CN113521032B - Preparation method and application of bone targeting nano-reagent containing glaucocalyxin A - Google Patents
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Abstract
The invention provides a preparation method and application of a bone targeting nano-reagent containing glaucocalyxin A, belonging to the field of biological materials. The bone targeting nanometer reagent containing glaucocalyxin A is prepared by coating glaucocalyxin A in CHO-PEG 2k ‑(ALN) 2 The copolymer was obtained. CHO-PEG 2k ‑(ALN) 2 The preparation method of the copolymer comprises the following steps: reacting N-tert-butyloxycarbonyl ethylenediamine with methyl acrylate at 35-45 ℃ for 42-54h to obtain hyperbranched molecules; reacting the hyperbranched molecules under the condition of pH8.0-9.0 to obtain hyperbranched ethylenediamine cross-linked molecules; reacting the sodium alendronate with hyperbranched ethylenediamine cross-linked molecules to obtain a compound 4; and dissolving the compound 4 in a saturated solution of hydrogen chloride and methanol, and reacting to obtain the copolymer. The nano reagent has bone tumor targeting property and better tumor treatment effect, the preparation method is simple, and the obtained nano particles have better size uniformity and can be applied to the preparation of medicines for treating osteosarcoma.
Description
Technical Field
The invention belongs to the field of biomedicine and biological materials, and particularly relates to a preparation method and application of a glaucocalyxin A-containing bone targeting nano-reagent.
Background
Osteosarcoma is the most common one of primary malignant bone tissue tumors of teenagers, and has the advantages of high malignancy degree, strong invasiveness, very rapid disease progression and extremely high mortality rate. At present, the clinical treatment method for osteosarcoma mainly comprises traditional methods such as surgical treatment, chemotherapy, radiotherapy and the like and comprehensive treatment. The diseased part of osteosarcoma is in bone tissue, the tumor margin is unclear, and the focus can not be completely removed by surgical treatment, so that the tumor recurs and is transferred; the conventional medicament has poor targeting property, the medicament is difficult to reach bone tissues, so that the curative effect is low, the treatment target is single, and the multi-medicament resistance can be caused after long-term use. At present, no medicine specially used for treating osteosarcoma exists, so that the development of a new medicine which can inhibit the proliferation and metastasis of osteosarcoma and can actively target bone tissues is urgently needed.
Disclosure of Invention
The invention aims to provide a nano reagent containing glaucocalyxin A, which has bone tumor targeting and better tumor treatment effect.
The invention also aims to provide the preparation method of the bone targeting nano preparation, the method is simple to operate and low in cost, and the obtained nano particles are good in size uniformity.
The third purpose of the invention is to apply the bone targeting nano-agent containing the glaucocalyxin A in preparing the medicine for treating osteosarcoma.
A bone targeting nanometer reagent containing glaucocalyxin A is prepared by wrapping glaucocalyxin A in CHO-PEG 2k -(ALN) 2 Obtained in a copolymer, said CHO-PEG 2k -(ALN) 2 The copolymer has the following structural formula:
The invention also provides a preparation method of the bone targeting nano-agent containing glaucocalyxin A, which comprises the following steps: CHO-PEG 2k -(ALN) 2 Dissolving the copolymer and the glaucocalyxin A in tetrahydrofuran, dripping the mixture into water in a stirring state, removing the tetrahydrofuran, finally centrifuging, and taking supernatant fluid to obtain the bone targeting nanoparticles containing the glaucocalyxin A.
In the present invention, the CHO-PEG 2k -(ALN) 2 The copolymer was prepared as follows:
(1) Reacting N-tert-butyloxycarbonyl ethylenediamine and methyl acrylate for 42-54h at 35-45 ℃ in a nitrogen atmosphere under a stirring state to obtain hyperbranched molecules;
(2) Reacting the hyperbranched molecules under the condition that the pH value is 8.0-9.0 to obtain hyperbranched ethylenediamine cross-linked molecules;
(3) Reacting sodium alendronate with hyperbranched ethylenediamine cross-linked molecules to obtain a compound 4, wherein the structural formula is as follows:
(4) Dissolving the compound 4 in a saturated solution of hydrogen chloride and methanol, and reacting at room temperature for 6-10h to obtain CHO-PEG 2k -(ALN) 2 A copolymer.
