Layer-by-layer self-assembly nano hydrogel boron carrying agent with controlled drug release and application thereof

Abstract

The invention discloses a drug controlled release layer-by-layer self-assembly nano hydrogel boron carrying agent and application thereof, and belongs to the technical field of biomedicine. According to the method, calcium alginate nano-particles, calcium alginate nano-particles modified by polyallylamine hydrochloride, calcium alginate nano-particles modified by boric acid and polyallylamine hydrochloride, and calcium alginate nano-particles modified by polyallylamine hydrochloride, boric acid and polyallylamine hydrochloride are prepared in sequence. According to the invention, boric acid is loaded into the layer-by-layer self-assembled nano hydrogel material, so that intelligent controlled release can be realized, and the material is applied to boron neutron capture therapy. The material has good water solubility, good dispersity in an aqueous solution, small size and uniform particle size. The material also has good biocompatibility, intelligent and accurate release control is realized, and important technical progress is brought to cancer treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a drug-controlled-release layer-by-layer self-assembled nano hydrogel boron carrier and application thereof. Background Art

[0002] Malignant tumors seriously threaten human life and health, and the diagnosis and treatment of malignant tumors are of great significance. Boron neutron capture therapy (BNCT) is a radiotherapy method for malignant tumors. Its basic principle is to use the reaction between neutrons and specific boron isotopes (such as boron 10). After neutrons irradiate boron-rich tumor cells, boron will capture neutrons to produce alpha particles and lithium ions. These high-energy particles can effectively kill tumor cells. Because alpha particles and lithium ions have strong biological effects, they can cause fatal cell damage within an extremely short distance (usually a few microns). This characteristic enables BNCT to effectively and accurately kill tumor cells with less damage to surrounding healthy cells.

[0003] With the continuous research on boron compounds, new and highly effective boron-containing drugs may appear in the future, which can not only better target tumor cells, but also improve the distribution and release characteristics in the body. This will greatly improve the effect and safety of treatment, making BNCT more effective in fighting various types of tumors. BNCT has positive clinical effects on malignant tumors, especially in glioblastoma and melanoma, which may prompt medical institutions to include BNCT in standard treatment plans. In addition, the advancement of neutron source technology will make the implementation of BNCT more flexible and convenient for promotion in more hospitals. Combination with personalized medicine will also optimize treatment plans, improve effects and reduce side effects. Finally, policy support and financial investment are also increasing. The attention and investment of governments and scientific research institutions in the field of cancer treatment have further enhanced its application prospects in cancer treatment.

[0004] Various promising boron compounds have been studied for BNCT, but these boron carriers have problems such as low targeting, high toxicity, low boron content and lack of active uptake mechanism. Since hydrogels are usually made of natural or synthetic polymers, they have good biocompatibility and are compatible with biological tissues, which can reduce side effects during treatment. At the same time, the use of hydrogels can improve the distribution of drugs in tumors, especially hydrogels that can intelligently control release to enhance the concentration of boron in tumor cells, thereby improving the overall efficacy of BNCT. In addition, hydrogels can be applied locally at the tumor site to avoid the side effects of systemic administration and improve the safety and effectiveness of treatment. Hydrogels derived from natural polymers also have good biodegradability. Among them, alginate is a natural hydrophilic polysaccharide that is commonly used to prepare hydrogels. Alginate is one of the most abundant biosynthetic materials, mainly derived from brown algae and bacteria. Alginate has a wide range of application value as a biomaterial, especially as a support matrix or delivery system for tissue repair and regeneration. Hydrogel microspheres loaded with boron drugs prepared by chelating alginate with calcium ions not only have high biocompatibility but also have biodegradability. Through in situ drug delivery, the boron drug loaded in the hydrogel is accumulated near the tumor and then irradiated with neutrons to achieve the effect of treating the tumor. The preparation process of alginate gel is simple, low-cost, and has highly adjustable mechanical properties. In addition, alginate has the ability to retain a large amount of liquid, which makes it very suitable as a fixed matrix for various applications, such as delivering drugs, genes or cells for tissue engineering and regenerative therapy. Hydrogels have been widely used in many biomedical fields, including wound dressings, artificial organs, drug carriers, etc. Alginate nanohydrogels can accurately release boron atoms at specific tumor sites through intelligent control to improve the therapeutic effect of BNCT and minimize the impact on healthy tissues, and have great potential value. Summary of the invention

