CN113521032A - Preparation method and application of bone targeting nano-reagent containing glaucocalyxin A - Google Patents

Preparation method and application of bone targeting nano-reagent containing glaucocalyxin A Download PDF

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CN113521032A
CN113521032A CN202110809252.9A CN202110809252A CN113521032A CN 113521032 A CN113521032 A CN 113521032A CN 202110809252 A CN202110809252 A CN 202110809252A CN 113521032 A CN113521032 A CN 113521032A
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glaucocalyxin
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张琪
朱健伟
马博
赵昂
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Nanjing Jishu Pharmaceutical Technology Co ltd
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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-PEG2k‑(ALN)2The copolymer was obtained. CHO-PEG2k‑(ALN)2The 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

Preparation method and application of bone targeting nano-reagent containing glaucocalyxin A
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, rapid disease progression and extremely high death 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 the application of the bone targeting nano-agent containing the glaucocalyxin A in the preparation of the drugs for treating osteosarcoma.
A bone targeting nanometer reagent containing glaucocalyxin A is prepared by extracting glaucocalyxin AThe A is coated on CHO-PEG2k-(ALN)2Obtained in a copolymer, said CHO-PEG2k-(ALN)2The copolymer has the following structural formula:
Figure BDA0003167598170000021
Figure BDA0003167598170000022
wherein n is 1000-5000.
The invention also provides a preparation method of the bone targeting nano-agent containing glaucocalyxin A, which comprises the following steps: CHO-PEG2k-(ALN)2Dissolving 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-PEG2k-(ALN)2The 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:
Figure BDA0003167598170000023
(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-PEG2k-(ALN)2A 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 20-28 h.
The invention also provides application of the glaucocalyxin A-containing bone targeting nano-reagent in preparation of a medicine for treating osteosarcoma.
Compared with the prior art, the main advantages of the invention comprise 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: the gene is a flow-type double-staining pattern of the glaucocalyxin bone-targeting nanoparticles for inducing apoptosis of human osteosarcoma cells, a Contrl group is a blank control, the cells are almost free from apoptosis, and compared with GLA (10 mu M) of a glaucocalyxin aqueous solution administration group, the apoptosis degree of the glaucocalyxin bone-targeting nanoparticles group (GLA-CHO) and the glaucocalyxin bone-targeting nanoparticles group (GLA-CHO-ALN) is remarkably improved.
FIG. 5: 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, 18.72mmol) was dissolved in 100ml of methanol, and 12.89g of methyl acrylate (149.76mmol) was added dropwise to the solution and reacted at 40 ℃ for 48 hours under stirring in 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(18.31mmol) as a viscous colorless clear oil in a yield of 97.8%. The reaction formula is as follows:
Figure BDA0003167598170000041
(2) synthesis of hyperbranched ethylenediamine cross-linked molecule
6.01g of Compound 1(18.08mmol) 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(12.03mmol) in 66.52% yield. The mass spectrum of compound 2 is shown in figure 1. The reaction formula is as follows:
Figure BDA0003167598170000042
(3) condensation reaction of hyperbranched ethylenediamine cross-linked molecule and sodium alendronate
7.08g of sodium alendronate (21.78mmol) was added to 20ml of deionized water to form a suspension. To the suspension was added dropwise a 0.1M aqueous solution of sodium hydroxide until the pH was 8.5, at which time the suspension became a clear solution. In a nitrogen atmosphere at room temperature, 1.52g of Compound 2(4.99mmol) was dissolved in 6ml of a DMF solution of N-hydroxysuccinimide (NHS) (NHS content: 2.30g, DMF dry) and stirred for 1h, then 3.83g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (EDC, 19.98mmol) and 2.02g of triethanolamine (TEA, 20mmol) were added and reacted for 12h with stirring. Washing the reaction product with saturated aqueous sodium bicarbonate solution, collecting the aqueous layer, and then using CH2Cl2Extraction was performed 2 times, each time for CH2Cl2In an amount of 5 ml, collecting all organic phases and evaporating under reduced pressure, passing the residue through a silica gel column (eluent is a mixture of dichloromethane and methanol in a volume ratio of 15: 1)Solution) to yield 1.81g of compound 3(3.63mmol) as a clear liquid in 72.75% yield.
1.81g of Compound 3 was dissolved in acetonitrile (20mL), 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 per 15 minutes. The pH was maintained at 8.5 with 1M NaOH solution prior to each addition. After the addition of compound 3, the reaction was carried out at room temperature under stirring for 12 hours, followed by rotary evaporation to give compound 4(2.20g, 2.71mmol, 74.66%). The reaction formula is as follows:
Figure BDA0003167598170000051
(4)CHO-PEG2k-(ALN)2synthesis of copolymer
