Preparation and application of macromolecular vesicle hydrogel drug carrier
Technical Field
The invention belongs to the field of new materials, and particularly relates to preparation and application of macromolecular vesicle composite hydrogel.
Background
Treatment of many diseases is accomplished by drugs. In this process, too low a concentration of drug does not play a therapeutic role; too high a concentration of the drug may cause side effects and even damage to normal tissues and organs. In addition, the therapeutic effect depends on whether the drug can be maintained at the affected site for a sufficient time. In order to improve the therapeutic effect of the drug, the concentration and administration frequency of the drug are often increased, but excessive drug damages normal tissues and organs.
The hydrogel is a macromolecular network structure which swells in water, has better biocompatibility, is soft and elastic after absorbing water, and is not easy to cause tissue damage. In addition, the hydrogel has a water-soluble environment similar to that of extracellular matrix, so that the hydrogel has certain bionic characteristics, and the characteristics cause wide attention in the field of drug delivery. Conventional hydrogels are formed by free radical polymerization or copolymerization of some hydrophilic monomers to form a hydrogel network. However, as a drug carrier, the hydrogel must have drug loading and controlled release capabilities. Because conventional hydrogels lack ligands for drug interaction, they have limited drug loading and controlled release capabilities.
Macromolecular vesicles composed of bilayers composed of hydrophilic and hydrophobic chains have been studied for a long time as a model for mimicking biological membranes, and in comparison with natural cell membranes composed of phospholipid bilayers and macromolecules such as proteins, the polymersomes can mimic many of their life-related properties, for example: permeability, osmotic response, intramembrane synthesis, protein assembly, and the like. At present, most of the macromolecular vesicles are obtained by self-assembly of block copolymers, and because the block copolymer composition units have higher molecular weight, compared with liposome vesicles, the macromolecular vesicles are more stable and firm, and are easy to apply to biological systems. Among them, it is also not used in the field of drug delivery. The characteristic of the vesicle wall of the macromolecular vesicle provides natural controllable conditions for the controlled release of the drug. In addition, the size of the macromolecular vesicles ranges from tens of nanometers to several micrometers, the macromolecular vesicles have high specific surface area, and a large amount of drugs can be adsorbed and loaded. Then, the polymer vesicles are usually in the form of a solution, and lack a fixed shape, which makes it difficult to achieve site-specific drug release.
disclosure of Invention
In consideration of the respective advantages of the hydrogel drug carrier and the macromolecular vesicles, and in order to overcome the defects of the traditional hydrogel drug carrier and the traditional macromolecular vesicle drug carrier, the invention aims to introduce the macromolecular vesicles into hydrogel to prepare the composite hydrogel drug carrier with drug controlled release capacity, and provides a novel composite hydrogel drug carrier containing the macromolecular vesicles for treating diseases and controlling drug release.
In order to achieve the above purpose, the invention firstly uses a macromolecule-monomer pair method to assemble the macromolecular vesicles, and the assembled macromolecular vesicles are composed of two parts, wherein the first part is biological macromolecules Heparin (HEP) or Chondroitin Sulfate (CS) with sulfonate and carboxylate, and the second part is poly diethylaminoethyl methacrylate (pDAA) short chains with tertiary amino. In aqueous solution, acid radicals on heparin or chondroitin and tertiary amino on short chains are combined together due to electrostatic action and show hydrophobic property, so that a hydrophobic layer of the vesicle is formed, and a macromolecular chain segment without electrostatic action still shows hydrophilic property to form a hydrophilic surface layer of the vesicle; and mixing the aqueous solution containing the macromolecular vesicles with hydroxyethyl methacrylate (HEMA) monomers, adding a photoinitiator I2959 to initiate polymerization to form a composite hydrogel drug carrier, and finally loading the drug into the hydrogel drug carrier in an adsorption mode.
