TiO2 nanoparticles as a carrier of botulinum toxin type A and their preparation method

Abstract

The application discloses a TiO2 nanoparticle as a botulinum toxin type A carrier and a preparation method of the TiO2 nanoparticle. The TiO2 nanoparticle is used as a botulinum toxin type A carrier for bladder perfusion and comprises the following steps: S1, synthesis of urchin-like nanoparticles; S2, preparation of a liposome film; and S3, preparation of drug-loaded nanoparticles. The urchin-like nanostructure is used as a drug carrier to deliver botulinum toxin, so that efficient intracavitary drug administration can be realized, and the nano-carrier technology can be applied to the clinic, thereby providing a safer and more effective treatment method for OAB patients.

Description

Technical Field

[0001] This invention relates to the technical field of drug-loaded nanoparticles, and more specifically, to a TiO2 nanoparticle used as a carrier for type A botulinum toxin for bladder instillation and its preparation method. Background Technology

[0002] Overactive bladder (OAB) is a syndrome characterized by urinary urgency, often accompanied by urinary frequency and nocturia, with or without urge incontinence. Studies indicate that OAB affects the urinary health of 12% to 17% of the global population. A 2017 domestic epidemiological survey showed a prevalence of 23.9% in people over 40 years of age, with the prevalence increasing with age. OAB severely impairs urinary function, and if left untreated, it can lead to serious complications such as vesicoureteral reflux, hydronephrosis, and even renal failure, impacting both quality of life and survival. Intravesical injection of botulinum toxin is an effective treatment for OAB. Because the molecular weight of the commonly used botulinum toxin type A is 150 kDa, it cannot diffuse across the bladder epithelial barrier to reach the submucosal nerve plexus in solution to exert its effect. Therefore, in clinical practice, botulinum toxin type A is administered directly by injection into the submucosal or muscle layer. However, botulinum toxin type A injection therapy has many limitations, such as drug leakage or uneven distribution, bleeding, pain, and infection risks, which limit its clinical application.

[0003] Intravesical drug delivery, or bladder instillation, is a commonly used drug administration method in urology, offering unique anatomical advantages. The drug solution, injected into the bladder via the urethra, acts directly on the epithelial cells, facilitating dissolution, diffusion, and transmembrane absorption, while avoiding the severe side effects of systemic administration. However, the physiological characteristics of the bladder and the physiological barriers of its mucosa, such as the urinary environment, urine flushing, and the bladder permeability barrier (BPB), limit intravesical drug retention and permeability, resulting in low bioavailability as drug components cannot effectively penetrate into the submucosa or muscle layer to exert their effects. This is especially true for large molecule drugs like botulinum toxin type A, which cannot be diffused and absorbed via bladder instillation in the form of ordinary solvents.

[0004] Nanoparticle drug carriers encapsulate drugs within nanoscale particles, thereby improving the solubility of hydrophobic drugs, enhancing their pharmacokinetics, increasing drug concentration at the lesion site, and overcoming drug resistance. This ultimately improves therapeutic efficacy and reduces drug toxicity. Furthermore, the application of nanoparticle-loaded drugs in bladder instillation further enhances drug retention and transmembrane absorption efficiency, improving bioavailability. However, bladder physiology and the bladder permeability barrier remain major obstacles in nanoparticle design. Therefore, improving botulinum toxin nanoparticle carriers, prolonging their retention time in the bladder, enhancing their permeability, and improving botulinum toxin bioavailability are urgent problems and highly challenging research directions.

[0005] Existing studies have reported the use of liposomes, thermosensitive polymer hydrogels, and hyaluronic acid-phosphatidylethanolamine as drug delivery carriers for bladder instillation of botulinum toxin in clinical trials or animal experiments. All three have demonstrated some therapeutic efficacy in clinical or animal studies, with liposomes being the most extensively studied. Randomized, double-blind clinical trials have shown that a single instillation of liposomal botulinum toxin can effectively relieve urinary frequency, but it cannot improve urge incontinence, and the effect only lasts for one month. Analysis based on the anatomical and physiological structure of the bladder reveals that the urinary environment, urine flow, and the presence of the bladder permeability barrier are the main factors limiting drug permeation and utilization within the bladder. Current drug delivery systems have not overcome the inherent bladder barrier's obstruction of drugs, failing to effectively penetrate into the submucosa or muscle layer to exert their effects. Therefore, all of the above nanocarriers suffer from low drug utilization and poor transmembrane transport capabilities, resulting in limited therapeutic effects of botulinum toxin, and their delivery methods require further improvement. Summary of the Invention

[0006] To address the aforementioned technical problems in related technologies, this invention provides TiO2 nanoparticles as a carrier of type A botulinum toxin and a method for their preparation, which can solve the above problems.

