CN113413490B - Ultrasonic-responsive composite hydrogel and preparation method and application thereof - Google Patents
Ultrasonic-responsive composite hydrogel and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an ultrasonic-responsive composite hydrogel and a preparation method and application thereof, belonging to the technical field of medicines. The ultrasonic responsive composite hydrogel is obtained by crosslinking sodium alginate and thioketal ethylene diamine, adding titanium dioxide, and then reacting with calcium chloride; the invention is based on covalent crosslinking between SA and tK and on SA and Ca2+The ionic crosslinking of the hydrogel can maintain the hydrogel structure together, so that the effect of blocking the vas deferens/oviduct is realized; the thioketal bonds among the molecules of the composite hydrogel have an active oxygen cutting response function, and the thioketal bonds are broken by triggering titanium dioxide through ultrasound, so that the noninvasive degradation and fertility recovery functions are realized. The ultrasonic response composite hydrogel can be used for non-invasive reversible recovery of male/female fertility and is also suitable for reversible contraception of male and female animals.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to an ultrasonic responsive composite hydrogel and a preparation method and application thereof.
Background
Contraception is an important and less studied hot problem, particularly male contraception which cannot be satisfactorily achieved by the existing male contraception methods such as vasectomy and condoms. On one hand, vasectomy is a permanent contraceptive method, has poor reversibility, and even if the operation can be carried out for recovery, the effect after the operation is not satisfactory; on the other hand, condoms are the most widely used contraceptive tools for men in sexual life, but the quality of the sexual life caused by the condoms is poor, so that the use will be poor for many men, and even if the condoms are used, the condoms are easy to damage due to improper methods, so that the contraception fails, and the accidental pregnancy rate is increased; moreover, the female contraception also has the problems of operation recovery and poor reversibility. Therefore, in view of the problems faced by contraception, the search for a novel reversible contraception method which is safe and effective, has high acceptability and can recover fertility at any time according to the desire of people has become a practical problem to be urgently solved clinically.
Disclosure of Invention
The invention aims to provide an ultrasonic-responsive composite hydrogel and a preparation method and application thereof, so as to solve the problems in the prior art, realize effective blocking of vas deferens/oviduct, realize recanalization of vas deferens/oviduct after the action of in vitro ultrasonic and ensure good reversible contraception effect.
In order to achieve the purpose, the invention provides the following scheme:
the invention aims to provide an ultrasonic-responsive composite hydrogel which is prepared by crosslinking sodium alginate and thioketone ethylenediamine, adding titanium dioxide, and reacting with calcium chloride.
The invention also aims to provide a preparation method of the ultrasonic-responsive composite hydrogel, which comprises the following steps:
(1) mixing a sodium alginate aqueous solution with thioketal ethylene diamine, and reacting to obtain an SA-tK solution;
(2) adding titanium dioxide into the SA-tK solution to react to obtain SA-tK @ TiO2A solution;
(3) mixing SA-tK @ TiO2And mixing the solution with calcium chloride to obtain the ultrasonic response composite hydrogel.
Sodium Alginate (SA), a by-product of extraction of iodine and mannitol from brown algae such as kelp or gulfweed, is a natural macromolecule with good tissue affinity and little irritation to surrounding tissues, and is formed by connecting β -D-mannuronic acid (M) and α -L-guluronic acid (α -L-guluronic acid, G) by a (1 → 4) bond. It has a-COO-group in its molecule, when it is towards seaWhen calcium ion is added to an aqueous solution of sodium alginate, Na in the G unit+Exchange with these divalent cations and convert the sodium alginate solution to gel. A time-controlled, injectable, white, homogeneous hydrogel was formed. It is closer to living tissue than conventionally synthesized biomaterials, has a surface that is less prone to cell and protein adhesion, and is similar in nature to extracellular matrix components. Through various structural modifications, the sodium alginate hydrogel can have more functions and can be clinically transformed and applied.