In the present invention, the reaction conditions in step (2) are as follows: the reaction temperature is room temperature, and the reaction is carried out for 4 to 8 hours under the stirring state.
In the invention, the reaction time in the step (3) is 20-28h.
The invention also provides application of the glaucocalyxin A-containing bone targeting nano-reagent in preparation of a medicament for treating osteosarcoma.
Compared with the prior art, the main advantages of the invention include the following aspects: (1) The bone targeting nano particle prepared by the invention has definite structure and simple synthesis process. (2) The bone targeting nanoparticle particles prepared by the invention have good water solubility, uniform particle size and better bone tumor targeting property. (3) The bone targeting nano-reagent containing glaucocalyxin A prepared by the invention has excellent anti-tumor effect and biological safety, has low toxic and side effects, and has good application prospect when being used as a novel anti-osteosarcoma targeting medicament.
Drawings
Figure 1 mass spectrum of key intermediate compound 2.
FIG. 2 is a TEM image of glaucocalyxin A bone-targeting nanoparticles, illustrating that the prepared nanoparticles are between 60-120 nm.
FIG. 3: is a cell activity diagram of treating osteosarcoma cells by the glaucocalyxin A monomer and the bone targeting nano reagent.
FIG. 4 is a schematic view of: the gene is a flow-type double-staining pattern of the glaucocalyxin A bone-targeting nanoparticles for inducing apoptosis of human osteosarcoma cells, a Contrl group is a blank control, the cells hardly undergo apoptosis, and compared with GLA (10 mu M) of a glaucocalyxin A aqueous solution administration group, the apoptosis degree of the glaucocalyxin A nanoparticle group (GLA-CHO) and the glaucocalyxin A bone-targeting nanoparticle group (GLA-CHO-ALN) is remarkably improved.
FIG. 5 is a schematic view of: is a result graph of the glaucocalyxin A bone targeting nano reagent for treating osteosarcoma tumors in nude mice.
Detailed Description
Example 1 preparation of bone-targeting nanomaterial
(1) Synthesis of hyperbranched molecules
3g of N-Boc-ethylenediamine (N-t-butoxycarbonylethylenediamine, 18.72 mmol) was dissolved in 100ml of methanol, and 12.89g of methyl acrylate (149.76 mmol) was added dropwise to the solution and reacted at 40 ℃ for 48 hours with stirring under a nitrogen atmosphere. The reaction product was dried under reduced pressure at 50 ℃ by removing the solvent and excess methyl acrylate with a rotary evaporator to give 6.08g of Compound 1 (18.31 mmol) as a viscous colorless clear oil in a yield of 97.8%. The reaction formula is as follows:
(2) Synthesis of hyperbranched ethylenediamine cross-linked molecule
6.01g of Compound 1 (18.08 mmol) was charged into a round-bottomed flask, dissolved in 6ml of tetrahydrofuran, and then added with 0.1M aqueous sodium hydroxide solution to adjust the pH to 8.5, followed by reaction at room temperature (10-30 ℃ C.) with stirring for 6 hours. After the reaction was completed, the pH of the mixture was adjusted to 6.0 with 1M hydrochloric acid. The solvent was removed under reduced pressure, the residue was dissolved in 20mL of methanol and cooled in an ice-water bath, then the filtrate was filtered and the solvent was removed by rotary evaporation to give 3.66g of white foam compound 2 (12.03 mmol) in 66.52% yield. The mass spectrum of compound 2 is shown in figure 1. The reaction formula is as follows:
(3) Condensation reaction of hyperbranched ethylenediamine cross-linked molecule and sodium alendronate
7.08g of sodium alendronate (21.78 mmol) was added to 20ml of deionized water to form a suspension. To the suspension was added dropwise a 0.1M aqueous sodium hydroxide solution until the pH was 8.5, at which time the suspension became a transparent solution. 1.52g of Compound 2 (4.99 mmol) are dissolved in 6ml of a DMF solution of N-hydroxysuccinimide (NHS) (NHS content 2.30g, DMF dry) under nitrogen at room temperature, stirred for 1h, then 3.83g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 19.98 mmol) and 2.02g of triethanolamine (TEA, 20 mmol) are added and reacted for 12h with stirring. Washing the reaction product with saturated aqueous sodium bicarbonate solution, collecting the aqueous layer, and then using CH 2 Cl 2 2 times of extraction, each time for CH 2 Cl 2 The amount of (5 ml) was used, all the organic phases were collected and evaporated under reduced pressure, and the residue was purified by means of a silica gel column (eluent was a mixed solution of dichloromethane and methanol in a volume ratio of 15).
