A casein-based natural polymer microcapsule with NIR responsiveness and a preparation method and application thereof

Abstract

The application belongs to the technical field of drug carriers, and particularly relates to a preparation method and application of casein-based natural high-molecular microcapsules with NIR responsiveness. The application introduces hollow copper sulfide nanoparticles into high-molecular microcapsules to synthesize a drug delivery carrier with NIR responsiveness, which can simultaneously deliver hydrophilic drugs and hydrophobic drugs, and the drug loading rates are 90.14% and 72.16% respectively. Experiments show that the carrier can precisely control the release of drugs under specific pH and near-infrared light irradiation, thereby improving the efficiency of drug treatment and reducing the side effects on normal tissues.

Description

Technical Field

[0001] This invention belongs to the field of drug carrier technology, specifically relating to a casein-based natural polymer microcapsule with NIR responsiveness, its preparation method, and its application. Background Technology

[0002] The combined treatment of photothermal therapy and chemotherapy has attracted much attention due to its good therapeutic effects and is considered one of the most popular cancer treatment methods. Dual drug loading not only reduces drug dosage and toxicity, but also enhances therapeutic efficacy through synergistic effects of the two drugs, thus becoming a hot topic in drug delivery research. Natural polymer microcapsule drug delivery systems not only efficiently protect and transport drugs, but also possess the advantages of natural biomaterials, such as biodegradability and low cytotoxicity, significantly improving the biocompatibility of the materials.

[0003] Currently, research on drug delivery via microcapsules largely focuses on polymer microcapsule systems. For example, some studies, such as those by Nehru S et al. (Nehru S, Guru A, Pachaiappan R, et al. Co-encapsulation and release of apigenin and ascorbic acid in polyelectrolyte multilayer capsules for targeted polycystic ovary syndrome[J]. Int J Pharm, 2024, 651: 123749), used polyallylamine hydrochloride (PAH) and dextran (DS) in a layer-by-layer self-assembly process as the wall material for hollow microcapsules with a size of 4±0.5 μm. Hydrophobic AP and hydrophilic AC molecules can be co-encapsulated and released into the PAH / DS microcapsule system to alleviate polycystic ovary syndrome (PCOS). The encapsulation efficiency of both AP and AC is approximately 20%. This dual drug delivery system can co-deliver two natural compounds, effectively interacting with ovarian cells, reducing cell damage, and normalizing PCOS. However, due to the synthetic polymer wall, the microcapsules exhibit poor degradability and large size, resulting in a lack of targeted therapy with chemotherapy alone. Sharma V et al. (Sharma V, Vijay J, Ganesh MR, et al. Multilayer capsules capsulating nimbin and doxorubicin for cancer chemo-photothermal therapy[J]. Int J Pharm, 2020, 582: 119350) used polyallylamine hydrochloride (PAH) and polymethacrylic acid (PMA) as microcapsule walls and incorporated gold nanorods to encapsulate and release the hydrophilic drug DOX and the hydrophobic drug NB for cancer treatment. The hollow capsules had a size of 4.5 ± 0.5 μm, and their morphology underwent a structural transformation under 808 nm laser irradiation. The encapsulation efficiency of both DOX and NB was 30%, demonstrating the combined chemotherapy-photothermal therapy. However, the polymer wall material resulted in poor biocompatibility, large microcapsule size, and low drug loading.

[0004] US10780057(B2) discloses a method for preparing casein-based silica-loaded dual-drug microcapsules. Dual-drug casein microcapsules are prepared via interfacial polymerization, with drug-loaded casein serving as the outer shell material and silica as the inner shell material. The prepared microcapsules can load drugs not only in the internal cavity but also within the casein shell. These dual-drug microcapsules hold promise for reducing the frequency of drug administration and improving therapeutic efficacy in disease treatment. However, the following problems exist: the preparation process affects biocompatibility, and single-drug chemotherapy may be accompanied by high drug dose toxicity and low absorption rates, increasing the risk of side effects.

