CN110755638A - Bone-targeting drug carrier and preparation method and application thereof - Google Patents
Bone-targeting drug carrier and preparation method and application thereof Download PDFInfo
- Publication number
- CN110755638A CN110755638A CN201911047436.5A CN201911047436A CN110755638A CN 110755638 A CN110755638 A CN 110755638A CN 201911047436 A CN201911047436 A CN 201911047436A CN 110755638 A CN110755638 A CN 110755638A
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- CN
- China
- Prior art keywords
- bone
- drug
- drug carrier
- cyclodextrin
- targeting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
The invention discloses a bone-targeting drug carrier, which comprises β -cyclodextrin serving as a drug-encapsulated main body and aspartic acid hexapeptide serving as a target and having bone targeting property, wherein the aspartic acid hexapeptide is connected with β -cyclodextrin through a flexible chain.
Description
Technical Field
The invention relates to the technical field of targeted drug carriers, in particular to a bone-targeted drug carrier and a preparation method and application thereof.
Background
The incidence of bone diseases is increasing day by day and is highly regarded by people, and the bone diseases mainly comprise osteoporosis, osteitis deformans, bone metastasis tumor, primary and secondary bone tumors, osteoarthritis and the like. The main component of the bone tissue is hydroxyapatite. Most of the calcium in the human body is present in the bones and accounts for about 99% of the total calcium content. Therefore, the bone tissue has biological specificity, such as low bleeding amount, high density and poor permeability, which brings great difficulty to clinical medication. In the traditional administration mode, the medicine is difficult to reach the pathological part according to an ideal state, and the defects of low curative effect and great adverse reaction are common defects of the medicine for treating the bone disease. To solve the problem, a bone targeting drug carrier can be developed, so that some drugs with large side effects and capable of treating bone diseases can directly reach a diseased part to exert drug effects.
The existing micromolecule hydrophobic antibacterial drugs capable of treating bone diseases, such as norfloxacin and the like, are orally taken when the bone diseases are treated, have a series of problems of slow effect, high systemic blood concentration and the like, particularly cause the drugs to be transported to the whole body along with body fluid or be metabolized too fast and the like when the drugs are directly applied to wounds during operation, and the like.
Disclosure of Invention
The invention aims to provide a bone-targeting drug carrier which can improve the solubility of the hydrophobic small-molecule drugs and accurately transport the hydrophobic small-molecule drugs to bone lesion sites for treating bone diseases.
The invention also aims to provide a preparation method of the bone targeting drug carrier.
The invention is realized by the following technical scheme: a bone-targeting drug carrier has a chemical structure shown in the following formula:
according to the structure of the compound, the drug carrier (β -CD-mPEG-D6) selects β -cyclodextrin (β -CD) as a main body of a drug-coated drug, aspartic acid hexapeptide (D6) is used as a target, and the specific structure is that β -cyclodextrin is connected with aspartic acid hexapeptide (D6) through polyethylene glycol (PEG).
β -cyclodextrin is a general name of a series of cyclic oligosaccharides produced by amylose under the action of β -cyclodextrin glucosyltransferase produced by bacillus, generally contains 6-12D-glucopyranose units, β -cyclodextrin molecule has a slightly tapered hollow cylindrical three-dimensional annular structure, in the hollow structure, the upper outer end (larger open end) is composed of secondary hydroxyl groups of C2 and C3, the lower end (smaller open end) is composed of primary hydroxyl groups of C6, has hydrophilicity, and a hydrophobic region is formed in the cavity due to the shielding effect of C-H bonds.
Aspartic acid oligopeptides have been applied to the study of various bone disease drugs as targets for bone targeting. And the aspartic acid hexapeptide, the aspartic acid heptapeptide and the aspartic acid octapeptide are taken as targets, target drugs indicated by the drugs are combined, HA adsorption is performed respectively, absorption, in vitro pharmacokinetics and pharmacodynamics experiments are performed for testing, and finally, the aspartic acid hexapeptide is taken as a bone target to obtain an optimal result, so that a bone target is achieved.
