CN106039316A - An osteoblast targeted vector constructed based on a polypeptide and preparation and applications thereof - Google Patents
An osteoblast targeted vector constructed based on a polypeptide and preparation and applications thereof Download PDFInfo
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- CN106039316A CN106039316A CN201610288361.XA CN201610288361A CN106039316A CN 106039316 A CN106039316 A CN 106039316A CN 201610288361 A CN201610288361 A CN 201610288361A CN 106039316 A CN106039316 A CN 106039316A
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
Abstract
The invention relates to an osteoblast targeted vector constructed based on a polypeptide. The targeted vector comprises a target head and a support. The target head is the polypeptide. The grafting mass ratio of the target head is 5-10%, and the mole grafting ratio is 2-6%. The targeted vector is prepared by subjecting the polypeptide and a vector to carboxylic acid covalent linking through polyethylene glycol, and is used for filling medicines treating bone fractures, osteoporosis, osteoarthritis, rheumatoid arthritis or tumor bone metastasis. Compared with the prior art, the targeted vector has osteoblast targeting performance, high bone tissue affinity and efficient medicine delivery capability, and is capable of enhancing osteogenesis effects, reducing medicine using amounts, and reducing cell toxicity and side effects. The targeted vector provides an important medicine delivery tool for future osteoporosis treatment.
Description
Technical field
The present invention relates to technical field of biological material, be specifically related to a kind of based on polypeptide build osteoblast targeting vector and
Preparation and application.
Background technology
Bone is the connective tissue of a kind of high special in higher vertebrate body, it provide the supporting function to body and
Muscle attachment area.It is also the main source of inorganic ions, and plays an active part in internal calcium phosphorus balance.The normal development of skeleton
Bone is needed constantly to absorb and rebuild with maintenance.Under physiological status, this balance is to coordinate, osteocyte, osteoclast and one-tenth
The function of osteocyte is normal, and the integrity of bone amount and microstructure is maintained at a stable state.But, this bone remodeling loses
Weighing apparatus is that numerous osteopathia includes osteoporosis, Pei Jiteshi disease, osteoporosis, metastatic bone cancer, rheumatoid arthritis, bone
The major reason of marrow inflammation etc..
As a complicated living tissue, bone is mainly by three kinds of different types of cells: osteoblast, osteoclast and bone
Cell.Osteoblast is bone formation cell, derives from the osteoprogenitor cells of local, is responsible for synthetic bone substrate.Osteoclast is bone
Absorbing cell, core is big, derives from multiple hemopoietic tissue.The cell of both types exists only in the mineralized bone matrix of bone surface,
And osteocyte is positioned at bone matrix, and can absorb and form bone matrix.The characteristic being connected by synapse due to it.The outer base of osteocyte
Matter is made up of organic and height mineralising hydroxyapatite.Along with deepening continuously, in recent years of bone biology research
The medicine development of bone metabolism disease, the conduct such as bone formation surface, bone resorption surface, osteoclast, bone matrix
New therapy target includes research in.Although some research product has been applied to the exploitation of medicine, but specific to bone and cell receptor,
Most of medicines newly developed do not possess bone specificity.
Osteoporosis (Osteoporosis) be a kind of reduced by the unbalance bone amount that causes of bone metabolism, skeleton fine structure
Occur to destroy caused skeleton fragility and the systemic disease of fracture easily occurs.Osteoporosis is important in a world wide
Health problem.Along with increasing the weight of of aged tendency of population, the number of patients of osteoporosis dramatically increases.At present, the whole world there are about 2
Hundred million people suffer from osteoporosis, and its sickness rate has leapt to the 7th of commonly encountered diseases.Wherein the prevalence of postmenopausal women is higher.65
The women fracture occurred frequently that year is above, has a strong impact on the quality of life of person in middle and old age, health and life-span.
