CN106039316B - A kind of osteoblast targeting vector and its preparation and application based on polypeptide building - Google Patents
A kind of osteoblast targeting vector and its preparation and application based on polypeptide building Download PDFInfo
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- CN106039316B CN106039316B CN201610288361.XA CN201610288361A CN106039316B CN 106039316 B CN106039316 B CN 106039316B CN 201610288361 A CN201610288361 A CN 201610288361A CN 106039316 B CN106039316 B CN 106039316B
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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 present invention relates to a kind of osteoblast targeting vector based on polypeptide building, which includes target head and bracket, and the target head is polypeptide, and the quality grafting rate of target head is 5~10%, and mole grafting rate is 2~6%；The targeting vector is that polypeptide and carrier are prepared by the covalent linkage of poly- diethyl polyol carboxylic acid, for loading treatment fracture, osteoporosis, osteoarthritis, rheumatoid arthritis or the drug of bone metastaes.Compared with prior art, the present invention has osteoblast targeting, height bone tissue compatibility and high potency drugs delivery capability, can be good at enhancing skeletonization effect, reduces dosage, reduces cytotoxicity and side effect.Important drugs carrier tool will be provided for the following osteoporosis therapy.
Description
Technical field
The present invention relates to technical field of biological material, and in particular to it is a kind of based on polypeptide building osteoblast targeting vector and its
Preparation and application.
Background technique
Bone is a kind of connective tissue of high special in higher vertebrate body, it provide to the supporting function of body and
Muscle attachment area.It is also the main source of inorganic ions, and plays an active part in intracorporal calcium phosphorus balance.The normal development of bone
Bone is needed constantly to absorb and rebuild with maintenance.Under physiological status, it is this balance be coordinate, osteocyte, osteoclast and at
The function of osteocyte is normal, and the integrality of bone amount and microstructure is maintained at a stable state.However, this bone remodeling loses
Weighing apparatus is that numerous bone diseases include osteoporosis, Pei Jiteshi disease, osteoporosis, metastatic carcinoma of bone, rheumatoid arthritis, bone
The major reason of marrow inflammation etc..
The living tissue complicated as one, bone is mainly by three kinds of different types of cells: osteoblast, osteoclast and bone
Cell.Osteoblast is bon e formation cell, the osteoprogenitor cells from part, is responsible for synthesis bone matrix.Osteoclast is bone
Cell is absorbed, core is big, derives from a variety of hematopoietic tissues.The cell of both types exists only in the mineralized bone matrix of bone surface,
And osteocyte is located in bone matrix, and can absorb and form bone matrix.The characteristic being connected due to it by cynapse.The outer base of osteocyte
Matter is made of the hydroxyapatite of organic matter and height mineralising.With deepening continuously for bone biology research, in recent years
The therapeutic agent of bone metabolic disease continues to develop, the conduct such as bon e formation surface, bone resorption surface, osteoclast, bone matrix
New therapy target is included in research.Although some research products have been applied to the exploitation of drug, specific to bone and cell receptor,
Most of therapeutic agents newly developed do not have bone specificity.
Osteoporosis (Osteoporosis) is a kind of by the unbalance caused bone amount reduction of bone metabolism, bone fine structure
The systemic disease that bone fragility caused by destroying occurs and easily fractures.Osteoporosis is important in a world wide
Health problem.With the exacerbation of aging of population, the number of patients of osteoporosis is dramatically increased.Currently, there are about 2 in the whole world
Hundred million people suffer from osteoporosis, and disease incidence has leapt to the 7th of common disease.Wherein the illness rate of postmenopausal women is higher.65
Year old or more the high-incidence fracture of women, seriously affect the quality of life, health and service life of person in middle and old age.