The tradition uses aspartic acid hexapeptide as the target, all directly combines aspartic acid hexapeptide with the medicine directly, carry out direct modification to the medicine, make the medicine have bone targeting, but there is very big limitation, on the one hand, not all medicines can all combine with aspartic acid medicine, form stable bone targeting medicine, on the other hand, the medicine preparation process that combines with aspartic acid hexapeptide is probably comparatively complicated, synthetic product is uncontrollable, produce more by-products, it is difficult to get rid of impurity, obtain the finished product targeting medicine of higher purity, can't be used for industrial production.
The application adopts a new technical thought, the β -cyclodextrin is modified by using the aspartic acid hexapeptide, and then the hydrophobic drug micromolecules are entrapped by utilizing the cavity inside the β -cyclodextrin by utilizing the structural characteristic of β -cyclodextrin, so that the drug entrapping range of the drug carrier can be widened, and the hydrophobic drug micromolecules can be entrapped by using the drug carrier.
The specific synthetic route of the bone targeting drug carrier is as follows:
the application prepares to directly graft the aspartic acid hexapeptide into β -cyclodextrin molecules, although the success is achieved, the number of successfully synthesized drug carriers is few, the yield is extremely low, and the β -cyclodextrin molecules are directly combined with the aspartic acid hexapeptide to have too short chain length, so that the targeting effect of the aspartic acid hexapeptide is influenced to a certain extent.
In the process of preparing the drug carrier, in order to improve the yield of the finished drug carrier, reduce byproducts and ensure the single reaction, three modified raw materials are particularly adopted:
modified β -Cyclodextrin in which one hydroxyl group is replaced by an amino group (β -CD-NH)2):
Modified aspartic acid hexapeptide (DDDDDD-SH) with mercapto added to the end of amino;
and modified polyethylene glycol (NHS-mPEG-MAL) with maleimide at one end and succinamide at the other end.
The synthetic raw materials of the three drug carriers can be obtained by purchasing finished products.
The synthesis route ensures the unicity of the reaction, simultaneously promotes the yield of finished drug carriers, basically does not generate side reactions, generates additional impurities and can remove the impurities through dialysis, and can obtain finished drug carriers with higher purity.
According to the synthetic route, the specific preparation method is as follows:
β -CD (1.5mM) was dissolved in PBS, argon was used for oxygen scavenging for 30min, NHS-mPEG-MAL (1mM) solution was added dropwise to β -CD solution, the mixture was stirred at room temperature for 1 hour, D6(1mM) solution was added dropwise to the solution and stirred at room temperature overnight, the product was collected and dialyzed for 3 days, and then lyophilized to a white solid powder (β -CD-mPEG-D6).
The specific process of using the prepared drug carrier to entrap drugs comprises the steps of dissolving β -CD-mPEG-D6(1mM) in UP water, adding hydrophobic micromolecule drug (1mM) powder, heating to 50 ℃, stirring for 1 hour after the hydrophobic micromolecule drug is dissolved, closing and heating, stirring until the temperature is room temperature, collecting products, freezing and drying into solid powder, wherein the solid powder is the drug carrier entrapping the hydrophobic micromolecule drug, and the hydrophobic micromolecule drug is entrapped in a β -cyclodextrin cavity structure.
The invention also provides a specific application of the bone targeting drug carrier, and the bone targeting drug carrier is prepared into a bone targeting drug, and comprises the drug carrier, wherein the hydrophobic small molecule drug for treating bone diseases or preventing bone infection is encapsulated inside β -cyclodextrin in the drug carrier.
Wherein, the hydrophobic small molecule drug can be drugs used for treating bone diseases, such as norfloxacin, cefazolin, compound sulfamethoxazole, imipenem and the like.
Bone diseases that can be treated include osteoporosis, bone metastases, primary and secondary bone tumors, osteoarthritis, bone infections, osteitis deformans, osteomyelitis, and other bone diseases.
