Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the pH response type antibacterial slow-release gel which has long effective time and has the effect of preventing cervical cancer while inhibiting bacteria and viruses.
Specifically, the invention provides a pH response type antibacterial slow-release gel, which comprises the following components in parts by weight:
the core material of the paclitaxel slow release microcapsule is modified mesoporous silica loaded paclitaxel, and the wall material is a crosslinked chitosan-gelatin composite wall material;
the mass percentage ratio of the core material to the wall material is 1: (2-4);
the modified mesoporous silica is nano hollow mesoporous silica which is subjected to amination modification and polyacrylic acid grafting modification in sequence.
The octylphenol polysaccharide is a main active ingredient for killing HPV, and comprises at least one of octylphenol polyether-9, octylphenol polyether-10, octylphenol polyether-11, octylphenol polyether-12, octylphenol polyether-13, octylphenol polyether-16, octylphenol polyether-20, octylphenol polyether-25, octylphenol polyether-30, octylphenol polyether-33, octylphenol polyether-40 and octylphenol polyether-70. The octabenan is a natural active microecological preparation, and can make cell contents leak through destroying cell membranes of bacteria and envelope materials of viruses, provide other components to enter cells so as to perform a killing function, kill 6 major 20 pathogens and male sperms, protect genital organs from being affected by various bacteria, viruses, fungi, chlamydia, mycoplasma and trichomonas, and is safe and nontoxic, and can be used for a long time, so that the octabenan has a better inhibiting and killing function on HPV viruses. Meanwhile, by means of holes formed by the rupture of the octabenan membrane, benzalkonium chloride can quickly enter the pathogenic microorganism coating slurry to further destroy genetic materials of the pathogenic microorganism coating slurry so as to play a role in killing.
Benzalkonium chloride (Benzalkonium Chloride), which is a cationic surfactant, belongs to a non-oxidizing bactericide, has broad-spectrum and high-efficiency bactericidal capacity, can kill any contacted pathogenic microorganism within 1 minute, and can play a role in bacteriostasis and antivirus, including HPV, HSV-2 and the like which can induce cervical cancer.
Melatonin, which is a gel product mainly acting as a strong antioxidant and scavenger, eliminates free radicals and oxidants such as oxygen, nitrogen and the like, eliminates metabolic products or foreign substances of body cells in time, stabilizes and cleans the intravaginal environment, avoids inducing new canceration due to persistent bad stimulus, has an anticancer effect itself, and simultaneously has the effect of strongly inhibiting tumorigenesis, development, transformation and metastasis, besides HPV infection, unstable and persistent intravaginal environment with pollutants such as metabolic waste, foreign substances brought by sexual life and the like is another important cause of cervical cancer occurrence. In the invention, melatonin is used as a strong antioxidant and scavenger, and can instantly remove free radicals such as oxygen, nitrogen and the like, oxidants, metabolites of organism cells and foreign substances, so as to stabilize and clean the environment in the vagina and avoid the possibility of inducing precancerous lesions due to lasting bad stimulus.
Paclitaxel: stabilize microtubules and retard cancer cell division, differentiation, metastasis and invasion. The slow released taxol microcapsule of the present invention has pH response function, nanometer modified mesoporous silica negative taxol as core material and crosslinked chitosan-gelatin as composite wall material. The modified mesoporous silica has improved loading capacity to paclitaxel and remarkably improved water solubility, and in addition, the modified mesoporous silica has pH responsiveness.
The carbomer sodium and the hydroxypropyl methylcellulose can form a cross-linked framework, so that the active ingredients in the gel can stay in the body for a long time, a slow-release medium for the medicine is provided, the time of the medicine exertion is prolonged, and the local medicine concentration is prevented from being too high.
Further, the thickener is hydroxypropyl methylcellulose, the humectant is carbomer sodium, the solvent comprises an aqueous solvent and an oily solvent, the aqueous solvent is double distilled water, and the oily solvent is glycerin.
The double distilled water is obtained by utilizing the difference of the volatility of each component in the liquid mixture, vaporizing the mixture, re-condensing the vapor to obtain distilled water, vaporizing the obtained distilled water, and re-condensing to obtain double distilled water, thereby removing most of pollutants. In the double distilled water preparation process, although volatile impurities such as carbon dioxide, ammonia and some organic matters cannot be removed, inorganic salts, organic matters, microorganisms, soluble gases and volatile impurities in water are extremely low through twice distillation, and especially, the preparation of the final gel is sterile and the heat-generating source is removed.