The drug for the treatment of osteoporosis is divided into three classes at present: 1) inhibiting bone resorption drug, such as Diphosphonate, estrogen
Deng；2) promoting bone growing drug, such as parathormone, fluoride；3) two-way function drug: vitamin K, strontium salt etc..Due to
Bone tissue has the features such as big, the local blood supply of hardness is not abundant, poor permeability and physiological and biochemical procedure are special, by general administration
Approach is difficult to make drug transport to lesions position.These drugs often pass through Formulations for systemic administration, and increasing dosage could be in bone tissue
In reach effective treatment concentration.Since drug lacks bone specificity, the therapeutic index of drug is not only reduced, and can be to disease
The non-bone tissue of people or organ cause unnecessary toxic side effect, such as the nearest report of the long-term side-effects of hormone replacement therapy
Display was reduced, bone loss or even serious side effects can for representing drug parathormone using more than 2 years curative effects
There are nervous symptoms, kidney failure etc..Therefore drug targeting is delivered to by bone tissue or osteoblast by drug delivery system, thus
Increase therapeutic effect, and it is particularly significant to reduce toxic side effect.Currently, according to the bone target of the targeting research to hydroxyapatite
It include: Tetracyclines, bis phosphoric acid salt, growth factor class and polypeptide to delivery system.
Tetracycline is used as Bone targeting target head as antimicrobial, cannot eliminate itself biological effect, and have side effect
And effect is caused to be restricted；The Bone targeting delivery system constructed by diphosphonate, can be rich in bone surface and dentin surface
Collection, is difficult to exclude；Growth factor has natural bone taxis, but has half-life short, mutability, and excessive application may have
The disadvantages of genotoxic potential and carcinogenicity.
Summary of the invention
It is good, malicious that it is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of Targeting Performances
Property and Small side effects based on polypeptide building osteoblast targeting vector and its preparation and application.
The purpose of the present invention can be achieved through the following technical solutions: a kind of osteoblast targeting based on polypeptide building
Carrier, the targeting vector include target head and bracket, and the target head is covalently attached to the bracket by poly- diethyl polyol carboxylic acid, described
Target head be polypeptide, the quality grafting rate of target head is 5~10%, and mole grafting rate is 2~6%.
The polypeptide is made of two kinds of amino acid of serine and asparatate, and 2~50 ammonia are contained in the polypeptide
The number ratio of base acid, the serine and asparatate is (0.25~4): 1.
The polypeptide includes serine and asparatate, and the sequence of the polypeptide is preferably Ser Asp Ser Ser
Asp, wherein Ser represents serine, and Asp represents asparatate.
Tooth and bone tissue increase as the highest organ of mineralization degree in human body wherein important promotion mineralization protein has
Bone amount treats the potentiality of osteoporosis.Ser Asp Ser Ser Asp is selected from dentin phosphoprotein (DPP) primary structure, the egg
Casamino acid, which puts in order, characteristic Ser Asp and Ser Ser Asp repetitive sequence.The sequence is selected to have following original
Cause: (1) DPP derives from the highest sclerous tissues of human body mineralization degree, tooth, and expression position is the dentine of mineralising, bone tissue
In also have expression；(2) DPP is the highest extracellular matrix protein of expression quantity in dentine, also there is significant expression in alveolar bone；
(3) its dentine of mouse and bone tissue mineralising that DPP lacks obviously are inhibited, and tooth is thinning, and absorption of alveolar bone is obvious；(4)
Protein chemistry, cytology and histology experiment confirm that amino acid Asp is the amino acid of highly acidic in DPP, lead to Ser
Asp Ser Ser Asp acidity is higher, can specifically bind the hydroxyapatite on bone tissue surface, hence it is evident that promotes into tooth cell
With the maturation and secretion mineralized dentin matrix of osteoblast.As more special mineralising regulatory protein, in the development and mineralising of dentine
It plays an important role in the process.(5) in vitro study in vivo finds Ser Asp Ser Ser Asp polypeptide, can be by thin with skeletonization
Cellular surface specific proteins periostin (periostin) combines, and endocytosis enters cell, so that selectively targeted act on into
Osteocyte.
The bracket is polyurethane bracket, and the bracket is 60~120 nanometers, is in oblate spheroid under Electronic Speculum, can wrap up and pass
Send small nut acids drug.The existing good physical mechanical property of polyurethane material, and there is good blood compatibility and biology
Compatibility, thus it is widely used in field of biomedicine.It has excellent anticoagulation function and biocompatibility, and toxotest is multiple
The medical requirement of core, the effect of no aberration inducing, no allergic reaction and excellent toughness, elasticity, wear-resisting moisture-proof, corrosion resistance and
Thermal stability, and after being prepared into nanosphere can effectively carrying medicament, target organ is acted on by blood circulation or target is thin
Born of the same parents.
