e. OVX+ISO2 groups femoral inferior segment RUNX2, PPAR- γ immunofluorescences expression intensity is higher than OVX+
ISO1 groups, difference has statistical significance (p<0.05) 2, Fig. 5, are shown in Table.
The Isopsoralen liposome of the Bone targeting of table 2 modification to removal ovary C57/BL6 mouse femur hypomere RUNX2 and
PPAR- γ expression influences (means standard deviation, n=6)
* for OVX+ISO2 compared with OVX+ISO1 groups, p<0.05；+ for Sham groups compared with OVX groups, p<0.05.
1.4.2 result of the test of the drug candidate to osteoporosis animal model：
Fat cell has Fatty toxicity to Gegenbaur's cell, fat cell into the state that fat breaks up can influence Gegenbaur's cell into
Bone ability and differentiation capability.
The Isopsoralen lipid physical efficiency of Bone targeting modification significantly inhibits the increasing of fat cell in ovariectomized mouse ossis
Plus, i.e., OVX+ISO2 groups adipocyte number is less than OVX+ISO1 groups, and difference has statistical significance (p<0.05).(table 3)
Influence of the Isopsoralen liposome of the Bone targeting of table 3 modification to C57/BL6 mouse femur hypomere fat cell amounts
(means standard deviation, n=6)
* for OVX+ISO compared with OVX groups, p<0.05；+ for Sham groups compared with OVX groups, p<0.05.
Scanned and pointed out by the Micro-CT scanning (Micro-CT) to each group C57/BL6 mouse femur hypomeres, Bone targeting modification
Isopsoralen liposome therapeutic can be obviously improved due to femoral inferior segment bone loss caused by removal ovary, i.e. OVX+ISO2 groups bone
Trabecular thickness (Tb.Th), bone volume/cumulative volume (BV/TV), bone trabecula quantity (Tb.N) are more than OVX+ISO1 groups, and difference has
Statistical significance (p<0.05)；And OVX+ISO2 groups TBV (Tb.Sp) is less than OVX+ISO1 groups, difference has statistics
Meaning (p<0.05) 3,4, Fig. 6, are shown in Table.
Influence (mean value ± of the Isopsoralen liposome of the Bone targeting of table 4 modification to C57/BL6 mouse femur hypomere bone amount
Standard deviation, n=6)
* for OVX+ISO2 compared with OVX+ISO1 groups, p<0.05；+ for Sham groups compared with OVX groups, p<0.05.
To sum up, liposome of the invention can effectively protect Isopsoralen as pharmaceutical carrier, improve Isopsoralen
Bioavilability, compared to simple Isopsoralen, there is more excellent therapeutic effect to osteoporosis.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of Isopsoralen liposome of Bone targeting modification, it is characterized in that, it is to be made up of the raw material of following parts by weight：
4 ~ 6 parts of lecithin, 4 ~ 6 parts of cholesterol, 0.2 ~ 0.4 part of Isopsoralen, 0.4 ~ 0.6 part of vitamin E, cholesterol-poly- second
4 ~ 6 parts of glycol-two banks, 0.08 ~ 0.12 part of glucose.
2. liposome as claimed in claim 1, it is characterized in that：The Isopsoralen liposome of Bone targeting modification, be by
What following mass concentration raw material was made：
Lecithin 5mgmL-1, cholesterol 5mgmL-1, Isopsoralen 0.3mgmL-1, vitamin E 0.5mgmL-1、
CPEG-two banks 5mgmL-1, glucose 0.1mgmL-1。
3. the preparation method of the Isopsoralen liposome of the Bone targeting modification described in claim 1 or 2, it is characterized in that, including
Following steps：
（1）Lecithin, cholesterol, Isopsoralen and vitamin E are weighed according to setting ratio to be dissolved in chloroformic solution, are then adopted
Uniform film is obtained with film dispersion method；
（2）PBS and CPEG-two banks are added into film again, are mixed in 20 ~ 30 DEG C of ultrasounds, filtering
Film, that is, obtain liposome turbid liquor, adds the osmotic pressure that glucose adjusts the liposome turbid liquor, obtains Bone targeting modification
Isopsoralen liposome.
4. preparation method as claimed in claim 3, it is characterized in that：Step（1）In, the rotary temperature of the film dispersion method is
30~45℃。
5. preparation method as claimed in claim 4, it is characterized in that：The rotary temperature of the film dispersion method is 30 DEG C.
6. preparation method as claimed in claim 3, it is characterized in that：Step（2）In, ultrasonic temperature is 20 ~ 30 DEG C.
7. preparation method as claimed in claim 3, it is characterized in that：Step（2）In, the addition ratio of PBS and lecithin
Example is 5mL：4 ~ 6mg, preferably 1mL：1mg.
8. preparation method as claimed in claim 3, it is characterized in that：Step（2）In, ultrasonic power is 100 ~ 200W, ultrasound
Selection of time is in 15-20min.
9. preparation method as claimed in claim 3, it is characterized in that：Step（2）In, the detailed process of filter membrane is：Cross successively
0.8 and 0.45um filter membrane.
10. the Isopsoralen liposome of the Bone targeting modification described in claim 1 or 2 is preparing treatment osteoporosis agents
In application.
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| US20100040546A1 (en) * | 2008-08-13 | 2010-02-18 | Searete Llc, A Limited Liability Corporation Of The State Of Delaware | Biological targeting compositions and methods of using the same |
| WO2013044734A1 (en) * | 2011-09-28 | 2013-04-04 | 中国科学院深圳先进技术研究院 | Bone targeted liposome and preparation method therefor |
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| US20100040546A1 (en) * | 2008-08-13 | 2010-02-18 | Searete Llc, A Limited Liability Corporation Of The State Of Delaware | Biological targeting compositions and methods of using the same |
| WO2013044734A1 (en) * | 2011-09-28 | 2013-04-04 | 中国科学院深圳先进技术研究院 | Bone targeted liposome and preparation method therefor |
| CN104274407A (en) * | 2013-07-08 | 2015-01-14 | 广东医学院 | Tanshinol lipidosome bone-targeting pharmaceutical preparation and preparation method thereof |
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