CN116059181A - A nano drug delivery system - Google Patents

Abstract

The invention provides a nano-drug delivery system, which is characterized in that: the nano capsule is constructed by self-assembling the polylactic acid-glycolic acid copolymer, the hydrophobic small molecule drug E2 and the vitamin D and is used for delivering the hydrophobic drug. The nano-drug delivery system not only can solve the problem of poor blood circulation effect caused by hydrophobicity, but also can load different drugs and various functional materials simultaneously, and is used for drug synergism to achieve better treatment effect.

Description

A nano drug delivery system

technical field

The invention relates to the field of biomedicine, in particular to a nano drug delivery system.

Background technique

In recent years, nanodrug delivery system (Nanodrug delivery system, NDDS) has been widely used in bioimaging, disease diagnosis and treatment. At the same time, how to improve the biodiversity of nanodrug delivery system has also been widely studied.

For hydrophobic drugs, poor circulation in the body fluid environment usually results in only a small amount of drug being able to act on the target site, resulting in poor therapeutic effect. And it may lead to the risk of side effects caused by acting on non-target sites.

Contents of the invention

The present invention aims to overcome the above-mentioned defects, and mainly aims at hydrophobic drugs. The construction can not only solve the problem of poor blood circulation caused by hydrophobicity, but also can simultaneously load different drugs and various functional materials for drug synergy, achieving Drug delivery system with better therapeutic effect.

The invention provides a nano drug delivery system, characterized in that:

Nanocapsules were constructed by self-assembly of polylactic acid-glycolic acid copolymer, hydrophobic small molecule drug E2 and vitamin D for the delivery of hydrophobic drugs.

Further, a nano-drug delivery system provided by the present invention is also characterized in that:

The shell layer of the polylactic acid-glycolic acid copolymer is also loaded with localization factors.

The positioning factor is preferably IR780, which is a dye that can be used in clinical practice, and its fluorescence spectrum is in the near-infrared region, which has a good tissue penetration depth and is conducive to positioning imaging.

Further, a nano-drug delivery system provided by the present invention is also characterized in that:

The surface of the above-mentioned polylactic acid-glycolic acid copolymer is modified with functional factors having a targeting effect based on biocoupling. The specific synthetic route is shown in Figure 1.

Furthermore, a nano-drug delivery system provided by the present invention is also characterized in that the specific preparation method is as follows:

S1. Adding the positioning factor to the polylactic acid-glycolic acid copolymer stroke mixture;

S2. Under the condition of 20-60°C, the liquid in the mixture is removed to form a film;

S3. Prefabrication of hydrophobic small molecule drugs and vitamin D into oil;

S4. Dissolving the film of S2 in a solvent, mixing it with the oil of S3, and circulating ultrasound for 5-20 minutes at a temperature below 10°C to obtain nanoparticles;

S5. For the nanoparticles of S4, no target product is obtained after at least one settling, centrifugation, and washing.

The dosage of each of the above components is adjusted based on the needs of the product components.

In the present invention, the preferred ratio relationship is also provided as follows:

Polylactic acid-glycolic acid copolymer: the mass ratio of positioning factor is 100-20000:1;

Polylactic acid-glycolic acid copolymer: the mass ratio of VD is 1-20:1;

The mass ratio of polylactic acid-glycolic acid copolymer: hydrophobic small molecular drug is 1:0.1-10.

Further, a nano-drug delivery system provided by the present invention is also characterized in that:

The above-mentioned hydrophobic small molecule drug E2 can also be replaced with at least one of the following drugs:

A. Drugs used to promote osteogenesis;

B. Drugs that promote mineralization of osteoblasts;

C. Drugs that promote the expression of ALP, Runx2, and IBSP;

D. Drugs that promote the expression of OCN and OPN.

Further, a nano-drug delivery system provided by the present invention is also characterized in that:

The above functional factors are bone tissue targeting molecules.

Further, a nano-drug delivery system provided by the present invention is also characterized in that:

The above-mentioned nano drug delivery system is applied to the preparation of drugs for promoting osteogenic differentiation.

Further, a nano-drug delivery system provided by the present invention is also characterized in that:

The above nano drug delivery system is applied to the preparation of drugs that promote the expression of ALP, Runx2, and IBSP.