The preparation method of the bone targeting drug comprises the following specific processes:
dissolving the bone-targeting drug carrier in UP water, adding hydrophobic micromolecule drug capable of treating bone diseases, wherein the mass ratio of the drug carrier to the hydrophobic micromolecule drug is 1:1, heating the drug carrier and the hydrophobic micromolecule drug in water bath, heating the drug carrier and the hydrophobic micromolecule drug to 50 ℃, stirring the drug carrier and the hydrophobic micromolecule drug for 1 hour after the hydrophobic micromolecule drug powder is dissolved, stopping heating the drug carrier and the hydrophobic micromolecule drug, stirring the drug carrier and the hydrophobic micromolecule drug until the temperature reaches the room temperature, collecting the product, and freeze-drying the product to obtain solid.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the drug carrier is an β -cyclodextrin carrier modified by aspartic acid hexapeptide, hydrophobic drugs which are difficult to be encapsulated, such as antibacterial drugs, anti-inflammatory drugs, anti-cancer drugs and the like can be loaded into a cavity structure of β -cyclodextrin, the hydrophilicity of the hydrophobic drugs is favorably improved, calcium ions of hydroxyapatite which is the most main component in bones are chelated, and thus targeting is performed, and the hydrophobic drugs encapsulated in the inner cavity of β -cyclodextrin can be used for accurately and efficiently treating bone diseases;
(2) in the preparation process of the drug carrier, the raw materials after special modification are adopted to carry out fixed reaction, side reactions are basically not generated, and unnecessary impurities are not generated, so that the yield of the drug carrier and the purity of the product can be greatly improved;
(3) the raw materials adopted by the invention are sold as finished products, the whole preparation process of the drug carrier and the process of carrying out hydrophobic drug entrapment by using the drug carrier are simple, the reaction condition is mild, complex reaction equipment is not needed, the industrial production conversion is easy to carry out, and the method has wide application prospect and value.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 shows the CD-D of a drug carrier of the present invention at a wavelength of 220nm6A concentration standard curve graph;
FIG. 2 is a fluorescence image of a control group in an HA specific adsorption experiment of the present invention;
FIG. 3 is a fluorescence image of an experimental group in an HA specific adsorption experiment according to the present invention;
FIG. 4 is an infrared spectrum of a sample and a control group according to the present invention;
FIG. 5 is a graph showing the bacteriostatic effect of the sample and the control group on Escherichia coli in the present invention;
FIG. 6 is a graph showing the bacteriostatic effect of the sample and the control group on Staphylococcus aureus in the present invention;
FIG. 7 is a graph showing the effect of the bone-targeting drug carrier of the present invention on cytotoxicity.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto, and various substitutions and alterations can be made without departing from the technical idea of the present invention as described above, according to the common technical knowledge and the conventional means in the field.
The present invention will be described in further detail with reference to the following examples for the purpose of making clear the objects, process conditions and advantages of the present invention, which are given by way of illustration only and are not intended to be limiting of the present invention.
Example 1:
the embodiment provides a bone-targeting drug carrier, which comprises β -cyclodextrin serving as a drug-encapsulated main body and aspartic acid hexapeptide serving as a target and having bone targeting property, wherein the aspartic acid hexapeptide is connected with β -cyclodextrin through a flexible chain.
Wherein the β -cyclodextrin is connected with only one aspartic acid hexapeptide through a flexible chain, and the flexible chain is polyethylene glycol.
The preparation method of the bone-targeting drug carrier comprises the following steps:
(1) 1.5mM modified β -cyclodextrin (β -CD) containing one amino group was dissolved in PBS buffer and deoxygenated using argon for 30 min;
(2) then, 1mM polyethylene glycol (NHS-mPEG-MAL) solution with maleimide at one end and succinamide at the other end is added dropwise, stirred for 1 hour at room temperature, added with 1mM modified aspartic acid hexapeptide (D6) with sulfhydryl at one end and stirred for overnight at room temperature;
(3) collecting the product, dialyzing for 3 days, and freeze-drying to obtain white solid powder (β -CD-mPEG-D6).
The yield thereof was 89%.
Example 2:
this example was conducted to quantify HA tablets with respect to the drug carrier (β -CD-mPEG-D6) prepared in the above example.
The specific experimental process is as follows:
1. a standard curve of CD-D6 was prepared: preparing a CD-D6 sample with the concentration of 1mg/mL, carrying out full spectrum scanning on the CD-D6 sample by using an enzyme-labeling instrument after the sample is subjected to gradient dilution, and obtaining the maximum absorption wavelength of CD-D6 to be 220 nm; the OD value of the CD-D6 sample after gradient dilution is detected by setting the wavelength of the microplate reader to be 220nm, and a standard curve chart of CD-D6 is drawn, as shown in FIG. 1.