In the field of acoustic catalysis, nano TiO2As the acoustic catalyst, it has the advantages of stability, high efficiency, no toxicity and low cost, but the nano TiO has the advantages of high stability, high efficiency, no toxicity and low cost2The acoustic response range of (2) is narrow, the loss of active ingredients is large, and the application of ultrasound is not facilitated. To nanometer TiO2The surface modification can enlarge the sound wave absorption range and improve the sound catalysis efficiency, and in addition, the polymer with better biocompatibility is utilized to carry out nano TiO2The surface is modified to enhance the stability in vivo. The invention firstly prepares functional high molecular polymers with good biocompatibility and high colloid stability, and takes the polymers as surface modifier and TiO as sound-sensitive agent2And carrying out surface modification to realize reversible degradation of the hydrogel.
Further, the concentration of the sodium alginate aqueous solution is 1.0% -5.0%; the molar ratio of the thioketal ethylene diamine to the sodium alginate is 500: 1.
Further, the mass-to-volume ratio of the titanium dioxide to the SA-tK solution is (1.0-2.0) mg:1000 ul.
Further, the calcium chloride is in the form of aqueous solution, and the concentration is 10-50 mg/ml; the SA-tK @ TiO2The volume ratio of the solution to the calcium chloride aqueous solution was 1: 1.
Further, the concentration of the calcium chloride aqueous solution is 10-30 mg/ml.
Further, the method comprises the step of adding a carboxyl activator and a polypeptide condensing agent into the sodium alginate aqueous solution for reaction before mixing the sodium alginate aqueous solution and the thioketone ethylene diamine.
The carboxyl activating agent is 1-ethyl- (3,3' -dimethylaminopropyl) carbodiimide (EDC);
the polypeptide condensing agent is N-hydroxysuccinimide (NHS).
Further, the molar ratio of the sodium alginate to the carboxyl activator to the polypeptide condensing agent is 2:1: 1; the reaction time is not shorter than 10 min.
The invention also aims to provide application of the ultrasonic-responsive composite hydrogel in preparation of a birth control medicament.
Further, the birth control drug is a reversible birth control drug.
The invention discloses the following technical effects:
the invention discloses an ultrasonic degradable composite hydrogel for reversible contraception, which mainly comprises Sodium Alginate (SA), thioketone ethylenediamine (tK) and calcium chloride (CaCl)2) And titanium dioxide (TiO)2) Nanoparticles are composed of four components, through covalent cross-linking between SA and tK and SA and Ca2+The hydrogel structure is maintained by ionic crosslinking, the effect of blocking the vas deferens/oviduct is realized, the gelling and remote noninvasive degradation functions are realized, and the hydrogel structure with high elasticity can be formed in situ at the vas deferens/oviduct; the thioketone bond between the molecules of the compound hydrogel has the function of active oxygen cutting response, and the compound TiO in the gel2The nano particles can generate a large amount of active oxygen molecules under the action of ultrasonic waves to promote the thioketone bonds of the tK molecules to break, so that the ionic crosslinking of the hydrogel is broken, the overall balance of the gel is broken, the solution is finally converted into solution, the solution is discharged out of a body through the vas deferens/oviducts, the aims of noninvasive vas deferens/oviducts recanalization and fertility recovery are fulfilled, the ultrasonic degradable hydrogel is rapidly formed in situ in the vas deferens/oviducts, and reversible vas deferens/oviducts blocking contraception is carried out.
The functional group is grafted on the basis of sodium alginate, and the functional high molecular polymer with better biocompatibility is constructed, so that safe, noninvasive, reversible and efficient contraception is realized.
The invention firstly prepares functional high molecular polymer with good biocompatibility and high colloidal stability, and uses the polymers to polymerizeThe substance is a surface modifier and realizes the purpose of sensitivity to sound agent TiO2The surface modification of the nano TiO material realizes the reversible degradation of the hydrogel and further expands the range of nano TiO2Application in the biomedical field.