Further, the antibacterial slow-release gel also comprises an osmotic pressure regulator, wherein the osmotic pressure regulator is at least one selected from sodium chloride salt and potassium chloride salt, and the osmotic pressure of the antibacterial slow-release gel is 250-340 mOsm/kg.H2 O。
While normal gel osmotic pressure is beneficial to the comfort of long-term in vivo retention of the gel, too low a crystalline osmotic pressure has its adverse aspects. The osmotic pressure regulator enables the gel of the invention to have a crystalline osmotic pressure balanced with body fluid. On the one hand, under the condition of no damage, breakage, ulcer or erosion, the gel of the invention is injected into the vagina immediately to about two minutes, and the vaginal wall and the cervix can contact local epithelial cells of the gel to possibly generate stress reaction, so that a user has short-term discomfort; on the other hand, in the case of injury, breakage, ulcer or erosion, the gel of the present invention is injected into vagina immediately to about 10 minutes, and the vaginal wall and cervix can contact local epithelial cells of the gel and other tissue cells which are in clearance and communication connection with the epithelial cells, exposed smooth muscle cells which are not completely covered by the epithelium, somatic and visceral sensory neurons are distributed on the vaginal wall, the peri-process of the uterine opening and the cervix, and visceral motor neurons govern the vaginal wall, the uterine opening and the exercise branch of the cervix, etc., water fluctuation and electrolyte surge can occur due to rapid change of the osmotic pressure of crystals inside and outside the cell membrane, potassium ion outflow and/or sodium ion inflow are stimulated, so that membrane potential depolarization occurs, and stress reaction occurs. Therefore, naCl or KCl salt component with physiological concentration is added into the gel to ensure that the osmotic pressure of the crystal is at the ion level required for maintaining the resting potential of cells, and the gel has no stimulation to the vagina and maintains the due resting potential of the vaginal wall, cervical epithelium, smooth muscle, nerve endings and the like no matter whether damage, ulcer or erosion exists when the gel is injected into the vagina, so that stress reaction is avoided.
Further, the preparation method of the taxol slow-release microcapsule comprises the following steps:
(1) Preparing a core material: adding modified mesoporous silica into an organic solvent for ultrasonic dispersion, adding taxol, magnetically stirring, centrifuging, repeatedly washing the centrifuged precipitate, and freeze-drying to obtain the core material;
(2) Heating gelatin solution to 50-60deg.C, adding glycerol, and dispersing and emulsifying for 20-30s;
(3) Adding the crosslinked chitosan solution, and stirring;
(4) Adding the core material, adding water while stirring, adding sodium hydroxide solution after dilution, adjusting the pH to 5.9-6.20, and reacting for 20-30min;
(5) Cooling, adding glutaraldehyde solution, standing and solidifying;
(6) Repeatedly cleaning and drying to obtain the taxol slow-release microcapsule.
The organic solvent is any one of dimethyl sulfoxide, methylene dichloride and acetone, and the mass percentage ratio of the modified mesoporous silica to the taxol is 1: (0.6-1.2).
Further, the preparation method of the modified mesoporous silica comprises the following steps:
step 1: amination modification of nano hollow mesoporous silica
1.1, adding nano hollow mesoporous silica into absolute ethyl alcohol for ultrasonic dispersion;
1.2 heating to 80-95 ℃, continuously introducing argon, adding gamma-aminopropyl triethoxysilane, and magnetically stirring;
1.3, repeatedly cleaning the precipitate with anhydrous ethanol after centrifugation, and freeze-drying to obtain nano-scale hollow mesoporous silica subjected to amination modification;
step 2: polyacrylic acid grafting modification
2.1, adding the nano-scale hollow mesoporous silica subjected to amination modification and polyacrylic acid into water, and magnetically stirring;
2.2 adding a composite initiator solution, reacting for 5-10min, adding an N, N-methylene bisacrylamide solution, and magnetically stirring under the protection of nitrogen;
and 2.3, centrifuging, repeatedly flushing the centrifuged precipitate, and drying to obtain the modified mesoporous silica.
Mesoporous silica nanometer is used as a relatively mature nanometer material for research, has the advantages of controllable generation, large specific surface area, excellent biocompatibility, easy surface functionalization modification, targeted drug delivery and the like, and plays a great role and wide prospect in a drug delivery system.