Further, a nano-drug delivery system provided by the present invention is also characterized in that:

The above-mentioned nano drug delivery system is applied to the preparation of drugs for promoting mineralization of osteoblasts.

Further, a nano-drug delivery system provided by the present invention is also characterized in that:

The above nano drug delivery system is applied to the preparation of drugs that promote the expression of OCN and OPN.

Action and effect of the present invention:

In the research of the present invention, based on the advantages of poly(lactic-co-glycolic acid, PLGA) having good biocompatibility, safety and targeted modification, it is applied to the drug carrier superior.

Through self-assembly and soybean oil droplets dissolved with hydrophobic small molecule drugs, such as: estradiol (17β-estradiol, E ₂ ), and vitamin D (Vitamin D, VD) to form nanocapsules for hydrophobic drugs deliver.

In addition, the present invention also loads localization factors, such as luminescent dye IR780, in the polymer shell PLGA by means of co-precipitation, thereby realizing real-time localization and tracking of nano-medicines.

In order to further realize the precise release capability of the nano-drug delivery system, the present invention also utilizes the carboxylic acid functional groups on the surface of PLGA to target bone tissue molecules through bioconjugation, such as Alendronate Sodium (ADA) modified On the surface of nanoparticles, the targeting effect of nanomedicine is realized.

Description of drawings

Figure 1, a schematic diagram of the synthesis route of the nano-drug delivery system provided in this example;

TEM and DLS characterization diagrams of the system of Fig. 2, the present embodiment component;

Fig. 3, the UV characterization diagram of the system of the component of the present embodiment;

Fig. 4, the confocal imaging figure of the system of the component of the present embodiment;

Fig. 5, the ALP dyeing experiment result of the system of the present embodiment component;

Fig. 6, the ALP activity experiment result of the system of the present embodiment component;

Fig. 7, the PCR experiment result of the system of the present embodiment component;

Fig. 8, the result of the osteogenic mineralization experiment of the system of the components of the present embodiment;

Fig. 9. The results of immunohistochemical staining of the osteoblast differentiation system of the components of this example after 14 days.

Detailed ways

1. Preparation of nano drug delivery system

As shown in Figure 1, the specific experimental steps of the nano-drug delivery system provided in this example are:

1. Preparation solution: PLGA (purchased from Bailingwei, average molecular weight 50W): 5mg dissolved in 200uL dichloromethane——①

IR780 (purchased from Bailingwei): 0.005mg dissolved in 20uL ethanol——②

VD (purchased from sigma): 0.5mg dissolved in 20uL ethanol——③

E ₂ (purchased from sigma): 4mg dissolved in 20uL ethanol——④

2. Take 10uL ② and add it to solution ①. The obtained mixed solution was subjected to rotary evaporation at 40° C. to obtain a green film. The resulting film was dried overnight in a fume hood. A dye-loaded polymer material is obtained.

3. Dissolve ③ and ④ in 1 mL of soybean oil for later use to obtain mixed oil droplets of the two drugs.

4. Re-dissolve the above-mentioned dye-loaded polymer in 200 uL of dichloromethane, add 1.26 mL of 5% PVA solution, and 50 uL of mixed oil droplets. The above mixture was placed in an ice-water bath, and under the action of 80W ultrasound, a cycle strategy of 3 seconds of ultrasound on and 7 seconds of ultrasound pause was used for 10 minutes.

5. After 4 hours of static precipitation, the obtained nanoparticle aqueous solution was washed and separated by a centrifuge. .

6. The above-mentioned nanoparticles were configured into a 1 mg/mL solution, and 10 uL of 2 mM EDC aqueous solution was added for carboxyl activation. After 10 minutes, 100 uL of 1 mg/mL ADA (purchased from Bailingwei) aqueous solution was added. After reacting at room temperature for 4 hours, centrifuge and wash at a speed of 15000r/10min to remove excess ADA in the solution and prepare the final product at 1mg/mL.

2. Experimental method

2.1. Transmission Electron Microscopy (TEM) Characterization:

Take the E2+VD@PLGAIR780-ADA/E2@PLGAIR780-ADA aqueous solution with a solid content of 1%, dilute it 100 times, take 10 μL and drop it on the copper grid, and use TEM to characterize it after natural drying. The instrument model is HT7700. The voltage is 100kV.