A kind of preparation method of the osteoblast targeting vector as described above based on polypeptide building, mainly includes following
Step:
(1) preparation of target head: by 12 channel semi-automatic polypeptide synthesizers in proportion by amino acid condensation at target head, to
With；
(2) preparation of bracket: by LDI and MEDA with 1:(0.5~1) molar ratio be dissolved in acetone, carry out hybrid reaction,
Then PEG and PCL is added, the ratio between the mole of PEG, PCL of addition and the mole of LDI of the addition are PEG:PCL:
LDI=(0.05~0.25): (0.01~0.05): (0.2~2), after reaction be added dehydrated alcohol reacted, addition it is anhydrous
The ratio between mole of ethyl alcohol and the LDI is (0.1~10): (1), obtaining thick PU liquid, petroleum ether then is added in thick PU liquid
It is precipitated, sediment is dissolved in acetone by filtering, and dialysis obtains PU nano-micelle liquid after centrifugation, for use；
(3) synthesis of targeting vector: target head obtained by step (1) is dissolved in deionized water, is then added and the matter such as target head
The EDC and NHS of amount carry out the activation of target peptide, PU nano-micelle liquid obtained by step (2) are then added, wherein the quality and PU of target head
The volume ratio of nano-micelle liquid is 1:(1~10), it dialyses after standing up to the osteoblast targeting vector.
In above-mentioned steps, LDI is lysine diisocyanate, MEDA are as follows: N methyldiethanol amine, PEG are polyethylene glycol
Dihydric alcohol, PCL are polycaprolactone diols, and EDC is 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride, and NHS is
N-hydroxysuccinimide.
The reaction temperature of step (2) described LDI, MEDA is 50~100 DEG C, and the reaction time is 20~30h；
The reaction temperature of the PEG, PCL are 50~100 DEG C, and the reaction time is 70~80h；
The reaction temperature of the dehydrated alcohol is 50~100 DEG C, and the reaction time is 40~50h；
The ratio between volume of the petroleum ether of the addition and thick PU liquid is 1:(0.2~0.5).
The temperature of step (3) described activation is 2~10 DEG C, and activation 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 ingredient of the target head only include two kinds of amino acid just
There can be good Bone targeting, therefore small peptide may be implemented to complete to target, therefore the basis that targeting is accomplished
On, it can satisfy the feature of small peptide.
A kind of application of the osteoblast targeting vector as described above based on polypeptide building, the targeting vector is for filling
Fill out the drug for the treatment of fracture, stock rickets, osteoporosis, osteoarthritis, rheumatoid arthritis or bone metastaes.
The mass ratio of the targeting vector and drug is 1:(5~10%).
Its main component is Ser amino acid and Asp amino acid, is got up according to a certain percentage with sequential connection.Using the target
To vehicle delivery small nucleic acids drug can efficiently by the target cell osteoblast of small nucleic acids drug delivery to characteristic, and its
The effect of promoting bone growing and bone apposition clearly, can treat including bone diseases such as osteoporosises caused by OVX.
Compared with prior art, the beneficial effects of the present invention are embodied in following several respects:
(1) target head of the invention derives from the native protein DPP with high mineralization regulating power, has very high targeting
Property, drug can accurately be sent into osteocyte, reduce side effects of pharmaceutical drugs；
(2) target head of the invention uses small peptide, and synthesis is simple, and production cost is low；
(3) bracket used in the present invention is the polyurethane material with good hydrophily, bioactivity and degradability, this
Material has very strong plasticity and hypotoxicity, and will not be enriched in vivo, ensure that Drug safety.
(4) preferred target head of the invention derives from the native protein DPP with high mineralization regulating power, promotes biological mine
Change ability is clear, meanwhile, reduce side effects of pharmaceutical drugs.