[0005] The following technical problems are recorded in the background technology: Boric acid and its derivatives are the first generation of boron drugs for BNCT treatment, but the clinical use of boric acid for BNCT treatment is a failure. The reason is that boric acid has low bioavailability and limited absorption effect of tumor cells, which may lead to the inability to achieve the ideal boron concentration during treatment, thereby reducing the efficacy. Secondly, the distribution and metabolism of boric acid in the body are uneven, which may cause certain toxicity in normal tissues, especially when used for a long time. However, hydrogel, a natural biomaterial, has excellent biocompatibility and can control the release rate of drugs to keep the concentration of boron compounds in the tumor area at an ideal level. In addition, local administration of hydrogel can reduce the side effects caused by systemic administration. At present, there is still a blank state for hydrogel materials loaded with boric acid, and there is no relevant research at home and abroad. The present invention develops a layer-by-layer self-assembled nanohydrogel material, and loads boric acid therein, especially capable of intelligently controlling release, and is applied to boron neutron capture therapy (BNCT). The material has good water solubility, good dispersibility in aqueous solution, small size, and uniform particle size. The material also has good biocompatibility and intelligent and precise controlled release, bringing important technological advances to cancer treatment.

[0006] The present invention is achieved through the following technical solutions: A drug controlled-release layer-by-layer self-assembled nano-hydrogel boron carrier is prepared according to the following steps: Step 1: preparing calcium alginate nanoparticles; Step 2: preparing polyallylamine hydrochloride modified calcium alginate nanoparticles; Step 3: preparing calcium alginate nanoparticles modified with boric acid and polyallylamine hydrochloride; Step 4: Prepare calcium alginate nanoparticles modified with polyallylamine hydrochloride, boric acid and polyallylamine hydrochloride, i.e., the final product is a layer-by-layer self-assembled nanohydrogel boron carrier for controlled drug release.

[0007] In the above technical solution, in step 1, the preparation method of the calcium alginate nanoparticles is as follows: Add 3% by mass, 1.5g of sodium alginate, 1.5g of isooctane and 0.034g of sorbitan trioleate to a 50mL centrifuge tube, and ultrasonically emulsify the mixture for 60 minutes. Then, add 0.1g of isooctane and 0.2g of polyoxyethylene sorbitan trioleate to the mixture for ultrasonic emulsification for 90 minutes. After ultrasonic emulsification, add 10ml of CaCl2 with a mass fraction of 10% to each 10ml of the mixture, and stir well for 60 minutes at a speed of 1500rpm. After stirring, wash three times with deionized water by centrifugation, collect the lower precipitate, and obtain calcium alginate nanoparticles.

[0008] In the above technical solution, in step 2, the preparation method of the polyallylamine hydrochloride-modified calcium alginate nanoparticles is as follows: Prepare a polyallylamine hydrochloride solution containing 0.1M CaCl2 at a concentration of 2mg / mL. Take 1ml each time and add it to the centrifuge tube containing 100μL of the calcium alginate nanoparticles prepared in step 1. Fully adsorb for 60 minutes, then centrifuge to remove the unadsorbed supernatant electrolyte solution, and repeatedly centrifuge and wash with deionized water for 3 times. After washing, collect the precipitate and freeze-dry it to obtain polyallylamine hydrochloride modified calcium alginate nanoparticles.