The medicine for the treatment of osteoporosis is divided three classes at present: 1) suppression bone resorption medicine, such as Diphosphonate, estrogen
Deng；2) promoting bone growing medicine, such as parathyroid hormone, fluoride etc.；3) two-way function medicine: vitamin K, strontium salt etc..Due to
Osseous tissue has hardness, and greatly, locally blood supply is abundant, poor permeability and the feature such as physiological and biochemical procedure is special, by administration
Approach is difficult to make transport of drug to lesions position.These medicines are usually through Formulations for systemic administration, and increasing using dosage could be at osseous tissue
In reach effectively to treat concentration.Owing to medicine lacks bone specificity, not only reduce the therapeutic index of medicine, and can be to disease
The non-bone tissue of people or organ cause unnecessary toxic and side effects, such as the nearest report of the long-term side-effects of Hormone Replacement Therapy
Display, as a example by representing medicine parathyroid hormone, applies and reduces more than 2 years curative effects, and bone loss, even serious side effects can
There are nervous symptoms, renal failure etc..Therefore by drug delivery system, drug targeting is delivered to osseous tissue or osteoblast, thus
Increase therapeutic effect, and it is particularly significant to reduce toxic and side effects.At present, according to the bone target of the targeting research to hydroxyapatite
Include to delivery system: Tetracyclines, bis phosphoric acid salt, somatomedin class and many peptides.
Tetracycline is as antimicrobial drug, as Bone targeting target head, it is impossible to eliminate itself biological effect, and have side effect
And cause effect to be restricted；The Bone targeting delivery system built by diphosphonate, can be rich at bone surface and dentin surface
Collection, is difficult to get rid of；Somatomedin has natural bone tropism, but has the half-life short, changeableness, and excess application may have
The shortcoming such as genotoxic potential and carcinogenecity.
Summary of the invention
Defect that the purpose of the present invention is contemplated to overcome above-mentioned prior art to exist and provide a kind of Targeting Performance good, malicious
Property and side effect little based on polypeptide build osteoblast targeting vector and preparation and application.
The purpose of the present invention can be achieved through the following technical solutions: a kind of osteoblast targeting built based on polypeptide
Carrier, this targeting vector includes target head and support, and described target head is covalently attached to described support by poly-diethanol carboxylic acid, described
Target head be polypeptide, the quality percent grafting of target head is 5～10%, and mole percent grafting is 2～6%.
Described polypeptide is made up of serine and two kinds of aminoacid of aspartic acid, containing 2～50 ammonia in described polypeptide
Base acid, the number of described serine and aspartic acid ratio is (0.25～4): 1.
Described polypeptide includes that serine and aspartic acid, the sequence of described polypeptide are preferably Ser Asp Ser Ser
Asp, wherein, Ser represents serine, and Asp represents aspartic acid.
Tooth and osseous tissue are as the highest organ of mineralization degree in human body, and the most important promotion mineralization protein has increase
Bone amount, treats osteoporotic potentiality.Ser Asp Ser Ser Asp is selected from dentin phosphoprotein (DPP) primary structure, this egg
Casamino acid has put in order distinctive Ser Asp and Ser Ser Asp repetitive sequence.This sequence is selected to have following former
Cause: (1) DPP derives from the sclerous tissues that human body mineralization degree is the highest, tooth, and it expresses position is the dentin of mineralising, osseous tissue
In also have expression；(2) extracellular matrix protein that during DPP is dentin, expression is the highest, also has notable expression in alveolar bone；
(3) DPP lacks its dentin of mice and osseous tissue mineralising are substantially suppressed, and tooth is thinning, and frontal resorption is obvious；(4)
Protein chemistry, cytology and histology experiment confirm, in DPP, aminoacid Asp is the aminoacid of highly acidic, causes Ser
Asp Ser Ser Asp acidity is higher, it is possible to the hydroxyapatite on specific binding osseous tissue surface, hence it is evident that promote into tooth cell
With osteoblastic maturation and secretion mineralized dentin matrix.As more special mineralising regulation albumen, in Dentinal growth and mineralising
During play an important role.(5) vivo and vitro research finds Ser Asp Ser Ser Asp polypeptide, it is possible to by thin with skeletonization
Cellular surface specific proteins periostin (periostin) combines, and endocytosis enters cell, thus selectively targeted acts on into
Osteocyte.
Described support is polyurethane support, and described support is 60～120 nanometers, in oblate spheroid under Electronic Speculum, can wrap up and passing
Send small nut acids medicine.The existing good physical and mechanical properties of polyurethane material, has again good blood compatibility and biology
The compatibility, thus it is widely used in biomedical sector.It has excellent anticoagulation function and biocompatibility, and toxotest is multiple
The medical requirement of core, without aberration inducing effect, without anaphylaxis and excellent toughness, elasticity, wear-resisting moisture-proof, corrosion resistance and
Heat stability, and after being prepared as Nano microsphere can carrying medicament effectively, act on target organ by blood circulation or target be thin
Born of the same parents.