[0005] Therefore, developing a dual drug delivery system utilizing natural polymer microcapsules, combining chemotherapy with photothermal therapy, is of great significance for improving drug efficacy and developing new treatment strategies. Summary of the Invention

[0006] The present invention aims to provide a casein-based natural polymeric microcapsule with NIR responsiveness, and its preparation method is a second objective. A third objective is to provide the application of the aforementioned natural polymeric microcapsule in the preparation of polymeric microcapsule drug carriers. The preparation method of this invention features a simple operation process and high drug loading efficiency. The prepared polymeric microcapsules exhibit good biocompatibility and biodegradability. By introducing hollow copper sulfide nanoparticles into the polymeric microcapsules, an NIR-responsive drug delivery carrier is synthesized, capable of simultaneously delivering hydrophilic and hydrophobic drugs, with drug loading rates reaching 90.14% and 72.16%, respectively. Experiments show that this carrier can precisely control drug release under near-infrared light irradiation, improving the efficiency of drug therapy and reducing side effects on normal tissues.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] A casein-based natural polymer microcapsule with NIR responsiveness, wherein the capsule wall is composed of natural polymer materials casein and chitosan, the template is calcium carbonate, and polyelectrolyte spheres are obtained by layer-by-layer self-assembly of chitosan and casein. The natural polymer microcapsule is endowed with NIR response by introducing hollow copper sulfide nanoparticles as a photothermal agent, and the natural polymer microcapsule is obtained by etching with acetic acid.

[0009] Preferably, the natural polymer microcapsules can simultaneously load both hydrophilic and hydrophobic drugs.

[0010] Based on a unified inventive concept, the preparation method of the casein-based natural polymer microcapsules with NIR responsiveness includes the following steps:

[0011] S1. Calcium carbonate microspheres were obtained by doping calcium carbonate with sodium polystyrene sulfonate (PSS) and used as microcapsule sacrificial templates.

[0012] S2. A polyelectrolyte sphere is obtained by depositing a natural polymer layer of casein (CN) and chitosan (CS) on a calcium carbonate sphere.

[0013] S3. Hollow copper sulfide nanoparticles (CuS) are introduced onto the above electrolyte spheres.

[0014] S4. The calcium carbonate template was removed by etching with acetic acid to obtain hollow CN / CS / CN / CS@CuS polymer microcapsules.

[0015] Preferably, in step S1, the calcium carbonate microspheres are prepared using the following method:

[0016] S11. Dissolve 0.1 M sodium carbonate solution and 0.1 M calcium nitrate solution in sodium polystyrene sulfonate aqueous solution (10 mg / mL) to obtain solution A and solution B;

[0017] S12. Mix solution A and solution B. Stir the mixture at 800-1200 rpm for 20-35 min at 25-35 ℃. Then centrifuge at 2500-3500 rpm for 2-3 min and wash with deionized water more than twice to obtain PSS-doped calcium carbonate microspheres.

[0018] Preferably, in step S2, the polyelectrolyte spheres are prepared by the following method: the obtained CaCO3 microspheres are sequentially immersed in polyallylamine hydrochloride, casein, chitosan, casein and chitosan solution (1 mg / mL, pH 6.0), each layer is deposited for 10-18 min, centrifuged at 2800-3200 rpm for 1-3 min, and washed twice with 0.4-0.6 M NaCl solution (pH 6.0) to remove unadsorbed polyelectrolytes.

[0019] Preferably, in step S4, the CaCO3 core is removed by etching with 15-22 mL of 0.1 M, pH 4.5 glacial acetic acid solution at a rotation speed of 220-280 rpm and an etching time of 20-28 min.

[0020] This invention further discloses the application of the aforementioned NIR-responsive casein-based natural polymeric microcapsules in the preparation of polymeric microcapsule drug carriers, and a method for preparing dual-drug loading using the aforementioned polymeric microcapsules, comprising the following steps:

[0021] 1) Casein and hydrophobic drugs are pre-combined, and the hydrophobic drugs are first introduced into the hydrophobic core of casein, and then introduced onto the microcapsules;

[0022] 2) By utilizing the electrostatic interaction between casein and hydrophilic drugs, hydrophilic drugs can be introduced into microcapsules that are already loaded with hydrophobic drugs, thus achieving dual drug loading.

[0023] Preferably, the hydrophobic drug is curcumin (CUR). The specific operation of step 1) is as follows: casein and curcumin are pre-combined, and 0.3-0.6 mL of 1 mg / mL curcumin ethanol solution is added to 3-6 mL of 1 mg / mL sodium caseinate solution (pH 6.0). The mixture is stirred at 400-800 rpm for 1-2 h to complete the CUR loading.

[0024] Preferably, the hydrophilic drug is doxorubicin (DOX). The specific operation of step 2) is as follows: using the electrostatic interaction between casein and doxorubicin, add 0.3 mL of 2 mg / mL DOX aqueous solution to the dispersion of curcumin microcapsules, and stir at 28-32 °C and 200-300 rpm for 10-14 h to complete the DOX loading.