Firstly, the mesoporous silica of the invention is subjected to amination modification, so that the drug carrying capacity of the mesoporous silica is further improved, and the water solubility of the taxol is improved, so that the taxol can fully play a role. After the polyacrylic acid is grafted and modified, the taxol slow-release microcapsule has pH responsiveness. The pH responsiveness mainly depends on the stimulation of the environmental pH to change the conformation of the material, so as to achieve the controlled release of the drug. The inside of which often carries a pair H+ Or OH (OH)- The sensitive ionizable groups or the chemical bonds which can be broken under specific conditions can change the form of the material or break the chemical bonds under specific pH conditions or ionic strength, so that the pH intelligent responsive release of the medicine is realized. The pH value of the microenvironment is changed when infectious diseases and malignant tumors occur in the human body, and the pH gradient difference of the microenvironment provides possibility for targeted drug delivery. The pH value of healthy vagina is about 3.8-4.4, and when inflammation or other diseases occur, the pH value of vagina is raised, and the pH value of extracellular environment where inflammation or cancer cells are located is 5.7-7.0, becauseBy utilizing the characteristic, the invention prepares the pH response slow release gel by utilizing the characteristic that polyacrylic acid receives protons at low pH value and deprotonates and swells at neutral and high pH values.
Further, the composite initiator solution is a mixed solution of ammonium persulfate and sodium bisulphite, wherein the mass percentage ratio of the ammonium persulfate to the sodium bisulphite in the mixed solution is (1.8-2.5): 1.
further, the mass percentage ratio of the crosslinked chitosan to the gelatin is 1: (0.8-1.2).
Further, the preparation method of the crosslinked chitosan comprises the following steps: dissolving chitosan in glacial acetic acid, magnetically stirring, adding a cross-linking agent solution, adjusting the pH of the solution to 4.8-5.2, reacting for 5-8h, centrifuging, repeatedly washing the centrifuged precipitate, and drying to obtain the cross-linked chitosan.
Further, the cross-linking agent is at least one selected from phytic acid and sodium hexametaphosphate, and the mass percentage ratio of the cross-linking agent to the chitosan is 1: (1-3.5).
Among the many natural materials, chitosan is a positively charged polysaccharide linked by beta- (1, 4) glycosidic bond, and has wide application in the aspects of self-assembled composite materials, biopolymer membranes, drug delivery vehicles and the like due to good biocompatibility and degradability and physical and chemical property stability. After the chitosan is crosslinked, the microstructure of the chitosan is changed, so that the controlled release performance of the hydrogel is affected. Therefore, the microcapsule prepared by the phytic acid and the sodium hexametaphosphate crosslinked chitosan has higher encapsulation rate and lower release rate, and the slow release performance of the microcapsule is optimized.
On the other hand, the preparation method of the pH response type antibacterial slow-release gel comprises the following steps:
s1: preparing taxol slow-release microcapsules;
s2: mixing octoxynol and benzalkonium chloride with an aqueous solvent to obtain a mixture A;
s3: mixing melatonin and paclitaxel slow release microcapsules with an aqueous solvent to mixture B;
s4: mixing a humectant with an osmotic pressure regulator to obtain a mixture C;
s5: and adding the mixture A, the mixture B, the mixture C and the thickening agent into an oily solvent, and stirring to obtain the antibacterial slow-release gel.
The invention has the advantages that:
1) The invention uses the combination of the octoxynol and the benzalkonium bromide as the main active ingredients for killing HPV, and after the pathogenic microorganisms indicate that holes are formed, the benzalkonium chloride can quickly enter the pathogenic microorganism coating slurry to further destroy genetic materials to play a role in killing, so that the invention has the effects of strong bacteriostasis, antivirus and keeping vagina clean.
2) The invention has the effect of preventing cervical by adding proper amount of paclitaxel and combining melatonin. The paclitaxel is added into the gel by using the mode of loading the paclitaxel by the modified mesoporous silica and coating the crosslinked chitosan-gelatin composite layer, the hollow mesoporous silica is subjected to amination modification, the drug carrying capacity is improved, the water solubility of the paclitaxel is improved, the paclitaxel slow-release microcapsule has pH responsiveness after being subjected to polyacrylic acid grafting modification, the paclitaxel slow-release microcapsule is released slowly when the pH value of vagina is increased under the condition of inflammation or other diseases, the embedding rate of the drug is improved by using the crosslinked chitosan, and the slow-release performance of the microcapsule is enhanced, so that the paclitaxel slow-release microcapsule has excellent slow-release performance and pH responsiveness, and can instantly clear free radicals such as oxygen and nitrogen, oxidizing agents, metabolites of organism cells and foreign substances by combining melatonin as strong antioxidants, so that the environment in the vagina is stabilized and cleaned, the possibility of inducing precancerous lesions due to lasting bad stimulus is avoided, and the effect of preventing cervical cancer is played.