The preparation method of a kind of osteoblast targeting vector built based on polypeptide as mentioned above, mainly includes following
Step:
(1) preparation of target head: in proportion amino acid condensation is become by 12 passage semi-automatic polypeptide synthesizers target head, treats
With；
(2) preparation of support: by LDI and MEDA with 1:(0.5～1) mol ratio be dissolved in acetone, carry out hybrid reaction,
Being subsequently adding PEG and PCL, the mole of PEG, PCL of addition is PEG:PCL with the ratio of the mole of the LDI of described addition:
LDI=(0.05～0.25): (0.01～0.05): (0.2～2), adds dehydrated alcohol and reacts after reaction, addition anhydrous
Ethanol and the ratio of the mole of described LDI are (0.1～10): (1), obtain thick PU liquid, then add petroleum ether in thick PU liquid
Precipitating, filter and precipitate is dissolved in acetone, after being centrifuged, dialysis obtains PU nano-micelle liquid, stand-by；
(3) synthesis of targeting vector: step (1) gained target head is dissolved in deionized water, is subsequently adding matter first-class with target
EDC and NHS of amount carries out the activation of target peptide, is subsequently adding step (2) gained PU nano-micelle liquid, the quality of its head that hits and PU
The volume ratio of nano-micelle liquid is 1:(1～10), dialyse after standing and i.e. obtain described osteoblast targeting vector.
In above-mentioned steps, LDI is lysinediisocyanate, and MEDA is: N methyldiethanol amine, and PEG is Polyethylene Glycol
Dihydroxylic alcohols, PCL is polycaprolactone diols, and EDC is 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, and NHS is
N-hydroxy-succinamide.
The reaction temperature of step (2) described LDI, MEDA is 50～100 DEG C, and the response time is 20～30h；
The reaction temperature of described PEG, PCL is 50～100 DEG C, and the response time is 70～80h；
The reaction temperature of described dehydrated alcohol is 50～100 DEG C, and the response time is 40～50h；
The petroleum ether of described addition is 1:(0.2～0.5 with the ratio of the volume of thick PU liquid).
The temperature of step (3) described activation is 2～10 DEG C, and soak time is 5～20min；
Dwell temperature is 2～10 DEG C, and time of repose is 10～20h.
Because the length of polypeptide target head determines the complexity of synthesis, the composition of this target head just only comprises two kinds of aminoacid
Can there is good Bone targeting, therefore can realize small peptide and just can complete targeting, the basis that therefore targeting is accomplished
On, it is possible to meet the feature of small peptide.
The application of a kind of osteoblast targeting vector built based on polypeptide as mentioned above, described targeting vector is used for filling
Fill out treatment fracture, stock rickets, osteoporosis, osteoarthritis, rheumatoid arthritis or the medicine of bone metastaes.
The mass ratio of described targeting vector and medicine is 1:(5～10%).
It is mainly composed of Ser aminoacid and Asp aminoacid, according to a certain percentage and be linked in sequence.Apply this target
Efficiently small nucleic acids medicine can be delivered to the target cell osteoblast of characteristic, and its to the small nucleic acids medicine of vehicle delivery
The effect of promoting bone growing and bone apposition clearly, can be treated and include the osteopathias such as osteoporosis that OVX causes.
Compared with prior art, beneficial effects of the present invention is embodied in following several respects:
(1) the target head of the present invention derives from the native protein DPP with high mineralization regulating power, has the highest targeting
Property, medicine accurately can be delivered in osteocyte, decrease side effects of pharmaceutical drugs；
(2) the target head of the present invention uses small peptide, and synthesis is simple, and production cost is low；
(3) support used by the present invention is the polyurethane material with good hydrophilic, biological activity and degradability, this
Material has the strongest plasticity and hypotoxicity, and will not be enriched with in vivo, it is ensured that Drug safety.
(4) the preferred target head of the present invention derives from the native protein DPP with high mineralization regulating power, and it promotes biological ore deposit
Change ability is clear and definite, meanwhile, decreases side effects of pharmaceutical drugs.