The invention adopts the following specific technical scheme:
a preparation method of a macromolecule vesicle hydrogel drug carrier comprises the following steps:
the method comprises the following steps: preparation of high molecular polymer solution
adding polysaccharide molecules with negative charges and diethylaminoethyl methacrylate (DEA) solution into an open reaction vessel, adding distilled water, stirring until the solution is transparent and uniform, adding redox initiator to initiate polymerization, stopping stirring after the reaction is finished, and introducing oxygen to inhibit polymerization;
Step two: preparation of macromolecular vesicle solution
Adding small-molecular-weight amino acid into the polymer solution obtained in the first step, crosslinking the vesicles with carbodiimide EDC, slightly adjusting the pH value to 5.5-6, and reacting at room temperature for a period of time, wherein the mass ratio of the carbodiimide EDC to the amino acid is 1: 1; standing the obtained solution for 1-2 hours, and removing precipitates to obtain a macromolecular vesicle solution;
Step three: preparation of macromolecular vesicle composite hydrogel
Mixing the macromolecular vesicle aqueous solution with hydroxyethyl methacrylate HEMA monomer according to a certain proportion, adding a certain amount of photoinitiator I2959, uniformly stirring, pouring into a mould, reacting in an ultraviolet reactor of 365nm for a period of time, soaking in water, and demoulding to obtain the macromolecular vesicle-containing composite hydrogel;
step four: loading the medicine into the macromolecular vesicle composite hydrogel.
In the first step, the redox initiator is ammonium persulfate/tetramethylethylenediamine, potassium persulfate/tetramethylethylenediamine or vitamin C/hydrogen peroxide, wherein the molar number of the oxidant and the reducing agent is equal.
in the first step, the polysaccharide molecule is heparin HEP or chondroitin sulfate CS.
in the first step, the concentration of heparin HEP is 1-5 mg/ml, the molar ratio of polysaccharide molecules to monomer DEA is 1: 100-1: 500, the concentration of an initiator is 2 mM-10 mM, the reaction time is 0.5-5 h, and the reaction temperature is 20-60 ℃;
in the second step, the molar ratio of the polysaccharide structural unit in the polymer solution to the amino acid with small molecular weight is 1: 5-5: 1, and the reaction time is 2-6 h;
In the third step, the volume ratio of the macromolecular vesicle aqueous solution to the HEMA monomer is 1: 5-4: 1, the dosage of the photoinitiator is 0.01-1% w/v, and the reaction time is 0.2-2 h.
In the first step, the concentration of heparin HEP is preferably 2-3 mg/ml, the molar ratio of polysaccharide molecules to monomer DEA is preferably 1: 200-1: 400, the concentration of an initiator is preferably 3 mM-7 mM, the reaction time is 1-3h, and the reaction temperature is 20-60 ℃;
In the second step, the molar ratio of the polysaccharide structural unit in the polymer solution to the amino acid with small molecular weight is preferably 1: 4-1: 1, and the reaction time is preferably 3-5 h;
In the third step, the volume ratio of the macromolecular vesicle aqueous solution to the hydroxyethyl methacrylate HEMA monomer is preferably 1: 4-2: 1, the dosage of the photoinitiator is preferably 0.05-0.5% w/v, and the reaction time is preferably 0.5-1.5 h.
The method for loading the medicine into the macromolecular vesicle composite hydrogel comprises the following steps: soaking the hydrogel in 1.5-3 mg/ml of drug solution to allow the drug to permeate into the hydrogel network; or the dosage of the drug is 1-5 mg/g by copolymerizing the monomer and the drug.
The invention also provides an application of the macromolecular vesicle hydrogel drug carrier in ophthalmic drugs, and the technical scheme is as follows:
And soaking the macromolecular vesicle composite hydrogel in 1.5-3 mg/ml of eye drops to allow the drug to permeate into the hydrogel network.
the eye medicine is norfloxacin, ofloxacin, puerarin and cyclosporine.
Advantageous effects
the performance of the obtained product meets the basic requirements of hydrogel drug carriers and can control the release of drugs. Has great social benefit and economic benefit. The specific properties are as follows:
(1) The composite hydrogel sheet has no obvious deformation after demoulding and swelling.
