Floating protein microbubble loaded with BCG (Bacillus Calmette Guerin) and application of floating protein microbubble in perfusion therapy of bladder cancer

Abstract

The invention relates to a BCG (Bacillus Calmette Guerin)-loaded floating protein microbubble and application thereof in perfusion therapy of bladder cancer. The invention discloses a BCG (Bacillus Calmette Guerin)-loaded floating protein microbubble. The BCG-loaded floating protein microbubble comprises a shell structure and an inner-layer gas nucleus structure, the shell structure is a cross-linked protein layer skeleton, the surface of the cross-linked protein layer skeleton is modified with hexadecyl trimethyl ammonium bromide, and BCG is loaded on the surface of the cross-linked protein layer skeleton under the action of the BCG and the hexadecyl trimethyl ammonium bromide. The gas filled in the inner layer can realize stable floating of the protein microvesicles in the bladder; the outer layer is loaded with the bacillus calmette-guerin vaccine, and controllable release of the bacillus calmette-guerin vaccine can be achieved under the condition of natural degradation or ultrasonic stimulation; meanwhile, gas in the microbubbles and a crosslinked protein shell form an ultrasonic impedance difference interface, and visualization of drug carrier distribution under ultrasound is achieved. Therefore, the floating protein microbubble can enhance the local immune stimulation effect in the bladder by prolonging the residence time and the controllable release characteristic of the BCG, and an innovative solution integrating in-situ long-time residence, visual monitoring, drug controlled release and long-acting treatment is provided for bladder cancer perfusion immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and in particular relates to floating protein microbubbles loaded with BCG and application thereof in bladder cancer perfusion therapy. Background Art

[0002] Bladder cancer is one of the most common urinary tract malignancies worldwide, with over 420,000 new cases annually. Approximately 70% of newly diagnosed patients are non-muscle invasive bladder cancer (NIBIC). The standard treatment for NBIBC is transurethral bladder tumor resection combined with postoperative intravesical instillation. Based on disease progression risk stratification, the standard postoperative treatment for patients with intermediate- and high-risk NBIBC is intravesical BCG immunotherapy. BCG activates the host's Th1 immune response, inducing antigen-presenting cells to capture BCG antigens and present them to CD4+ and CD8+ T lymphocytes, promoting the differentiation of cytotoxic T cells and ultimately achieving tumor cell clearance. Intravesical BCG immunotherapy significantly reduces the recurrence rate and risk of progression in patients with NBIBC. Its efficacy depends on the effective residence time of BCG in the bladder. However, clinical practice has shown that after intravesical instillation, BCG is rapidly excreted in the urine, resulting in a limited duration of effective action within the bladder and severely limiting its immune activation efficiency.

[0003] In order to prolong the residence time of BCG drugs in the bladder, researchers have proposed a variety of long-term delivery carriers in the bladder, mainly including the following two categories: (1) Adhesive hydrogel delivery carriers: Hydrogels that can be loaded with drugs are prepared by bioadhesive materials (such as chitosan and hyaluronic acid) and adhered to the surface of the bladder epithelium to achieve long-term drug retention. Although this type of carrier can prolong the drug release time, long-term adhesion may stimulate the bladder wall, leading to inflammatory reactions and abnormal urodynamics, and even induce bladder fibrosis. (2) Magnetic field responsive delivery carriers: Drugs are loaded through magnetic nanoparticles and targeted retention is achieved with the help of an external magnetic field. However, the use of this carrier depends on complex external equipment, and the clinical application scenarios are limited. In addition, both of the above-mentioned carriers are difficult to achieve precise control of drug release and lack real-time monitoring methods. Therefore, it is urgent to develop a new type of long-term drug delivery carrier in the bladder that integrates long-term in situ residence, visual monitoring, drug controlled release and long-term treatment.