Accompanying drawing explanation
Fig. 1 is the composition principle schematic diagram of the present invention；
Fig. 2 a is embodiment 1 targeting vector picture of pattern under transmission electron microscope；
Fig. 2 b is embodiment 1 targeting vector potential image of targeting vector under transmission electron microscope；
Fig. 3 a is that embodiment 1 hits the UV-VIS spectrophotometry test result of head；
Fig. 3 b is the UV-VIS spectrophotometry test result of carrier in embodiment 1；
Fig. 3 c is the testing result figure of the percent grafting of targeting vector in embodiment 1；
Fig. 4 is embodiment 1 targeting vector targeting experimental result picture at osteoblast surfaces；
Fig. 5 is that internal osteoblast orients Bone targeting carrier the result schematic diagram；
Fig. 6 is into the intracorporeal organ distribution experiments result schematic diagram of osteoblast targeting vector；
Fig. 7 is the performance test results that embodiment 1 targeting vector prevents RNA from degrading；
Fig. 8 skeletonization targeting vector increases the design sketch of bone amount.
Detailed description of the invention
Elaborating embodiments of the invention below, the present embodiment is carried out under premised on technical solution of the present invention
Implement, give detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following enforcement
Example.
Embodiment 1
A kind of osteoblast targeting vector built based on polypeptide, this targeting vector includes target head and support, target head and
Frame target head and support are covalently bound by poly-diethanol carboxylic acid (PEG-COOH).Its target head quality connects a rate and is about 6%, and target head rubs
You connect a rate is 3%.Target head is the polypeptide being made up of serine and aspartic acid, and wherein the sequence of this polypeptide is Ser Asp
Ser Ser Asp, wherein, Ser represents serine, and Asp represents aspartic acid；Support is polyurethane support.
Above-mentioned targeting vector, its synthetic method is as it is shown in figure 1, include following step:
(1) preparation of target head: use 12 passage semi-automatic polypeptide synthesizers, applies amino acid condensation order method of attachment
Synthesis target head, presses Ser-Asp-by serine (Fmoc-Asp (otBu)-OH) and aspartic acid (Fmoc-Ser (Tbu)-OH)
Ser-Ser-Asp is linked in sequence, and obtains target head Ser Asp Ser Ser Asp, stand-by；
(2) preparation of support: the first step, the synthesis of PU: 7.85g LDI, 2.98g MEDA and 89.17g acetone are in airtight
Mixing in reaction bulb, 60 DEG C of vibrations seal reaction 24 hours.Take reacted solution 49.98g, add 4.44g PEG, 0.58g
PCL and 45g acetone (gross mass is 100g), mixed liquor adds 0.5g dehydrated alcohol, nothing in 60 DEG C of oscillating reactionss after 72 hours
60 DEG C of oscillating reactionss of water-stop 48 hours, can obtain PU liquid.Second step, PU purify: be slowly added in 100mL petroleum ether
35mL thick PU liquid, petroleum ether precipitation, stand to filter and to obtain PU precipitation, PU is precipitated vacuum drying and obtains and slightly purify PU.PU is dissolved in
The PU acetone soln of the third 10%, PU acetone solution and acetone be 1:9 by volume, are 1:1 by PU acetone diluent and deionized water
Ratio mixing, low-temperature and high-speed is centrifuged 10min, 15000g；Supernatant dialysis acetone after Li Xin, treats that the liquid in bag filter becomes
Become milky can obtain PU nano-micelle liquid, after PU Micellar Solution Which Is is freeze-dried, seal freezen protective.
(3) synthesis of targeting vector: skeletonization target peptide Ser Asp Ser Ser Asp is dissolved in 1mL deionized water, then adds
Enter EDC and NHS with the quality such as target peptide and carry out the activation of target peptide, 4 DEG C hatch after 10min again toward in target peptide activation mixed liquor by one
Fixed mass volume ratio (peptide masses: PU liquid amasss ≈ 1:5) adds PU nano-micelle liquid, and PU target peptide mixed liquor 4 DEG C reacted
Night.After reaction terminates, with deionized water reactant liquor dialysed (MW=3500) to remove the impurity such as inorganic salt, after dialysis i.e.
The PU nano-micelle liquid (Ser Asp-PU) of target peptide coupling can be obtained, after lyophilization, seal freezen protective.
Being scanned above-mentioned targeting vector with electron-microscope scanning, its result is as shown in Figure 2 a and 2 b.
The percent grafting of the targeting vector of the employing UV-VIS spectrophotometry above-mentioned gained of test, test result such as Fig. 3 a,
Shown in Fig. 3 b and Fig. 3 c, result shows: the percent grafting of this targeting vector is 6.8%.