(2) The equilibrium water content of the composite hydrogel is between 45 and 55 percent, and has no obvious difference with the traditional hydrogel.
(3) the storage modulus of the composite hydrogel is 6-9 x 10 4, the loss modulus is 6-20 x 10 3, and the storage modulus and the loss modulus slightly increase with the increase of the concentration of the macromolecular vesicles, but no obvious difference exists.
(4) The ofloxacin obtained from the composite hydrogel by a soaking method has the advantage that the loading capacity of the ofloxacin is obviously increased along with the increase of the content of the macromolecular vesicles. In addition, as the concentration of the loaded drug increases, the loading of the drug in the hydrogel also increases.
Drawings
FIG. 1 is an ofloxacin drug loading curve for a macromolecule vesicle hydrogel.
FIG. 2 is the ofloxacin release curve of the macromolecular vesicle hydrogel at a drug concentration of 0.26%.
FIG. 3 is the ofloxacin release curve of the macromolecular vesicle hydrogel at a drug concentration of 0.66%.
FIG. 4 is the ofloxacin release curve of the macromolecular vesicle hydrogel at a drug concentration of 1.26%.
Wherein the content of the first and second substances,
1-curve of example 1, 2-curve of example 2, 3-curve of example 3, 1-curve of comparative example 1
Detailed Description
the following examples are intended to illustrate the practice of the present invention.
Example 1
Adding 25mg of HEP and 100 mu L of DEA solution into an open reaction vessel (beaker), adding 10mL of distilled water, stirring until the solution is transparent and uniform, adding 5mM of vitamin C, after completely dissolving, adding 5mM of hydrogen peroxide, initiating polymerization at 60 ℃ for 2h, stopping stirring, and introducing oxygen to inhibit polymerization after the reaction is finished. Then 22mg of alanine and 22mg of EDC are added, the pH value is slightly adjusted to 5.5-6, and the reaction is carried out for 4h at room temperature. And standing the obtained solution for 1-2 hours, and removing the precipitate to obtain the macromolecular vesicle solution. The effective particle size of the resulting vesicles was 218.0, and the polydispersity was 0.075.
Mixing the macromolecular vesicle aqueous solution and a HEMA monomer according to a ratio of 4:1, adding 0.05% w/v photoinitiator (I2959), uniformly stirring, pouring into a mold, reacting in an ultraviolet reactor with a wavelength of 365nm for 45min, soaking in water, and demolding to obtain the macromolecular vesicle-containing composite hydrogel.
the drug loading and release profiles are shown as red lines in fig. 1-4.
Example 2
Adding 25mg of HEP and 100 mu L of DEA solution into an open reaction vessel (beaker), adding 10mL of distilled water, stirring until the solution is transparent and uniform, adding 5mM of vitamin C, after completely dissolving, adding 5mM of hydrogen peroxide, initiating polymerization at 60 ℃ for 2h, stopping stirring, and introducing oxygen to inhibit polymerization after the reaction is finished. Then 22mg of alanine and 22mg of EDC are added, the pH value is slightly adjusted to 5.5-6, and the reaction is carried out for 4h at room temperature. And standing the obtained solution for 1-2 hours, and removing the precipitate to obtain the macromolecular vesicle solution. The effective particle size of the resulting vesicles was 218.0, and the polydispersity was 0.075.
mixing the macromolecular vesicle aqueous solution and a HEMA monomer according to a ratio of 2:1, adding 0.05% w/v photoinitiator (I2959), uniformly stirring, pouring into a mold, reacting in an ultraviolet reactor of 365nm for 45min, soaking in water, and demolding to obtain the macromolecular vesicle-containing composite hydrogel.
The drug loading and release profiles are shown as blue lines in fig. 1-4.