The invention can realize that the composite hydrogel is stably retained in the vas deferens/oviduct for a long time and continuously blocks the vas deferens/oviduct to form a hydrogel barrier, thereby preventing sperms/ova from being discharged through the vas deferens/oviduct and realizing effective contraception. Meanwhile, the ultrasonic responsive hydrogel does not generate obvious side effect in vivo, can stably exist in vivo, can play a role in lasting contraception, and is an ideal material for contraception.
The ultrasonic response composite hydrogel can be used for non-invasive reversible recovery of male/female fertility and is also suitable for reversible contraception of male and female animals.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a schematic diagram of the design of an ultrasound-responsive composite hydrogel of the present invention;
FIG. 2 is a fluorescence measurement result of the composite hydrogel material ultrasonically generating active oxygen;
FIG. 3 is a graph showing the effect of simulating the ultrasonic degradation function of a hydrogel in vitro;
FIG. 4 is a graph showing quantitative variation of gel mass for each group;
FIG. 5 shows the gel composition of example 1, PBS buffer, and SA-tK-Ca2+@TiO2Respectively treating the components before and after gel ultrasonic degradation, and performing cell treatment under microscopeObserving;
FIG. 6 shows the experimental verification of the gel composition and SA-tK-Ca of example 1 using CCK82+@TiO2The biotoxicity result of the components before and after gel ultrasonic degradation on cells;
FIG. 7 shows the oestrus cycle of female SD rats and mating monitoring after male and female caging;
FIG. 8 is a schematic illustration of hydrogel permanently and stably occluding vas deferens at an animal level and self-clearing and restoring fertility under ultrasound;
figure 9 is a graph of the effect of long-term stable blocking of vas deferens and self-clearance and restoration of fertility under ultrasound verified at animal level.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
FIG. 1 is a schematic diagram of the design of an ultrasound-responsive composite hydrogel of the present invention.
The ultrasonic responsive composite hydrogel mainly comprises four components, namely Sodium Alginate (SA), thioketal ethylene diamine (tK) and calcium chloride (CaCl)2) And titanium dioxide (TiO)2) And (3) nanoparticles. By covalent crosslinking between SA and tK and SA and Ca2+The ionic crosslinking of the hydrogel can maintain the hydrogel structure together, block the vas deferens/oviduct and realize the birth control function. The thioketone bonds among the molecules of the composite hydrogel have an active oxygen cutting response function, and after the in vitro ultrasonic action, the ultrasonic response molecules TiO in the composite hydrogel have the function of cutting response2The nano particles generate a large amount of active oxygen to promote the thioketone bond to break and the gel to be dispersed, so that noninvasive vasectomy/oviduct recanalization is realized, and the fertility is recovered.
Firstly, SA and tK molecules form SA-tK between fluid and gel states in vitro through intermolecular covalent crosslinking, and then TiO is added2Uniformly mixing the nano particles into SA-tK to obtain SA-tK @ TiO2Subsequently, SA-tK @ TiO2With CaCl2The solution is simultaneously injected into the vas deferens in situ to quickly form hydrogel, block the vas deferens, stop sperms and effectively realize contraception.
The composite hydrogel of the invention keeps a stable gel state in the coexistence of various molecules.
When the ultrasonic responsive composite hydrogel is used for treatment, the hydrogel can be injected in situ to block the vas deferens/oviduct, so that the sperms/ova are prevented from being discharged, and contraception is realized. After the in vitro ultrasonic treatment, the gel is degraded and automatically discharged out of the body, and the purpose of non-invasive reversible fertility recovery can be realized.
The preferred administration mode of the invention is in-situ injection and retrograde perfusion of the urinary tract; further preferred is administration by in situ injection.
