CN114392270A - Chitosan microsphere, preparation method and application thereof - Google Patents

Chitosan microsphere, preparation method and application thereof Download PDF

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CN114392270A
CN114392270A CN202111585150.XA CN202111585150A CN114392270A CN 114392270 A CN114392270 A CN 114392270A CN 202111585150 A CN202111585150 A CN 202111585150A CN 114392270 A CN114392270 A CN 114392270A
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chitosan
acryloyl
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郭平
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Suzhou Hao Microbial Medical Technology Co ltd
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Abstract

The invention discloses a chitosan microsphere which is formed by polymerizing an acryloyl chitosan intermediate through a cross-linking agent and a charge modifier, and comprises the following steps: (1) dissolving a chitosan derivative in water, adding an acid-binding agent, and reacting with an acryloyl chloride compound to obtain an acryloyl chitosan intermediate; (2) dissolving the intermediate of the acryloyl chitosan in water, and adding a cross-linking agent, a charge modifier and persulfate to obtain a polymer monomer solution; and adding the polymer monomer solution into the oil phase mixture, and adding an initiator to perform polymerization reaction to obtain the porous chitosan microspheres. Through water-in-oil reverse phase polymerization, the degradation period of the chitosan microsphere is prolonged to 1-12 months, and meanwhile, groups capable of loading different kinds of charge drugs are introduced into the microsphere, so that the drug loading capacity of the microsphere is improved, and the drug loading range of the microsphere is expanded.

Description

Chitosan microsphere, preparation method and application thereof
Technical Field
The invention belongs to the field of pharmaceutical chemicals, relates to a high polymer material application technology, and particularly relates to chitosan microspheres, a preparation method and application thereof.
Background
Chitosan (Chitosan), the chemical name of which is beta- (1,4) -2-amino-2-deoxy-D-glucan, is a linear polymerization product of Chitosan deacetylation and is also the only basic aminopolysaccharide existing in the biology world. Chitosan forms various intramolecular and intermolecular hydrogen bonds through the interaction of hydroxyl groups and amino groups distributed on a macromolecular chain, and a crystallization area is easy to form, so that the chitosan is not dissolved in water, an alkali solution and a common oil phase solvent and can only be dissolved in a dilute acid solution, and the modification application of the chitosan is greatly limited.
The carboxymethyl chitosan is a product of carboxymethyl of chitosan, the introduction of carboxymethyl destroys the secondary structure of chitosan molecule, so that the crystallinity is greatly reduced, and simultaneously, the sodium carboxylate is dissolved in water, so that the carboxymethyl chitosan is soluble in water. Carboxymethyl chitosan is an important water-soluble chitosan derivative, has good biocompatibility and biodegradability, good lubricating property and viscoelasticity, has physical and chemical properties similar to those of amino polysaccharide in joints, and is applied to the treatment of arthritis. Also, carboxymethyl chitosan and hyaluronic acid are used for preparing drug-loaded embolism microspheres for interventional embolism treatment of malignant tumors. In addition, carboxymethyl chitosan is easily processed into nanoparticles, making it more suitable for drug delivery. However, the degradation period of carboxymethyl chitosan in vivo is short, and after carboxymethyl substitution, amino groups on a macromolecular chain are consumed, so that the load capacity of the carboxymethyl chitosan on a charged drug is reduced. Therefore, the development of prolonging the degradation period of the chitosan microsphere and expanding the drug loading range of the chitosan microsphere is very necessary.
Osteoarthritis (ostoarthritis OA) is a degenerative joint disease that causes joint pain, deformity and dysfunction, and is the fourth most disabling disease in humans. The OA is mostly generated in middle-aged and elderly people, the total prevalence rate of primary OA of people over 40 years old in China is as high as 46.3%, and the prevalence rate of primary OA of people over 60 years old in China is 62.2%. Currently there is a lack of specific methods for treating OA, and existing treatments aim to relieve pain, improve or restore joint function, improve patient quality of life, slow disease progression and correct deformities, including basic treatments (health education, exercise therapy, physical therapy and mobility assistance support) and the use of topical non-steroidal anti-inflammatory drugs (NSAIDs). Glucocorticoid injected into joint cavity can also quickly relieve pain and improve joint function, but if the glucocorticoid is used for many times for a long time, the loss of joint cartilage mass is accelerated, and the clinical application is careful.