[0004] In recent years, microbubble-based drug delivery vehicles have attracted widespread attention due to their unique physicochemical properties. Microbubbles are usually composed of a gas core (such as air, fluorocarbon gas) and a coating material (such as protein, lipid or polymer). Their density is lower than that of body fluids, and they can form a stable floating layer on the liquid surface through the buoyancy effect. In addition, microbubbles can be loaded with drugs and achieve controlled release of drugs through material degradation or external stimulation (such as ultrasound). In the intravesical application scenario, floating microbubbles have the following potential advantages: (1) They float on the urine during the bladder filling period, avoiding direct contact with the bladder wall and reducing adhesion-related side effects such as mechanical stimulation of the bladder; (2) During urination, they are retained in the bladder trigone as the liquid level drops, avoiding loss with urine, significantly reducing the drug excretion rate; (3) The gas core allows microbubbles to be used as ultrasound contrast agents to visualize the drug delivery process. Patent CN115737589A discloses adhesive drug microspheres comprising an outer layer, an intermediate layer, and an inner layer; the outer layer is an adhesive layer composed of a positively charged hydrophilic polymer material, the intermediate layer and the inner layer are respectively a cross-linked shell layer and a porous inner layer composed of a water-soluble protein or a water-soluble polymer, the porous interior being filled with gas and used to load the drug. When the microspheres are loaded with drugs for treating bladder diseases, bladder instillation therapeutic microsphere preparations can be obtained, which can be used in the fields of tumor targeting or image enhancement. However, due to the large size of BCG, it cannot be directly incubated into the microbubbles through a concentration gradient like other small molecule drugs, nor can it be simply released through a concentration gradient like other small molecule drugs. Currently, there are no reports of floating protein microbubbles loaded with BCG and applied to bladder cancer immunoperfusion therapy. Summary of the Invention

[0005] The object of the present invention is to overcome the above-mentioned defects and shortcomings in the prior art and provide a floating protein microbubble loaded with BCG, comprising an outer shell structure and an inner gas core structure; the outer shell structure is a cross-linked protein layer, the outer shell surface is modified with hexadecyltrimethylammonium bromide, and the BCG is loaded on the surface of the floating protein microbubble through hexadecyltrimethylammonium bromide; the inner layer is filled with gas.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned BCG-loaded floating protein microbubbles.

[0007] The third object of the present invention is to provide the use of the above-mentioned BCG-loaded floating protein microbubbles in the preparation of bladder cancer instillation therapeutic drugs.

[0008] The fourth object of the present invention is to provide a preparation of floating protein microbubbles loaded with the above-mentioned BCG vaccine.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions: The present invention provides a floating protein microbubble loaded with BCG, comprising an outer shell structure and an inner gas core structure; the outer shell structure is a cross-linked protein layer skeleton, the surface of the cross-linked protein layer skeleton is modified with hexadecyltrimethylammonium bromide, and the BCG is loaded on the surface of the cross-linked protein layer skeleton through the reaction with hexadecyltrimethylammonium bromide.

[0010] BCG Vaccine is a live vaccine made from attenuated bovine tuberculosis bacteria. BCG bacteria are hydrophobic and carry a negative surface charge. The combined effects of hydrophobicity and negative charge, as well as BCG's limited duration of action in the bladder, can affect its mucosal adsorption and immune activation during intravesical instillation therapy. Therefore, the present invention first prepared floating protein microbubbles surface-modified with cetyltrimethylammonium bromide. Cetyltrimethylammonium bromide is a common surfactant currently used primarily for emulsification and stabilization of hydrophobic chemotherapy drugs (e.g., in the construction of small molecule drug carriers such as paclitaxel). However, the present invention demonstrates that cetyltrimethylammonium bromide can be adapted to the properties of negatively charged active drugs, allowing BCG to be directly loaded onto the surface of floating protein microbubbles via cetyltrimethylammonium bromide. This solves the problem of BCG's large size preventing smooth microbubble entry and exit, and effectively retaining BCG in the bladder. The microbubbles described in the present invention not only possess BCG loading capacity but also enable real-time monitoring of drug carrier distribution under ultrasound.

[0011] Furthermore, the particle size of the microbubbles is 60 to 140 μm. The appropriate particle size ensures that the floating protein microbubbles loaded with BCG can be injected into the bladder through the urethra, avoiding the risk of urethral obstruction.