[0009] In the above technical solution, in step 3, the preparation method of the calcium alginate nanoparticles modified with boric acid and polyallylamine hydrochloride is as follows: Prepare a 20 mg / ml boric acid solution, take the polyallylamine hydrochloride-modified calcium alginate nanoparticles prepared in step 2 and add them to the boric acid solution for full adsorption for 24 hours. Then centrifuge to remove the unabsorbed boric acid solution, and repeatedly centrifuge and wash with deionized water for 3 times. After washing, the calcium alginate nanoparticles modified with boric acid and polyallylamine hydrochloride are obtained; In the above technical solution, in step 4, the preparation method of the calcium alginate nanoparticles modified by polyallylamine hydrochloride, boric acid and polyallylamine hydrochloride is as follows: The calcium alginate nanoparticles modified with boric acid and polyallylamine hydrochloride prepared in step 3 are further mixed with a polyallylamine hydrochloride solution containing 0.1M CaCl2 at a concentration of 2 mg / mL, and fully adsorbed for 60 minutes. Then centrifugal washing is performed, the unadsorbed supernatant solution is removed, and washing is repeated 3 times. After washing, calcium alginate nanoparticles modified with polyallylamine hydrochloride, boric acid and polyallylamine hydrochloride are obtained.

[0010] Application of a layer-by-layer self-assembled nanohydrogel boron carrier for controlled drug release in boron neutron capture therapy.

[0011] Beneficial effects: (1) Precisely controlled release: By adjusting the interactions between layers and the polymer properties, the release rate and time of boron can be precisely controlled to ensure that sufficient boron is present in tumor cells during neutron beam irradiation.

[0012] (2) Enhanced targeting: Nanohydrogels can be administered locally to achieve tissue targeting; highly specific affinity molecule modification on the surface can efficiently target tumor cells, thereby reducing the impact on normal cells.

[0013] (3) Biocompatibility and safety: Use biocompatible materials to reduce adverse reactions in the body.

[0014] This technology provides a new drug delivery strategy for BNCT, which may greatly enhance the therapeutic effect and fight against refractory tumors such as melanoma, glioma, osteosarcoma, head and neck tumors, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the synthesis steps of the present invention.

[0016] Figure 2 This is a schematic diagram of a scanning electron microscope (SEM) of the nanoparticles of the present invention.

[0017] Figure 3 Schematic diagram of dynamic light scattering (DLS) of the nanoparticles of the present invention.

[0018] Figure 4 Schematic diagram of the Zeta potential of the layer-by-layer self-assembly of the nanoparticles of the present invention.

[0019] Figure 5 It is the infrared spectrum of the nanoparticles of the present invention.

[0020] Figure 6 The particle size distribution changes of the nanoparticles after standing in different solutions for 7 days.

[0021] Figure 7 This is a visual schematic diagram of nanoparticles in different solutions.

[0022] Figure 8 The survival rate of B16-F10 cells after co-culture with nanoparticles of different concentrations for 24 hours.

[0023] Fig. 9 Schematic diagram of hemolysis of nanoparticles and statistical diagram of hemolysis rate.

[0024] Fig.10 This is the cell fluorescence imaging of indocyanine green (ICG)-labeled nanoparticles after co-culture with B16-F10 cells.

[0025] Fig.11 The fluorescence statistics of indocyanine green (ICG)-labeled nanoparticles after co-culture with B16-F10 cells.

[0026] Fig.12 Schematic diagram of the fluorescence distribution of indocyanine green (ICG)-labeled nanoparticles in mice. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0028] Example A layer-by-layer self-assembled nanohydrogel boron carrier for controlled drug release, such as Figure 1 The synthesis steps are as follows: Step 1. Add 1.5 g of sodium alginate, 1.5 g of isooctane and 0.034 g of sorbitan trioleate (span 85) with a mass fraction of 3% to a 50 mL centrifuge tube, and ultrasonically emulsify the mixture for 60 minutes. Then, add 0.1 g of isooctane and 0.2 g of polyoxyethylene sorbitan trioleate (Tween 85) to the mixture for ultrasonic emulsification for 90 minutes. After ultrasonic emulsification, add 10 ml of CaCl2 with a mass fraction of 10% to every 10 ml of the mixture, and stir at a speed of 1500 rpm for 60 minutes. After stirring, wash the mixture three times with deionized water by centrifugation, collect the lower precipitate, and obtain calcium alginate nanoparticles (product 1).