3) The invention uses carbomer sodium and hydroxypropyl methylcellulose to ensure that active ingredients in the gel can stay in the body for a long time, provide a medium for slow release of the medicine, thereby prolonging the time of the medicine exertion and avoiding overhigh local medicine concentration.
4) The osmotic pressure of the antibacterial slow-release gel is 250-340 mOsm/kg.H2 O, the osmotic pressure of the colloid is beneficial to the long-time in-vivo retention of the gel,and avoid producing uncomfortable sense, avoid producing stress reaction, influence the gel effect.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to examples.
The pH response type antibacterial slow-release gel comprises the following components in parts by weight:
the octylphenol polysaccharide comprises at least one of octylphenol polyether-9, octylphenol polyether-10, octylphenol polyether-11, octylphenol polyether-12, octylphenol polyether-13, octylphenol polyether-16, octylphenol polyether-20, octylphenol polyether-25, octylphenol polyether-30, octylphenol polyether-33, octylphenol polyether-40 and octylphenol polyether-70. The humectant is carbomer sodium, the thickener is hydroxypropyl methylcellulose, the solvent comprises aqueous solvent and oily solvent, the aqueous solvent is double distilled water, and the oily solvent is glycerol.
The core material of the taxol slow-release microcapsule is modified mesoporous silica loaded taxol, and the wall material is a crosslinked chitosan-gelatin composite wall material; wherein the modified mesoporous silica is nano hollow mesoporous silica which is sequentially subjected to amination modification and polyacrylic acid grafting modification, and the crosslinked chitosan is chitosan of crosslinked phytic acid or sodium hexametaphosphate. The mass percentage ratio of the core material to the wall material is 1: (2-4); the mass percentage ratio of the mesoporous silica to the taxol is 1: (0.6-1.2), the mass percentage ratio of the crosslinked chitosan to the gelatin is 1: (0.8-1.2).
The antibacterial sustained-release gel also comprises osmotic pressure regulator, wherein the osmotic pressure regulator is at least one selected from sodium chloride salt and potassium chloride salt, and the osmotic pressure regulator is added to make the osmotic pressure of the antibacterial sustained-release gel be 250-340 mOsm/kg.H2 O。
A preparation method of pH response type antibacterial slow-release gel comprises the following steps:
s1: preparing taxol slow release microcapsules:
s1.1 preparation of core Material
(1) Preparation of modified mesoporous silica
1. Amination modification
1.1, adding nano hollow mesoporous silica into absolute ethyl alcohol, and performing ultrasonic dispersion for 20-30min;
1.2 heating to 80-95 ℃, continuously introducing argon, adding gamma-aminopropyl triethoxysilane, and magnetically stirring for 5-7h;
1.3 centrifuging at 11000-13000rpm for 25-35min, repeatedly cleaning with anhydrous ethanol, and lyophilizing to obtain amination-modified nanoscale hollow mesoporous silica;
2. polyacrylic acid grafting modification
2.1, adding the nano-scale hollow mesoporous silica subjected to amination modification and polyacrylic acid into water, and magnetically stirring for 2-4 hours at 40-50 ℃;
2.2, the mass percent ratio of the added components is (1.8-2.5): 1, reacting ammonium persulfate and sodium bisulphite composite initiator solution for 5-10min, adding N, N-methylene bisacrylamide solution with the mass fraction of 6-8%, and magnetically stirring for 6-8h under the protection of nitrogen;
2.3 centrifuging for 20-30min at 9000-11000rpm, repeatedly washing the centrifuged precipitate, and drying to obtain modified mesoporous silica;
(2) Adding modified mesoporous silica into an organic solvent, performing ultrasonic dispersion for 5-10min, adding taxol, and magnetically stirring for 10-12h, wherein the organic solvent is any one of dimethyl sulfoxide, dichloromethane and acetone;
(3) Centrifuging at 10000-12000rpm for 15-25min, repeatedly washing the centrifuged precipitate, and lyophilizing to obtain core material;
s1.2, heating a fully swelled gelatin solution with the mass fraction of 20-25% to 50-60 ℃, adding glycerol, and shearing for 20-30S by a high shearing dispersion emulsifying machine at 10000-12000 rpm;