Preparation of ultrasonic responsive composite hydrogel
Example 1
An ultrasonic-responsive composite hydrogel is prepared by the following steps:
(1) weighing Sodium Alginate (SA) and putting into a serum bottle filled with distilled water to prepare a uniform 1.0% (w/V) SA solution;
(2) adding EDC and NHS into a reaction system at a dosage of the SA monomer and the molar ratio of 1-ethyl- (3,3' -dimethylaminopropyl) carbodiimide (EDC) to N-hydroxysuccinimide (NHS) of 2:1, and stirring for more than 10min, thereby activating carboxyl on SA molecules, catalyzing and stabilizing the reaction process of SA and thioketal ethylene diamine (tK);
(3) in a molar ratio of SA monomer to tK molecule of 500: adding tK molecules into the solution at a dose of 1 and continuously stirring for at least 6 hours to prepare an SA-tK solution, and refrigerating at the temperature of-20 ℃ for later use;
the structure of SA-tK is shown as formula 1:
(4) TiO is added into SA-tK solution at room temperature according to the proportion of 2.0mg to 1000ul2Stirring the nano particles for more than 10min, mixing uniformly to prepare the SA-tK @ TiO2Refrigerating the solution at-20 ℃ for later use;
(5) at room temperature, adding SA-tK @ TiO2With CaCl2Mixing the aqueous solution (with the concentration of 30mg/ml) according to the volume ratio of 1:1 to obtain the ultrasonic-responsive composite hydrogel SA-tK-Ca2+@TiO2(formula 2).
Example 2
An ultrasonic-responsive composite hydrogel is prepared by the following steps:
(1) weighing Sodium Alginate (SA) and putting into a serum bottle filled with distilled water to prepare a uniform 5.0% (w/V) SA solution;
(2) adding EDC and NHS into a reaction system at a dosage of the SA monomer and the molar ratio of 1-ethyl- (3,3' -dimethylaminopropyl) carbodiimide (EDC) to N-hydroxysuccinimide (NHS) of 2:1, and stirring for more than 10min, thereby activating carboxyl on SA molecules, catalyzing and stabilizing the reaction process of SA and thioketal ethylene diamine (tK);
(3) in a molar ratio of SA monomer to tK molecule of 500: adding tK molecules into the solution at a dose of 1 and continuously stirring for at least 6 hours to prepare an SA-tK solution, and refrigerating at the temperature of-20 ℃ for later use;
(4) TiO is added into SA-tK solution at room temperature according to the proportion of 1.5mg to 1000ul2Stirring the nano particles for more than 10min, mixing uniformly to prepare the SA-tK @ TiO2Refrigerating the solution at-20 ℃ for later use;
(5) at room temperature, adding SA-tK @ TiO2With CaCl2Mixing the aqueous solution (50mg/ml) according to the volume ratio of 1:1 to obtain the ultrasonic-responsive composite hydrogel SA-tK-Ca2+@TiO2。
Example 3
An ultrasonic-responsive composite hydrogel is prepared by the following steps:
(1) weighing Sodium Alginate (SA) and putting into a serum bottle filled with distilled water to prepare a uniform 2.0% (w/V) SA solution;
(2) adding EDC and NHS into a reaction system at a dosage of the SA monomer and the molar ratio of 1-ethyl- (3,3' -dimethylaminopropyl) carbodiimide (EDC) to N-hydroxysuccinimide (NHS) of 2:1, and stirring for more than 10min, thereby activating carboxyl on SA molecules, catalyzing and stabilizing the reaction process of SA and thioketal ethylene diamine (tK);
(3) in a molar ratio of SA monomer to tK molecule of 500: adding tK molecules into the solution at a dose of 1 and continuously stirring for at least 6 hours to prepare an SA-tK solution, and refrigerating at the temperature of-20 ℃ for later use;
(4) TiO is added into the SA-tK solution at room temperature according to the proportion of 1.0mg to 1000ul2Stirring the nano particles for more than 10min, mixing uniformly to prepare the SA-tK @ TiO2Refrigerating the solution at-20 ℃ for later use;
(5) at room temperature, SA-tK @ TiO2With CaCl2Mixing the aqueous solution (10mg/ml) according to the volume ratio of 1:1 to obtain the ultrasonic-responsive composite hydrogel SA-tK-Ca2+@TiO2。
Second, functional verification of ultrasonic generation of active oxygen by material
And (3) verifying the function of the gel for exciting the active oxygen under the ultrasonic action by using a fluorescence spectrophotometer and an active oxygen detection probe DCFH.