By fluorescent labeling on above-mentioned target headband, then allow mice and people take this targeting vector, and mice and people are carried out
Following the trail of, as shown in Figure 4, result shows result, and Ser Asp Ser Ser Asp can reach and periostin (periostin) by polypeptide
Location, illustrates that Ser Asp Ser Ser Asp in conjunction with periostin, and can enter osteoblast altogether.
After vehicle delivery FITC fluorescent labeling small nucleic acids described in mouse tail vein, sections observation, result is as it is shown in figure 5, tie
Fruit display, fluorescin is specific enrichment in osteoblast, illustrates that this carrier has into osteoblast targeting.
After vehicle delivery FITC fluorescent labeling small nucleic acids described in mouse tail vein injection, observed by living imaging instrument, its
As shown in Figure 6, result shows result, and fluorescence signal is close in soft tissue organs, and in osseous tissue, experimental group is apparently higher than sky
Vehicle group, illustrates that this carrier has Bone targeting effect.
By upper for filling in the targeting vector of above-mentioned gained siRNA, internal injection.And increase contrast groups, direct hormone
SiRNA, tests siRNA degraded situation in vivo, and its result, as it is shown in fig. 7, result shows, utilizes the present invention to load
SiRNA degradation speed in vivo is less so that siRNA can have the more time to make contact to osteocyte, increases having of siRNA
Effect property.
By the inhibitor of upper for filling in the targeting vector of above-mentioned gained miRNA214, internal injection, targeting intervenes estrogen
Lacking the osteoporosis caused, after injecting January, osteoporosis mice bone amount substantially increases, and result as shown in Figure 8, illustrates this
Carrier can deliver miRNA and treat osteoporosis.
Embodiment 2
A kind of osteoblast targeting vector built based on polypeptide, this targeting vector includes target head and support, target head and
Frame is covalently bound by poly-diethanol carboxylic acid (PEG-COOH).Its target head quality connects a rate and is about 5%,.Target head is by serine
With the polypeptide of aspartic acid composition, wherein the sequence of this polypeptide is Ser Asp, and wherein, Ser represents serine, and Asp represents sky
Aspartic Acid；Support is polyurethane support.
Above-mentioned targeting vector, its synthetic method such as, including following step:
(1) preparation of target head: use 12 passage semi-automatic polypeptide synthesizers, applies amino acid condensation order method of attachment
Synthesis target head, presses Ser-Asp by serine (Fmoc-Asp (otBu)-OH) and aspartic acid (Fmoc-Ser (Tbu)-OH) suitable
Sequence connects, and obtains target head Ser Asp, stand-by；
(2) preparation of support: the first step, the synthesis of PU: the MEDA of LDI and 0.5mol of 1mol is dissolved in acetone, puts
Mixing in confined reaction bottle, vibration seals reaction, and reaction temperature is 50 DEG C, and the response time is 20h.It is subsequently adding 0.05mol
The PCL of PEG and 0.01mol, mix oscillating reactions, reaction temperature is 50 DEG C, and the response time is 70h, adds 0.1mol's
Dehydrated alcohol, oscillating reactions, reaction temperature is 50 DEG C, and the response time is 40h, can obtain thick PU liquid.Second step, PU purify:
Add the petroleum ether of 5 times that gained thick PU liquid amasss, precipitate, obtain and slightly purify PU.It is dissolved in acetone by slightly purifying PU, centrifugal
Rear dialysis can obtain PU nano-micelle liquid, preserves after lyophilization.
(3) synthesis of targeting vector: be dissolved in 1mL deionized water by skeletonization target peptide Ser Asp, adds and the matter such as target peptide
EDC and NHS of amount carries out the activation of target peptide, after 2 DEG C hatch 20min, then adds PU nano-micelle in target peptide activation mixed liquor
Liquid, its quality that hits: PU nano-micelle liquid=1:5, PU target peptide mixed liquor 2 DEG C reaction 20h.After reaction terminates, use deionization
Water reactant liquor is dialysed (MW=3500) to remove the impurity such as inorganic salt, after dialysis the PU nanometre glue of target peptide coupling
Bundle liquid (Ser Asp-PU), seals freezen protective after lyophilization.