s1.3, preparing crosslinked chitosan: dissolving chitosan in glacial acetic acid with the mass fraction of 1-1.5%, magnetically stirring at 20-30 ℃ for 0.5-1h, adding a cross-linking agent solution with the concentration of 0.8-1.5%, adjusting the pH of the solution to 4.8-5.2, reacting for 5-8h, centrifuging at the speed of 8000-10000r/min for 10-15min, repeatedly washing the centrifuged precipitate, and drying to obtain cross-linked chitosan;
s1.4, adding a 2-2.5% mass fraction crosslinked chitosan solution preheated at 50-60 ℃ and stirring at 400-500rpm for 10-15min;
s1.5, adding the core material, adding distilled water with the temperature of 50-60 ℃ while stirring, adding sodium hydroxide solution with the mass fraction of 1.5-2%, adjusting the pH to 5.9-6.2, and continuously reacting for 20-30min at the temperature of 50-60 ℃;
s1.6, cooling to 15-25 ℃, adding glutaraldehyde solution with the mass fraction of 4.5-5.5%, standing and solidifying for 25-30min at 30-35 ℃ after 5-10 min;
s1.7, repeatedly cleaning and drying to obtain the taxol slow-release microcapsule;
s2: mixing and stirring the octoxynol and the benzalkonium chloride with an aqueous solvent to obtain a mixture A, wherein the stirring speed is 200-300r/min, and the stirring time is 30-40min;
s3: mixing melatonin and taxol slow release microcapsules with an aqueous solvent, and stirring to obtain a mixture B, wherein the stirring speed is 500-600r/min, and the stirring time is 30-40min;
s4: mixing and stirring the humectant and the osmotic pressure regulator to obtain a mixture C, wherein the stirring speed is 400-500r/min, and the stirring time is 30-40min;
s5: adding the mixture A, the mixture B, the mixture C and the thickening agent into an oily solvent, and stirring to obtain the antibacterial slow-release gel, wherein the stirring speed is 400-500r/min, and the stirring time is 30-40min.
And (3) after stirring the steps S2, S3 and S4, standing for 10-12h, wherein the volume ratio of the solvents in the steps S2, S3 and S4 is (2-3): (2-3): 1.
example 1
The pH response type antibacterial slow-release gel comprises the following components in parts by weight:
the octylphenol polysaccharide is octylphenol polyether-10, octylphenol polyether-30 and octylphenol polyether-33 with the mass ratio of 1:1:1. The humectant is carbomer sodium, the thickener is hydroxypropyl methylcellulose, and the solvent is aqueous solvent double distilled water, and the oily solvent is glycerol.
The core material of the taxol slow-release microcapsule is modified mesoporous silica loaded taxol, and the wall material is a crosslinked chitosan-gelatin composite wall material; wherein the modified mesoporous silica is nano hollow mesoporous silica which is sequentially subjected to amination modification and polyacrylic acid grafting modification, and the crosslinked chitosan is chitosan of crosslinked phytic acid or sodium hexametaphosphate. The mass percentage ratio of the core material to the wall material is 1:3, a step of; the mass percentage ratio of the mesoporous silica to the taxol is 1:1, the mass percentage ratio of the crosslinked chitosan to the gelatin is 1:1.
the antibacterial slow-release gel also comprises an osmotic pressure regulator, wherein the osmotic pressure regulator is sodium chloride salt, and the osmotic pressure of the antibacterial slow-release gel is 300 mOsm/kg.H2 O。
A preparation method of pH response type antibacterial slow-release gel comprises the following steps:
s1: preparing taxol slow release microcapsules:
s1.1 preparation of core Material
(1) Preparation of modified mesoporous silica
1. Amination modification
1.1, adding nano hollow mesoporous silica into absolute ethyl alcohol, and performing ultrasonic dispersion for 25min;
1.2 heating to 90 ℃, continuously introducing argon, adding gamma-aminopropyl triethoxysilane, and magnetically stirring for 6 hours;
1.3 centrifuging at 12000rpm for 30min, repeatedly cleaning with anhydrous ethanol, and freeze drying to obtain amination modified nanoscale hollow mesoporous silica;
2. polyacrylic acid grafting modification
2.1, adding the nano-scale hollow mesoporous silica subjected to amination modification and polyacrylic acid into water, and magnetically stirring for 3 hours at 40 ℃;
2.2, the mass percent ratio of the added materials is 2:1, reacting ammonium persulfate and sodium bisulphite composite initiator solution for 8min, adding N, N-methylene bisacrylamide solution with the mass fraction of 7.5%, and magnetically stirring for 6h under the protection of nitrogen;
2.3 centrifuging at 10000rpm for 25min, repeatedly washing the centrifuged precipitate, and drying to obtain modified mesoporous silica;