2.2. Dynamic Light Scattering (DLS) characterization:

Take the E2+VD@PLGAIR780-ADA/E2@PLGAIR780-ADA aqueous solution with a solid content of 1%, dilute it 100 times, take 1mL and place it in a cuvette, which is completed by ZetasizerZS-90 from Malvern Company in the United Kingdom. The test uses 633nm red The light acts as the excitation light, and the angle between the incident light and the detector is 90°.

2.3. Ultraviolet-Visible (UV-vis) absorption spectrum characterization:

After using the same deionized water to set the baseline, take the E2+VD@PLGAIR780-ADA/E2@PLGAIR780-ADA/PLGAIR780 aqueous solution with a solid content of 1%, and put it in the cuvette, which is completed by the UV spectrometer Lambda 750, and the scanning speed is 1nm/ s, scanning range 850nm-250nm.

2.4. Confocal imaging:

Firstly, 1×108 osteoblasts were incubated with serum-containing incubation medium for proliferation, and the incubator conditions were 37°C, 5% CO2. When the cell density was 5×108, it was trypsinized and transferred to a confocal culture dish, and 2 mL of serum-free incubation medium was added. Then 20 μL of E2+VD@PLGAIR780-ADA/E2@PLGAIR780-ADA/PLGAIR780 aqueous solution with a solid content of 1% was added, and after 12 hours of attachment, the medium was sucked away and the cells were washed with PBS. Cell fluorescence imaging was done using Olympus FV1000 confocal.

2.5. Alkaline phosphatase staining:

Passage the osteoblasts in a 12-well cell culture plate. When the cell density reaches about 60-70%, replace the osteogenic differentiation medium and add DMSO, PLGAIR780-ADA, E2, E2@PLGAIR780-ADA, E2+ according to the grouping VD@PLGAIR780-ADA, the final concentration in each well is 10-7. The differentiation medium was replaced every 3 days, and corresponding treatment drugs were added. On the seventh day of differentiation, aspirate the culture medium, wash 3 times with PBS, and then add 4% paraformaldehyde to fix at room temperature for 15-20min. Wash off excess fixative with PBS. Prepare BCIP/NBT staining working solution according to the volume of 500ul per well to ensure that the sample can be fully covered. Incubate at room temperature in the dark for 30 minutes until the color develops to the expected depth. To remove BCIP/NBT staining, wash with distilled water 1-2 times to terminate the color reaction. Cell staining was observed with a microscope.

2.6.ALP activity:

Passage the osteoblasts in a 24-well plate. When the cell density reaches about 60-70%, replace the osteogenic differentiation medium and add DMSO, PLGAIR780-ADA, E2, E2@PLGAIR780-ADA, E2+VD@ according to the group PLGAIR780-ADA, the final concentration in each well is 10-7. The differentiation medium was replaced every 3 days, and corresponding treatment drugs were added. On the seventh day of differentiation, aspirate the culture medium, wash with PBS 3 times, add RIPA protein lysate, place on ice for 20min, scrape off the cells, and suck into the centrifuge tube. Centrifuge at 12000rpm at 4°C for 10min, and suck out the supernatant for later use.

Refer to the table below to set up blank control wells, standard wells and sample wells using a 96-well plate. The volumes of standard products are 4, 8, 16, 24, 32 and 40 microliters respectively, and 50 microliters of samples can be added directly. If the activity of alkaline phosphatase in the sample is too high, the amount of sample can be reduced or diluted properly before measurement.

Gently blow and mix with a pipette tip, or use a shaker to mix.

Incubate at 37°C for 5-10 minutes. (Note: When the activity of alkaline phosphatase in the sample to be tested is low, the incubation time can be extended to 30 minutes)

Add 100 μl of reaction stop solution to each well to stop the reaction. At this point, wells with standards or alkaline phosphatase activity will appear in different shades of yellow.

Absorbance was measured at 405 nm. If 405nm cannot be measured, the absorbance can also be detected in the range of 400-415nm. If it cannot be determined immediately, the determination can be completed within several hours, and the developed yellow color is stable within several hours.

Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard to the standard wells of the 96-well plate, and add the standard dilution to make up to 20 μl, which is equivalent to the concentration of the standard being 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5mg/ml.

Add an appropriate volume of sample to the sample wells of the 96-well plate. If the sample is less than 20μl, add standard diluent to make up to 20μl. Note the sample volume.