Viscosupplementation (viscosupplementation) is another commonly used treatment that achieves short-term pain relief, improved joint function, and reduced analgesic drug use by injecting Hyaluronic Acid (HA) through the joint cavity. Although HA is effective and safe in treating OA, and is shown to relieve symptoms and improve joint function, its effectiveness is soon lost because HA is degraded in vivo to monosaccharides.
The chitosan substance and HA are both from natural animals and plants and have polysaccharide structures, and in vitro experiments show that the chitosan or the modified chitosan can promote the synthesis of cartilage extracellular matrix, reduce inflammatory reaction and regulate cartilage cell metabolism. It has viscoelastic properties similar to hyaluronic acid, and can be used as a supplementary ingredient for joint fluid to slow the progression of arthritis. The chitosan hydrogel can provide an ideal three-dimensional environment for the proliferation of chondrocytes and the secretion of extracellular matrix, so that the phenotype and the function of the chondrocytes can be maintained. But also has the problems of short degradation period, frequent injection and the like. The hydrogel microspheres prepared by using the chitosan material can achieve the effects of relieving joint pain for a long time, protecting joint structures and improving joint functions by delaying the degradation rate and prolonging the viscoelasticity.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a chitosan microsphere and a preparation method thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in a first aspect, the invention discloses chitosan microspheres, which are prepared by polymerizing an acryloyl chitosan intermediate through a cross-linking agent, a charge modifier and an initiator, wherein the initiator comprises a first initiator and a second initiator.
The invention discloses a preparation method of chitosan microspheres, which comprises the steps of dissolving an acryloyl chitosan intermediate in water, adding a cross-linking agent, a charge modifier and a first initiator to obtain a polymer monomer solution; and adding the polymer monomer solution into the oil phase mixture, and adding a second initiator to perform polymerization reaction to obtain the porous chitosan microsphere.
In certain embodiments, the acrylated chitosan intermediate is prepared by the steps of: dissolving the chitosan derivative in water, adding an acid-binding agent, and reacting with an acryloyl chloride compound to obtain an acryloyl chitosan intermediate.
In certain embodiments, the chitosan derivative includes, but is not limited to, one or more of carboxymethyl chitosan, hydroxypropyl chitosan, hydroxyethyl chitosan.
In certain embodiments, the acid scavenger includes, but is not limited to, one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, pyridine, triethylamine.
In certain embodiments, the acryloyl chloride compound includes, but is not limited to, one or more of acryloyl chloride, methacryloyl chloride, ethacryloyl chloride.
In certain embodiments, the crosslinking agent includes, but is not limited to, one or more of N, N' -methylenebisacrylamide, ethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, hydroxyethyl methacrylate.
In certain embodiments, the charge modifying agent comprises a compound with an acidic functional group or a compound with a basic functional group.
In certain embodiments, the compound with an acidic functional group includes, but is not limited to, one or more of sodium methallyl sulfonate, sodium 2-acrylamido-2-methylpropane sulfonate, sodium methacrylate, and 3-sulfopropyl methacrylate.
In certain embodiments, the compound with a basic functionality includes, but is not limited to, one or more of allyl (2-aminoethyl) carbamate hydrochloride, N- (2-aminoethyl) acrylamide hydrochloride, ethyl 2- (dimethylamino) methacrylate, ethyl 2- (diethylamino) methacrylate.
In certain embodiments, the first initiator is selected from persulfates including, but not limited to, one or more of potassium persulfate, sodium persulfate, ammonium persulfate; the second initiator is selected from tetramethylethylenediamine.
Wherein: the combination mode of the first initiator and the second initiator is selected, namely persulfate and tetramethylethylenediamine are selected to form an initiation system, and the persulfate alone can also be decomposed to generate free radicals to initiate polymerization reaction, but the reaction speed is relatively slow. Therefore, tetramethylethylenediamine is generally added to accelerate the generation of radicals from persulfate, thereby accelerating the initiation of polymerization.