(2) Adding the modified mesoporous silica into dimethyl sulfoxide, performing ultrasonic dispersion for 7min, adding paclitaxel, and magnetically stirring for 11h;
(3) Centrifuging at 11000rpm for 20min, repeatedly washing the centrifuged precipitate, and freeze drying to obtain core material;
s1.2, heating a fully swelled gelatin solution with the mass fraction of 23% to 55 ℃, adding glycerol, and shearing for 25S by a high-shearing dispersion emulsifying machine at 11000 rpm;
s1.3, preparing crosslinked chitosan: dissolving chitosan in glacial acetic acid with the mass fraction of 1.5%, magnetically stirring at 25 ℃ for 1h, adding sodium hexametaphosphate solution with the concentration of 1.0%, adjusting the pH of the solution to 4.9, reacting for 6h, centrifuging at 10000r/min for 10min, repeatedly washing the centrifuged precipitate, and drying to obtain crosslinked chitosan;
s1.4, adding a cross-linked chitosan solution with the mass fraction of 2.5 percent which is preheated at the preheating temperature of 55 ℃ and stirring for 15 minutes at 500 rpm;
s1.5, adding the core material, adding distilled water with the temperature of 55 ℃ while stirring, adding sodium hydroxide solution with the mass fraction of 2%, adjusting the pH value to be 5.9, and continuously reacting for 25min at the temperature of 55 ℃;
s1.6, cooling to 15 ℃, adding glutaraldehyde solution with mass fraction of 5.5%, standing for curing for 30min at 30 ℃ after 10 min;
s1.7, repeatedly cleaning and drying to obtain the taxol slow-release microcapsule;
s2: mixing and stirring the octoxynol and the benzalkonium chloride with an aqueous solvent to obtain a mixture A, wherein the stirring speed is 200r/min, and the stirring speed is 30min;
s3: mixing melatonin and taxol slow release microcapsules with an aqueous solvent, stirring to obtain a mixture B, wherein the stirring speed is 600r/min, and stirring is carried out for 30min;
s4: mixing and stirring the humectant and the osmotic pressure regulator to obtain a mixture C, wherein the stirring speed is 400r/min, and the stirring speed is 30min;
s5: adding the mixture A, the mixture B, the mixture C and the thickening agent into an oily solvent, and stirring to obtain the antibacterial slow-release gel, wherein the stirring speed is 500r/min, and the stirring speed is 30min.
And (3) after stirring in the steps S2, S3 and S4, standing for 12 hours, wherein the volume ratio of the solvents in the steps S2, S3 and S4 is 2:2:1.
example 2
This example differs from example 1 in that the gel has different parts of components, specifically including:
example 3
This example differs from example 1 in that the gel has different parts of components, specifically including:
example 4
This example differs from example 1 in that the octoxynol comprises octylphenol polyether-11 and octylphenol polyether-25 in a mass ratio of 7:1.
Example 5
This example differs from example 1 in that it was prepared by the following steps:
a preparation method of pH response type antibacterial slow-release gel comprises the following steps:
s1: preparing taxol slow release microcapsules:
s1.1 preparation of core Material
(1) Preparation of modified mesoporous silica
1. Amination modification
1.1, adding nano hollow mesoporous silica into absolute ethyl alcohol, and performing ultrasonic dispersion for 25min;
1.2 heating to 90 ℃, continuously introducing argon, adding gamma-aminopropyl triethoxysilane, and magnetically stirring for 6 hours;
1.3 centrifuging at 12000rpm for 30min, repeatedly cleaning with anhydrous ethanol, and freeze drying to obtain amination modified nanoscale hollow mesoporous silica;
2. polyacrylic acid grafting modification
2.1, adding the nano-scale hollow mesoporous silica subjected to amination modification and polyacrylic acid into water, and magnetically stirring for 3 hours at 40 ℃;
2.2, the mass percent ratio of the added materials is 2:1, reacting ammonium persulfate and sodium bisulphite composite initiator solution for 8min, adding N, N-methylene bisacrylamide solution with the mass fraction of 7.5%, and magnetically stirring for 6h under the protection of nitrogen;
2.3 centrifuging at 10000rpm for 25min, repeatedly washing the centrifuged precipitate, and drying to obtain modified mesoporous silica;
(2) Adding the modified mesoporous silica into dimethyl sulfoxide, performing ultrasonic dispersion for 7min, adding paclitaxel, and magnetically stirring for 11h;
(3) Centrifuging at 11000rpm for 20min, repeatedly washing the centrifuged precipitate, and freeze drying to obtain core material;