[0025] This invention develops a novel drug delivery system combining chemotherapy and photothermal therapy by using a natural polymer as the capsule wall and incorporating a near-infrared response mechanism. This carrier can simultaneously deliver both hydrophobic and hydrophilic drugs and exhibits further responsiveness under near-infrared light irradiation, making it suitable for drug delivery and targeted therapy.

[0026] Specifically, this invention constructs a natural polymer microcapsule co-loaded with a hydrophilic drug and a hydrophobic drug, with the capsule wall composed of casein and chitosan. Due to the amphiphilic structure of casein, the hydrophobic drug is pre-complexed with casein, utilizing hydrophobic interactions to encapsulate the hydrophobic drug molecules within the hydrophobic core of the casein micelles, thereby significantly improving the water solubility and bioavailability of the hydrophobic drug. Simultaneously, the hydrophobic drug-loaded casein layer is located on the surface of the microcapsule, imparting a negative charge to the microcapsule and providing strong support for the efficient loading of the positively charged hydrophilic drug. Simultaneous delivery of the hydrophilic drug DOX and the hydrophobic drug CUR achieves loading rates of 90.14% and 72.16%, respectively. Furthermore, the introduction of copper sulfide into the system endows the natural polymer microcapsule with photothermal responsiveness, enabling it to release drugs via NIR.

[0027] Compared with the prior art, the technical advantages of the present invention are as follows:

[0028] 1. This invention uses natural polymer materials casein and chitosan as capsule walls, and prepares polymer microcapsules using a template method and layer-by-layer self-assembly to ensure good biocompatibility and biodegradability.

[0029] 2. This invention introduces hollow copper sulfide as a photothermal agent, and combines it with chemotherapy and photothermal therapy to develop a novel polymer microcapsule carrier. This carrier exhibits further responsiveness under near-infrared light irradiation and is suitable for drug delivery and targeted therapy.

[0030] 3. The natural polymer microcapsules prepared by this invention can deliver two types of drugs together, with a high drug loading rate and synergistic enhancement of the two drugs, thereby improving the therapeutic effect. Attached Figure Description

[0031] Figure 1 SEM image of CN / CS / CN / CS@CuS / CN microcapsules;

[0032] Figure 2 UV-Vis-NIR absorption spectrum of CuS;

[0033] Figure 3 Zeta potential diagram during the self-assembly process of CN / CS / CN / CS@CuS / CN microcapsules;

[0034] Figure 4 CuS, CN / CS / CN / CS@CuS / CN, CN / CS / CN / CS / CN and deionized water at 0.8 W / cm 2 Temperature change curve under NIR laser irradiation;

[0035] Figure 5 CuS and CN / CS / CN / CS@CuS / CN at 0.8 W / cm 2 Photothermal conversion capability under near-infrared laser irradiation;

[0036] Figure 6 NIR-responsive drug release from DOX-loaded CN / CS / CN / CS@CuS / CN;

[0037] Figure 7 NIR-responsive drug release from CN / CS / CN / CS@CuS / CN loaded with CUR;

[0038] Figure 8 Drug release response of DOX-loaded CN / CS / CN / CS@CuS / CN under different media and laser irradiation conditions;

[0039] Figure 9 Drug release response of CUR-loaded CN / CS / CN / CS@CuS / CN under different media and laser irradiation conditions;

[0040] Figure 10 . Evaluation of the toxicity of CN / CS / CN / CS@CuS / CN microcapsules to EA.hy926 cells;

[0041] Figure 11 SEM image of the microcapsule wall consisting of 3 layers (2 layers of casein and 1 layer of chitosan);

[0042] Figure 12 SEM image of the microcapsule wall consisting of 5 layers (3 layers of casein and 2 layers of chitosan). Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments.

[0044] It should be noted that the preparation method of hollow copper sulfide nanoparticles involved in this invention and its embodiments is as follows: First, copper chloride (CuCl2) solution is mixed with hexadecyltrimethylammonium bromide (CTAB) aqueous solution for reaction; then 5 mL of L-ascorbic acid (AA) solution is added to continue the reaction; subsequently, sodium hydroxide (NaOH) solution and thioacetamide (TAA) solution are added to the reaction system to continue the reaction. After the reaction is completed, centrifugation and washing are performed to remove excess unreacted substances, and the hollow copper sulfide (CuS) nanoparticles of this invention can be obtained.