Example 3
adding 25mg of HEP and 100 mu L of DEA solution into an open reaction vessel (beaker), adding 10mL of distilled water, stirring until the solution is transparent and uniform, adding 5mM of vitamin C, after completely dissolving, adding 5mM of hydrogen peroxide, initiating polymerization at 60 ℃ for 2h, stopping stirring, and introducing oxygen to inhibit polymerization after the reaction is finished. Then 22mg of alanine and 22mg of EDC are added, the pH value is slightly adjusted to 5.5-6, and the reaction is carried out for 4h at room temperature. And standing the obtained solution for 1-2 hours, and removing the precipitate to obtain the macromolecular vesicle solution. The effective particle size of the resulting vesicles was 218.0, and the polydispersity was 0.075.
Mixing the macromolecular vesicle aqueous solution and a HEMA monomer according to a ratio of 1:1, adding 0.05% w/v photoinitiator (I2959), uniformly stirring, pouring into a mold, reacting in an ultraviolet reactor of 365nm for 45min, soaking in water, and demolding to obtain the macromolecular vesicle-containing composite hydrogel.
The drug loading and release profiles are shown as powder lines in fig. 1-4.
Example 4
Adding 100mg of CS and 100 mu L of DEA solution into an open reaction vessel (beaker), adding 10mL of distilled water, stirring until the solution is transparent and uniform, adding 5mM of vitamin C, adding 5mM of hydrogen peroxide after completely dissolving, initiating polymerization at 60 ℃ for 2h, stopping stirring, and introducing oxygen to inhibit polymerization after the reaction is finished. Then 22mg of lysine and 22mg of EDC are added, the pH value is slightly adjusted to 5.5-6, and the reaction is carried out for 4h at room temperature. And standing the obtained solution for 1-2 hours, and removing the precipitate to obtain the macromolecular vesicle solution. The vesicle obtained had an effective particle diameter of 209.0 and a polydispersity of 0.097.
Mixing the macromolecular vesicle aqueous solution and a HEMA monomer according to a ratio of 4:1, adding 0.05% w/v photoinitiator (I2959), uniformly stirring, pouring into a mold, reacting in an ultraviolet reactor with a wavelength of 365nm for 45min, soaking in water, and demolding to obtain the macromolecular vesicle-containing composite hydrogel.
example 5
Adding 100mg of CS and 100 mu L of DEA solution into an open reaction vessel (beaker), adding 10mL of distilled water, stirring until the solution is transparent and uniform, adding 5mM of vitamin C, adding 5mM of hydrogen peroxide after the solution is completely dissolved, initiating polymerization at 60 ℃ for 2h, stopping stirring, introducing oxygen to inhibit polymerization after the reaction is finished, standing the obtained solution for 1-2 h, and removing precipitates to obtain the macromolecular vesicle solution. The effective particle diameter of the obtained vesicle is 180.5, and the polydispersity index is 0.083.
Mixing the macromolecular vesicle aqueous solution and a HEMA monomer according to a ratio of 2:1, adding 0.05% w/v photoinitiator (I2959), uniformly stirring, pouring into a mold, reacting in an ultraviolet reactor of 365nm for 45min, soaking in water, and demolding to obtain the macromolecular vesicle-containing composite hydrogel.
Example 6
Adding 25mg of HEP and 100 mu L of DEA solution into an open reaction vessel (beaker), adding 10mL of distilled water, stirring until the solution is transparent and uniform, adding 5mM of vitamin C, adding 5mM of hydrogen peroxide after complete dissolution, initiating polymerization at 60 ℃ for 2h, stopping stirring, introducing oxygen to inhibit polymerization after the reaction is finished, standing the obtained solution for 1-2 hours, and removing precipitates to obtain the macromolecular vesicle solution. The effective particle diameter of the resulting vesicle was 157.7, and the polydispersity was 0.147.
Mixing the macromolecular vesicle aqueous solution and a HEMA monomer according to a ratio of 1:1, adding 0.05% w/v photoinitiator (I2959), uniformly stirring, pouring into a mold, reacting in an ultraviolet reactor of 365nm for 45min, soaking in water, and demolding to obtain the macromolecular vesicle-containing composite hydrogel.