DCFH itself does not fluoresce, however in the presence of active oxygen, it can be oxidized to fluorescent substance DCF, the fluorescence intensity of which is proportional to the level of active oxygen. The reactive oxygen species level can be obtained by detecting the fluorescent signal of DCF.
The specific operation method comprises the following steps: DCFH probe solution was mixed with the composite hydrogel SA-tK-Ca prepared in example 1 in a small beaker2+@TiO2After mixing, the mixed solution was administered at 2.25w/cm2The ultrasonic treatment of power, the experimental result shows, as the ultrasonic time lengthens, the fluorescence intensity of the solution gradually increases, proving that the composite hydrogel can gradually generate active oxygen molecules under the ultrasonic stimulation, and the generation speed of the active oxygen is gradually increased as the ultrasonic time lengthens (figure 2). Finally, the composite hydrogel is proved to generate active oxygen under the ultrasonic stimulation.
Thirdly, simulating the ultrasonic degradation function of the gel in vitro
In order to verify the feasibility of the ultrasonic noninvasive fertility restoration strategy, an in vitro simulation experiment is performed. Mixing SA-tK @ TiO2With CaCl2The solution was injected into a polyethylene plastic hose so that it formed a hydrogel within the tube. One group was then given 2.25w/cm2And (3) carrying out ultrasonic treatment with power, stopping ultrasonic treatment for 30s every 5min, observing the gel state and weighing the mass of the residual gel. The gel is gradually degraded along with the prolonging of the ultrasonic process, the gel is completely degraded after 30min, and the gel is not obviously changed when the control group is not subjected to the ultrasonic treatmentAnd (4) transforming.
The specific operation method comprises the following steps:
the polyethylene plastic hose is used for simulating the vas deferens, and the ultrasonic noninvasive fertility recovery strategy is verified in vitro. The SA-tK @ TiO prepared in example 1 was taken2With CaCl2The aqueous solution (concentration 30mg/ml) was simultaneously injected into a polyethylene plastic hose at a volume ratio of 1:1, and left to stand for 3min, and gel was formed and stably remained in the hose. Subsequently, the experimental group (Ultrasound +) used 2.25w/cm2Ultrasonic treatment with power, the control group (ultrasonic-) is placed at room temperature and is not treated, the gel of the experimental group is gradually degraded along with the extension of the ultrasonic process, the gel is completely degraded after 30min, the gel of the control group has no obvious change (figure 3), and the curve of the gel residual mass time is shown in figure 4.
Fourthly, safety verification of hydrogel on cellular level
The cells selected in the experiment are human umbilical vein endothelial cell HUVEC and mouse fibroblast L929 cell line. The gel composition of example 1, PBS buffer and SA-tK-Ca were mixed2+@TiO2The components before and after gel ultrasonic degradation are respectively treated with cells, and the cells are observed under a microscope, and the components have no obvious influence on the cell growth (figure 5). Gel (-) in FIG. 5 is SA-tK-Ca without sonication2+@TiO2Gel group, Gel (+) is SA-tK-Ca after ultrasonic treatment2+@TiO2And (4) gel group.
CCK8 experiment verified the gel composition of example 1 and SA-tK-Ca2+@TiO2The components before and after gel sonication were not significantly toxic to cell growth (figure 6). Gel (-) in FIG. 6 is SA-tK-Ca without sonication2+The Gel group of @ TiO2, Gel (+) is SA-tK-Ca which has been treated with ultrasound2+@ TiO2 gel group.
All the experiments prove that the hydrogel and all the components thereof have good safety to cells.