[0012] Furthermore, the gas core size of the inner gas core structure is 60-120 μm.

[0013] Furthermore, the protein of the cross-linked protein layer includes but is not limited to bovine serum albumin, bovine hemoglobin, gelatin or a complex thereof.

[0014] Furthermore, the cross-linking agent used in the cross-linked protein layer includes but is not limited to phthaloyl chloride or glutaraldehyde.

[0015] Furthermore, the types of gases filled in the inner gas core structure include but are not limited to air, sulfur hexafluoride, oxygen, perfluoropropane or mixed gases.

[0016] Furthermore, the BCG loading rate on the surface of the BCG-loaded floating protein microbubbles is 65% to 75%.

[0017] The present invention also provides a method for preparing the above-mentioned microbubbles, comprising the following steps: S1. The protein aqueous solution is prepared into droplets by emulsification; S2. adding a crosslinking agent to the droplets obtained in step S1 to crosslink to obtain microspheres having a crosslinked protein shell; S3. The microspheres obtained in step S2 are freeze-dried, vacuumed, and filled with gas to obtain floating protein microbubbles containing gas in the inner layer; S4. The surface of the floating protein microbubbles obtained in step S3 is modified with cetyltrimethylammonium bromide, and then incubated with BCG to obtain floating protein microbubbles loaded with BCG.

[0018] Furthermore, the surfactant used in the emulsification method in step S1 includes but is not limited to Span 85.

[0019] Furthermore, the emulsification method in step S1 is specifically to mix the protein solution in an organic solvent containing a surfactant, and the droplet size (60-140 μm) can be controlled by adjusting the speed of the homogenizer (200-500 rpm).

[0020] Furthermore, the concentration of the protein aqueous solution is 5 to 30 wt%.

[0021] Furthermore, the concentration of the surfactant is 3 to 12 wt%.

[0022] Furthermore, in step S4, the added mass ratio of the floating protein microbubbles whose surfaces are modified with hexadecyltrimethylammonium bromide to the BCG vaccine is 1.8-2.2:1.

[0023] The floating protein microbubbles loaded with BCG provided by the present invention are filled with gas and have a density lower than that of urine, thereby being able to float on the urine in the bladder; the outer shell is a cross-linked protein layer, which has a certain mechanical strength and is not easily deformed. It can also prevent the gas in the internal gas core structure from escaping, thereby prolonging the floating time of the protein microbubbles in the urine. The upper surface of the outer shell is modified with hexadecyltrimethylammonium bromide, and the BCG is loaded on the surface of the floating protein microbubbles through hexadecyltrimethylammonium bromide. The long-term release of the BCG is achieved through the natural degradation of the microbubbles or under ultrasonic stimulation conditions, so that it plays a therapeutic role in bladder cancer infusion. In addition, the gas core structure inside the microbubbles and the protein cross-linked shell form an ultrasonic impedance difference interface, and also have the function of ultrasonic visualization, which can realize real-time monitoring and dynamic tracking of bladder infusion drug carriers under ultrasound. The appropriate particle size ensures that the floating protein microbubbles loaded with BCG can be injected into the bladder through the urethra, avoiding the risk of urethral obstruction.

[0024] During implementation, the BCG-loaded floating protein microbubble formulation is retrogradely injected into the bladder through the urethra. The microbubble formulation resides in the bladder for a long time through a physical flotation mechanism, remaining within the bladder wall during urination and preventing excretion. When the urine is refilled, the microbubble formulation resumes its floating state. By extending the BCG's residence time and enabling controlled release, the floating protein microbubble formulation enhances the local immune stimulation within the bladder, providing an innovative solution for instillation immunotherapy of bladder cancer that combines long-term in situ retention, visual monitoring, controlled drug release, and long-term treatment.

[0025] The present invention also provides the use of the above-mentioned BCG-loaded floating protein microbubbles in the preparation of bladder cancer perfusion therapeutic drugs.

[0026] The present invention also provides a preparation comprising the above-mentioned floating protein microbubbles loaded with BCG.