1.81g of Compound 3 was dissolved in acetonitrile (20 mL), and the solution was divided into four portions, and 1mL of a solution of 50mL of a 30mM aqueous solution of sodium alendronate was added every 15 minutes. The pH was maintained at 8.5 with 1M NaOH solution prior to each addition. After the addition of the compound 3, the reaction was carried out at room temperature under stirring for 12 hours, followed by rotary evaporation to obtain compound 4 (2.20g, 2.71mmol, 74.66%). The reaction formula is as follows:
(4)CHO-PEG 2k -(ALN) 2 synthesis of copolymer
Compound 4 (2.20g, 2.71mmol) was dissolved in a saturated solution of HCl-MeOH, and the reaction was stirred at room temperature for 8h, followed by rotary evaporation to give 1.35g of Compound 5 (1.90 mmol) with a yield of 70.11%. The reaction formula is as follows:
1.25 grams of CHO-PEG 2k -COOH (Mw =2000,0.5 mmol) with 0.23g NHS (2 mmol) and 0.38g EDC (2 mmol) dissolved in anhydrous DMF (6 mL) and stirred for 1h to activate the carboxylic acid moiety. 1.35g of Compound 5 (1.90 mmol) are dissolved in 4ml of anhydrous DMF, 2.02g of TEA (20 mm) are added and after stirring to homogeneity, the solution containing CHO-PEG prepared as described above is added 2k The reaction is carried out for 24h in a solution of-COOH at room temperature (10-30 ℃) with stirring. The resulting solution was poured into dialysis bags (MWCO 1000 Da) and dialyzed against deionized water for 72 hours to remove other unreacted molecules. Finally, freeze drying is carried out to obtain the target product CHO-PEG 2k -(ALN) 2 Wherein ALN is an abbreviation for sodium alendronate. PEG is an abbreviation for polyethylene glycol. The reaction formula is as follows:
Example 2 preparation of glaucocalyxin A bone-targeting nanoparticles
100mg of CHO-PEG prepared in example 1 were weighed 2k -(ALN) 2 Dissolving the copolymer and 10mg of glaucocalyxin A in 10ml of tetrahydrofuran, slowly dripping the mixture into 10ml of water which is quickly stirred (1000 revolutions per minute) at the speed of 20 drops per minute, adding nitrogen and stirring for 120 minutes to remove the tetrahydrofuran in the solution, finally centrifuging, and taking supernatant fluid to obtain the glaucocalyxin A bone targeting nanoparticles with the concentration of 1 mg/ml.
The morphology and the size of the glaucocalyxin bone targeting nanoparticles are observed by a transmission electron microscope, and as shown in a figure 2, the result shows that the nanoparticles are spherical, the size is uniform, the particle size is 60-120nm, and the particle size is less than 200nm, so that the glaucocalyxin bone targeting nanoparticles can enter tumor tissues through strong penetration and long retention (EPR) effects.
Example 3 Glaucocalyxin A bone-targeting nanoparticles inhibiting human osteosarcoma cell viability
The glaucocalyxin A bone targeting nanoparticles with the glaucocalyxin A concentration of 1mg/ml are diluted by deionized water until the glaucocalyxin A concentration is 1, 2, 5, 10, 20 and 50 mu g respectively.