[0007] To achieve the above-mentioned technical objectives, the technical solution of the present invention is implemented as follows: A method for preparing TiO2 nanoparticles as a carrier of type A botulinum toxin includes the following steps: S1. Synthesis of sea urchin-like nanoparticles: First, sodium titanate powder is prepared from TiO2 powder. Then, sodium titanate peak particles are prepared from sodium titanate powder. TiO2 peak particles are then prepared from sodium titanate peak particles. The TiO2 peak particles are collected, and smaller TiO2 peak particles are separated by sedimentation. S2. Preparation of liposome membranes: 2 mg palmitoyl oleoyl phosphatidylcholine was dissolved in 10 ml chloroform, gently shaken to mix, and then treated with ultrasound for 10 s. The organic solvent was then removed by evaporation on a rotary evaporator to obtain POPC liposome membrane. S3. Preparation of drug-loaded nanoparticles: Type A botulinum toxin crystals, prepared sea urchin-like nanoparticles, and prepared liposome films were dissolved together in PBS and stirred at room temperature to obtain self-assembled botulinum toxin-loaded sea urchin nanoparticles.

[0008] Furthermore, step S1 also includes: S1-1. Disperse 1g of TiO2 powder in 40 ml of NaOH aqueous solution with a molar concentration of 5mol / L, stir for 30 minutes, then transfer to a fluorine-lined stainless steel autoclave, heat at 120℃ for 24h, then obtain the precipitate by centrifugation, wash with deionized water until pH is 10.5, and finally vacuum dry at 60℃ to obtain sodium titanate powder. S1-2. Mix 19 ml of 1 mol / L NaOH aqueous solution with 1.25 ml of 30% hydrogen peroxide, then disperse 0.1 g of sodium titanate powder in the mixture. Stir the resulting suspension magnetically and treat it with ultrasound for 30 minutes. Then transfer it to a stainless steel autoclave lined with Teflon and heat it at 150°C for 8 hours. Obtain the precipitate by centrifugation, wash it with deionized water until the pH is 7, and finally vacuum dry it at 60°C to produce sodium titanate spike particles. S1-3. Sodium titanate spike particles were treated with an excess of HNO3 solution with a molar concentration of 0.05 mol / L under stirring to generate hydrogen titanate spinel particles. Then, the hydrogen titanate spike particles were washed five times with deionized water and calcined at 400℃ for 1 h to generate TiO2 spike particles.

[0009] Furthermore, the TiO2 powder in step S1-1 is Degussa P25.

[0010] Furthermore, the sodium titanate powder in step S1-1 is a one-dimensional nanostructure bundle of sodium titanate.

[0011] Furthermore, in step S1, separating the smaller TiO2 peak particles by sedimentation includes: resuspending the collected TiO2 peak particles in 1 ml of sterile water, and then pouring them into a 15 ml centrifuge tube containing 9 ml of sterile water for natural sedimentation. After sedimentation for 30 minutes, the TiO2 peak particles located on the upper layer of sterile water in the centrifuge tube are collected.

[0012] Furthermore, the smaller TiO2 spike particles are TiO2 spiny microspheres, which have a spiny sea urchin-like structure. Before use, the TiO2 spiny microspheres need to be washed five times with sterile water, sterilized by high pressure, and stored at 4°C.

[0013] Furthermore, all operations in steps S1 to S3 are performed in a non-toxic and sterile environment.

[0014] A TiO2 nanoparticle as a carrier of botulinum toxin type A, wherein the TiO2 nanoparticle is used as a carrier of botulinum toxin type A for bladder instillation.

[0015] The beneficial effects of this invention are as follows: By using sea urchin-like nanostructures as drug carriers to deliver botulinum toxin, efficient intravesical drug delivery can be achieved, and it is expected that this nanocarrier technology can be applied in clinical practice, providing a safer and more effective treatment method for OAB patients. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] The present invention will now be described in further detail with reference to the accompanying drawings.

[0018] Figure 1 This is a schematic diagram illustrating the synthesis of sea urchin-like TiO2 nanoparticles according to an embodiment of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0020] like Figure 1 As shown, the present invention discloses a TiO2 nanoparticle as a carrier of type A botulinum toxin and its preparation method. The TiO2 nanoparticle is used as a carrier of type A botulinum toxin for bladder instillation, including the synthesis of sea urchin-like nanoparticles, the synthesis of liposome films, and the mixing of type A botulinum toxin with the two to form a composite.