[0045] Example 1

[0046] The preparation method of a casein-based natural polymer microcapsule with NIR responsiveness according to this embodiment includes the following steps:

[0047] S1. Calcium carbonate microspheres were obtained by doping calcium carbonate with sodium polystyrene sulfonate (PSS) and used as microcapsule sacrificial templates. The specific steps are as follows:

[0048] S11. Dissolve 0.1 M sodium carbonate (Na2CO3) solution and 0.1 M calcium nitrate (Ca(NO3)2) solution respectively in 10 mg / mL sodium polystyrene sulfonate (PSS) aqueous solution to obtain solution A and solution B;

[0049] S12. Under vigorous stirring, Na2CO3 solution A was rapidly injected into Ca(NO3)2 solution B, and the resulting mixture was allowed to stand at 30℃ for 30 min. Subsequently, PSS-doped calcium carbonate (CaCO3) microspheres were obtained by centrifugation (3000 rpm, 30 min) and washing three times with deionized water.

[0050] S2. Polyelectrolyte spheres are obtained by alternately depositing casein (CN) and chitosan (CS) natural polymer layers on calcium carbonate spheres. The specific steps are as follows:

[0051] The obtained CaCO3 microspheres were sequentially immersed in polyallylamine hydrochloride (PAH), casein (CN), chitosan (CS), casein (CN), and chitosan (CS) solutions (1 mg / mL, pH 6.0). After each deposition lasting 15 min, the microspheres were washed twice with 0.5 M NaCl solution (pH 6.0) to remove unadsorbed polyelectrolytes and obtain polyelectrolyte spheres.

[0052] S3. Introduce hollow copper sulfide nanoparticles onto the above electrolyte spheres;

[0053] The polyelectrolyte spheres were then blended with 2 mg of hollow CuS nanoparticles and reacted for 15 min to complete the CuS coating.

[0054] S4. Remove the calcium carbonate template using acetic acid etching to obtain hollow CN / CS / CN / CS@CuS polymer microcapsules. The specific steps are as follows:

[0055] The CaCO3 sacrificial template was removed by etching with 20 mL of 0.1 M, pH 4.5 glacial acetic acid solution at a speed of 250 rpm for 25 min to obtain hollow CN / CS / CN / CS@CuS microcapsules.

[0056] In this embodiment, the hollow copper sulfide nanoparticles were prepared as follows: 1 mL of 0.2 M copper chloride (CuCl2) solution was mixed with 100 mL of 0.13 M hexadecyltrimethylammonium bromide (CTAB) aqueous solution, and the reaction temperature was maintained at 30°C. The temperature of the reaction system was raised to 60°C, and 5 mL of 0.1 M L-ascorbic acid (AA) solution was added, and the reaction was stirred continuously. Subsequently, 10 mL of 0.2 M sodium hydroxide (NaOH) solution and 1 mL of 0.6 M thioacetamide (TAA) solution were added to the reaction system. The reaction mixture was stirred continuously at 30°C for 3 days. After the reaction was completed, excess unreacted substances were removed by centrifugation (8000 rpm, 20 minutes) and two washings (for washing, an appropriate amount of deionized water was added to the centrifuged copper sulfide, and centrifugation was performed again at 8000 rpm for 20 minutes, which counted as one washing). Hollow copper sulfide (CuS) nanoparticles were finally obtained.

[0057] Example 2

[0058] The NIR-responsive casein-based natural polymer microcapsules and their preparation method in this embodiment differ from those in Example 1 in that:

[0059] In step S12, the resulting mixture is stirred at 1200 rpm for 20 min at 25°C and then centrifuged at 2500 rpm for 3 min.

[0060] In step S2, after each deposition for 10 min, centrifuge at 2800 rpm for 3 min, and wash twice with 0.4M NaCl solution;

[0061] In step S4, the CaCO3 sacrificial template was removed by etching with 15 mL of 0.1 M, pH 4.5 glacial acetic acid solution at a speed of 220 rpm for 28 min.

[0062] The rest is the same as in Example 1.

[0063] Example 3

[0064] The NIR-responsive casein-based natural polymer microcapsules and their preparation method in this embodiment differ from those in Example 1 in that:

[0065] In step S12, the resulting mixture is stirred at 800 rpm for 35 min at 35°C and then centrifuged at 3500 rpm for 2 min.