Detailed description of the invention
Fig. 1 is composition principle schematic diagram of the invention；
Fig. 2 a is the picture of 1 targeting vector of embodiment pattern under transmission electron microscope；
Fig. 2 b is the potential image of 1 targeting vector of embodiment targeting vector under transmission electron microscope；
Fig. 3 a is the ultraviolet-visible spectrophotometry test result of target head in embodiment 1；
Fig. 3 b is the ultraviolet-visible spectrophotometry test result of carrier in embodiment 1；
Fig. 3 c is the testing result figure of the grafting rate of targeting vector in embodiment 1；
Fig. 4 is targeting experimental result picture of 1 targeting vector of embodiment in osteoblast surfaces；
Fig. 5 is that internal osteoblast orients Bone targeting carrier verification result schematic diagram；
Fig. 6 is the intracorporeal organ distribution experiments result schematic diagram at osteoblast targeting vector；
Fig. 7 is the performance test results that 1 targeting vector of embodiment prevents RNA from degrading；
The effect picture of Fig. 8 skeletonization targeting vector increase bone amount.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
A kind of osteoblast targeting vector based on polypeptide building, which includes target head and bracket, target head and branch
Frame target head and bracket are covalently attached by poly- diethyl polyol carboxylic acid (PEG-COOH).It is about 6% that its target head quality, which connects branch rate, and target head is rubbed
It is 3% that you, which connect branch rate,.Target head is the polypeptide being made of serine and asparatate, and wherein the sequence of the polypeptide is Ser Asp
Ser Ser Asp, wherein Ser represents serine, and Asp represents asparatate；Bracket is polyurethane bracket.
Above-mentioned targeting vector, synthetic method as shown in Figure 1, including the following steps:
(1) preparation of target head: 12 channel semi-automatic polypeptide synthesizers are used, using amino acid condensation order connection method
Target head is synthesized, serine (Fmoc-Asp (otBu)-OH) and asparatate (Fmoc-Ser (Tbu)-OH) are pressed into Ser-Asp-
Ser-Ser-Asp is linked in sequence to get target head Ser Asp Ser Ser Asp, for use；
(2) preparation of bracket: the synthesis of the first step, PU: 7.85g LDI, 2.98g MEDA and 89.17g acetone are in closed
It is mixed in reaction flask, 60 DEG C of oscillation sealings are reacted 24 hours.The solution 49.98g after answering is negated, 4.44g PEG, 0.58g is added
PCL and 45g acetone (gross mass 100g), mixed liquor add 0.5g dehydrated alcohol, nothing after oscillating reactions 72 hours in 60 DEG C
The oscillating reactions 48 hours of 60 DEG C of water-stop, can be obtained PU liquid.Second step, PU purification: it is slowly added in 100mL petroleum ether
The thick PU liquid of 35mL, petroleum ether precipitation, standing filter to obtain PU precipitating, and PU precipitating vacuum drying is obtained and slightly purifies PU.PU is dissolved in
The third 10% PU acetone soln, PU acetone solution and acetone are 1:9 by volume, are 1:1 by PU acetone dilution and deionized water
Ratio mixing, low-temperature and high-speed be centrifuged 10min, 15000g；Supernatant dialysis acetone after centrifugation, becomes to the liquid in bag filter
It can be obtained PU nano-micelle liquid at milky, seal freezen protective after PU Micellar Solution Which Is are freeze-dried.
(3) synthesis of targeting vector: skeletonization target peptide Ser Asp Ser Ser Asp is dissolved in 1mL deionized water, then plus
Enter the activation for carrying out target peptide with the EDC and NHS of the quality such as target peptide, presses one into target peptide activation mixed liquor again after 4 DEG C of incubation 10min
PU nano-micelle liquid is added in fixed mass volume ratio (peptide masses: PU liquid product ≈ 1:5), and 4 DEG C of PU target peptide mixed liquor reacted
Night.After reaction, (MW=3500) is dialysed to remove the impurity such as inorganic salts, after dialysis i.e. to reaction solution with deionized water
The PU nano-micelle liquid (Ser Asp-PU) that target peptide coupling can be obtained, seals freezen protective after freeze-drying.
Above-mentioned targeting vector is scanned with electron-microscope scanning, result is as shown in Figure 2 a and 2 b.