Quantitative adsorption experiment of HA: the HA disc is placed in a pore plate, 1mL of CD-D6 sample with the concentration of 1mg/mL is placed on the HA disc by a pipette gun, the HA disc is placed in a refrigerator at 4 ℃ for 24 hours, and 100 mu L of liquid is taken for OD value detection with the wavelength of 220 nm. Performing concentration conversion by using a CD-D6 standard curve to obtain HA adsorption quantity of 0.08mg/cm2。
As shown in FIG. 1, the adsorption of HA by CD-D6 was 0.08mg/cm 2.
Example 3:
this example was conducted on the drug carrier (β -CD-mPEG-D6) prepared in the above example, and subjected to an in vitro hydroxyapatite adsorption experiment.
The experimental principle is as follows:
for the fluorescence of β -cyclodextrin, β -cyclodextrin was used to encapsulate amantadine labeled with the fluorescent label FICT, which was chosen because amantadine was the best matched to the cavity of β -cyclodextrin.
The specific experimental process is as follows:
1. ADA was labeled with FITC:
respectively adding 5mgAd-NH21.6mgF-NHS, 1m1 anhydrous DMSO, 50. mu.L anhydrous DIPEA were added to a 1.5ml glass bottle and shaken overnight at 25 ℃ and 700rpm in the dark. Separating and purifying the product by High Performance Liquid Chromatography (HPLC), and drying by a freeze concentration centrifugal drier;
2. F-Ada was loaded into β -CD and CD-D6, respectively;
3. adding 50 μ L of β -CD and CD-D6 solution of 1mM into 50 μ L of 1mM ADA-F, respectively, performing shaking incubation on the mixed solution at 25 ℃ and 300rpm in the dark for 5h, and performing freeze drying to obtain F-A-CD and F-A-CD-D6 samples;
4. F-A-CD-D6 was adsorbed onto HA discs and fluorescence detected:
respectively preparing 2mg/mLF-A-CD and F-A-CD-D6 solutions, placing the hydroxyapatite sheet in a 24-hole plate, adding 1mL of the solution, standing for 24h, washing for 3 times by using ultrapure water, and carrying out a laser confocal test with excitation light of 488 nm.
The experimental conclusion is that as shown in fig. 2 and fig. 3, no fluorescent substance is found in fig. 2, which indicates that F-A-CD HAs no adsorption effect on Hydroxyapatite (HA) and HAs no targeting property, and the obvious fluorescent substance is found in fig. 3, which indicates that aspartic acid hexapeptide is successfully combined with β -cyclodextrin, can be specifically combined with HA and HAs targeting property.
Example 4:
in this example, the drug carrier prepared in the above example is used to entrap a hydrophobic small molecule drug, and in this example, the hydrophobic small molecule drug is preferably Norfloxacin (NFX).
The norfloxacin is a third-generation quinolone antibacterial agent, can block the action of DNA gyrase of pathogenic bacteria in a digestive tract, blocks the DNA replication of bacteria, and has an inhibiting effect on the bacteria, and is extremely slightly dissolved in water, so that the norfloxacin is extremely difficult to be used as a bactericidal drug for treating bone infection.
The process of encapsulating norfloxacin using the above pharmaceutical carrier is as follows:
β -CD-mPEG-D6(1mM) was dissolved in UP water, NFX (1mM) powder was added, the temperature was raised to 50 ℃ and stirred for 1 hour after NFX was dissolved in water, heating was turned off and stirring was continued until room temperature, and the product was collected and freeze-dried to a pale yellow solid powder.
Wherein the preparation process comprises heating in water bath.
The yield was 90%.
Example 5:
in this example, characterization tests were performed on the prepared drug carrier and the drug carrier encapsulating norfloxacin:
β -cyclodextrin (β -CD), polyethylene glycol (m-PEG), aspartic acid hexapeptide (D6), a drug carrier (β -CD-m-PEG-D6), Norfloxacin (NFX) and a bone targeting drug (β -CD-m-PEG-D6-NFX) encapsulated by the test drug carrier by taking norfloxacin as a drug model are respectively shown in figure 4.