Compound 4(2.20g, 2.71mmol) was dissolved in saturated HCl-MeOH and the reaction stirred at room temperature for 8h, then rotary evaporated to give 1.35g of Compound 5(1.90mmol) in 70.11% yield. The reaction formula is as follows:
Figure BDA0003167598170000052
1.25 grams of CHO-PEG2k-COOH (Mw ═ 2000, 0.5mmol) with 0.23g NHS (2mmol) and 0.38g EDC (2mmol) dissolved in anhydrous DMF (6mL) and stirred for 1h to activate the carboxylic acid moiety. 1.35g of Compound 5(1.90mmol) are dissolved in 4ml of anhydrous DMF, 2.02g of TEA (20mm) are added and after stirring to homogeneity, the solution containing CHO-PEG prepared as described above is added2kThe 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 1000Da) 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-PEG2k-(ALN)2Wherein ALN is an abbreviation for sodium alendronate. PEG is an abbreviation for polyethylene glycol. The reaction formula is as follows:
Figure BDA0003167598170000061
Figure BDA0003167598170000062
where n is 2000.
Example 2 preparation of glaucocalyxin A bone-targeting nanoparticles
100mg of CHO-PEG prepared in example 1 were weighed2k-(ALN)2Dissolving 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 through 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 × 103～1×104The 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 the drugs intervene 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. According to absorbance valueCalculating the inhibition rate of the glaucocalyxin A bone-targeted nano particles and the glaucocalyxin A monomers on the activity of osteosarcoma cells, and calculating half inhibition concentration IC50The value is obtained. A cell viability inhibition map was plotted as shown in fig. 3. IC of glaucocalyxin A bone targeting nanoparticles50IC of aqueous solution of glaucocalyxin A with value of 15.27 mug/mL5018.85. mu.g/mL. IC of glaucocalyxin bone-targeted nanoparticles compared to glaucocalyxin monomers50The content is remarkably reduced, which shows that the glaucocalyxin A bone targeting nanoparticles can better inhibit tumor proliferation.
EXAMPLE 4 detection of Effect of Glaucocalyxin A bone-targeting nanoparticles on inducing apoptosis of human osteosarcoma cells by flow cytometry
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 nanoparticles 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 with deionized water.
The Annexin-PI double staining kit is adopted to detect the effect of the glaucocalyxin A bone targeting nanoparticles on inducing apoptosis of human osteosarcoma cells. Preparing human osteosarcoma 143B cells in logarithmic growth phase into 2 × 10 cells in 1640 culture medium6The cells were suspended at a concentration of 2 mL/mL in 6-well plates and cultured for 24 hours. Removing culture solution, 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, intervening for 24 hours, 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 at 2000rpm for 5min at 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 dyeing, uniformly mixing, keeping out of the sun, reacting for 15min, detecting by using a flow cytometer, and obtaining the glaucocalyxin bone targeting nanoparticles for inducing human bone to induce human bone, wherein the glaucocalyxin A bone targeting nanoparticles are obtainedThe flow-type double staining graph (fig. 4) of sarcoma cell apoptosis shows that the apoptosis degree (49.6% + 30.6%) of the glaucocalyxin bone targeting nanoparticle administration group is significantly improved compared with the apoptosis degree (29.4% + 23.1%) of the glaucocalyxin aqueous solution administration group, which indicates that the glaucocalyxin bone targeting nanoparticle can better promote osteosarcoma cell apoptosis.
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-syringe6At/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 volume3The drug delivery treatment is carried out and divided into 3 groups, a blank control group, a glaucocalyxin A monomer administration group (aqueous glaucocalyxin A solution), a glaucocalyxin A bone targeting nanoparticle administration group once every two days, and the drug delivery dose of each time of the glaucocalyxin A monomer administration group and the glaucocalyxin A bone targeting nanoparticle administration group is 5mg glaucocalyxin A/kg. 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 (6)

1. A bone targeting nanometer reagent containing glaucocalyxin A is prepared by wrapping glaucocalyxin A in CHO-PEG2k-(ALN)2Obtained in a copolymer, said CHO-PEG2k-(ALN)2The copolymer has the following structural formula:
Figure RE-FDA0003225499680000011
wherein n is 1000-5000.
2. A preparation method of a bone targeting nano-reagent containing glaucocalyxin A is characterized by comprising the following steps: CHO-PEG2k-(ALN)2Dissolving 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.
3. The method according to claim 2, wherein the CHO-PEG is added to the sample2k-(ALN)2The 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:
Figure RE-FDA0003225499680000012
(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-PEG2k-(ALN)2A copolymer.
4. The method according to claim 3, 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.
5. The process according to claim 4, wherein the reaction time in the step (3) is 20 to 28 hours.
6. The use of the glaucocalyxin A-containing bone-targeting nanoagent of claim 1 in the preparation of a medicament for treating osteosarcoma.
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