Application fluorescent labeling Ser Asp, in people with mouse bone-forming cell, Ser Asp can reach and periostin by polypeptide
(periostin) position altogether, illustrate that Ser Asp in conjunction with periostin, and can enter osteoblast.Observing under transmission electron microscope should
The size of targeting vector and shape characteristic.Described carrier PU-Ser Asp can wrap up siRNA and reduce the external degradation of siRNA
Speed.After vehicle delivery FITC fluorescent labeling small nucleic acids described in mouse tail vein, sections observation finds, fluorescin is thin at skeletonization
Specific enrichment in born of the same parents, illustrates that this carrier has into osteoblast targeting.Vehicle delivery described in mouse tail vein injection
After FITC fluorescent labeling small nucleic acids, by living imaging instrument it has been observed that fluorescence signal is close, at bone in soft tissue organs
In tissue, experimental group is apparently higher than empty carrier group, illustrates that this carrier has Bone targeting effect.
By the inhibitor of upper for filling in the targeting vector of above-mentioned gained miRNA214, the matter of the miRNA214 inhibitor of filling
Amount is 5%:1 with the mass ratio of targeting vector.Internal injection, targeting intervenes the osteoporosis that estrogen deficiency causes, injection
After January, osteoporosis mice bone amount substantially increases.Illustrate that this carrier can deliver miRNA and treat osteoporosis.
Embodiment 3
A kind of osteoblast targeting vector built based on polypeptide, this targeting vector includes target head and support, target head and
Frame is covalently bound by poly-diethanol carboxylic acid (PEG-COOH).Its target head quality percent grafting is about 6%.Target head be by serine and
The polypeptide of aspartic acid composition, a length of 20 aminoacid, wherein the sequence of this polypeptide is Ser Asp Asp Asp Asp
Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp (Ser:Asp=1:4), its
In, Ser represents serine, and Asp represents aspartic acid；Support is polyurethane support.
Above-mentioned targeting vector, its synthetic method such as, including following step:
(1) preparation of target head: use 12 passage semi-automatic polypeptide synthesizers, applies amino acid condensation order method of attachment
Synthesis target head, presses Ser-Asp by serine (Fmoc-Asp (otBu)-OH) and aspartic acid (Fmoc-Ser (Tbu)-OH) suitable
Sequence connects, and obtains target head, stand-by；
(2) preparation of support: the first step, the synthesis of PU: the MEDA of LDI and 1mol of 1mol is dissolved in acetone, is placed in
Mixing in confined reaction bottle, vibration seals reaction, and reaction temperature is 100 DEG C, and the response time is 30h.It is subsequently adding 1.25mol's
The PCL of PEG and 0.25mol, mixes oscillating reactions, and reaction temperature is 100 DEG C, and the response time is 80h, adds the nothing of 10mol
Water-ethanol, oscillating reactions, reaction temperature is 100 DEG C, and the response time is 50h, can obtain thick PU liquid.Second step, PU purify: add
Enter the petroleum ether of 2 times that gained thick PU liquid amasss, precipitate, obtain and slightly purify PU.It is dissolved in acetone, after being centrifuged by slightly purifying PU
Dialysis can obtain PU nano-micelle liquid, preserves after lyophilization.
(3) synthesis of targeting vector: be dissolved in 1mL deionized water by skeletonization target peptide Ser Asp, adds and the matter such as target peptide
EDC and NHS of amount carries out the activation of target peptide, after 10 DEG C hatch 5min, then adds PU nano-micelle in target peptide activation mixed liquor
Liquid, its quality that hits: PU nano-micelle liquid=1:1, PU target peptide mixed liquor 10 DEG C reaction 10h.After reaction terminates, use deionization
Water reactant liquor is dialysed (MW=3500) to remove the impurity such as inorganic salt, after dialysis the PU nanometre glue of target peptide coupling
Bundle liquid (Ser Asp-PU), seals freezen protective after lyophilization.
Application fluorescent labeling Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp Ser Asp Asp Asp
Asp Ser Asp Asp Asp Asp, in people with mouse bone-forming cell, Ser Asp Asp Asp Asp Ser Asp Asp
Asp Asp Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp can reach and periostin (periostin) by polypeptide
Location, illustrates Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp Ser altogether
Asp Asp Asp Asp in conjunction with periostin, and can enter osteoblast.The size of this targeting vector is observed under transmission electron microscope
And shape characteristic.Described carrier PU-Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp Ser Asp Asp Asp
Asp Ser Asp Asp Asp Asp can wrap up siRNA and reduce the external degradation speed of siRNA.Described in mouse tail vein
After vehicle delivery FITC fluorescent labeling small nucleic acids, sections observation finds, fluorescin is specific enrichment in osteoblast, explanation
This carrier has into osteoblast targeting.After vehicle delivery FITC fluorescent labeling small nucleic acids described in mouse tail vein injection,
By living imaging instrument it has been observed that fluorescence signal is close in soft tissue organs, in osseous tissue, experimental group is apparently higher than sky
Vehicle group, illustrates that this carrier has Bone targeting effect.