[0027] The present invention also provides a bladder cancer perfusion immunotherapy method based on floating protein microbubbles loaded with BCG, wherein the microbubbles are retrogradely injected into the bladder through the urethra, and the microbubbles float on the urine; the BCG is controllably released by natural degradation of the microbubbles or low-frequency ultrasound stimulation; and during the treatment process, the distribution of the drug carrier is monitored using a high-frequency ultrasound probe of a conventional clinical diagnostic ultrasound instrument.

[0028] Furthermore, the parameters of the low-frequency ultrasound stimulation are low frequency 0.1-2 MHz, mechanical index MI 0.2-0.7, duty cycle 5-30%, pulse repetition frequency 1-5 Hz, and sound pressure amplitude 0.05-0.8 MPa.

[0029] Furthermore, the parameter of the high-frequency ultrasonic monitoring is 7 to 15 MHz.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a floating protein microbubble loaded with BCG, comprising an outer shell structure and an inner gas-core structure; the outer shell structure is a cross-linked protein layer skeleton, the surface of the cross-linked protein layer skeleton is modified with hexadecyltrimethylammonium bromide, and the BCG is loaded on the surface of the cross-linked protein layer skeleton through the interaction with the hexadecyltrimethylammonium bromide; the inner layer is filled with gas. The protein cross-linked layer outer shell can prevent the gas in the internal gas-core structure from escaping, making the density of the microbubble lower than that of urine, and achieving stable floating of the protein microbubble in the bladder; the hexadecyltrimethylammonium bromide modified with the outer shell loads the BCG on the surface of the floating protein microbubble, and by regulating the mechanical strength of the microbubble outer shell, the microbubble is ruptured under natural degradation or ultrasonic stimulation conditions, thereby achieving controlled release of the BCG; the gas in the microbubble forms an ultrasonic impedance difference interface with the cross-linked protein shell, enabling the microbubble to be used as an ultrasound-enhanced contrast agent, realizing visualization of the distribution of the drug carrier under ultrasound. Therefore, the floating protein microbubbles described in the present invention can enhance the local immune stimulation effect in the bladder by prolonging the residence time and controlled release characteristics of BCG in the bladder, providing an innovative solution for bladder cancer perfusion immunotherapy that integrates long-term in situ residence, visual monitoring, controlled drug release and long-term treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a fluorescence micrograph of floating protein microbubbles loaded with BCG in their shells.

[0032] Figure 2 This is an optical image of floating protein microbubbles loaded with BCG floating above urine.

[0033] Figure 3 This is a graph showing the cumulative release curve of floating protein microbubbles loaded with BCG in urine.

[0034] Figure 4 Ultrasound image of floating protein microbubbles loaded with BCG retained in the rat bladder. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0036] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0037] Example 1 Preparation Method of Floating Protein Microbubbles Loaded with BCG in the Shell BCG was fluorescently stained using auramine O. A bovine serum albumin solution (1 mL, 10 wt%) was added to a toluene solution (20 mL) containing Span 85 (10 wt%) and emulsified using a homogenizer (500 rpm, 3 min) to form water-in-oil droplets. To the droplets, 1 mL of a supersaturated toluene solution containing terephthaloyl chloride was added and vortexed at 25°C for 5 min to crosslink the droplets, forming protein microspheres with a stable shell structure. After centrifugation (1000 rpm, 2 min), the resulting microsphere precipitate was washed 3–5 times with anhydrous ethanol and then washed three times with pure water to remove residual ethanol. The resulting microsphere precipitate was frozen at low temperature and then freeze-dried in a freeze dryer. The resulting floating protein microbubbles were sealed in a container, evacuated, and then filled with oxygen. Add 5 mL of 10 wt% cetyltrimethylammonium bromide solution to the container containing floating protein microbubbles and incubate on a shaker at room temperature (100 rpm) for 2 h. After incubation, wash the container three times with saline to obtain floating protein microbubbles surface-modified with cetyltrimethylammonium bromide. Weigh 20 mg of cetyltrimethylammonium bromide-modified floating protein microbubbles into a centrifuge tube, add 10 mg of lyophilized BCG powder, and incubate on a shaker at room temperature (100 rpm) for 2 h. After incubation, wash the container three times with saline and centrifuge (1000 rpm, 2 min) to isolate the floating protein microbubbles with BCG attached to their outer shell.