As shown in FIG. 4, the drug carrier (β -CD-m-PEG-D6) map contains characteristic peaks of β -cyclodextrin (β -CD), polyethylene glycol (m-PEG) and aspartic acid hexapeptide (D6), and 1650cm-1Has a stretching vibration peak of 1570cm-1The peak stretching vibration of (2) was enhanced by esterification of amide I (C-O) of NHS in NHS-mPEG-MAL with NH2 of β -CD to form amide II (N-H) of 1712cm-1The (C ═ C) characteristic peak of MAL disappeared because C ═ C of MAL had already undergone an addition reaction with a mercapto group.
In the bone targeting drug (β -CD-m-PEG-D6-NFX) pattern, it can be seen that similar to the characteristic peak of β -CD-m-PEG-D6, but no characteristic peak of NFX appears, and it can be proved that NFX is already entrapped in the interior of β -cyclodextrin.
According to the spectrum gaps of the components, the results show that β -CD-m-PEG-D6 and β -CD-m-PEG-D6-NFX are successfully prepared.
Example 6:
in this example, norfloxacin is used as a drug model to test the drug loading rate and the loading efficiency of the bone targeting drug carrier.
β -CD-mPEG-D6(1mM) was dissolved in UP water, NFX (1mM) powder was added, the temperature was raised to 50 ℃ and stirred for 1 hour after NFX was dissolved in water, heating was turned off and stirring was continued until room temperature, and the product was collected and freeze-dried to a pale yellow solid powder.
In this example, the norfloxacin concentration in the norfloxacin solution and the solution after the collection of the product was measured, and the measurement was calculated by comparing the absorbance (λ 278nm) in the solution with the drug standard curve.
In this embodiment, the drug loading rate and the drug loading rate of the composite nano-drug carrier are calculated according to the following formula:
drug loading (wt.%) is (mass of loaded drug/mass of carrier) x 100%;
the drug loading rate (%) × 100% (mass of loaded drug/total mass of drug).
Finally, the invention measures that the theoretical drug loading rate of the drug carrier in the embodiment is 10 (wt.%), the actual drug loading rate is 8.97 (wt.%), and the drug loading rate is as high as 89.7%.
Example 7:
in this embodiment, after norfloxacin is used as a drug model and encapsulated in the bone targeting drug (β -CD-m-PEG-D6-NFX), the release of norfloxacin is tested, which is specifically as follows:
2mg of the prepared β -CD-m-PEG-D6-NFX sample is weighed into 50mL PBS (pH7.4), placed into a constant temperature shaking table (37 ℃, 75rpm), and 100 μ L of the sample is sampled at the time points of 5 min, 15 min, 25 min, 30min, 45 min, 60 min, 90 min, 120 min, 150min and 180 min.
And detecting the absorbance OD value of the sample at 278nm by using a microplate reader, and comparing according to a standard curve of NFX concentration to obtain a corresponding concentration value.
In conclusion, NFX-CD-D6 was released to 80% at 20min and 100% at 150min, which corresponds to the release rate of β -cyclodextrin inclusion compound.
Example 8:
in the embodiment, norfloxacin is used as a drug model, and the bone targeting drug carrier is coated with norfloxacin to prepare a bone targeting drug (β -CD-m-PEG-D6-NFX) and then tested for in vitro antibacterial ability.
Experiment I, the bacteriostatic ability of the bone targeting drug (β -CD-m-PEG-D6-NFX) is judged according to the bacteriostatic circle.
The selected strains are as follows: escherichia coli and Staphylococcus aureus.
The specific experimental process is as follows:
1. the experimental raw materials are prepared and used,
the solid raw material mainly comprises a drug component and HA tablets, wherein the HA tablets are respectively placed in β -CD-m-PEG-D6-NFX, CD-D6 and blank solution with the concentration of 1mg/mL for 1 hour, then the HA tablets are taken out and washed with UP water for three times, and the solid raw material specifically comprises the HA tablets containing bone targeting drugs (β -CD-m-PEG-D6-NFX), the HA tablets only containing Norfloxacin (NFX), the HA tablets only containing drug carriers (β -CD-m-PEG-D6) and the HA tablets without any component (serving as blank control groups).