[0029] Step 2. Prepare a polyallylamine hydrochloride (PAH) solution containing 0.1M CaCl2. The concentration of PAH is 2mg / mL. Take 1ml each time and add it to 100μL of product 1 (>10 6 The PAH-modified calcium alginate nanoparticles (product 2) were obtained by centrifuging the PAH-modified calcium alginate nanoparticles to remove the unadsorbed supernatant electrolyte solution by centrifugation and washing the supernatant with deionized water for 3 times. After washing, the precipitate was collected and freeze-dried to obtain PAH-modified calcium alginate nanoparticles (product 2).

[0030] Step 3. Prepare a 20 mg / ml boric acid (BA) solution, take a certain amount of product 2 and add it to the boric acid solution for full adsorption for 24 hours. Then centrifuge to remove the unadsorbed boric acid solution, and wash it three times with deionized water. After washing, calcium alginate nanoparticles SA@(PAH-BA) (product 3) modified with BA and PAH are obtained.

[0031] Step 4. The SA@(PAH-BA) product 3 was mixed with a 2 mg / mL PAH solution and fully adsorbed for 60 minutes. Then, the mixture was centrifuged and washed to remove the unadsorbed supernatant solution, and the mixture was washed three times. After washing, calcium alginate nanoparticles modified by layer-by-layer self-assembly were obtained, namely SA@(PAH-BA-PAH) (final product).

[0032] Performance Verification: The layer-by-layer self-assembled nano-hydrogel material synthesized by this process has a small and uniform particle size, good monodispersity, and a particle size of about 310 nm. Figure 2 ) and dynamic light scattering DLS ( Figure 3 ) characterizes the size and morphology of the nanohydrogel. The positive and negative changes in the Zeta potential characterize the successful loading of the layer-by-layer self-assembly of nanomaterials ( Figure 4 ). Infrared spectrum at 2252cm -1 With 633cm -1 The characteristic peaks that appeared proved the successful loading of boric acid ( Figure 5 ). Inductively coupled plasma mass spectrometry (ICP-MS) detected that the boron content in the product reached 10.1%, which is higher than the BNCT therapeutic drug dihydroxyphenylalanine boron (5.2%). The synthesized nanohydrogel has good stability and water solubility ( Figure 6 and Figure 7 ).

[0033] After culturing the nano-hydrogel boron carrier and melanoma cells (B16-F10) for 24 hours, the cell survival rate was detected and it was found that when the boron concentration of the nanomaterial was 0-200ppm, the survival rate of B16-F10 cells was about 80%, with low toxicity ( Figure 8 ). Hemolytic experiments have shown that nano-hydrogels of different concentrations have good biocompatibility ( Fig. 9 ).

[0034] The nanohydrogel was labeled with indocyanine green (ICG), co-cultured with melanoma cells (B16-F10) and observed with confocal laser scanning microscopy (CLSM). The results showed that the nanohydrogel could be enriched in melanoma cells, and the optimal enrichment time was 24 hours ( Fig.10 Image J software was used to count the fluorescence values ​​and perform three-dimensional plots, and the fluorescence values ​​at 4 hours and 24 hours were counted ( Fig.11 ).

[0035] A B16-F10 subcutaneous tumor model was constructed, and the nanohydrogel was labeled with indocyanine green (ICG) and injected into the tumor. In vivo fluorescence detection was performed using a small animal imaging system (IVIS). The results showed the distribution of the nanohydrogel boron carrier in the tumor, and the drug release could be precisely controlled after the addition of EDTA ( Fig.12 ).