In some embodiments, the preparation method of the chitosan microsphere comprises the following specific operation steps:
(1) adding an acid-binding agent into an aqueous solution of 0.1-10% (w/v) of chitosan derivative, uniformly stirring, cooling to-5-10 ℃, dropwise adding an acryloyl chloride compound, and dialyzing and freeze-drying after the reaction to obtain an acryloyl chitosan intermediate;
(2) dissolving the intermediate of the acryloyl chitosan in the water, and then adding a cross-linking agent, a charge modifier and persulfate to obtain a polymer monomer solution; adding a surfactant into an oil phase solvent to form an oil phase mixture of 0.1-10% (w/v); adding a polymer monomer solution into the oil phase mixture, mechanically stirring and dispersing, and adding tetramethylethylenediamine to initiate polymerization reaction to obtain porous chitosan microspheres;
(3) and cleaning, screening, filling and drying the porous chitosan microspheres to obtain microsphere products.
In certain embodiments, the chitosan derivative, the acryloyl chloride compound, and the acid-binding agent in step (1) are present in a mass ratio of 10: (0.1-5): (0.1-4).
In certain embodiments, the surfactant in step (2) includes, but is not limited to, one or more or several of span 80, ABIL EM 90, polyoxyethylene fatty alcohol ether.
In certain embodiments, the oil phase solvent in step (2) includes, but is not limited to, one or more of liquid paraffin, n-hexane, petroleum ether.
In certain embodiments, the volume ratio of the polymer monomer solution to the oil phase mixture in step (2) is 1: (10-50).
In certain embodiments, the mass ratio of the acrylated chitosan intermediate, the cross-linking agent, the charge modifying agent, the persulfate, and the tetramethylethylenediamine in step (2) is 10: (0.1-1): (0.1-2): (0.01-0.05): (0.05-0.5).
In some embodiments, the chitosan microspheres prepared by the above preparation method have a particle size of 10 to 1000 μm.
In a third aspect, use of chitosan microspheres in a positively charged medicament, wherein: positively charged drugs include doxorubicin, epirubicin, or irinotecan hydrochloride.
In a fourth aspect, the use of chitosan microspheres in a negatively charged medicament, wherein: the negatively charged drug includes sodium dichlorophenolate or betamethasone sodium phosphate.
The invention has the following beneficial effects:
(1) according to the invention, through water-in-oil reverse phase polymerization, the degradation period of the chitosan microspheres is prolonged and can reach 1-12 months, and meanwhile, groups capable of loading different kinds of charge drugs are introduced into the microspheres, so that the drug loading capacity of the microspheres is improved, and the drug loading range of the microspheres is expanded.
(2) The chitosan microsphere prepared by the invention has good elasticity and flexibility, the compression deformation of the chitosan microsphere can reach more than 80%, and the chitosan microsphere has good softness. The microsphere can still keep better softness under the condition of good elasticity, can be used for treating osteoarthritis, provides an ideal three-dimensional environment for the proliferation of chondrocytes and the secretion of extracellular matrix, and achieves the aims of relieving joint pain for a longer time, protecting joint structure and improving joint function by delaying the degradation rate of chitosan hydrogel.
(3) The chitosan microsphere has the advantages of uniform and controllable particle size, good dispersibility, high product roundness, good biocompatibility and good stability, and can ensure that the particle size range is between 10 and 1000 mu m. The preparation process is mild, and the microsphere can be used for loading drugs with different charges, so that the stability of the microsphere preparation is improved, and the sustained release and controlled release of the drugs are realized.
Drawings
FIG. 1 is a graph showing the loading of betamethasone sodium phosphate by chitosan microspheres of example 1 of the present invention;
FIG. 2 is a drawing of a microsphere of chitosan microsphere loaded with betamethasone sodium phosphate according to example 1 of the present invention;
FIG. 3 is a graph showing the loading curve of doxorubicin onto chitosan microspheres of example 1 of the present invention;
FIG. 4 is a diagram of microspheres of chitosan microspheres loaded with doxorubicin of example 1 of the present invention;
FIG. 5 is a graph showing the degradation time of chitosan microspheres of example 1 of the present invention;
FIG. 6 is a friction force test chart of the chitosan microspheres of example 1 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
(1) Weighing 6g of carboxymethyl chitosan, adding the carboxymethyl chitosan into 100mL of deionized water, adding 10mL of sodium hydroxide solution with the mass concentration of 20%, uniformly stirring, cooling to-5-10 ℃, dropwise adding 2g of acryloyl chloride, reacting for 0.5h after dropwise adding is finished, dialyzing by using deionized water to remove impurities, and freeze-drying to obtain the intermediate of the acryloyl chitosan.