Comparative example 1
Mixing deionized water and HEMA monomer according to a ratio of 4:1, adding 0.05% w/v photoinitiator (I2959), stirring uniformly, pouring into a mould, reacting in an ultraviolet reactor of 365nm for 45min, soaking in water, and demoulding to obtain the composite hydrogel containing the macromolecular vesicles.
Comparative example 2
Mixing deionized water and HEMA monomer according to a ratio of 2:1, adding 0.05% w/v photoinitiator (I2959), stirring uniformly, pouring into a mould, reacting in an ultraviolet reactor of 365nm for 45min, soaking in water, and demoulding to obtain the composite hydrogel containing the macromolecular vesicles.
comparative example 3
Mixing deionized water and HEMA monomer according to a ratio of 1:1, adding 0.05% w/v photoinitiator (I2959), stirring uniformly, pouring into a mould, reacting in an ultraviolet reactor of 365nm for 45min, soaking in water, and demoulding to obtain the composite hydrogel containing the macromolecular vesicles.
The experimental methods in the specific examples and comparative examples are as follows:
Particle diameter and zeta potential
measuring by using a nano ZS multi-angle granularity and high-sensitivity Zeta potential analyzer, and measuring the particle size and Zeta potential of the macromolecular vesicle solution by using a dynamic light scattering principle
Determination of drug load:
Adding a certain amount of medicine into the macromolecular vesicle solution, dialyzing for 3 days, and measuring the medicine concentration by using an ultraviolet spectrophotometer to obtain the relative loading capacity of the macromolecular vesicles.
Determination of equilibrium moisture content:
A sample of the copolymer was placed in a sufficient amount of distilled water, swollen to a constant mass and taken out, the surface was carefully blotted with filter paper, and its mass M1(g) was weighed at room temperature, and then the copolymer was dried in a 60 ℃ dry box to a constant weight, and its mass M (g) was weighed. The equilibrium water content of the hydrogel was calculated according to the following formula: EWC (%) - (M1-M)/M1X 100%
Determination of drug load:
The prepared hydrogel is placed in a drug solution with a certain concentration and 1mL, and is loaded in a thermostatic water bath at 37 ℃ for 48h until the hydrogel is balanced. And (3) measuring the absorbance of the maximum absorption wavelength of the drug before and after loading of the drug by using an ultraviolet spectrophotometer, calculating the solubility of the drug through a standard curve, and calculating the amount of the drug loaded into the hydrogel through the volume difference of the solubilities.
Determination of drug Release amount:
and respectively placing the loaded hydrogel in PBS, placing the loaded hydrogel in a constant-temperature water bath at 37 ℃, detecting the absorbance of the drug concentration at a specific wavelength in different release time periods by using an ultraviolet spectrophotometer, and calculating the solubility of the drug by using a standard curve, thereby obtaining the cumulative release amount of the drug.
When the loading measurement of the drug ofloxacin was performed on the composite hydrogel of the macromolecular vesicles of examples 1 to 3 and comparative example 1 and a loading curve was plotted, it was found that: the loading of ofloxacin is obviously increased along with the increase of the content of the macromolecular vesicles. In addition, as the concentration of the loaded drug increases, the loading of the drug in the hydrogel also increases. This ensures the concentration of the drug during treatment.
when the drug release measurement of ofloxacin drug and the release curve plotting are performed on the composite hydrogel of the macromolecular vesicles of examples 1 to 3 and the comparative example, it can be found that: the drug is released from the hydrogel smoothly along with the prolonging of the time, and the release proportion of the drug is less and the total amount of the drug is increased along with the increase of the content of the macromolecular vesicles in the same time, so that the release time of the drug is prolonged on one hand, and on the other hand, the sufficient drug concentration ensures the treatment effect in the initial treatment stage.
Although the present invention has been described in conjunction with the above embodiments, the present invention is not limited to the above embodiments, and those skilled in the art can easily make modifications and variations thereto without departing from the true spirit and scope of the present invention.