[0021] The sea urchin-like nanoparticles were synthesized through a two-step hydrothermal reaction under optimized conditions, forming a spiny sea urchin-like structure from nanospheres, which was then used in subsequent synthesis processes. Specifically, 1g of TiO2 powder (Degussa P25) was dispersed in 40... In a 5 mol / L NaOH aqueous solution, the mixture was stirred for 30 minutes, then transferred to a fluorine-lined stainless steel autoclave and heated at 120°C for 24 hours. The precipitate was then obtained by centrifugation, washed with deionized water until the pH reached approximately 10.5, and finally vacuum-dried at 60°C to obtain sodium titanate powder (a one-dimensional nanostructure bundle of sodium titanate). Alternatively, 19 ml of a 1 mol / L NaOH aqueous solution was mixed with 1.25 ml of 30% hydrogen peroxide, and 0.1 g of sodium titanate powder was dispersed in the mixture. The resulting suspension was magnetically stirred and ultrasonically treated for 30 minutes, then transferred to a Teflon-lined stainless steel autoclave and heated at 150°C for 8 hours. The precipitate was obtained by centrifugation, washed with deionized water until the pH reached approximately 7, and finally vacuum-dried at 60°C to produce titanium dioxide. Sodium titanate spike particles were treated with an excess of 0.05 mol / L HNO3 solution under stirring to generate hydrogen titanate spinel particles. The hydrogen titanate spike particles were then washed five times with deionized water and calcined at 400℃ for 1 hour to generate TiO2 spike particles. The TiO2 spike particles were collected and resuspended in 1 ml of sterile water. This was then poured into a 15 ml centrifuge tube containing 9 ml of sterile water for natural sedimentation. After sedimentation for 30 minutes, the TiO2 spike particles in the upper layer of the sterile water (approximately 4 ml of TiO2 spike particles in the upper layer of sterile water) were collected. Smaller TiO2 spike particles were separated. These smaller TiO2 spike particles were washed five times with sterile water before use, sterilized by autoclaving, and stored at 4℃.

[0022] The synthesis of liposome membranes involved dissolving 2 mg of palmitoyl oleoyl phosphatidylcholine in 10 ml of chloroform, gently shaking to mix, and then treating with ultrasound for 10 seconds. The organic solvent was then removed by evaporation in a rotary evaporator to obtain POPC liposome membranes.

[0023] Type A botulinum toxin is mixed with the two to form a complex. The botulinum toxin crystals, the prepared sea urchin-like nanoparticles, and the prepared liposome film are dissolved together in PBS (37°C) and stirred at room temperature to dissolve, thus obtaining self-assembled botulinum toxin-loaded sea urchin nanoparticles.

[0024] It should be noted that all of the above operations are performed in a non-toxic and sterile environment.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing TiO2 nanoparticles loaded with botulinum toxin type A, characterized in that, The TiO2 nanoparticles serve as a carrier for type A botulinum toxin administered via bladder instillation. The sea urchin-like nanoparticles are synthesized via a two-step hydrothermal reaction under optimized conditions, forming a spiny sea urchin-like structure from nanospheres, which is then used in subsequent synthesis processes. The specific steps include: S1, Synthetic sea urchin-like nanoparticles: S1-1. 1g of TiO2 powder is dispersed in 40 ml of NaOH aqueous solution with a molar concentration of 5mol / L and stirred for 30 minutes. Then, it is transferred to a fluorine-lined stainless steel autoclave and heated at 120℃ for 24 hours. The precipitate is then obtained by centrifugation, washed with deionized water until the pH is 10.5, and finally vacuum dried at 60℃ to obtain sodium titanate powder. The sodium titanate powder is a one-dimensional nanostructure bundle of sodium titanate. S1-2. Mix 19 ml of 1 mol / L NaOH aqueous solution with 1.25 ml of 30% hydrogen peroxide, then disperse 0.1 g of sodium titanate powder in the mixture. Stir the resulting suspension magnetically and sonicate it for 30 minutes. Transfer the mixture to a Teflon-lined stainless steel autoclave and heat it at 150°C for 8 hours. Centrifuge the precipitate, wash it with deionized water until pH=7, and vacuum dry it at 60°C to obtain sodium titanate peak particles. S1-3. Sodium titanate spike particles were treated with an excess of HNO3 solution with a molar concentration of 0.05 mol / L under stirring to generate spinel hydrogen titanate particles. The particles were washed five times with deionized water and calcined at 400℃ for 1 h to obtain TiO2 spike particles. The TiO2 peak particles were resuspended in 1 ml of sterile water, added to a 15 ml centrifuge tube containing 9 ml of sterile water, and allowed to settle naturally for 30 minutes. The small-sized TiO2 peak particles in the supernatant were collected. The small-sized TiO2 spike particles are TiO2 spiny microspheres, exhibiting a spiny sea urchin-like structure; The TiO2 spiny microspheres were washed five times with sterile water and autoclaved, and stored at 4°C. S2. Preparation of liposome films: 2 mg palmitoyl oleoyl phosphatidylcholine was dissolved in 10 ml chloroform, shaken to mix, and sonicated for 10 s. The organic solvent was removed by rotary evaporation to obtain POPC liposome membrane. S3. Preparation of drug-loaded nanoparticles: Type A botulinum toxin crystals, the sea urchin-like nanoparticles, and the liposome film were dissolved together in PBS and stirred at room temperature to prepare self-assembled botulinum toxin-loaded sea urchin nanoparticles.

2. The method for preparing TiO2 nanoparticles loaded with botulinum toxin type A according to claim 1, characterized in that, The TiO2 powder in step S1-1 is Degussa P25.

3. The method for preparing TiO2 nanoparticles loaded with botulinum toxin type A according to claim 1, characterized in that, All operations in steps S1 to S3 are performed in a non-toxic and sterile environment.

4. A TiO2 nanoparticle loaded with botulinum toxin type A, obtained by the preparation method according to any one of claims 1 to 3, characterized in that, These TiO2 nanoparticles serve as a carrier for type A botulinum toxin administered via bladder instillation.

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