Add 200 μl of BCA working solution to each well, and place at 37°C for 20-30 minutes.

Use a microplate reader to measure the absorbance of A562, or other wavelengths between 540-595nm.

Calculate the protein concentration of the sample from the standard curve and the sample volume used.

ALP activity is defined as the amount of alkaline phosphatase required to hydrolyze para-nitrophenyl phosphate chromogenic substrate to produce 1 umolp-nitrophenol per minute under the condition of 25°C, which is defined as an enzyme activity unit.

2.7. Real-time fluorescent quantitative PCR:

Passage the osteoblasts in a 6-well plate. When the cell density reaches about 60-70%, replace the osteogenic differentiation medium and add DMSO, PLGAIR780-ADA, E2, E2@PLGAIR780-ADA, E2+VD@ according to the group PLGAIR780-ADA, the final concentration in each well is 10-7. The differentiation medium was replaced every 3 days, and corresponding treatment drugs were added. On the seventh day of differentiation, aspirate the medium, wash 3 times with PBS, add Trizol lysate, and aspirate the lysate. Put it on ice, add 200ul chloroform to every 1ml lysate, and shake to mix. 4°C, 12000rpm, centrifuge for 10min. The colorless liquid in the upper layer was sucked out and transferred to a clean centrifuge tube, and an equal volume of isopropanol was added, mixed upside down, and left to stand on ice for 10 minutes. Centrifuge at 12000rpm at 4°C for 15min. Aspirate the liquid, and add 1ml of 70% ethanol to wash the precipitate, a total of 2 times. Dry the residual ethanol and add 20ul DEPC water to dissolve the RNA. According to the concentration of RNA, it was reverse transcribed into cDNA for Q-PCR experiment.

Reagent volume SYBR green mix(2*) 5 Sample 1 Primer-forward 0.2 Primer-reverse 0.2 <![CDATA[H₂O]]> 3.6

The reaction system was prepared according to the above table, and the reaction conditions in the following table were tested.

2.8. Alizarin red staining:

Passage the osteoblasts in a 6-well plate. When the cell density reaches about 60-70%, replace the osteogenic differentiation medium and add DMSO, PLGAIR780-ADA, E2, E2@PLGAIR780-ADA, E2+VD@ according to the group PLGAIR780-ADA, the final concentration in each well is 10-7. The differentiation medium was replaced every 3 days, and corresponding treatment drugs were added. On the 14th day of differentiation, aspirate the medium and wash 3 times with PBS. Add an appropriate amount of 4% paraformaldehyde to each well, and fix at room temperature for 15-20min. After fixation, the fixative was aspirated, and the residual fixative was washed with PBS. Add 500ul Alizarin Red staining solution to each well and incubate at room temperature for 30min-1h in the dark. Aspirate the staining solution and wash off the residual staining solution with distilled water. Observe under a microscope.

2.9. Immunohistochemical staining:

Passage the osteoblasts in a 24-well plate. When the cell density reaches about 60-70%, replace the osteogenic differentiation medium and add DMSO, PLGAIR780-ADA, E2, E2@PLGAIR780-ADA, E2+VD@ according to the group PLGAIR780-ADA, the final concentration in each well is 10-7. The differentiation medium was replaced every 3 days, and corresponding treatment drugs were added. On the 14th day of differentiation, aspirate the medium and wash 3 times with PBS. Add an appropriate amount of 4% paraformaldehyde to each well, and fix at room temperature for 15-20min. After fixation, the fixative was aspirated, and the residual fixative was washed with PBS. Permeabilization solution was added to each well for 15 min at room temperature, and residual permeation solution was washed with PBS. Add 5% BSA to block at room temperature for 1 h. Osteopontin and Osteocalcin primary antibodies were added and incubated overnight at 4°C. The next day, the primary antibody was aspirated, and PBS was added to clear it, 5min*3 times. Add the corresponding secondary antibody, incubate at room temperature for 1 h, wash with PBS 3 times, 5 min each time. Prepare DAB chromogenic solution, add an appropriate amount of DAB chromogenic solution to each well, and develop color in the dark for 30 minutes. Add hematoxylin to stain the nuclei for 2 min, and wash off excess staining solution with distilled water. Observe under a microscope.