Test the grafting rate of above-mentioned resulting targeting vector using ultraviolet-visible spectrophotometry, test result such as Fig. 3 a,
Shown in Fig. 3 b and Fig. 3 c, the results showed that the grafting rate of the targeting vector is 6.8%.
By fluorescent marker on above-mentioned target head band, mouse and people is then allowed to take the targeting vector, and mouse and people are carried out
Tracking, as a result as shown in figure 4, the results show that Ser Asp Ser Ser Asp energy polypeptide reaches and periostin (periostin)
Common location illustrates that Ser Asp Ser Ser Asp can be in conjunction with periostin, and enters osteoblast.
After vehicle delivery FITC fluorescent marker small nucleic acids described in mouse tail vein, sections observation, as a result as shown in figure 5, knot
Fruit shows that fluorescin specific enrichment in osteoblast illustrates that the carrier has into osteoblast targeting.
After vehicle delivery FITC fluorescent marker small nucleic acids described in mouse tail vein injection, observed by living imaging instrument,
As a result as shown in fig. 6, experimental group is apparently higher than sky in bone tissue the results show that fluorescence signal is close in soft tissue organs
Vehicle group illustrates that the carrier has Bone targeting effect.
Upper siRNA, internal injection will be loaded in above-mentioned resulting targeting vector.And increase contrast groups, direct hormone
SiRNA, test siRNA degradation situation in vivo, result using the present invention as shown in fig. 7, the results show that loaded
The degradation speed of siRNA in vivo is smaller, allows siRNA to have more time contact osteoblasts, increase siRNA has
Effect property.
The inhibitor of upper miRNA214, internal injection will be loaded in above-mentioned resulting targeting vector, estrogen is intervened in targeting
Osteoporosis caused by shortage, after injecting January, osteoporosis mouse bone amount is obviously increased, as a result as shown in figure 8, explanation should
Carrier can deliver miRNA and treat osteoporosis.
Embodiment 2
A kind of osteoblast targeting vector based on polypeptide building, which includes target head and bracket, target head and branch
Frame is covalently attached by poly- diethyl polyol carboxylic acid (PEG-COOH).It is about 5% that its target head quality, which connects branch rate,.Target head is by serine
With the polypeptide of asparatate composition, wherein the sequence of the polypeptide is Ser Asp, wherein Ser represents serine, and Asp represents day
L-aminobutanedioic acid；Bracket is polyurethane bracket.
Above-mentioned targeting vector, synthetic method such as, including the following steps:
(1) preparation of target head: 12 channel semi-automatic polypeptide synthesizers are used, using amino acid condensation order connection method
Target head is synthesized, serine (Fmoc-Asp (otBu)-OH) and asparatate (Fmoc-Ser (Tbu)-OH) is suitable by Ser-Asp
Sequence connects to get target head Ser Asp, for use；
(2) preparation of bracket: the synthesis of the first step, PU: the MEDA of the LDI of 1mol and 0.5mol is dissolved in acetone, is set
It is mixed in confined reaction bottle, oscillation sealing reaction, reaction temperature is 50 DEG C, reaction time 20h.Then 0.05mol is added
PEG and 0.01mol PCL, mix oscillating reactions, reaction temperature be 50 DEG C, reaction time 70h adds 0.1mol's
Dehydrated alcohol, oscillating reactions, reaction temperature are 50 DEG C, reaction time 40h, can be obtained thick PU liquid.Second step, PU purification:
5 times of petroleum ether of the thick PU liquid product of gained is added, is precipitated, obtains and slightly purify PU.Thick purification PU is dissolved in acetone, is centrifuged
Dialysing afterwards can get PU nano-micelle liquid, save after freeze-drying.
(3) synthesis of targeting vector: skeletonization target peptide Ser Asp is dissolved in 1mL deionized water, is added and the matter such as target peptide
The EDC and NHS of amount carry out the activation of target peptide, after 2 DEG C of incubations 20min, then the addition PU nano-micelle into target peptide activation mixed liquor
Liquid, wherein target head quality: PU nano-micelle liquid=1:5,2 DEG C of reaction 20h of PU target peptide mixed liquor.After reaction, deionization is used
Water is dialysed (MW=3500) to reaction solution to remove the impurity such as inorganic salts, and the PU nanometre glue of target peptide coupling can be obtained after dialysis
Beam liquid (Ser Asp-PU), seals freezen protective after freeze-drying.