(2) Dissolving 5g of an intermediate of the acryloyl chitosan in 20ml of deionized water, and then adding 0.1g of N, N' -methylene bisacrylamide, 0.5g of (2-aminoethyl) allyl carbamate hydrochloride and 0.01g of potassium persulfate to prepare a polymer monomer solution; adding 5g of span 80 into 200mL of liquid paraffin to form an oil phase mixture; adding a polymer monomer solution into the oil phase mixture, mechanically stirring and dispersing, controlling the temperature to be 50 ℃, and then adding 0.1g of tetramethylethylenediamine to initiate polymerization for 6 hours to generate porous chitosan microspheres;
(3) sequentially cleaning the porous chitosan microspheres by using petroleum ether, acetone and deionized water; and then sieving the microspheres by using a screen, filling and drying to obtain microsphere products of 30-60 mu m.
Example 2
(1) Weighing 1.0g of hydroxypropyl chitosan, adding the hydroxypropyl chitosan into 100mL of deionized water, adding 10mL of pyridine solution with the mass concentration of 1%, uniformly stirring, cooling to-5-10 ℃, dropwise adding 0.1g of methacryloyl chloride, reacting for 0.5h after dropwise adding, dialyzing by using deionized water to remove impurities, and freeze-drying to obtain the intermediate of the acryloyl chitosan.
(2) Dissolving 1.0g of the intermediate of the acryloyl chitosan in 10ml of deionized water, and then adding 0.1g of polyethylene glycol dimethacrylate, 0.1g of 2-acrylamide-2-methylpropanesulfonic acid sodium salt and 0.01g of sodium persulfate to prepare a polymer monomer solution; adding 1g of polyoxyethylene fatty alcohol ether into 100mL of normal hexane to form an oil phase mixture; adding a polymer monomer solution into the oil phase mixture, mechanically stirring and dispersing, controlling the temperature to be 50 ℃, and then adding 0.05g of tetramethylethylenediamine to initiate polymerization for 6 hours to generate porous chitosan microspheres;
(3) sequentially cleaning the porous chitosan microspheres by using petroleum ether, acetone and deionized water; and then sieving the microspheres by using a screen, filling and drying to obtain microsphere products with the particle size of 70-100 microns.
Example 3
(1) Weighing 10g of hydroxyethyl chitosan, adding the hydroxyethyl chitosan into 100mL of deionized water, adding 20mL of triethylamine solution with the mass concentration of 20%, uniformly stirring, cooling to-5-10 ℃, dropwise adding 5g of ethyl acryloyl chloride, reacting for 1.0h after dropwise adding, dialyzing by using deionized water to remove impurities, and freeze-drying to obtain the intermediate of the acryloyl chitosan.
(2) Dissolving 10g of the intermediate of the acryloyl chitosan in 20ml of deionized water, and then adding 1.0g of hydroxyethyl methacrylate, 2.0g of 2- (diethylamino) ethyl methacrylate and 0.05g of ammonium persulfate to prepare a polymer monomer solution; adding 20g ABIL EM 90 into 250mL petroleum ether to form an oil phase mixture; adding a polymer monomer solution into the oil phase mixture, mechanically stirring and dispersing, controlling the temperature to be 50 ℃, and then adding 0.5g of tetramethylethylenediamine to initiate polymerization for 6 hours to generate porous chitosan microspheres;
(3) sequentially cleaning the porous chitosan microspheres by using petroleum ether, acetone and deionized water; and then sieving the microspheres by using a screen, filling and drying to obtain microsphere products of 200-300 mu m.