In addition, 1, 2, 5, 10, 20, 50 μ g/ml of glaucocalyxin A aqueous solution (glaucocalyxin A monomer) is prepared.
Taking 143B cells of human osteosarcoma cells in logarithmic growth phase, and culturing at 5 × 10 3 ～1×10 4 The density of each hole is inoculated in a 96-hole plate, each hole is 100 mu L, the glaucocalyxin A bone targeting nanoparticles and the glaucocalyxin A aqueous solution with different concentrations are respectively added into each hole, 6 multiple holes are arranged in parallel for each concentration of the drug, so as to investigate the inhibiting effect of each concentration of the drug on the activity of human osteosarcoma cells. After each drug intervenes for 24 hours, the influence of the glaucocalyxin A bone targeting nanoparticles and the glaucocalyxin A aqueous solution on the growth and the activity of osteosarcoma cells is evaluated by adopting a tetrazolium salt (MTT) colorimetric method. Calculating the inhibition rate of the glaucocalyxin A bone-targeted nanoparticles and the glaucocalyxin A monomers on the activity of osteosarcoma cells according to the absorbance value, and calculating the half inhibition concentration IC 50 The value is obtained. The cell viability inhibition map was plotted as shown in fig. 3. IC of glaucocalyxin A bone targeting nanoparticles 50 IC of aqueous solution of glaucocalyxin A with value of 15.27 mug/mL 50 18.85. Mu.g/mL. IC of glaucocalyxin bone-targeted nanoparticles compared to glaucocalyxin monomers 50 The content is remarkably reduced, which shows that the glaucocalyxin A bone targeting nanoparticles can better inhibit tumor proliferation.
Example 4 flow cytometry for detecting Effect of glaucocalyxin A bone-targeting nanoparticles on inducing apoptosis of human osteosarcoma cells
The glaucocalyxin A-containing nanoparticle with the glaucocalyxin A concentration of 10 mu g/mL is prepared according to the preparation method of the glaucocalyxin A bone-targeted nanoparticle with the glaucocalyxin A concentration of 1mg/mL in the embodiment 2, and the difference is that 100mg of PEG replaces the glaucocalyxin A bone-targeted nanoparticle, and finally deionized water is adopted to dilute the glaucocalyxin A bone-targeted nanoparticle to 10 mu g/mL.
The glaucocalyxin A bone targeting nano particle with the glaucocalyxin A concentration of 10 mu g/mL: the glaucocalyxin A bone targeting nanoparticles with the glaucocalyxin A concentration of 1mg/ml in example 2 are obtained by diluting the glaucocalyxin A bone targeting nanoparticles with deionized water.
Method for detecting apoptosis of human osteosarcoma cells induced by glaucocalyxin A bone-targeted nanoparticles by adopting Annexin-PI double staining kitThe function of (1). Preparing human osteosarcoma 143B cells in logarithmic growth phase into 2 × 10 cells in 1640 culture medium 6 The cells were suspended at a concentration of 2 mL/mL in 6-well plates and cultured for 24 hours. Removing culture solution, respectively adding 2mL of glaucocalyxin A aqueous solution containing 10 mu g/mL of glaucocalyxin A, glaucocalyxin A nanoparticles and glaucocalyxin A bone targeting nanoparticles into each hole, after intervening for 24 hours, respectively collecting dead cells in the supernatant of each hole, digesting and collecting cells at the bottom of the dish by pancreatin without EDTA, uniformly mixing the supernatant and the cells at the bottom of the dish, centrifuging for 5min at 2000rpm under the condition of 4 ℃, collecting the cells, adding 500 mu L of Binding Buffer suspension cells, adding 5 mu L of Annexin-V-FITC probe, uniformly mixing, adding 5 mu L of PI for staining, uniformly mixing, keeping out of the sun, reacting for 15min, detecting by using a flow cytometer, obtaining a flow double staining graph (figure 4) of inducing apoptosis of human osteosarcoma cells by using the glaucocalyxin A bone targeting nanoparticles, wherein the apoptosis degree (49.6% + 30.6%) of the glaucocalyxin A bone targeting nanoparticles in the administration group is obviously higher than that of the glaucocalyxin A bone targeting nanoparticles in the glaucocalyxin A aqueous solution administration group (29.4% + 23.1.6%), thereby indicating that the apoptosis degree of the glaucocalyxin A nanoparticles can be better promoted than that targeted apoptosis of the glaucocalyxin A cells in the glaucocalyxin A bone targeting nanoparticles.