Fifthly, the hydrogel realizes the functions of long-acting stable contraception and remote noninvasive ultrasonic self-clearing fertility recovery on the animal level
Animal experiments are carried out to further verify the long-acting stable contraception effect of the hydrogel and the effect of restoring fertility by remote noninvasive ultrasonic self-cleaning.
Experimental group SA-tK @ TiO prepared in example 12With CaCl2Aqueous solution (concentration 30mg/ml) was injected into rat vas deferens in situ at a volume ratio of 1:1, PBS was injected into rat vas deferens in control group 1, vas deferens ligation operation was performed on rats in control group 2, and each male mouse and female mouse were caged 6 days after the treatment, and their sexual behavior was observed and recorded (fig. 7). In the control group 1, mice were born 18 days after cage-closure, but no new mice were still present at day 54 in the experimental group, which proves that the hydrogel has a stable contraceptive effect, and then at day 54, after the ultrasonic treatment of the rats in vitro for 8min, the sexual behavior of the rats is continuously observed, and as a result, new mice also appear in the experimental group 18 days after ultrasonic treatment. The successful degradation of the hydrogel after ultrasonic treatment and the recanalization of the vas deferens are proved, and the fertility of the rat is restored. While the control group 2, which had been subjected to vasectomy, had no newborn mice at all times. FIG. 8 is a schematic illustration of hydrogel permanently and stably occluding vas deferens at an animal level and self-clearing and restoring fertility under ultrasound; fig. 9 is a graph of the effect of the hydrogel on animal level for verifying that the hydrogel in the test group can stably block vas deferens for a long time and can automatically clear and restore fertility under the action of ultrasound.
The above results all demonstrate that the composite hydrogel (SA-tK-Ca)2+@TiO2) Has long-acting and stable contraceptive effect, has remote noninvasive ultrasonic cleaning capability and realizes the effect of noninvasive fertility recovery.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (9)
1. The ultrasonic-responsive composite hydrogel is characterized in that the hydrogel is obtained by crosslinking sodium alginate and thioketone ethylene diamine, adding titanium dioxide, and then reacting with calcium chloride.
2. A method for preparing the ultrasound-responsive composite hydrogel according to claim 1, comprising the steps of:
(1) mixing a sodium alginate aqueous solution with thioketal ethylene diamine, and reacting to obtain an SA-tK solution;
(2) adding titanium dioxide into the SA-tK solution to react to obtain SA-tK @ TiO2A solution;
(3) mixing SA-tK @ TiO2And mixing the solution with calcium chloride to obtain the ultrasonic responsive composite hydrogel.
3. The method for preparing the ultrasound-responsive composite hydrogel according to claim 2, wherein the mass volume concentration of the sodium alginate aqueous solution is 1.0% -5.0%; the molar ratio of the thioketal ethylene diamine to the sodium alginate is 500: 1.
4. The method for preparing the ultrasound-responsive composite hydrogel according to claim 3, wherein the mass-to-volume ratio of the titanium dioxide to the SA-tK solution is (1.0-2.0) mg:1000 uL.
5. The method for preparing the ultrasound-responsive composite hydrogel according to claim 4, wherein the calcium chloride is in the form of an aqueous solution having a concentration of 10 to 50 mg/mL; the SA-tK @ TiO2The volume ratio of the solution to the calcium chloride aqueous solution was 1: 1.
6. The method for preparing the ultrasound-responsive composite hydrogel according to claim 2, further comprising a step of adding a carboxyl activator and a polypeptide condensing agent to the aqueous solution of sodium alginate before mixing the aqueous solution of sodium alginate with ethylenediamine thioketal to perform a reaction.
7. The method for preparing the ultrasound-responsive composite hydrogel according to claim 6, wherein the molar ratio of the sodium alginate to the carboxyl activator to the polypeptide condensing agent is 2:1: 1.
8. Use of the ultrasound-responsive composite hydrogel according to claim 1 for the preparation of a birth control medicament.
9. The use according to claim 8, wherein the birth control drug is a reversible birth control drug.
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