CN114652897B - Time-sequence-release double-drug-loading hydrogel and preparation method and application thereof - Google Patents

Abstract

The invention discloses a time-sequence release double-drug-loading hydrogel, which takes PBAE as a carrier material, and the material realizes the time-sequence release of vancomycin and total flavonoids of rhizoma drynariae by loading the vancomycin and forming the hydrogel with the total flavonoids of rhizoma drynariae to prepare the double-drug-loading bone repair material, so that the vancomycin and the total flavonoids of rhizoma drynariae can play a synergistic effect to better promote the generation of osteoblasts, thereby having application prospect in bone repair treatment.

Description

Time-sequence-release double-drug-loading hydrogel and preparation method and application thereof

Technical Field

The invention relates to a bone repair biomaterial, in particular to a time-sequence release double-drug-loading hydrogel and a preparation method thereof, and also relates to application of the hydrogel in preparing a bone repair material for promoting osteoblast generation.

Background

Bone defects refer to the occurrence or loss of bone mass, such as bone destruction and osteonecrosis. Clinically, the treatment of bone defects requires bone grafting or bone substitute material filling. The in vitro construction of a substitute material with physiological functions is the research focus of bone tissue engineering, the defective tissue can be repaired through the substitute material, so that the substitute material can compensate or replace related functional activities of tissues and organs, and meanwhile, the good substitute material not only has mechanical properties and biological properties similar to those of the bone tissue, but also has wide material sources, low price and easy acquisition, and can better meet clinical requirements. In a word, the bone repair material has good development and application prospects.

In the bone repair process, bacterial or microbial infection is easy to occur, the bone repair curative effect is reduced due to the infection, and therefore, the intravenous drip of antibiotics is clinically needed in the bone repair process. Vancomycin is a glycopeptide antibiotic, and mainly interferes with the synthesis of a bacterial cell wall by interfering with a key component peptidoglycan in the structure of the bacterial cell wall, so as to inhibit the generation of phospholipid and polypeptide in the cell wall. Infections following repair of bone damage are usually caused by endogenous skin flora or by exogenous bacteria, the most common pathogens being gram-positive bacteria such as staphylococcus aureus and staphylococcus epidermidis. Because vancomycin has low cytotoxicity and mainly aims at gram-positive bacteria, the vancomycin has obvious inhibition effect on staphylococcus aureus and staphylococcus epidermidis.

Rhizoma Drynariae, also known as rhizoma Drynariae and rhizoma Drynariae, is the rhizome of Epimedium plant of Drynariae, and has effects of strengthening bone, invigorating kidney, removing blood stasis and relieving pain. The rhizoma Drynariae total flavonoids, the main active ingredient in rhizoma Drynariae, has effects of promoting bone formation, inhibiting bone absorption, promoting bone angiogenesis, and inhibiting inflammatory reaction. In recent years, the total flavonoids of drynaria has made some progress in treating bone defects and fracture healing, knee osteoarthritis, osteoporosis and stretch bone formation, but no report of applying the total flavonoids of drynaria to bone repair materials is found.

Poly (beta-amino esters) (PBAE) is a synthetic polymer carrier material, the synthetic method is simple, the raw materials are cheap and easy to obtain, the PBAE has various structures, the structure of the polymer has branched chain or straight chain structures with different lengths, and simultaneously because the synthetic mode and the raw materials are different, the end capping groups of the polymer are different, thereby providing convenience for the structure modification and the expansion of the application range. Meanwhile, PBAE can be degraded into nontoxic small molecular products in vivo, accumulation in vivo can not occur, PBAE also has sensitivity to pH, and solubility can be improved by protonation of amino groups in the structure.