The liquid raw materials comprise bone targeting drug (β -CD-m-PEG-D6-NFX) prepared into solution with concentration of 1mg/mL by using culture medium, Norfloxacin (NFX) prepared into solution (the molar ratio is consistent with that of β -CD-m-PEG-D6-NFX), drug carrier (β -CD-m-PEG-D6) prepared into solution with concentration of 1mg/mL, and culture medium (used as blank control group).
2. Preparation of the culture Medium
Taking four bacterial culture dishes, wherein two of the bacterial culture dishes are filled with a culture medium suitable for growth and propagation of escherichia coli, and the other two of the bacterial culture dishes are filled with a culture medium suitable for growth and propagation of staphylococcus aureus.
3. Inoculation of bacteria and bacteriostatic test
Sterilizing each sample, inoculating escherichia coli in a culture dish suitable for growth and propagation of escherichia coli, placing one of solid raw materials in each culture medium, separately placing the solid raw materials, labeling, inversely incubating for 24 hours, and observing the solid raw materials; and digging four small holes in each culture medium inoculated with bacteria, uniformly dispersing the liquid raw materials, putting 80 mu L of the liquid raw materials into the holes, covering the culture dish cover and marking, incubating for 24 hours, and observing the culture dish.
The staphylococcus aureus is inoculated in a culture dish suitable for the growth and propagation of the staphylococcus aureus, the specific operation steps are consistent with the operation of escherichia coli, and repeated description is omitted here.
In conclusion, as can be seen from fig. 5(a) and fig. 6(a), first, norfloxacin and the drug carrier comprising norfloxacin had inhibitory effects on both escherichia coli and staphylococcus aureus; secondly, no drug carrier carrying norfloxacin has no antibacterial effect; thirdly, the inhibition effect of the drug carrier coated with norfloxacin on escherichia coli and staphylococcus aureus exceeds the inhibition effect of norfloxacin which is directly used, which shows that the drug carrier has synergy and gain effects on the inhibition effect of norfloxacin after the norfloxacin is coated; fourthly, the liquid norfloxacin and the liquid drug carrier coated with norfloxacin have better bacteriostatic effect than the bacteriostatic condition of the solid norfloxacin, in addition, the solid drug carrier coated with norfloxacin also has certain bacteriostatic effect, and the solid norfloxacin has almost no bacteriostatic effect.
According to the graphs in fig. 5(B) and fig. 6(B), firstly, only the drug carrier carrying norfloxacin HAs the inhibition zone for escherichia coli and staphylococcus aureus, and secondly, the norfloxacin HAs no inhibition effect, which indicates that norfloxacin drug is not combined with HA characteristically, and is not considered to exist on the surface of HA, so that β -CD-m-PEG-D6-NFX is successfully prepared, and can be used for specifically adsorbing and releasing norfloxacin to achieve the inhibition effect.
And the second experiment is that the bone targeting drug (β -CD-m-PEG-D6-NFX) is detected to have excellent in vitro antibacterial property.
The specific process comprises the steps of preparing a bone targeting drug (β -CD-m-PEG-D6-NFX) by using the encapsulated norfloxacin as an experimental group, directly using norfossa as a control group, and respectively testing the minimum inhibitory concentration of escherichia coli and staphylococcus aureus of each group.
The Minimum Inhibitory Concentrations (MIC) for E.coli and S.aureus are given in Table I and Table II:
TABLE Pair of Minimum Inhibitory Concentrations (MIC) of Escherichia coli
Note that the concentration of NFX was consistent with the molarity of β -CD-m-PEG-D6-NFX
TABLE Minimum Inhibitory Concentration (MIC) for Staphylococcus aureus
Note that the concentration of NFX was consistent with the molarity of β -CD-m-PEG-D6-NFX
In conclusion, as can be seen from tables one and two, the bone targeting drug provided in the present embodiment has superior antibacterial performance, even surpasses the in vitro antibacterial effect of norfloxacin, which indicates that the drug carrier has synergistic and beneficial effects on the antibacterial effect of norfloxacin after carrying norfloxacin.