[0036] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A drug controlled release layer-by-layer self-assembled nano-hydrogel boron carrier, characterized in that: Prepared according to the following steps: Step 1: preparing calcium alginate nanoparticles; Step 2: preparing polyallylamine hydrochloride modified calcium alginate nanoparticles; Step 3: preparing calcium alginate nanoparticles modified with boric acid and polyallylamine hydrochloride; Step 4: Prepare calcium alginate nanoparticles modified with polyallylamine hydrochloride, boric acid and polyallylamine hydrochloride, that is, the final product is a layer-by-layer self-assembled nanohydrogel boron carrier for controlled drug release.

2. The drug controlled release layer-by-layer self-assembled nano-hydrogel boron carrier according to claim 1, characterized in that In step 1, the preparation method of the calcium alginate nanoparticles is as follows: In a 50mL centrifuge tube, 1.5g of sodium alginate, 1.5g of isooctane and 0.034g of sorbitan trioleate with a mass fraction of 3% were added, and the mixture was ultrasonically emulsified for 60 minutes; then 0.1g of isooctane and 0.2g of polyoxyethylene sorbitan trioleate were added to the mixture for ultrasonic emulsification for 90 minutes; after the ultrasonic emulsification, 10ml of CaCl2 with a mass fraction of 10% was added to every 10ml of the mixture, and the mixture was stirred at a speed of 1500rpm for 60 minutes; after the stirring, the mixture was centrifuged and washed three times with deionized water, and the lower precipitate was collected to obtain calcium alginate nanoparticles.

3. The drug controlled release layer-by-layer self-assembled nano-hydrogel boron carrier according to claim 1, characterized in that: In step 2, the preparation method of the polyallylamine hydrochloride modified calcium alginate nanoparticles is as follows: Prepare a polyallylamine hydrochloride solution containing 0.1M CaCl2 at a concentration of 2mg / mL. Take 1ml each time and add it to the centrifuge tube containing 100μL of the calcium alginate nanoparticles product prepared in step 1, fully adsorb for 60 minutes, then centrifuge to remove the unadsorbed supernatant electrolyte solution, and repeatedly centrifuge and wash with deionized water for 3 times; after washing, collect the precipitate and freeze-dry it to obtain polyallylamine hydrochloride modified calcium alginate nanoparticles.

4. The layer-by-layer self-assembled nano-hydrogel boron carrier for controlled drug release according to claim 1, characterized in that: In step 3, the preparation method of the calcium alginate nanoparticles modified with boric acid and polyallylamine hydrochloride is as follows: Prepare a 20 mg / ml boric acid solution, take the polyallylamine hydrochloride-modified calcium alginate nanoparticles prepared in step 2 and add them to the boric acid solution for full adsorption for 24 hours; then centrifuge to remove the unabsorbed boric acid solution, and repeatedly centrifuge and wash with deionized water for 3 times; after washing, obtain boric acid and polyallylamine hydrochloride-modified calcium alginate nanoparticles.

5. The layer-by-layer self-assembled nano-hydrogel boron carrier for controlled drug release according to claim 1, characterized in that: In step 4, the preparation method of the polyallylamine hydrochloride, boric acid and polyallylamine hydrochloride modified calcium alginate nanoparticles is as follows: The calcium alginate nanoparticles modified with boric acid and polyallylamine hydrochloride prepared in step 3 were further mixed with a polyallylamine hydrochloride solution containing 0.1M CaCl2 at a concentration of 2 mg / mL and fully adsorbed for 60 minutes; then centrifuged and washed to remove the unadsorbed supernatant solution, and washed repeatedly for 3 times; after washing, calcium alginate nanoparticles modified with polyallylamine hydrochloride, boric acid and polyallylamine hydrochloride were obtained.

6. Use of the layer-by-layer self-assembled nanohydrogel boron carrier for controlled drug release according to any one of claims 1 to 5 in boron neutron capture therapy.

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