The PBAE is used as a carrier material, the vancomycin is loaded, and the nano-microspheres and the total flavonoids of the rhizoma drynariae form hydrogel, so that the double-drug-loaded bone repair material is prepared, the time-sequence release of the vancomycin and the total flavonoids of the rhizoma drynariae is realized, the vancomycin and the total flavonoids of the rhizoma drynariae can play a synergistic effect, the osteoblast generation is better promoted, and the double-drug-loaded bone repair material has a very good application prospect in bone repair treatment.

Disclosure of Invention

The invention aims to provide a time-sequence release double-drug-loading hydrogel, which takes PBAE as a carrier material, and realizes time-sequence release of two drugs by loading vancomycin and forming hydrogel with total flavonoid nano microspheres of rhizoma drynariae, so that the two drugs can play a synergistic effect to better promote osteoblast generation and carry out bone repair treatment.

The above purpose is realized by the following technical scheme:

a preparation method of a time-sequence release double-drug-loading hydrogel comprises the following steps:

1) Mixing natural polymer material solution containing rhizoma Drynariae total flavonoids as water phase with oil phase containing emulsifier, stirring, emulsifying, adding chemical cross-linking agent for solidification, centrifuging, washing precipitate, and drying to obtain rhizoma Drynariae total flavonoids nanometer microsphere;

2) Carrying out Michael addition reaction on PEG400DA, isobutylamine and vancomycin to obtain a vancomycin-loaded macromonomer, adding the drynaria total flavone nanospheres obtained in the step 1) into a reacted system, uniformly mixing, adding an initiator, water and a catalyst to enable the macromonomer to be chemically crosslinked to form hydrogel,

the natural polymer material is chitosan or sodium alginate or gelatin or Arabic gum;

the emulsifier is tween or span;

the oil phase is liquid paraffin or vegetable oil;

the chemical cross-linking agent is glutaraldehyde.

The initiator is Ammonium Persulfate (APS), and the catalyst is Tetramethylethylenediamine (TEMED).

Preferably, the content of the total flavonoids of the rhizoma drynariae in the water phase is 1-10%.

Preferably, the molar ratio of PEG400DA to isobutylamine is from 0.5 to 2:1.

preferably, the vancomycin accounts for 1-10% of the mass of the reaction system.

Preferably, the addition amount of the rhizoma drynariae total flavone nano-microspheres is 1-5% of the mass of the reacted system.

Preferably, the temperature of the Michael addition reaction is from 70 to 100 ℃.

According to one embodiment of the present invention, a preferred preparation method is as follows:

1) Preparation of drynaria total flavone nano-microsphere

Dispersing chitosan in dilute acetic acid water solution, swelling to prepare 1.5% chitosan solution; dissolving rhizoma Drynariae total flavonoids in chitosan solution to obtain chitosan solution containing 5% rhizoma Drynariae total flavonoids, and forming water phase; uniformly stirring 50mL of liquid paraffin and 1.5mL of Span-80 to form an oil phase; adding 10mL of water phase into the oil phase, stirring and emulsifying for 2h, then adding cross-linking agent glutaraldehyde, stirring and crosslinking for 1h, centrifuging, washing the precipitate, and drying to obtain rhizoma drynariae total flavonoids chitosan microspheres;

2) Preparation of vancomycin-loaded macromonomer solution

Reacting PEG400DA, isobutylamine and vancomycin at 85 ℃, wherein the molar ratio of the PEG400DA to the isobutylamine is 1.2:1, the vancomycin accounts for 4% of the mass of a reaction system, and a vancomycin-loaded macromonomer solution is obtained;

3) Preparation of double drug-loaded hydrogel system

Adding the microspheres prepared in the step 1) into the macromonomer solution prepared in the step 2) according to the addition amount of 3%, uniformly dispersing, adding an APS initiator, a TEMED catalyst and water, and performing ultrasonic treatment at room temperature to form hydrogel.

The time-sequence release double-drug-loading hydrogel obtained by the preparation method and the application of the hydrogel in preparing the bone repair material for promoting the generation of osteoblasts are also within the protection scope of the invention.