3. Experimental results:

3.1. System Characterization

A. From the TEM characterization diagram in Figure 2, it can be found that this example successfully constructed nano-droplets with drug-loaded mixed oil droplets as the core and PLGA as the outer layer, that is, the polymer PLGAIR780 loaded with functional dyes as the shell .

B. From the ultraviolet absorption spectrum (UV-Vis) of Figure 3, it can be found that: 780nm is the absorption of the dye IR780, and 280nm is the absorption of the drug estradiol and vitamin D.

3.2. Proof that nanoparticles can enter cells

As shown in Figure 4, the dyes were used to track and image the nanoparticles in real time, and the confocal microscopy demonstrated that the nanoparticles could enter the cells.

3.3. The role of nanomedicine in the process of osteogenic differentiation

As shown in Figure 5, ALP staining indicated that E2+VD@PLGAIR780-ADA could promote osteogenic differentiation, and the osteogenic effect was stronger than that of E2@PLGAIR780-ADA.

As shown in Figure 6, the ALP activity experiment showed that E2+VD@PLGAIR780-ADA could promote osteogenic differentiation, and the osteogenic effect was significantly stronger than that of E2@PLGAIR780-ADA.

As shown in Figure 7, PCR experiments showed that the expression of osteogenic markers ALP, Runx2, and IBSP was significantly increased in the E2+VD@PLGA _IR780 -ADA group, indicating that the nanomedicine can significantly promote osteogenic differentiation.

As shown in Figure 8, the osteogenic mineralization experiment, namely, the alizarin red staining experiment showed that E2+VD@PLGA _IR780 -ADA can significantly promote the mineralization of osteoblasts, and the effect is stronger than that of E2@PLGA _IR780 -ADA Group.

As shown in Figure 9, the results of immunohistochemical staining after 14 days of osteoblast differentiation indicated that the expression of OCN and OPN in cells was significantly increased after E2+VD@PLGA _IR780 -ADA treated cells, which indicated that E2+VD@PLGA _IR780 - ADA can promote osteoblast differentiation.

Claims (10)

1. A nano-drug delivery system, characterized by:

the nano capsule is constructed by self-assembling the polylactic acid-glycolic acid copolymer, the hydrophobic small molecule drug E2 and the vitamin D and is used for delivering the hydrophobic drug.

2. A nano-drug delivery system as in claim 1, wherein:

and a positioning factor is also loaded in the shell layer of the polylactic acid-glycolic acid copolymer.

3. A nano-drug delivery system as in claim 1, wherein:

the surface of the polylactic acid-glycolic acid copolymer is modified with functional factors with targeting effect based on biological coupling effect.

4. A nano-drug delivery system according to claim 2, wherein the specific preparation method is as follows:

s1, adding a positioning factor into a travel mixture of a polylactic acid-glycolic acid copolymer;

s2, removing liquid matters in the mixture at the temperature of 20-60 ℃ to form membranous matters;

s3, prefabricating the hydrophobic micromolecular medicaments and the vitamin D into oily matters;

s4, dissolving the membranous substance of the S2 in a solvent, mixing with oily substance of the S3, and circularly carrying out ultrasonic treatment at the temperature below 10 ℃ for 5-20min to obtain nano particles;

s5, carrying out at least one time of settling, centrifuging and washing on the nano particles of the S4 to obtain a target-free product.

5. A nano-drug delivery system according to any one of claims 1-3, wherein:

the hydrophobic small molecule drug E2 may be replaced with at least one of the following drugs:

A. a medicament for promoting osteogenesis;

B. a medicament for promoting mineralization of osteoblasts;

C. drugs that promote expression of ALP, runx2, IBSP;

D. drugs that promote the expression of OCN and OPN.

6.A nano-drug delivery system according to claim 3, wherein:

the functional factor is a bone tissue targeting molecule.

7. A nano-drug delivery system as in claim 6, wherein:

the nano-drug delivery system is applied to preparation of drugs for promoting osteogenic differentiation.

8. A nano-drug delivery system as in claim 6, wherein:

the nano-drug delivery system is applied to the preparation of drugs for promoting the expression of ALP, runx2 and IBSP.

9. A nano-drug delivery system as in claim 6, wherein:

the nano-drug delivery system is applied to the preparation of drugs for promoting the mineralization of osteoblasts.

10. A nano-drug delivery system as in claim 6, wherein:

the nano-drug delivery system is applied to preparing drugs for promoting the expression of OCN and OPN.

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