Method for improving doxorubicin loading capacity in Au-coated SiO2 nano-carrier with core-shell structure and application of doxorubicin loading capacity

Abstract

The invention relates to a method for improving doxorubicin loading capacity in a core-shell structure Au (at) m-SiO2 nano-carrier. The method comprises the following steps: mixing and incubating core-shell structure nano-particles of mesoporous silica coated gold nano-particles and drug doxorubicin hydrochloride in an aqueous solution, so that doxorubicin is adsorbed on the inner surfaces of mesopores; the product and Na2SO4 are mixed and incubated in an aqueous solution, so that the medicine adsorbed in silicon dioxide mesopores forms small nanocrystals; mixing the product with NaOH in an aqueous solution to promote drug molecules to be adsorbed on nanocrystals to form Dox aggregates and fill mesopores with the Dox aggregates; and aging the product, and centrifuging to remove free drugs, so that the loading capacity of adriamycin in the Au (at) m-SiO2 nano-carrier with the core-shell structure is finally improved. According to the method, the drug loading capacity and the encapsulation efficiency of the adriamycin can be greatly improved, and the adriamycin has the drug release characteristics of pH response and temperature response.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a method for increasing the loading amount of doxorubicin in a core-shell structured Au@SiO2 nanocarrier and an application thereof. Background Art

[0002] Doxorubicin (Dox) is a potent chemotherapy drug widely used in the treatment of various cancers. However, Dox has disadvantages such as severe acute toxicity (such as cardiotoxicity) and non-targeted drug delivery, which limit its clinical application. With the continuous development of the field of nanomedicine, a multifunctional nano-drug delivery platform using a gold / mesoporous silica core-shell structure has been gradually explored to overcome the above shortcomings. Among them, the core of the gold nanoparticle provides localized plasmon optical properties for drug tracing and controlled targeted release; the high specific surface area mesoporous silica shell is used to load more drugs and achieve sustained release of drugs and good biocompatibility.

[0003] However, in the currently reported gold / mesoporous silica core-shell nano-drug delivery platform, Dox is adsorbed on the inner surface of the mesoporous silica pores in the form of molecules or ions. After centrifugation to remove free Dox, a large amount of Dox remains in the supernatant, and only a small amount of drug is loaded into the nanocarrier, resulting in low drug loading and drug encapsulation efficiency (Zhenjiang Zhang et al. Mesoporous Silica-Coated Gold Nanorods as a Light-Mediated Multifunctional Theranostic Platform for Cancer Treatment. Adv. Mater. 2012, 24, 1418-1423. Yanyan Yu et al.).

[0004] Therefore, a processing method is needed to obtain a gold / mesoporous silica core-shell structured multifunctional nano-drug delivery platform with high doxorubicin loading to improve drug loading and encapsulation efficiency. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for increasing the doxorubicin loading capacity in a core-shell structured Au@SiO2 nanocarrier and its application.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for increasing the doxorubicin loading capacity in a core-shell Au@m-SiO2 nanocarrier, the method comprising the following steps:

[0008] (1) mixing and incubating the core-shell structured nanoparticles of mesoporous silica-coated gold nanoparticles and the drug doxorubicin hydrochloride in an aqueous solution;

[0009] (2) mixing the product of step (1) with Na2SO4 in an aqueous solution and incubating;

[0010] (3) mixing the product of step (2) with NaOH in an aqueous solution;

[0011] (4) The product of step (3) is aged and then centrifuged to remove free drugs, thereby ultimately increasing the loading amount of doxorubicin in the core-shell structure Au@m-SiO2 nanocarrier.

[0012] In the method of the present invention, the core-shell structure nanoparticles of mesoporous silica coated gold nanoparticles and the drug doxorubicin hydrochloride are mixed and incubated in an aqueous solution. During this process, doxorubicin is mainly adsorbed on the inner surface of the mesopores; after that, it is mixed and incubated with Na2SO4, so that the drug adsorbed in the silica mesopores forms small nanocrystals (2Dox-NH3 + +SO4 2- =(Dox-NH3)2SO4); subsequently, mixing with a NaOH solution promotes drug adsorption onto the nanocrystals, forming Dox aggregates that fill the mesopores. After drug loading, the solution can be centrifuged multiple times to remove free drug and return the pH to neutral. This method significantly increases doxorubicin loading and encapsulation efficiency, while also exhibiting pH- and temperature-responsive drug release.

[0013] Preferably, the size of the gold nanoparticles is 5-100 nm, for example, 5 nm, 10 nm, 15 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 100 nm, etc., but is not limited to the values listed above, and other values not listed within this range are also applicable. The gold particles can be spherical, rod-shaped, spindle-shaped, flake-shaped, etc.

[0014] Preferably, the shell thickness of the mesoporous silica is 1-100 nm, for example, it can be 1 nm, 2 nm, 5 nm, 10 nm, 20 nm, 50 nm, 70 nm, 80 nm or 100 nm, etc., but is not limited to the listed values, and other unlisted values within this numerical range are also applicable.