[0066] In step S2, after each deposition for 18 min, centrifuge at 3200 rpm for 1 min and wash twice with 0.6 M NaCl solution;

[0067] In step S4, the CaCO3 sacrificial template is removed by etching with 22 mL of glacial acetic acid solution at a speed of 280 rpm for 20 min.

[0068] The rest is the same as in Example 1.

[0069] Example 4

[0070] Taking Example 1 as an example, hollow CN / CS / CN / CS@CuS microcapsules were prepared using the hollow CN / CS / CN / CS@CuS microcapsules prepared therein: Casein solution (1 mg / mL, pH 6.0) was added to the CN / CS / CN / CS@CuS aqueous dispersion, and stirred at 30 ℃ and 300 rpm for 15 min. After centrifugation, the microcapsules were washed twice with deionized water to obtain hollow CN / CS / CN / CS@CuS / CN microcapsules.

[0071] Example 5

[0072] This embodiment discloses a method for preparing dual-drug loading using polymeric microcapsules from Example 1. The preparation method includes the following steps:

[0073] 1) Curcumin (CUR) loading: 0.4 mL of 1 mg / mL curcumin ethanol solution was added to 4 mL of 1 mg / mL sodium caseinate solution (pH 6.0), and stirred at 600 rpm for 2 h to obtain a curcumin-loaded casein solution. The premixed casein-curcumin solution was added to CN / CS / CN / CS@CuS aqueous dispersion, and stirred at 30 ℃ and 300 rpm for 15 min. The CUR-loaded microcapsules were collected by centrifugation.

[0074] 2) Loading of doxorubicin (DOX): 0.3 mL of DOX (2 mg / mL) aqueous solution was added to the dispersion of curcumin-loaded microcapsules and stirred at 30 °C and 250 rpm for 12 h to complete the DOX loading.

[0075] Effect Experiment

[0076] To illustrate the effectiveness of this invention, the sample prepared in Example 4 was used as an example, and its SEM and UV-Vis-NIR absorption spectra, Zeta potential, and photothermal properties were measured. The results are shown below. Figures 1-5 As shown; simultaneously, taking the drug-loaded sample prepared in Example 5 as an example, the drug loading rate and drug release rate of the nanocarrier of the present invention were measured, and the results are shown in [the table below]. Figure 6-9 As shown.

[0077] The calculation method for photothermal conversion efficiency is as follows:

[0078] After irradiating CuS and CN / CS / CN / CS@CuS / CN with a certain concentration for 1200 seconds and then cooling for 1200 seconds using a 0.8 W / cm² near-infrared laser, the photothermal conversion efficiency (η) was calculated using the following formula. The calculated photothermal conversion efficiencies (η) for CuS and CN / CS / CN / CS@CuS / CN were 48.14% and 26.86%, respectively.

[0079]

[0080]

[0081] In these two formulas, h and S represent the thermal conductivity coefficient and the surface area of ​​the container, respectively. max and T amb These represent the system's highest temperature and the ambient temperature, respectively. Q loss This refers to the thermal energy absorbed by the container and deionized water. I represents the near-infrared laser power, and A... 808 This indicates the absorbance of the sample at 808 nm. C D m D and τ s These correspond to the specific heat capacity and mass of deionized water, and the time constant of the system, respectively.

[0082] The method for determining drug loading rate is as follows:

[0083] After dialysis, the liquid outside the dialysis bag was collected, and the absorbance of DOX and CUR was measured using a UV-Vis spectrophotometer at wavelengths of 481 nm and 426 nm, respectively. Based on the measured absorbance values, the amount of free DOX, CUR, and drug loading efficiency in the dialysate could be calculated by comparing them with DOX and CUR standard curves. The drug loading efficiency can be calculated using the following formulas: DOX drug loading efficiency is 90.14%, and CUR drug loading efficiency is 72.16%.

[0084]

[0085] The drug release test method is as follows:

[0086] In the drug release experiment, the release medium was a mixture of phosphate-buffered saline (PBS, pH 7.4 or pH 6.0) and anhydrous ethanol at a volume ratio of 90:10 (v / v), with 1% Tween 80 added. The samples were irradiated using an 808 nm near-infrared (NIR) laser with a power density of 0.8 or 1.6 W / cm². The absorbance of the release medium outside the dialysis bag was measured at 481 nm and 426 nm wavelengths at regular intervals. Based on the standard curve relationship between absorbance and concentration, the concentrations of DOX and CUR were calculated, yielding the DOX and CUR release amounts at each time point. The drug release rate of the material under different conditions was calculated using the following formula, and a cumulative drug release curve was plotted to demonstrate the drug release behavior under different conditions, thus evaluating the drug release performance of the material.