s1.2, heating a fully swelled gelatin solution with the mass fraction of 23% to 55 ℃, adding glycerol, and shearing for 25S by a high-shearing dispersion emulsifying machine at 11000 rpm;
s1.3, preparing crosslinked chitosan: dissolving chitosan in glacial acetic acid with the mass fraction of 1.5%, magnetically stirring at 25 ℃ for 1h, adding sodium hexametaphosphate solution with the concentration of 1.0%, adjusting the pH of the solution to 4.9, reacting for 6h, centrifuging at 10000r/min for 10min, repeatedly washing the centrifuged precipitate, and drying to obtain crosslinked chitosan;
s1.4, adding a cross-linked chitosan solution with the mass fraction of 2.5 percent which is preheated at the preheating temperature of 55 ℃ and stirring for 15 minutes at 500 rpm;
s1.5, adding the core material, adding distilled water with the temperature of 55 ℃ while stirring, adding sodium hydroxide solution with the mass fraction of 2%, adjusting the pH value to be 5.9, and continuously reacting for 25min at the temperature of 55 ℃;
s1.6, cooling to 15 ℃, adding glutaraldehyde solution with mass fraction of 5.5%, standing for curing for 30min at 30 ℃ after 10 min;
s1.7, repeatedly cleaning and drying to obtain the taxol slow-release microcapsule;
s2: mixing and stirring the octoxynol and the benzalkonium chloride with an aqueous solvent to obtain a mixture A, wherein the stirring speed is 300r/min, and the stirring speed is 30min;
s3: mixing melatonin and taxol slow release microcapsules with an aqueous solvent, stirring to obtain a mixture B, wherein the stirring speed is 500r/min, and stirring is carried out for 30min;
s4: mixing and stirring the humectant and the osmotic pressure regulator to obtain a mixture C, wherein the stirring speed is 500r/min, and the stirring speed is 30min;
s5: adding the mixture A, the mixture B, the mixture C and the thickening agent into an oily solvent, and stirring to obtain the antibacterial slow-release gel, wherein the stirring speed is 400r/min, and the stirring speed is 30min.
And (3) after stirring in the steps S2, S3 and S4, standing for 12 hours, wherein the volume ratio of the solvents in the steps S2, S3 and S4 is 2:2:1.
example 6
This example differs from example 1 in that steps S2, S3, S4 are all stirred and then not allowed to stand, and the next step is directly performed.
Comparative example 1
The present comparative example differs from example 1 in that melatonin is not contained.
Comparative example 2
This comparative example differs from example 1 in that paclitaxel is added alone, not in the form of microcapsules.
Comparative example 3
This comparative example differs from example 1 in that it does not contain a paclitaxel slow-release microcapsule.
The following experimental tests were performed on examples 1 to 6 and comparative examples 1 to 3:
experiment 1 arsenic, mercury, lead content detection
Instrument apparatus: JA 5003N-type electronic balance; AFS-8220 type atomic fluorescence photometer for beijing ji day; shimadzu AA-6300C type atomic absorption photometer.
Reagent name, grade: nitric acid (GR), hydrochloric acid (GR), sodium hydroxide (GR), sodium borohydride (GR), thiourea (AR), ascorbic acid (GR), and pure water.
Standard solution and concentration: 100. Mu.g/L arsenic, 20. Mu.g/L mercury, 10mg/L lead.
The detection basis is as follows: the fourth chapter of cosmetic safety Specification (2015 edition) is 1.4 first method of arsenic, 1.2 first method of mercury, and 1.3 first method of lead.
Detection conditions: the temperature is 26 ℃ and the humidity is 62% RH.
The results are shown in the following table:
| group of | Arsenic (As) | Mercury | Lead |
| Example 1 | ＜0.010 | ＜0.0020 | ＜1.5 |
| Example 2 | ＜0.010 | ＜0.0020 | ＜1.5 |
| Example 3 | ＜0.010 | ＜0.0020 | ＜1.5 |
| Example 4 | ＜0.010 | ＜0.0020 | ＜1.5 |
| Example 5 | ＜0.010 | ＜0.0020 | ＜1.5 |
| Example 6 | ＜0.010 | ＜0.0020 | ＜1.5 |
| Comparative example 1 | ＜0.010 | ＜0.0020 | ＜1.5 |
| Comparative example 2 | ＜0.010 | ＜0.0020 | ＜1.5 |
| Comparative example 3 | ＜0.010 | ＜0.0020 | ＜1.5 |
From experiment 1, the antibacterial sustained-release gel prepared by the preparation method provided by the invention has extremely low arsenic, mercury and lead content, is safe to use and meets the requirements.
Experiment 2 stability determination
Instrument apparatus: JA5003N type electronic balance, shimadzu LC-20AT type liquid chromatograph, LHS-250HC-I type constant temperature and humidity box.