The invention has the beneficial effects that:

1) The hydrogel provided by the invention contains two medicines, the rhizoma drynariae total flavonoids are beneficial to promoting the generation of osteoblasts, the vancomycin can resist infection, and the combination of the two medicines is more beneficial to bone repair. The vancomycin is directly crosslinked in the hydrogel and is released along with the degradation of the hydrogel, so that the drug release speed is high, and the vancomycin can play an anti-infection role in the early treatment period; due to the slow release effect of the microspheres, the total flavonoids of the rhizoma drynariae are slowly released, so that the action time of bone repair is prolonged, and the curative effect of the bone repair is improved.

2) The PBAE is selected as a carrier, and the PBAE has the characteristics of biodegradability, hydrophobicity, low cytotoxicity, chemical modification diversity, structural diversity, derivative diversity and the like, not only meets the preparation conditions of the double-drug-loading hydrogel, but also enables the product to have better mechanical property and biocompatibility and can be better applied to in vivo bone repair.

3) The invention also has the advantages of wide material source, low price, simple process, suitability for large-scale production and the like.

Drawings

Fig. 1 is a particle size distribution diagram of the rhizoma drynariae total flavone nano-microspheres prepared in step 1).

Fig. 2 is a scanning electron microscope image of the rhizoma drynariae total flavone nano-microspheres prepared in step 1).

FIG. 3 is a schematic representation of the reaction principle (Michael addition) of a vancomycin-loaded macromonomer solution.

FIG. 4 is a comparison graph of infrared spectra of PEG400DA, vancomycin-loaded PBAE macromonomer and vancomycin.

FIG. 5 is a scanning electron microscope image of a double-loading hydrogel for repairing bone.

Fig. 6 is a schematic process route of the double-heavy-load medicinal bone repair hydrogel.

FIG. 7 is the release curve of vancomycin in the double-loading drug bone repair hydrogel.

FIG. 8 is the release curve of total flavone of drynaria in hydrogel for double-loading medicinal bone repair.

Detailed Description

Example 1

A bone repair hydrogel for promoting osteoblast generation is prepared by the following steps:

1) Preparation of drynaria total flavone nano-microsphere

Appropriate amount of chitosan was dispersed in 2% (volume) diluted acetic acid aqueous solution and swollen for 24h to make 1.5% (weight) chitosan solution. The total flavone of drynaria rhizome is dissolved in chitosan solution to prepare the chitosan solution containing 5 percent (by weight) of the total flavone of drynaria rhizome. Uniformly stirring 50mL of liquid paraffin and 1.5mL of Span-80 to form an oil phase, slowly adding 10mL of the chitosan solution, continuously stirring, emulsifying for 2h, then slowly adding 1mL of a 25% glutaraldehyde solution as a crosslinking agent, and stirring for crosslinking; centrifuging, removing supernatant, washing lower layer with petroleum ether, isopropanol and ultrapure water for 3 times, and lyophilizing to obtain rhizoma Drynariae total flavone nanometer microsphere.

As can be seen from figure 1, the particle size of the microsphere prepared by the invention is between 150 nm and 250nm, and the average particle size is about 200 nm; as can be seen from FIG. 2, the microspheres prepared by the invention have regular and flat shapes and are spherical.

2) Preparation of vancomycin-loaded macromonomer solution

Reacting PEG400DA, isobutylamine and vancomycin at 85 ℃ for 48 hours, wherein the molar ratio of the PEG400DA to the isobutylamine is 1.2:1, the vancomycin accounts for 4 percent (weight) of the reaction system, and the vancomycin-loaded macromonomer solution is obtained. The reaction principle of this step is shown in FIG. 3.

Fig. 4 is a comparison graph of infrared spectra of PEG400DA, vancomycin-loaded macromer and vancomycin, and it can be seen from the graph that the ratio of C = C/C = O content of reacted drug-loaded macromer/PEG 400DA is smaller, and the product is correct.