[0038] in .

[0039] The results are as follows Figure 1 As shown in FIG, BCG is loaded on the shell of floating protein microbubbles, wherein the loading rate of BCG is 72%. Figure 1 It can be seen that BCG is unevenly distributed on the surface of the microbubbles and does not cover the entire surface of the microbubbles. This is mainly because: ① Unlike ordinary drug solutions, BCG is a larger microorganism and cannot be evenly dispersed in the solution. Its size is also uneven and it cannot be evenly spread to all areas of the microbubble surface; ② The distribution of hexadecyltrimethylammonium bromide on the microbubble surface is not completely uniform. There may be local areas with high positive charge, and BCG is more inclined to bind to these areas.

[0040] Example 2 Preparation Method of Floating Protein Microbubbles Loaded with BCG in the Shell A gelatin solution (1 mL, 10 wt%) was added to a toluene solution (20 mL) containing Span 80 (10 wt%). The mixture was stirred and emulsified using a homogenizer (500 rpm, 3 min) to form water-in-oil droplets. Glutaraldehyde (1 mL) was added to the droplets, and cross-linking was performed by vortexing at 25°C for 5 min to form protein microspheres with a stable shell structure. After centrifugation (800 rpm, 2 min), the resulting microsphere precipitate was washed 3–5 times with anhydrous ethanol and then washed three times with pure water to remove residual ethanol. The resulting microsphere precipitate was frozen at low temperature and then freeze-dried in a freeze dryer. The resulting floating protein microbubbles were sealed in a container, evacuated, and then filled with perfluoropropane gas. Hexadecyltrimethylammonium bromide (5 mg / mL, 5 mL) was added to the container containing the floating protein microbubbles and incubated at room temperature for 2 h. After incubation, the cells were washed three times with saline to obtain floating protein microbubbles modified with cetyltrimethylammonium bromide. 20 mg of cetyltrimethylammonium bromide-modified floating protein microbubbles were weighed and placed in a centrifuge tube. 10 mg of BCG lyophilized powder was added and incubated for 2 h on a shaker at room temperature (100 rpm). After incubation, the cells were washed three times with saline and centrifuged (1000 rpm, 2 min) to isolate floating protein microbubbles loaded with BCG. The BCG loading efficiency was 68%.

[0041] Example 3 Study on the residence time of floating protein microbubbles loaded with BCG in the bladder The BCG-loaded floating protein microbubbles prepared in Example 1 were dispersed in 10 mL of urine and placed in a urine collection tube. The urine collection tube was then incubated in a 37°C water bath. The floating of the BCG-loaded floating protein microbubbles in the urine was recorded using a camera at different time points.

[0042] The results are as follows Figure 2 As shown in the figures, most of the microbubbles can float on the urine for more than 24 hours. After bladder immunoperfusion therapy, BCG is usually rapidly excreted with urine after 2 hours. This result shows that the floating protein microbubbles loaded with BCG of the present invention have high stability in urine and can prolong the residence time of BCG in the bladder.

[0043] Example 4 Study on the sustained release of BCG by floating protein microbubbles loaded with BCG in the shell during natural degradation BCG was fluorescently stained with auramine O. Fluorescently stained BCG-loaded floating protein microbubbles (120 mg, containing 30 mg BCG) were resuspended in 10 mL of urine, placed in a urine collection tube, and incubated in a 37°C water bath for 48 hours. Urine was collected at predetermined time points and observed and imaged under an inverted fluorescence microscope. Three mg of BCG was added to 1 mL of urine and observed and imaged under an inverted fluorescence microscope to obtain fluorescence images of a 3 mg / mL BCG concentration. Image J software was used to calculate the BCG fluorescence area in urine images at different time points and the fluorescence area of ​​3 mg / mL BCG. The cumulative release rate of BCG from the floating protein microbubbles was calculated, with the fluorescence area of ​​3 mg / mL BCG set as 100%.