Example 9:
in this example, the in vitro cytotoxicity test of the bone targeting drug carrier is as follows:
toxicity testing was performed using L929. Digesting cells by pancreatin, blowing, centrifuging, adding a culture medium, counting cells, adjusting the volume of a cell suspension, inoculating, adding 100 mu L of cell suspension into a 96-well plate, inoculating at the density of 104 cells/well, transferring to a cell culture box, incubating for 24h, and adding 200 mu L of a sterile sample: CD. D6, CD-D6, PEG, NFX and NFX-CD-D6, setting 6 parallel samples in each group, continuously incubating for 24h, adding 200 mu LAlamarBlue reagent, uniformly mixing, incubating for 4h, absorbing 200 mu L of detection solution from each hole, placing in a 96-well plate, and measuring the absorbance values of the detection solution at the wavelength of 570nm and 600nm by using an enzyme-labeling instrument. The absorbance is positively correlated with the number of active cells, so that the proliferation condition of the cells can be reflected. The specific calculation formula is as follows:
wherein Asample-570nm is the absorbance of a sample to be detected at 570nm, Asample-600nm is the absorbance of the sample to be detected at 600nm, and Ablank-570nm and Ablank-600nm are the absorbance of AlamarBlue detection solution of blank holes at 570nm and 600nm respectively.
As shown in FIG. 7, it can be seen from the histogram of the effect of different concentrations of CD-D6 and NFX-CD-D6 on the proliferation rate of cells that neither CD-D6 nor NFX-CD-D6 is cytotoxic.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. The bone-targeting drug carrier is characterized by comprising β -cyclodextrin serving as a drug-encapsulating main body and aspartic acid hexapeptide serving as a target and having bone targeting, wherein the aspartic acid hexapeptide is connected with β -cyclodextrin through a flexible chain.
2. The bone-targeted drug carrier of claim 1, wherein the β -cyclodextrin is linked to only one aspartic acid hexapeptide via a flexible chain.
3. The bone-targeting drug carrier of claim 1 or 2, wherein the flexible chain is polyethylene glycol.
4. The preparation method of the bone-targeting drug carrier according to any one of claims 1 to 3, comprising the following steps:
(1) dissolving modified β -cyclodextrin containing one amino group in a PBS buffer solution, and using argon to remove oxygen for protection for 30 min;
(2) then dripping the flexible chain raw material solution, stirring for 1 hour at room temperature, dripping the modified aspartic acid hexapeptide with one end being sulfhydryl, and stirring overnight at room temperature;
(3) collecting the product, dialyzing for 3 days, and freeze-drying to obtain white solid powder.
5. The method for preparing a bone-targeting drug carrier of claim 4, wherein the β -cyclodextrin, the flexible chain and the aspartic acid hexapeptide are added in a ratio of 1.5:1: 1.5.
6. The method for preparing a bone-targeting drug carrier according to claim 4 or 5, wherein the flexible chain material is polyethylene glycol with maleimide at one end and succinamide at the other end.
7. A bone-targeting drug, which comprises the drug carrier according to any one of claims 1 to 3, wherein the hydrophobic small-molecule drug for treating bone diseases or preventing bone infection is encapsulated inside the β -cyclodextrin in the drug carrier.
8. The bone-targeted drug of claim 7, wherein the hydrophobic small molecule drug comprises norfosapraxin, cefazolin, sulfamethoxazole, and imipenem.
9. The bone-targeted drug of claim 7, wherein the bone diseases to be treated include osteoporosis, bone metastases, primary and secondary bone tumors, osteoarthritis, bone infections, osteitis deformans, osteomyelitis.
10. The preparation method of the bone targeting drug according to any one of claims 7 to 9, wherein the specific process comprises:
dissolving the bone-targeting drug carrier of any one of claims 1 to 3 in UP water, adding a hydrophobic small-molecule drug capable of treating bone diseases, wherein the mass ratio of the drug carrier to the hydrophobic small-molecule drug is 1:1, heating the mixture in water bath, heating the mixture to 50 ℃, stirring the mixture for 1 hour after the hydrophobic small-molecule drug powder is dissolved, stopping heating, stirring the mixture until the mixture is at room temperature, collecting the product, and freeze-drying the product to obtain solid powder.
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CN114209890A (en) * | 2021-11-19 | 2022-03-22 | 浙江瑞谷生物科技有限公司 | Bone repair material with strong bone affinity and preparation method thereof |
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