3) Preparation of double drug-loaded hydrogel system

Adding the microspheres prepared in the step 1) into the macromonomer solution prepared in the step 2) according to the addition amount of 3%, uniformly dispersing, adding an APS initiator (accounting for 1.5% of the mass of the solution), a TEMED catalyst (accounting for 0.5% of the mass of the solution) and water, and carrying out ultrasonic treatment at room temperature for 10min to form hydrogel and using the hydrogel as it is.

Fig. 5 is a scanning electron microscope image of a double-loading medicinal bone repair hydrogel.

Fig. 6 is a schematic process route of a double-loading medicinal bone repair hydrogel.

Example 2

A double drug-loaded hydrogel is prepared by the following steps:

1) Preparation of rhizoma drynariae total flavone microsphere

Dispersing a proper amount of sodium alginate in 2% dilute acetic acid water solution, and swelling for 24h to prepare 2% sodium alginate solution. Dissolving rhizoma Drynariae total flavonoids in sodium alginate solution to obtain sodium alginate solution containing 8% rhizoma Drynariae total flavonoids. Uniformly stirring 50mL of liquid paraffin and 2mL of sodium alginate-pan-80 to form an oil phase, slowly adding 5mL of the sodium alginate solution, continuously stirring, emulsifying for 2 hours, then slowly adding 1mL of a 25% glutaraldehyde solution as a crosslinking agent, and stirring and crosslinking for 1 hour; centrifuging, removing supernatant, washing lower layer with petroleum ether, isopropanol and ultrapure water for 3 times, and lyophilizing to obtain rhizoma Drynariae total flavone nanometer microsphere.

2) Preparation of PBAE macromer solution

Reacting PEG400DA, isobutylamine and vancomycin at 75 ℃ for 48 hours, wherein the molar ratio of the PEG400DA to the isobutylamine is 1.6:1, the vancomycin accounts for 2 percent (weight) of the reaction system, and the vancomycin-loaded macromonomer solution is obtained.

3) Preparation of double drug-loaded hydrogel system

Adding the microspheres prepared in the step 1) into the macromonomer solution prepared in the step 2) according to the addition amount of 5%, uniformly dispersing, adding an APS initiator (accounting for 1.5% of the mass of the solution), a TEMED catalyst (accounting for 0.5% of the mass of the solution) and water (accounting for 2.5% of the mass of the solution), and performing ultrasonic treatment at room temperature for 10min to form hydrogel and using the hydrogel as it is.

Example 3

A double drug-loaded hydrogel is prepared by the following steps:

1) Preparation of rhizoma drynariae total flavone microsphere

Dispersing proper amount of chitosan in 2% dilute acetic acid water solution, swelling for 24 hr to obtain 1% chitosan solution. Dissolving rhizoma Drynariae total flavonoids in chitosan solution to obtain chitosan solution containing 3% rhizoma Drynariae total flavonoids. Uniformly stirring 50mL of soybean oil and 1mL of Tween 80 to form an oil phase, slowly adding 20mL of the chitosan solution, continuously stirring, emulsifying for 2h, slowly adding 1mL of a 25% glutaraldehyde solution as a crosslinking agent, and stirring for crosslinking for 1h; centrifuging, removing supernatant, washing lower layer with petroleum ether, isopropanol and ultrapure water for 3 times, and lyophilizing to obtain rhizoma Drynariae total flavone nanometer microsphere.

2) Preparation of PBAE macromer solution

Reacting PEG400DA, isobutylamine and vancomycin at 80 ℃ for 48 hours, wherein the molar ratio of the PEG400DA to the isobutylamine is 0.8:1, the vancomycin accounts for 6 percent of the reaction system, and the vancomycin-carrying macromonomer solution is obtained.