[0044] The results are as follows Figure 3 As shown, floating protein microbubbles loaded with BCG can continuously release BCG through the natural degradation of microbubbles.

[0045] Example 5 Distribution of floating protein microbubbles loaded with BCG in the bladder A saline suspension of BCG-loaded floating protein microbubbles (12 mg / mL, 0.1 mL) was instilled into the bladder of female SD rats through the urethra via the external flexible tube of a 24G pen-type intravenous catheter. Ultrasound imaging of the rat bladder was then performed using a small animal ultrasound imaging system at predetermined time points (0 h, 2 h, 4 h, 8 h, 12 h, and 24 h) to monitor the distribution of BCG-loaded floating protein microbubbles in the bladder.

[0046] The results are as follows Figure 4 As shown, floating protein microbubbles can float above the bladder of SD rats and achieve retention.

[0047] Example 6 Treatment of Bladder Tumors with Floating Protein Microbubbles Loaded with BCG SD rats with successfully established orthotopic bladder cancer models were randomly divided into three groups, each consisting of six rats: a saline group, a BCG group, and a BCG-loaded floating protein microbubble group. All three groups of rats were administered intravesically once weekly for four times. The saline group, serving as the control group, received 0.1 mL of saline. The BCG group received 0.3 mg / 0.1 mL of BCG via intravesical instillation. The BCG-loaded floating protein microbubble group received 1.2 mg / 0.1 mL of BCG-loaded floating protein microbubble (containing 0.3 mg of BCG) via intravesical instillation. Bladder tumor progression was monitored weekly throughout treatment using a small animal ultrasound imaging system.

[0048] The results showed that bladder tumors in the saline group grew rapidly, those in the BCG group increased slightly, and those in the BCG-loaded protein microbubble group gradually decreased. In summary, the BCG-loaded protein microbubble group had the best therapeutic effect, significantly outperforming both the BCG and saline control groups.

Claims

1. A floating protein microbubble loaded with BCG, characterized in that: It includes an outer shell structure and an inner gas core structure; the outer shell structure is a cross-linked protein layer skeleton, the surface of the cross-linked protein layer skeleton is modified with hexadecyltrimethylammonium bromide, and the BCG is loaded on the surface of the cross-linked protein layer skeleton through the reaction with hexadecyltrimethylammonium bromide.

2. The microbubble according to claim 1, characterized in that The particle size of the microbubbles is 60-140 μm.

3. The microbubble according to claim 1, characterized in that The protein of the cross-linked protein layer is selected from bovine serum albumin, bovine hemoglobin, gelatin or a complex thereof.

4. The microbubble according to claim 1, characterized in that The cross-linking agent used in the cross-linked protein layer is phthaloyl chloride or glutaraldehyde.

5. The microbubble according to claim 1, characterized in that The BCG loading rate on the surface of the BCG-loaded floating protein microbubbles is 65% to 75%.

6. The method for preparing microbubbles according to any one of claims 1 to 5, characterized in that: The steps include: S1. The protein aqueous solution is prepared into droplets by emulsification; S2. adding a crosslinking agent to the droplets obtained in step S1 to crosslink to obtain microspheres having a crosslinked protein shell; S3. The microspheres obtained in step S2 are freeze-dried, vacuumed, and filled with gas to obtain floating protein microbubbles containing gas in the inner layer; S4. The surface of the floating protein microbubbles obtained in step S3 is modified with cetyltrimethylammonium bromide, and then incubated with BCG to obtain floating protein microbubbles loaded with BCG.

7. The preparation method according to claim 6, characterized in that: The surfactant used in the emulsification method in step S1 is Span 85.

8. The preparation method according to claim 6, characterized in that: In step S4, the added mass ratio of the floating protein microbubbles whose surfaces are modified with hexadecyltrimethylammonium bromide to the BCG vaccine is 1.8-2.2:

1.

9. Use of the BCG-loaded floating protein microbubbles according to any one of claims 1 to 5 in the preparation of a drug for intravenous treatment of bladder cancer.

10. A preparation, characterized in that The preparation comprises the floating protein microbubbles loaded with BCG according to any one of claims 1 to 5.

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