Example 4
The chitosan microspheres prepared in example 1 were subjected to drug loading test on betamethasone sodium phosphate, and the test conditions and test results were as follows:
the betamethasone sodium phosphate and the chitosan microspheres with the particle size range of 30-60 mu m are mixed, sampling is carried out for 5min, 10min, 20min and 30min respectively, the content of the betamethasone sodium phosphate in the samples is measured by using HPLC (ultraviolet detection wavelength of 254nm), test data are shown in figure 1, and figure 1 shows that when the feeding ratio of the betamethasone sodium phosphate to the microspheres is 50mg/g, the drug loading efficiency of the microspheres loaded with the betamethasone sodium phosphate is up to more than 88% at 10min, which indicates that the chitosan microspheres prepared by the invention can rapidly load the betamethasone sodium phosphate.
Wherein: the microsphere drug loading efficiency (1-content of betamethasone sodium phosphate/amount of betamethasone sodium phosphate in sample) × 100%.
As can be seen from FIG. 2, the chitosan microspheres loaded with betamethasone sodium phosphate have uniform and controllable particle size and good dispersibility. The chitosan microspheres of the invention have good stability.
Example 5
The chitosan microspheres prepared in example 2 were subjected to a drug loading test on doxorubicin, and the test conditions and test results thereof were as follows:
dissolving doxorubicin in injection water to prepare a doxorubicin solution, mixing chitosan microspheres with the particle size range of 70-100 microns with the doxorubicin solution, sampling for 5min, 10min, 20min and 30min respectively, and determining the content of doxorubicin in the sample by using HPLC (ultraviolet detection wavelength of 254nm), wherein the test data is shown in figure 3, and figure 3 shows that when the dosage ratio of doxorubicin to microspheres is 50mg/g, the drug loading efficiency of microsphere-loaded doxorubicin is over 90% at 10min, which indicates that the chitosan microspheres prepared by the method can be used for rapidly loading doxorubicin.
Wherein: the microsphere drug loading efficiency (1-doxorubicin content/doxorubicin input in the sample) is 100%.
As can be seen from FIG. 4, the chitosan microspheres loaded with doxorubicin have uniform and controllable particle size and good dispersibility. The chitosan microspheres of the invention have good stability.
Example 6
The test method and test results of the degradation test of the chitosan microspheres are as follows:
chitosan microspheres having particle sizes ranging from 30 to 60 μm, 70 to 100 μm and 200 to 300 μm were added to a phosphate buffer (pH 7.4), degradation tests were performed in a constant temperature shaker at 37 ℃, and the degradation progress of the chitosan microspheres was judged according to the weight of the chitosan microspheres as described in table 1 below. Fig. 5 shows the degradation curves of chitosan microspheres with different particle size ranges, and fig. 5 shows that the larger the particle size range of the chitosan microspheres is, the longer the degradation time is. As can be seen, the chitosan microspheres of the invention have good biodegradability.
Table 1 shows the degradation schedule of chitosan microspheres according to the weight of the chitosan microspheres
Figure BDA0003426299730000071
Figure BDA0003426299730000081
Example 7
The external friction force test method and the test results of the chitosan microspheres are as follows:
mixing microspheres with different particle sizes and a PBS solution into a suspension, testing the friction force of the suspension by using a friction tester, wherein the test result is shown in FIG. 6, and as can be seen from FIG. 6, the larger the particle size of the microspheres is, the smaller the friction force is. The microsphere of the invention can still keep better softness under the condition of good elasticity. The good softness of the microspheres is mainly attributed to the fact that the number of cross-linked bonds in the microspheres is controlled, so that the microspheres are easy to deform and are softer after being stressed. Therefore, the microsphere can be used for treating osteoarthritis, provides an ideal three-dimensional environment for proliferation of chondrocytes and secretion of extracellular matrix, and achieves the aims of relieving joint pain for a long time, protecting joint structure and improving joint function by delaying the degradation rate of the chitosan hydrogel.
In summary, the following steps: the chitosan microsphere provided by the invention is mild in preparation process, the degradation period of the chitosan microsphere is prolonged through water-in-oil reverse phase polymerization reaction, and meanwhile, groups capable of loading different kinds of charge drugs are introduced into the microsphere, so that the drug loading capacity of the microsphere is improved, the drug loading range of the microsphere is expanded, the stability of the microsphere dosage form is improved, and the sustained release and controlled release of the drugs are realized.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept of the present invention, and these should be considered as within the scope of the present invention.