3) Preparation of double drug-loaded hydrogel system

Adding the microspheres prepared in the step 1) into the macromonomer solution prepared in the step 2) according to the addition of 1%, uniformly dispersing, adding an APS initiator, a TEMED catalyst and water, and carrying out ultrasonic treatment at room temperature for 10min to form hydrogel and using the hydrogel as it is.

Example 4

A double drug-loaded hydrogel is prepared by the following steps:

1) Preparation of rhizoma drynariae total flavone microsphere

Dispersing a proper amount of Arabic gum into 2% diluted acetic acid water solution, and swelling for 24h to prepare 1.2% Arabic gum solution. Dissolving rhizoma Drynariae total flavonoids in acacia gum solution to obtain acacia gum solution containing 10% rhizoma Drynariae total flavonoids. Uniformly stirring 50mL of liquid paraffin and 0.5mL of liquid paraffin-80 to form an oil phase, slowly adding 5mL of the Arabic gum solution, continuously stirring, emulsifying for 1 hour, then slowly adding 1.5mL of a 25% glutaraldehyde solution serving as a cross-linking agent, and stirring and crosslinking for 1 hour; centrifuging, removing supernatant, washing lower layer with petroleum ether, isopropanol and ultrapure water for 3 times, and lyophilizing to obtain rhizoma Drynariae total flavone nanometer microsphere.

2) Preparation of PBAE macromer solution

Reacting PEG400DA, isobutylamine and vancomycin at 90 ℃ for 48 hours, wherein the molar ratio of the PEG400DA to the isobutylamine is 2:1, the vancomycin accounts for 5 percent of the reaction system to obtain the vancomycin-loaded macromonomer solution.

3) Preparation of double drug-loaded hydrogel system

Adding the microspheres prepared in the step 1) into the macromonomer solution prepared in the step 2) according to the addition of 1%, uniformly dispersing, adding an APS initiator, a TEMED catalyst and water, and carrying out ultrasonic treatment at room temperature for 10min to form hydrogel and using the hydrogel as it is.

Test examples

(1) Release degree detection of double drug-loaded hydrogel

The hydrogels prepared in examples 1-4 were placed in Phosphate Buffered Saline (PBS) at pH 7.4, in a shaker (37 ℃,70 rpm), and the release medium was collected at specific times. Vancomycin hydrochloride and naringin are used for preparing reference substance solution, and the drug release amount of vancomycin and the total flavone of rhizoma drynariae is determined by HPLC method under the wavelength of 280nm and 284nm respectively. At each sampling point, the tubes were compensated with fresh PBS.

The test results are shown in fig. 7 and 8, and vancomycin is directly crosslinked in the hydrogel and is released along with the degradation of the hydrogel, wherein the test example 1 reaches a peak value in about 24 days. Due to the encapsulation effect of the microspheres, the release speed of the rhizoma drynariae total flavonoids is relatively slow, and reaches a peak value in about 50 days, wherein the drug release of the embodiment 1 is more sufficient. The two drugs have different release speeds, so that the time sequence release of the drugs is realized.

(2) Effect of double drug-loaded hydrogel on growth of mouse osteoblasts (MC 3T 3-E1)

The double-drug-loaded hydrogel prepared by the invention is co-cultured with MC3T3-E1 cells, and then CCK8 cell proliferation activity detection is carried out, wherein the specific operation method comprises the following steps:

sterilizing the hydrogel, soaking in DMEM medium, culturing at 37 deg.C for 36 hr and 72 hr, collecting the extractive solutions, and sterilizing.

Mouse osteoblasts (MC 3T 3-E1) were cultured in a medium containing 10% fetal bovine serum, 50U/ml penicillin and 50U/ml streptomycin. The culture medium is replaced every 3 days, the culture contains 5% CO at 37 deg.C ₂ Cultured in a cell culture box.