EXAMPLE 5 therapeutic Effect of Glaucocalyxin A bone-targeting nanoparticles on in situ tumors of osteosarcoma in nude mice
The nude mouse is anesthetized by intramuscular injection, after the pain sense of the nude mouse disappears, a longitudinal incision of 1cm is cut at the upper end of the right hind limb tibia of the nude mouse by a surgical scissors under a microscope for 8 times of surgery, the skin is cut, and the upper end of the tibia is exposed. The bone cavity was passed through with a 1mL syringe in the tibial direction and then pulled out, and 5. Mu.L of 143B cells (concentration: 2X 10) from the human osteosarcoma cells were aspirated with a micro-syringe 6 At/5 μ L) was injected into the tibia, and the incision was closed with surgical sutures after gradually withdrawing the suture until 5 μ L was completed. The whole operation process is completed in a clean bench.
When the tumor grows to about 100mm in volume 3 Performing administration treatment, and dividing into 3 groups, blank control group, glaucocalyxin A monomer administration group (glaucocalyxin A water solution), glaucocalyxin A bone targeting nanoparticle administration group once every two days, glaucocalyxin A monomer administration group and glaucocalyxin A monomer administration groupThe dosage of the calyxin A bone targeting nanoparticle administration group is 5mg of glaucocalyxin A/kg in each administration. On day 21 of dosing, tumors were sacrificed, and tumor volumes were measured and calculated. The results are shown in FIG. 5. Through calculation, compared with a blank control group, the tumor volume of the glaucocalyxin bone targeting nanoparticle administration group is reduced by 68%, and the tumor volume of the glaucocalyxin monomer administration group is reduced by 23%. The treatment effect of the glaucocalyxin A bone targeting nanoparticle administration group is better than that of the glaucocalyxin A monomer administration group.
Claims (3)
1. A preparation method of a bone targeting nano-reagent containing glaucocalyxin A is characterized by comprising the following steps: CHO-PEG 2k -(ALN) 2 Dissolving the copolymer and the glaucocalyxin A in tetrahydrofuran, dripping the mixture into water in a stirring state, removing the tetrahydrofuran, centrifuging, and taking supernatant to obtain the bone targeting nanoparticles containing the glaucocalyxin A, wherein the CHO-PEG is used for preparing the bone targeting nanoparticles containing the glaucocalyxin A 2k -(ALN) 2 The copolymer has the following structural formula:
wherein n is 1000-5000; the CHO-PEG 2k -(ALN) 2 The copolymer was prepared as follows:
(1) Reacting N-tert-butyloxycarbonyl ethylenediamine and methyl acrylate for 42-54h at 35-45 ℃ in a nitrogen atmosphere under a stirring state to obtain hyperbranched molecules;
(2) Reacting the hyperbranched molecules under the condition that the pH value is 8.0-9.0 to obtain hyperbranched ethylenediamine cross-linked molecules;
(3) Reacting sodium alendronate with hyperbranched ethylenediamine cross-linked molecules to obtain a compound 4, wherein the structural formula is as follows:
(4) Dissolving the compound 4 in a saturated solution of hydrogen chloride and methanol, and reacting for 6-10h at room temperature to obtain CHO-PEG 2k -(ALN) 2 A copolymer.
2. The method according to claim 1, wherein the reaction conditions in the step (2) are as follows: the reaction temperature is room temperature, and the reaction is carried out for 4 to 8 hours under the stirring state.
3. The process according to claim 2, wherein the reaction time in the step (3) is 20 to 28 hours.
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