By the inhibitor of upper for filling in the targeting vector of above-mentioned gained miRNA214, the matter of the miRNA214 inhibitor of filling
Amount is 10%:1 with the mass ratio of targeting vector.Internal injection, targeting intervenes the osteoporosis that estrogen deficiency causes, injection
After January, osteoporosis mice bone amount substantially increases.Illustrate that this carrier can deliver miRNA and treat osteoporosis.
Embodiment 4
A kind of osteoblast targeting vector built based on polypeptide, this targeting vector includes target head and support, target head and
Frame is covalently bound by poly-diethanol carboxylic acid (PEG-COOH).Its target head quality percent grafting is about 10%, and target head is by serine
With the polypeptide of aspartic acid composition, this polypeptide has 50 aminoacid, and wherein serine with the number ratio of aspartic acid is
The sequence of=4:1 wherein this polypeptide is as follows:
Ser Ser Ser Ser Asp Ser Ser Ser Ser Asp Ser Ser Ser Ser Asp Ser Ser
Ser Ser Asp Ser Ser Ser Ser Asp Ser Ser Ser Ser Asp Ser Ser Ser Ser Asp Ser
Ser Ser Ser Asp Ser Ser Ser Ser Asp Ser Ser Ser Ser Asp, wherein, Ser represents serine,
Asp represents aspartic acid；Support is polyurethane support.
Above-mentioned targeting vector, its synthetic method such as, including following step:
(1) preparation of target head: use 12 passage semi-automatic polypeptide synthesizers, applies amino acid condensation order method of attachment
Synthesis target head, presses Ser-Asp by serine (Fmoc-Asp (otBu)-OH) and aspartic acid (Fmoc-Ser (Tbu)-OH) suitable
Sequence connects, and obtains target head, stand-by；
(2) preparation of support: the first step, the synthesis of PU: the MEDA of LDI and 0.7mol of 1mol is dissolved in acetone, puts
Mixing in confined reaction bottle, vibration seals reaction, and reaction temperature is 75 DEG C, and the response time is 24h.It is subsequently adding 0.025mol
The PCL of PEG and 0.005mol, mix oscillating reactions, reaction temperature is 75 DEG C, and the response time is 72h, adds the nothing of 5mol
Water-ethanol, oscillating reactions, reaction temperature is 75 DEG C, and the response time is 48h, can obtain thick PU liquid.Second step, PU purify: add
Enter the petroleum ether of 3 times that gained thick PU liquid amasss, precipitate, obtain and slightly purify PU.It is dissolved in acetone, after being centrifuged by slightly purifying PU
Dialysis can obtain PU nano-micelle liquid, preserves after lyophilization.
(3) synthesis of targeting vector: be dissolved in 1mL deionized water by skeletonization target peptide Ser Asp, adds and the matter such as target peptide
EDC and NHS of amount carries out the activation of target peptide, after 4 DEG C hatch 10min, then adds PU nano-micelle in target peptide activation mixed liquor
Liquid, its quality that hits: PU nano-micelle liquid=1:5, PU target peptide mixed liquor 4 DEG C reaction 12h.After reaction terminates, use deionization
Water reactant liquor is dialysed (MW=3500) to remove the impurity such as inorganic salt, after dialysis the PU nanometre glue of target peptide coupling
Bundle liquid (Ser Asp-PU), seals freezen protective after lyophilization.
Application fluorescent labeling Ser Asp, in people with mouse bone-forming cell, target head reaches and periostin (periostin)
Location, illustrates that target head in conjunction with periostin, and can enter osteoblast altogether.Described carrier PU-Ser Ser Ser Ser Asp
SiRNA can be wrapped up and reduce the external degradation speed of siRNA.Vehicle delivery FITC fluorescent labeling small nut described in mouse tail vein
After acid, sections observation finds, fluorescin is specific enrichment in osteoblast, illustrates that this carrier has into osteoblast targeting
Effect.After vehicle delivery FITC fluorescent labeling small nucleic acids described in mouse tail vein injection, by living imaging instrument it has been observed that glimmering
Optical signal is close in soft tissue organs, and in osseous tissue, experimental group is apparently higher than empty carrier group, illustrates that this carrier has bone target
To effect.