Reagent name, grade: 0.1mol/L ammonium acetate buffer solution and acetonitrile.
Standard solution and concentration: 100. Mu.g/L benzalkonium chloride standard solution.
The detection basis is as follows: the disinfection technical Specification 2.2.3.2.1 in 2002 and the cosmetic safety technical Specification (2015) 4.4 benzalkonium chloride.
Instrument conditions: chromatographic column, 250 x 4.6mm,5 μm CN column; column temperature, 25 ℃; sample volume, 20 μl; a flow rate of 1.0mL/min; a detection wavelength, an ultraviolet detector (260 nm); mobile phase, 0.1mol/L ammonium acetate buffer (ph=5.0) +acetonitrile=50+50.
Sample storage conditions: stored at 37℃for 3 months.
Detection conditions: the temperature is 27 ℃ and the humidity is 60% RH.
The results are shown in the following table:
the antibacterial slow-release gel prepared by the invention is stable, and the reduction rate of active ingredients in the gel is low.
Experiment 3 bacteriostasis experiment
Test strain: coli (ATCC 25922), staphylococcus aureus (ATCC 6538), candida albicans (ATCC 10231), the above strains were algebraically 3-5 generations and the bacterial suspension was formulated with 0.03 mol/LPBS.
Graduated pipettes (0.1 mL, 1.0mL, 5.0mL, 10.0 mL), and the like.
The detection method comprises the following steps: the test was carried out with reference to annex C4 of GB15979-2002 hygienic Standard for Disposable hygienic products, with the sample "gel" stock solution, for 2min, 5min, 10min and 20min, and the test repeated 3 times. The test temperature was 24 ℃.
The results of the bacteriostasis rate are shown in the following table:
the slow-release antibacterial gel has good antibacterial effect and slow-release effect, and the antibacterial rate is still more than 99.0% after the gel is used for 10 minutes.
Experiment 4 inhibition of tumor cells
The concentration was 5X 104 Inoculating each/mL human cervical cancer HeLa cell strain into 6-well plate, culturing in DMEM (10% fetal bovine serum) medium for 24 hr, providing 10 test groups, adding the gels provided in the inventive examples 1-7 and comparative examples 1-3, respectively, and final concentration of 1.8X10-5 M. After 24h incubation, the culture medium was discarded, washed 3 times with PBS, and after 20min fixation with 4% paraformaldehyde per well, the liquid was aspirated, washed with PBS, glycerol-blocked, and the viability of HeLa cells was examined, and the results are shown in the following table.
| Group of | Survival of HeLa cells (%) |
| Example 1 | 21.73±1.15 |
| Example 2 | 23.48±1.14 |
| Example 3 | 24.45±0.92 |
| Example 4 | 22.47±0.87 |
| Example 5 | 21.43±1.14 |
| Example 6 | 25.08±0.74 |
| Comparative example 1 | 33.37±0.81 |
| Comparative example 2 | 37.04±0.91 |
| Comparative example 3 | 46.04±1.21 |
From the results of examples and comparative examples, it can be seen that the present invention has a better inhibitory effect on cancer cells, and from the results of examples and comparative example 2, it can be seen that the paclitaxel slow-release microcapsules used in the present invention have a greatly enhanced inhibitory effect on cancer cells than paclitaxel alone.
Experiment 5 drug accumulation Release Rate of sustained Release antibacterial gel at different pH values
3 parts of 15mg paclitaxel slow release microcapsules are weighed and respectively added into PBS buffer solutions with 5ml of pH values of 4, 5 and 6, and slow release is carried out at 37 ℃. Taking 3ml of supernatant after 10 hours, measuring the absorbance of the solution, simultaneously adding 3ml of fresh PBS buffer solution with corresponding pH value, keeping the total volume of the solution unchanged, and finally calculating the cumulative release rate of the drug, wherein the result is shown in the following table:
note that: the drug release rate in this experiment was the maximum drug release rate.
As can be seen from the accumulated drug release rate at different pH values, the drug release rate is lower when the pH value is lower, and the drug release rate is improved along with the gradual increase of the pH value, so that the taxol slow-release microcapsule has pH responsiveness.
In conclusion, the gel has the effects of strong bacteriostasis and antivirus, and keeping vagina clean, and the effect of preventing cervix is achieved by adding a proper amount of taxol slow-release microcapsules and combining melatonin.
The foregoing description of the preferred embodiments of the present invention has been presented for purposes of clarity and understanding, and is not intended to limit the invention to the particular embodiments disclosed, but is intended to cover all modifications, alternatives, and improvements within the spirit and scope of the invention as outlined by the appended claims.