Using fluorescent marker Ser Asp, in people and mouse bone-forming cell, Ser Asp energy polypeptide reaches and periostin
(periostin) common location illustrates that Ser Asp can be in conjunction with periostin, and enters osteoblast.Observation should under transmission electron microscope
The size and shape characteristic of targeting vector.The carrier PU-Ser Asp can wrap up siRNA and reduce the external degradation of siRNA
Speed.After vehicle delivery FITC fluorescent marker small nucleic acids described in mouse tail vein, sections observation is found, fluorescin is thin in skeletonization
Specific enrichment in born of the same parents illustrates that the carrier has into osteoblast targeting.Vehicle delivery described in mouse tail vein injection
After FITC fluorescent marker small nucleic acids, by living imaging instrument it has been observed that fluorescence signal is close in soft tissue organs, in bone
Experimental group is apparently higher than empty carrier group in tissue, illustrates that the carrier has Bone targeting effect.
The inhibitor of upper miRNA214, the matter of the miRNA214 inhibitor of filling will be loaded in above-mentioned resulting targeting vector
Amount and the mass ratio of targeting vector are 5%:1.Osteoporosis caused by estrogen deficiency, injection are intervened in internal injection, targeting
After January, osteoporosis mouse bone amount is obviously increased.Illustrate that the carrier can deliver miRNA and treat osteoporosis.
Embodiment 3
A kind of osteoblast targeting vector based on polypeptide building, which includes target head and bracket, target head and branch
Frame is covalently attached by poly- diethyl polyol carboxylic acid (PEG-COOH).Its target head quality grafting rate is about 6%.Target head be by serine and
The polypeptide of asparatate composition, length are 20 amino acid, and wherein the sequence of the 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),
In, Ser represents serine, and Asp represents asparatate；Bracket is polyurethane bracket.
Above-mentioned targeting vector, synthetic method such as, including the following steps:
(1) preparation of target head: 12 channel semi-automatic polypeptide synthesizers are used, using amino acid condensation order connection method
Target head is synthesized, serine (Fmoc-Asp (otBu)-OH) and asparatate (Fmoc-Ser (Tbu)-OH) is suitable by Ser-Asp
Sequence connects to get target head, for use；
(2) preparation of bracket: the synthesis of the first step, PU: the MEDA of the LDI of 1mol and 1mol is dissolved in acetone, is placed in
It is mixed in confined reaction bottle, oscillation sealing reaction, reaction temperature is 100 DEG C, reaction time 30h.Then it is added 1.25mol's
The PCL of PEG and 0.25mol mixes oscillating reactions, and reaction temperature is 100 DEG C, and reaction time 80h adds the nothing of 10mol
Water-ethanol, oscillating reactions, reaction temperature are 100 DEG C, reaction time 50h, can be obtained thick PU liquid.Second step, PU purification: add
2 times of petroleum ether for entering the thick PU liquid product of gained, is precipitated, obtains and slightly purify PU.Thick purification PU is dissolved in acetone, after centrifugation
It dialyses and can get PU nano-micelle liquid, saved after freeze-drying.
(3) synthesis of targeting vector: skeletonization target peptide Ser Asp is dissolved in 1mL deionized water, is added and the matter such as target peptide
The EDC and NHS of amount carry out the activation of target peptide, after 10 DEG C of incubations 5min, then the addition PU nano-micelle into target peptide activation mixed liquor
Liquid, wherein target head quality: PU nano-micelle liquid=1:1,10 DEG C of reaction 10h of PU target peptide mixed liquor.After reaction, deionization is used
Water is dialysed (MW=3500) to reaction solution to remove the impurity such as inorganic salts, and the PU nanometre glue of target peptide coupling can be obtained after dialysis
Beam liquid (Ser Asp-PU), seals freezen protective after freeze-drying.