[0087]

[0088] Figure 1 This is a SEM image of the CN / CS / CN / CS@CuS / CN drug carrier from Example 4. Figure 1 As can be seen, the microcapsules exhibit a rough surface, a hollow interior, and a double-concave disk structure, with a complete morphology. This indicates that natural polymer microcapsules with NIR response have been successfully prepared.

[0089] Figure 2 This is the UV-Vis-NIR absorption spectrum of CuS, derived from... Figure 2 It can be seen that hollow copper sulfide nanoparticles have excellent near-infrared response. This excellent near-infrared response enables CuS nanoparticles to effectively convert light energy into heat energy under near-infrared light irradiation, thereby achieving a highly efficient photothermal effect.

[0090] Figure 3This is a Zeta potential diagram of the self-assembly process of CN / CS / CN / CS@CuS / CN microcapsules, generated by... Figure 3 It is evident that the initial CaCO3 particles exhibit strong negative charge due to PSS doping. After depositing a positively charged PAH on the CaCO3 surface, the particle zeta potential increases. Subsequent assembly of a CN / CS / CN / CS natural polymer layer and hollow CuS results in a regular alternating change in particle potential. Etching with acetic acid removes a large amount of CaCO3, leading to a significant reduction in the negative charge on the particle surface. Since CuS is positively charged in the second outermost layer, it can electrostatically assemble with the negatively charged outermost CN layer. The assembled microcapsules retain a negative charge, thus enhancing the binding force with DOX.

[0091] Figure 4 It is CuS, CN / CS / CN / CS@CuS / CN, CN / CS / CN / CS / CN and deionized water at 0.8 W / cm 2 The temperature change curve under NIR laser irradiation, by Figure 4 It was found that after irradiation with near-infrared laser (0.8 W / cm²) for 60 minutes, the temperatures of CuS and CN / CS / CN / CS@CuS / CN increased by 14°C and 12.9°C, respectively, while the temperatures of CN / CS / CN / CS / CN and deionized water increased by only 9.1°C and 8.2°C, respectively. This indicates that the addition of CuS endows CN / CS / CN / CS@CuS / CN with excellent photothermal properties.

[0092] Figure 5 Temperature curves of CuS and CN / CS / CN / CS@CuS / CN under 0.8 W / cm² near-infrared laser irradiation for 1200 seconds and cooling for 1200 seconds are shown. The photothermal conversion efficiencies (η) of CuS and CN / CS / CN / CS@CuS / CN are calculated to be 48.14% and 26.86%, respectively.

[0093] Figure 6 This refers to the NIR-responsive drug release of the DOX-loaded CN / CS / CN / CS@CuS / CN in Example 5 of this invention at pH 6.0, achieved by... Figure 6 It can be seen that the cumulative release rate of DOX in 48 h without laser irradiation is 33.32%, the release of DOX under 0.8 W / cm² laser irradiation reaches 56.32%, and under 1.6 W / cm² laser irradiation, the drug release rate increases rapidly, and the cumulative release of DOX reaches 64.66%, indicating that the material has obvious NIR-responsive drug release performance.

[0094] Figure 7This refers to the NIR-responsive drug release of the CUR-loaded CN / CS / CN / CS@CuS / CN in Example 5 of the present invention at pH 6.0, by... Figure 7 It can be seen that the cumulative release rate of CUR in 48 h without laser irradiation is 24.24%, the release amount of CUR reaches 34.6% under the condition of 0.8 W / cm² laser irradiation, and the drug release rate increases rapidly under the condition of 1.6 W / cm² laser irradiation, with the cumulative release amount of CUR reaching 54.62%, indicating that the material has obvious NIR-responsive drug release performance.

[0095] Figure 8 This invention, in Example 5, describes the drug release response of DOX-loaded CN / CS / CN / CS@CuS / CN under different media and laser irradiation conditions. Figure 8 It can be seen that under pH 7.4 and without laser irradiation, the release of DOX is relatively low, with a cumulative release rate of 22.92% over 48 hours. Under near-infrared laser irradiation, the release of DOX is 42.29% within 12 hours, while without laser irradiation, the drug release only increases by 4.66% over 12 hours. After another 12 hours of near-infrared laser irradiation, the release rapidly increases to 63.24%. This phenomenon further demonstrates the NIR response characteristics of the CN / CS / CN / CS@CuS / CN material.