By the inhibitor of upper for filling in the targeting vector of above-mentioned gained miRNA214, the matter of the miRNA214 inhibitor of filling
Amount is 7.5%:1 with the mass ratio of targeting vector.Internal injection, targeting intervenes the osteoporosis that estrogen deficiency causes, note
After penetrating January, osteoporosis mice bone amount increases.Illustrate that this carrier can deliver miRNA and treat osteoporosis.
Claims (9)
1. the osteoblast targeting vector built based on polypeptide, it is characterised in that this targeting vector includes target head and support,
Described target head is covalently attached to described support by poly-diethanol carboxylic acid, and described target head is polypeptide, the quality percent grafting of target head
It is 5～10%.
A kind of osteoblast targeting vector built based on polypeptide the most according to claim 1, it is characterised in that described
Polypeptide is made up of serine and two kinds of aminoacid of aspartic acid, containing 2～50 aminoacid in described polypeptide, and described serine
It is (0.25～4) with the number ratio of aspartic acid: 1.
A kind of osteoblast targeting vector built based on polypeptide the most according to claim 2, it is characterised in that described
Polypeptide includes 3 serines and 2 aspartic acids, and the sequence of described polypeptide is Ser Asp Ser Ser Asp, wherein, Ser
Representing serine, Asp represents aspartic acid.
A kind of osteoblast targeting vector built based on polypeptide the most according to claim 2, it is characterised in that described
Support is polyurethane support, and described support is oblate spheroid, and the particle diameter of described support is 60～120nm.
5. a preparation method for the osteoblast targeting vector built based on polypeptide as described in Claims 1 to 4 is arbitrary, it is special
Levying and be, the method mainly includes following step:
(1) preparation of target head: in proportion amino acid condensation is become target head by 12 passage semi-automatic polypeptide synthesizers, stand-by；
(2) preparation of support: by LDI and MEDA with 1:(0.5～1) mol ratio be dissolved in acetone, carry out hybrid reaction, then
Adding PEG and PCL, the mole of PEG, PCL of addition is PEG:PCL:LDI=with the ratio of the mole of the LDI of described addition
(0.05～0.25): (0.01～0.05): (0.2～2), adds dehydrated alcohol and reacts after reaction, the dehydrated alcohol of addition
It is (0.1～10) with the ratio of the mole of described LDI: 1, obtains thick PU liquid, in thick PU liquid, then add petroleum ether sink
Forming sediment, filter and precipitate is dissolved in acetone, after being centrifuged, dialysis obtains PU nano-micelle liquid, stand-by；
(3) synthesis of targeting vector: step (1) gained target head is dissolved in deionized water, is subsequently adding and target first-class quality
EDC and NHS carries out the activation of target peptide, is subsequently adding step (2) gained PU nano-micelle liquid, the quality of its head that hits and PU nanometer
The volume ratio of Micellar Solution Which Is is 1:(1～10), dialyse after standing and i.e. obtain described osteoblast targeting vector.
The preparation method of a kind of osteoblast targeting vector built based on polypeptide the most according to claim 5, its feature
Being, the reaction temperature of step (2) described LDI, MEDA is 50～100 DEG C, and the response time is 20～30h；
The reaction temperature of described PEG, PCL is 50～100 DEG C, and the response time is 70～80h；
The reaction temperature of described dehydrated alcohol is 50～100 DEG C, and the response time is 40～50h；
The petroleum ether of described addition is 1:(0.2～0.5 with the ratio of the volume of thick PU liquid).
The preparation method of a kind of osteoblast targeting vector built based on polypeptide the most according to claim 5, its feature
Being, the temperature of step (3) described activation is 2～10 DEG C, and soak time is 5～20min；
Dwell temperature is 2～10 DEG C, and time of repose is 10～20h.
8. an application for the osteoblast targeting vector built based on polypeptide as described in Claims 1 to 4 is arbitrary, its feature exists
In, described targeting vector be used for loading treatment fracture, stock rickets, osteoporosis, osteoarthritis, rheumatoid arthritis or
The medicine of bone metastaes.
The application of a kind of osteoblast targeting vector built based on polypeptide the most according to claim 8, it is characterised in that
The mass ratio of described targeting vector and medicine is 1:(5～10%).
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