Using fluorescent marker Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp Ser Asp Asp Asp
Asp Ser Asp Asp Asp Asp, in people and mouse bone-forming cell, Ser Asp Asp Asp Asp Ser Asp Asp
Asp Asp Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp energy polypeptide reaches and periostin (periostin)
Common location illustrates Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp Ser Asp Asp Asp Asp Ser
Asp Asp Asp Asp can be in conjunction with periostin, and enters osteoblast.The size of the targeting vector is observed under transmission electron microscope
And shape characteristic.The 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 marker small nucleic acids, sections observation discovery, fluorescin specific enrichment in osteoblast, explanation
The carrier has into osteoblast targeting.After vehicle delivery FITC fluorescent marker 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, experimental group is apparently higher than sky in bone tissue
Vehicle group illustrates that the carrier has Bone targeting effect.
The inhibitor of upper miRNA214, the matter of the miRNA214 inhibitor of filling will be loaded in above-mentioned resulting targeting vector
Amount and the mass ratio of targeting vector are 10%:1.Osteoporosis caused by estrogen deficiency, injection are intervened in internal injection, targeting
After January, osteoporosis mouse bone amount is obviously increased.Illustrate that the carrier can deliver miRNA and treat osteoporosis.
Embodiment 4
A kind of osteoblast targeting vector based on polypeptide building, which includes target head and bracket, target head and branch
Frame is covalently attached by poly- diethyl polyol carboxylic acid (PEG-COOH).Its target head quality grafting rate is about 10%, and target head is by serine
With the polypeptide of asparatate composition, which has 50 amino acid, and wherein the number of serine and asparatate ratio is
The sequence of=4:1 wherein the 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 asparatate；Bracket is polyurethane bracket.
Above-mentioned targeting vector, synthetic method such as, including the following steps:
(1) preparation of target head: 12 channel semi-automatic polypeptide synthesizers are used, using amino acid condensation order connection method
Target head is synthesized, serine (Fmoc-Asp (otBu)-OH) and asparatate (Fmoc-Ser (Tbu)-OH) is suitable by Ser-Asp
Sequence connects to get target head, for use；
(2) preparation of bracket: the synthesis of the first step, PU: the MEDA of the LDI of 1mol and 0.7mol is dissolved in acetone, is set
It is mixed in confined reaction bottle, oscillation sealing reaction, reaction temperature is 75 DEG C, and the reaction time is for 24 hours.Then 0.025mol is added
PEG and 0.005mol PCL, mix oscillating reactions, reaction temperature be 75 DEG C, reaction time 72h adds the nothing of 5mol
Water-ethanol, oscillating reactions, reaction temperature are 75 DEG C, reaction time 48h, can be obtained thick PU liquid.Second step, PU purification: add
3 times of petroleum ether for entering the thick PU liquid product of gained, is precipitated, obtains and slightly purify PU.Thick purification PU is dissolved in acetone, after centrifugation
It dialyses and can get PU nano-micelle liquid, saved after freeze-drying.
(3) synthesis of targeting vector: skeletonization target peptide Ser Asp is dissolved in 1mL deionized water, is added and the matter such as target peptide
The EDC and NHS of amount carry out the activation of target peptide, after 4 DEG C of incubations 10min, then the addition PU nano-micelle into target peptide activation mixed liquor
Liquid, wherein target head quality: PU nano-micelle liquid=1:5,4 DEG C of reaction 12h of PU target peptide mixed liquor.After reaction, deionization is used
Water is dialysed (MW=3500) to reaction solution to remove the impurity such as inorganic salts, and the PU nanometre glue of target peptide coupling can be obtained after dialysis
Beam liquid (Ser Asp-PU), seals freezen protective after freeze-drying.
Using fluorescent marker Ser Asp, in people and mouse bone-forming cell, target head reaches and periostin (periostin)
Common location illustrates that target head can be in conjunction with periostin, and enters osteoblast.The carrier PU-Ser Ser Ser Ser Asp
SiRNA can be wrapped up and reduce the external degradation speed of siRNA.Vehicle delivery FITC fluorescent marker small nut described in mouse tail vein
After acid, sections observation discovery, fluorescin specific enrichment in osteoblast illustrates that the carrier has and targets at osteoblast
Effect.It is by living imaging instrument it has been observed that glimmering after vehicle delivery FITC fluorescent marker small nucleic acids described in mouse tail vein injection
Optical signal is close in soft tissue organs, and experimental group is apparently higher than empty carrier group in bone tissue, illustrates that the carrier has bone target
To effect.