[0096] Figure 9 This invention, in Example 5, describes the drug release response of CUR-loaded CN / CS / CN / CS@CuS / CN under different media and laser irradiation conditions. Figure 9 It can be seen that under pH 7.4 and without laser irradiation, the release of CUR is relatively low, with a cumulative release rate of 30.81% over 48 hours. Under near-infrared laser irradiation, the release of CUR is 32.53% within 12 hours, while without laser irradiation, the drug release only increases by 3.27% over 12 hours. After another 12 hours of near-infrared laser irradiation, the release rapidly increases to 52.71%. This phenomenon further demonstrates the NIR response characteristics of the CN / CS / CN / CS@CuS / CN material.

[0097] Figure 10 This is an evaluation of the cytotoxicity of the CN / CS / CN / CS@CuS / CN microcapsules of Example 4 of the present invention on EA.hy926 cells. The results showed that even at a concentration of 100 μg / mL, the cells still had more than 85% cell viability, indicating that the CN / CS / CN / CS@CuS / CN microcapsules have low cytotoxicity.

[0098] Comparative Experiment 1: The Influence of the Number of Capsule Wall Layers on the Invention

[0099] In this comparative experiment, under the same etching conditions, the number of capsule wall layers affects the material shape, such as... Figure 11 and Figure 12 , Figure 11 and Figure 12 The samples were derived from the control group and the present invention (as in Example 4). The control group, CN / CS / CN@CuS, was created by reducing the number of capsule layers compared to the present invention, specifically by removing the outermost casein (CN) layer and the chitosan (CS) layer before the introduction of hollow copper sulfide nanoparticles. Specifically, the CaCO3 microspheres obtained in Example 1 were sequentially immersed in polyallylamine hydrochloride (PAH), casein (CN), chitosan (CS), and casein (CN) before being directly etched with hollow copper sulfide nanoparticles. Results showed that... Figure 11 The sample with two layers of casein and one layer of chitosan had a capsule wall that was too thin, resulting in severe microcapsule collapse and unstable material shape. Figure 12 The microcapsules composed of a 3-layer casein and 2-layer chitosan capsule wall obtained by this invention exhibit a regular capsule-like structure with an intact shape. Therefore, this invention further prefers 3 layers of casein and 2 layers of chitosan as the microcapsule wall.

[0100] Comparative Experiment 2: The Influence of Etching Conditions on the Invention

[0101] In this comparative experiment, the number of etching attempts and the amount of etching solution were increased based on Example 1. The results showed that etching with 25 mL of acetic acid solution in two sessions for 45 min was as effective as etching with 20 mL of acetic acid solution in a single session for 25 min. However, more etching attempts and longer etching times damaged the microcapsule structure. Therefore, this invention selects 15-22 mL of acetic acid solution for 20-28 min, and further optimizes 20 mL of acetic acid solution for 25 min.

[0102] Comparative Experiment 3: The Influence of Curcumin Loading Order on the Invention

[0103] This comparative experiment mainly studies the effect of curcumin loading order on the curcumin loading rate of the present invention. Experimental sample preparation:

[0104] Sample of Experiment 1 (i.e., the present invention): Based on the electrolyte balls of Example 1, curcumin was loaded separately according to the drug loading method of the present invention (see Example 5), and the drug loading rate of the obtained single-drug microcapsules was determined.

[0105] Control 1 sample: The preparation method is the same as above, except that the electrolyte spheres are first coated with a casein layer, then copper sulfide is added, then etched, and finally curcumin is loaded separately.

[0106] Control 2 sample: The preparation method is the same as above. The electrolyte ball is first etched, then casein loaded with curcumin is added, and finally copper sulfide is added.

[0107] The drug loading rate of the three groups of samples were measured. The results showed that the drug loading rate of the sample of the present invention when loaded with curcumin alone was 78.75%, while the drug loading rates of control 1 and control 2 were 71.85% and 78.05%, respectively. This indicates that the drug loading efficiency is the highest when using curcumin that is pre-bound to casein for loading. Considering that the drug will be lost when performing dual loading of drugs in the later stage, the present invention uses the last step to load curcumin that is pre-bound to casein onto microcapsules.