Claims (9)

1. The chitosan microsphere is characterized by being formed by polymerizing an acryloyl chitosan intermediate through a cross-linking agent, a charge modifier and an initiator, wherein the initiator comprises a first initiator and a second initiator.
2. The preparation method of chitosan microspheres of claim 1, wherein the intermediate of acrylated chitosan is dissolved in water, and a cross-linking agent, a charge modifier, and a first initiator are added to obtain a polymer monomer solution; and adding the polymer monomer solution into the oil phase mixture, and adding a second initiator to perform polymerization reaction to obtain the porous chitosan microsphere.
3. The preparation method of chitosan microspheres of claim 2, wherein said intermediate of acrylated chitosan is prepared by the following steps: dissolving the chitosan derivative in water, adding an acid-binding agent, and reacting with an acryloyl chloride compound to obtain an acryloyl chitosan intermediate.
4. The method for preparing chitosan microspheres according to claim 2, wherein the chitosan derivatives include but are not limited to one or more of carboxymethyl chitosan, hydroxypropyl chitosan, hydroxyethyl chitosan; acid-binding agents include, but are not limited to, one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, pyridine, triethylamine; acryloyl chloride compounds include, but are not limited to, one or more of acryloyl chloride, methacryloyl chloride, ethacryloyl chloride; the cross-linking agent includes but is not limited to one or more of N, N' -methylene bisacrylamide, ethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, hydroxyethyl methacrylate; the charge modifier comprises a compound with an acidic functional group or a compound with a basic functional group, wherein the compound with an acidic functional group comprises one or more of but not limited to sodium methacrylate sulfonate, sodium 2-acrylamide-2-methyl propane sulfonate, sodium methacrylate and 3-sulfopropyl methacrylate, and the compound with a basic functional group comprises one or more of but not limited to allyl (2-aminoethyl) carbamate hydrochloride, N- (2-aminoethyl) acrylamide hydrochloride, ethyl 2- (dimethylamino) methacrylate and ethyl 2- (diethylamino) methacrylate; the first initiator is selected from persulfates including but not limited to one or more of potassium persulfate, sodium persulfate, and ammonium persulfate; the second initiator is selected from tetramethylethylenediamine.
5. The preparation method of the chitosan microspheres of claim 2, comprising the following steps:
(1) adding an acid-binding agent into an aqueous solution of 0.1-10% (w/v) of chitosan derivative, uniformly stirring, cooling to-5-10 ℃, dropwise adding an acryloyl chloride compound, and dialyzing and freeze-drying after the reaction to obtain an acryloyl chitosan intermediate;
(2) dissolving the intermediate of the acryloyl chitosan in the water, and then adding a cross-linking agent, a charge modifier and persulfate to obtain a polymer monomer solution; adding a surfactant into an oil phase solvent to form an oil phase mixture of 0.1-10% (w/v); adding a polymer monomer solution into the oil phase mixture, mechanically stirring and dispersing, and adding tetramethylethylenediamine to initiate polymerization reaction to obtain porous chitosan microspheres;
(3) and cleaning, screening, filling and drying the porous chitosan microspheres to obtain microsphere products.
6. The preparation method of chitosan microspheres of claim 5, wherein the mass ratio of the chitosan derivative, the acryloyl chloride compound and the acid-binding agent in step (1) is 10: (0.1-5): (0.1-4).
7. The method for preparing chitosan microspheres according to claim 5, wherein the surfactant in step (2) includes but is not limited to one or more of span 80, ABIL EM 90, polyoxyethylene fatty alcohol ether; the oil phase solvent comprises one or more of liquid paraffin, n-hexane and petroleum ether; the volume ratio of the polymer monomer solution to the oil phase mixture is 1: (10-50); the mass ratio of the intermediate of the acryloyl chitosan to the cross-linking agent to the charge modifier to the persulfate to the tetramethyl ethylene diamine is 10: (0.1-1): (0.1-2): (0.01-0.05): (0.05-0.5).
8. Use of chitosan microspheres according to claim 1, wherein the positively charged drug comprises doxorubicin, epirubicin or irinotecan hydrochloride.
9. The use of chitosan microspheres of claim 1, wherein the negatively charged drug comprises sodium dichlorophenolate or betamethasone sodium phosphate.
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