The inhibitor of upper miRNA214, the matter of the miRNA214 inhibitor of filling will be loaded in above-mentioned resulting targeting vector
Amount and the mass ratio of targeting vector are 7.5%:1.Osteoporosis caused by estrogen deficiency, note are intervened in internal injection, targeting
After penetrating January, osteoporosis mouse bone amount increases.Illustrate that the carrier can deliver miRNA and treat osteoporosis.
Claims (7)
1. a kind of osteoblast targeting vector based on polypeptide building, which is characterized in that the targeting vector includes target head and bracket,
The target head is covalently attached to the bracket by poly- diethyl polyol carboxylic acid, and the target head is polypeptide, the quality grafting rate of target head
It is 5~10%；
The polypeptide is made of two kinds of amino acid of serine and asparatate, and 2~50 amino acid are contained in the polypeptide,
The number of the serine and asparatate ratio is (0.25~4): 1；
The bracket is polyurethane bracket, and the bracket is in oblate spheroid, and the partial size of the bracket is 60~120nm.
2. a kind of osteoblast targeting vector based on polypeptide building according to claim 1, which is characterized in that described
Polypeptide includes 3 serines and 2 asparatates, and the sequence of the polypeptide is SDSSD, wherein S represents serine, and D is represented
Asparatate.
3. a kind of preparation method of the osteoblast targeting vector based on polypeptide building as described in claim 1~2 is any, special
Sign is that this method is main including the following steps:
(1) preparation of target head: by 12 channel semi-automatic polypeptide synthesizers in proportion by amino acid condensation at target head, for use；
(2) preparation of bracket: by LDI and MEDA with 1:(0.5~1) molar ratio be dissolved in acetone, carry out hybrid reaction, then
PEG and PCL is added, the ratio between the mole of PEG, PCL of addition and the mole of LDI of the addition are PEG:PCL:LDI=
(0.05~0.25): (0.01~0.05): (0.2~2) is added dehydrated alcohol and is reacted after reaction, the dehydrated alcohol of addition
It is (0.1~10) with the ratio between the mole of the LDI: 1, thick PU liquid is obtained, petroleum ether is then added in thick PU liquid and is sunk
It forms sediment, sediment is dissolved in acetone by filtering, and dialysis obtains PU nano-micelle liquid after centrifugation, for use；
(3) synthesis of targeting vector: target head obtained by step (1) is dissolved in deionized water, is then added and target first-class quality
EDC and NHS carry out the activation of target peptide, and PU nano-micelle liquid obtained by step (2) is then added, wherein the quality of target head with PU nanometers
The volume ratio of Micellar Solution Which Is is 1:(1~10), it dialyses after standing up to the osteoblast targeting vector.
4. a kind of preparation method of osteoblast targeting vector based on polypeptide building according to claim 3, feature
It is, the reaction temperature of step (2) described LDI, MEDA is 50~100 DEG C, and the reaction time is 20~30h；
The reaction temperature of the PEG, PCL are 50~100 DEG C, and the reaction time is 70~80h；
The reaction temperature of the dehydrated alcohol is 50~100 DEG C, and the reaction time is 40~50h；
The ratio between volume of the petroleum ether of the addition and thick PU liquid is 1:(0.2~0.5).
5. a kind of preparation method of osteoblast targeting vector based on polypeptide building according to claim 3, feature
It is, the temperature of step (3) described activation is 2~10 DEG C, and activation time is 5~20min；
Dwell temperature is 2~10 DEG C, and time of repose is 10~20h.
6. a kind of application of the osteoblast targeting vector based on polypeptide building as described in claim 1~2 is any, feature exist
In, the targeting vector for load treatment fracture, stock rickets, osteoporosis, osteoarthritis, rheumatoid arthritis or
The drug of bone metastaes.
7. a kind of application of osteoblast targeting vector based on polypeptide building according to claim 6, which is characterized in that
The mass ratio of the targeting vector and drug is 1:(5~10%).
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