[0108] The present invention has been described in detail above through specific embodiments and examples, but these are not intended to limit the invention. Many modifications and improvements can be made by those skilled in the art without departing from the principles of the invention, and these should also be considered within the scope of protection of the present invention.

Claims

1. A casein-based natural polymer microcapsule with NIR responsiveness, characterized in that, The capsule wall is composed of natural polymer materials casein and chitosan, and the template is calcium carbonate. Polyelectrolyte spheres are obtained by layer-by-layer self-assembly of chitosan and casein. The natural polymer microcapsules are given NIR response by introducing hollow copper sulfide nanoparticles in the photothermal agent, and the natural polymer microcapsules are obtained by etching with acetic acid.

2. The casein-based natural polymer microcapsules with NIR responsiveness according to claim 1, characterized in that, This natural polymer microcapsule can simultaneously load both hydrophilic and hydrophobic drugs.

3. The method for preparing NIR-responsive casein-based natural polymer microcapsules according to claim 1 or 2, characterized in that, Includes the following steps: S1. Calcium carbonate microspheres were obtained by doping calcium carbonate with sodium polystyrene sulfonate (PSS) and used as microcapsule sacrificial templates. S2. Polyelectrolyte particles are obtained by alternately depositing casein (CN) and chitosan (CS) natural polymer layers on calcium carbonate spheres. S3. Hollow copper sulfide nanoparticles (CuS) are introduced onto the above electrolyte spheres. S4. Use acetic acid etching to remove the calcium carbonate template to obtain hollow CN / CS / CN / CS@CuS polymer microcapsules.

4. The method for preparing casein-based natural polymer microcapsules with NIR responsiveness according to claim 3, characterized in that, In step S1, the calcium carbonate microspheres are prepared using the following method: S11. Dissolve sodium carbonate solution and calcium nitrate solution in sodium polystyrene sulfonate aqueous solution to obtain solution A and solution B, respectively; S12. Mix solution A and solution B, stir the resulting mixture at 800-1200 rpm for 20-35 min at 25-35℃, centrifuge at 2500-3500 rpm for 2-3 min, and wash with deionized water more than twice to obtain PSS-doped calcium carbonate microspheres.

5. The method for producing NIR-responsive casein-based natural polymer microcapsules according to claim 3, characterized in that, In step S2, the polyelectrolyte spheres are prepared by the following method: the obtained CaCO3 microspheres are sequentially immersed in polyallylamine hydrochloride, casein, chitosan, casein and chitosan solutions, and after each deposition for 10-18 min, they are centrifuged at 2800-3200 rpm for 1-3 min, and washed twice with 0.4-0.6 M NaCl solution to remove unadsorbed polyelectrolytes.

6. The method for producing NIR-responsive casein-based natural polymer microcapsules according to claim 3, characterized in that, In step S4, the CaCO3 sacrificial template is removed by etching with 15-22 mL of 0.1 M, pH 4.5 glacial acetic acid solution at a speed of 220-280 rpm for 20-28 min.

7. The application of the casein-based natural polymeric microcapsules with NIR responsiveness as described in claim 1 or 2 in the preparation of polymeric microcapsule drug carriers.

8. A method for preparing dual-drug loading using polymeric microcapsules as described in claim 1 or 2, characterized in that, Includes the following steps: 1) Casein and hydrophobic drugs are pre-combined. The hydrophobic drugs are first introduced into the hydrophobic core of casein, and then introduced onto the polymer microcapsules. 2) By utilizing the electrostatic interaction between casein and hydrophilic drugs, hydrophilic drugs can be introduced into polymer microcapsules that are already loaded with hydrophobic drugs, thus achieving dual drug loading.

9. The method for preparing polymeric microcapsules for dual drug loading according to claim 8, characterized in that, The hydrophobic drug is curcumin (CUR); the specific operation of step 1) is as follows: add 0.3-0.6 mL of 1 mg / mL curcumin ethanol solution to 3-6 mL of 1 mg / mL sodium caseinate solution (pH 6.0), stir at 400-800 rpm for 1-2 h to complete CUR loading.

10. The method for preparing polymeric microcapsules for dual drug loading according to claim 8, characterized in that, The hydrophilic drug is doxorubicin (DOX). The specific operation of step 2) is as follows: using the electrostatic interaction between casein and doxorubicin, add 0.3 mL of 2 mg / mL DOX aqueous solution to the dispersion of curcumin-loaded microcapsules, and stir at 28-32 ℃ and 200-300 rmp for 10-14 h to complete the DOX loading.

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