Observing 80-90% cell adherent growth under microscope, digesting the cell with pancreatin to obtain cell suspension, counting, and diluting to density of 2 × 10 ⁴ Per ml, then adding 100. Mu.L of cell suspension per well in a 96-well plate, 6 duplicate wells per group, placing in an incubator for pre-incubation for 24 hours (at 37 ℃,5% ₂ Conditions of (2) leaching the leaching solution of each example for different time periods in a 96-well plate, incubating the plate in an incubator for 48 hours, adding 10. Mu.L of CCK8 solution to each well, incubating the plate in the incubator for 4 hours, and detecting the OD value of the plate by a microplate reader. The results are shown in Table 1.

A blank group and the following control groups were set simultaneously:

control group 1: the hydrogel does not contain rhizoma Drynariae total flavonoids. PEG400DA, isobutylamine and vancomycin are reacted and then directly subjected to chemical crosslinking to form hydrogel, and the preparation conditions are the same as those in example 1.

Control group 2: the hydrogel does not contain vancomycin. And reacting PEG400DA with isobutylamine, adding the drynaria total flavone nano-microspheres, and then carrying out chemical crosslinking to form hydrogel, wherein the preparation conditions are the same as those in example 1.

Control group 3: rhizoma Drynariae total flavonoids are not prepared into microsphere. The preparation conditions of the rhizoma drynariae total flavonoids directly dispersed in the vancomycin-loaded macromonomer solution and forming hydrogel are the same as those of the example 1.

Control group 4: the hydrogel did not contain both drugs. After the reaction of PEG400DA and isobutylamine, the hydrogel is formed by direct chemical crosslinking.

TABLE 1 Effect of 0h lixiviation of Dual drug loaded hydrogels on (MC 3T 3-E1) cell growth

As can be seen from the experimental results, the cell proliferation of the control group 4 was close to that of the blank group because no drug was added. In the control group 3, the drynaria total flavonoids are directly dispersed in the hydrogel, so that the total flavonoids are basically dissolved out after 36 hours, and the total flavonoids content cannot be obviously increased even if the leaching time is continuously increased, so that the OD values in two time periods are close. Control 1 did not contain drynaria total flavonoids and control 2 did not contain vancomycin, and thus although osteoblast proliferation was promoted, the OD values were significantly lower than those of examples 1-4 (P < 0.05).

Claims (3)

1. A preparation method of a time-sequence release double-drug-loading hydrogel is characterized by comprising the following steps:

1) Preparation of drynaria total flavone nano-microsphere

Dispersing chitosan in dilute acetic acid water solution, and swelling to prepare 1.5% chitosan solution; dissolving rhizoma Drynariae total flavonoids in chitosan solution to obtain chitosan solution containing 5% rhizoma Drynariae total flavonoids, and forming water phase; uniformly stirring 50mL of liquid paraffin and 1.5mL of Span-80 to form an oil phase; adding 10mL of water phase into the oil phase, stirring and emulsifying for 2h, adding a cross-linking agent glutaraldehyde, stirring and crosslinking for 1h, centrifuging, washing the precipitate, and drying to obtain the drynaria total flavone nano-microspheres;

2) Preparation of vancomycin-loaded macromonomer solution

Reacting PEG400DA, isobutylamine and vancomycin at 85 ℃, wherein the molar ratio of the PEG400DA to the isobutylamine is 1.2:1, the vancomycin accounts for 4% of the mass of the reaction system to obtain a vancomycin-loaded macromonomer solution;

3) Preparation of double drug-loaded hydrogel system

Adding the microspheres prepared in the step 1) into the macromonomer solution prepared in the step 2) according to the addition amount of 3%, uniformly dispersing, adding an APS initiator, a TEMED catalyst and water, and performing ultrasonic treatment at room temperature to form hydrogel.

2. The time-release double-drug-loaded hydrogel obtained by the preparation method according to claim 1.

3. The use of the time-release double drug-loaded hydrogel of claim 2 in the preparation of a bone repair material that promotes osteoblast generation.

Images