[0015] Preferably, the concentration of the core-shell structured nanoparticles in the aqueous solution in step (1) is 0.01-10 nmol / L, for example, 0.01 nmol / L, 0.02 nmol / L, 0.05 nmol / L, 0.1 nmol / L, 0.2 nmol / L, 0.5 nmol / L, 1 nmol / L, 2 nmol / L, 5 nmol / L or 10 nmol / L, etc., but is not limited to the listed values, and other values not listed within this numerical range are also applicable.

[0016] Preferably, the concentration of doxorubicin hydrochloride in the solution in step (1) is 0.01-3 mg / mL, for example, 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL or 3 mg / mL, etc., but is not limited to the listed values, and other values not listed within this numerical range are also applicable.

[0017] Preferably, the temperature of the mixed incubation in step (1) is 10-35°C, for example, it can be 10°C, 12°C, 15°C, 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 35°C, etc., and the time is not less than 10 minutes, for example, it can be 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 5 hours, etc., but is not limited to the listed values, and other values not listed within this numerical range are also applicable.

[0018] Preferably, during the mixed incubation in step (1), any one or at least two of stirring treatment, ultrasonic treatment, oscillation treatment or mixer treatment are also performed.

[0019] Preferably, the concentration of Na2SO4 in the aqueous solution in step (2) is 0.1-50mmol / L, for example, it can be 0.1mmol / L, 0.2mmol / L, 1mmol / L, 2mmol / L, 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L, 25mmol / L, 30mmol / L, 35mmol / L, 40mmol / L, 45mmol / L or 50mmol / L, but is not limited to the listed values. Other values not listed within this numerical range are also applicable, more preferably 5-25mmol / L.

[0020] When the concentration of Na2SO4 in the aqueous solution is selected to be 0.1-50 mmol / L, especially 5-25 mmol / L, the drug loading amount and encapsulation efficiency of doxorubicin hydrochloride can be higher.

[0021] Preferably, the temperature of the mixed incubation in step (2) is 10-35°C, for example, it can be 10°C, 12°C, 15°C, 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 35°C, etc., and the time is not less than 10 minutes, for example, it can be 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 5 hours, etc., but is not limited to the listed values, and other values not listed within this numerical range are also applicable.

[0022] Preferably, during the mixed incubation in step (2), any one or at least two of stirring treatment, ultrasonic treatment, oscillation treatment or mixer treatment are also performed.

[0023] Preferably, the concentration of NaOH in the aqueous solution in step (3) is 0.1-3 mmol / L, for example, 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, 0.4 mmol / L, 0.5 mmol / L, 0.7 mmol / L, 1 mmol / L, 1.5 mmol / L, 2 mmol / L, or 3 mmol / L, but is not limited to the values listed above. Other values not listed within this range are also applicable. More preferably, it is 1-2.5 mmol / L.

[0024] When the concentration of NaOH in the aqueous solution is selected to be 0.1-3 mmol / L, especially 1-2.5 mmol / L, the drug loading amount and encapsulation efficiency of doxorubicin hydrochloride can be higher.

[0025] Preferably, the aging temperature in step (4) is 10-35°C, for example, it can be 10°C, 12°C, 15°C, 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 35°C, etc., and the time is not less than 48h, for example, 48h, 60h, 72h, 84h, 96h, 108h, 120h, 132h, 144h, 156h, 168h, etc., but is not limited to the listed values, and other values not listed within the numerical range are also applicable.

[0026] Preferably, during the aging process of step (4), any one or at least two of stirring treatment, ultrasonic treatment, oscillation treatment or mixer treatment are performed.

[0027] Preferably, the number of centrifugation in step (4) is 1-5 times, for example, it can be 1 time, 2 times, 3 times, 4 times or 5 times, each time independently carried out at 8000-15000 rpm (for example, 8000 rpm, 9000 rpm, 10000 rpm, 12000 rpm, 13000 rpm, 15000 rpm, etc.) for 1-10 min (for example, 1 min, 3 min, 5 min, 6 min, 7 min, 8 min, 10 min, etc.).

[0028] In a second aspect, the present invention provides an application of the method described in the first aspect in preparing a core-shell structured Au@m-SiO2 nano drug delivery system loaded with doxorubicin hydrochloride.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] In the method disclosed herein, core-shell nanoparticles composed of mesoporous silica-coated gold nanoparticles are mixed with the drug doxorubicin hydrochloride in an aqueous solution and incubated to allow the doxorubicin to adsorb onto the inner surfaces of the mesopores. The mixture is then mixed with sodium sulfate (NaSO) to form small nanocrystals of the drug adsorbed within the silica mesopores. Subsequently, the mixture is mixed with sodium hydroxide (NaOH) to encourage drug molecules to adsorb onto the nanocrystals, forming Dox aggregates that fill the mesopores. After drug loading, the mixture can be centrifuged multiple times to remove free drug and return the pH to neutral. This method significantly increases the drug loading and encapsulation efficiency of doxorubicin and exhibits pH- and temperature-responsive drug release properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the extinction spectrum of different Dox concentrations;

[0032] Figure 2 is the linear fitting graph of Dox extinction at 480 nm;

[0033] Figure 3 This is a TEM image of the gold / mesoporous silica core-shell structure model particles used in the present invention;

[0034] Figure 4 This is a physical image and extinction spectrum of the supernatant and substrate after the sample was centrifuged three times in Example 1;

[0035] Figure 5 1 is an element imaging test result diagram of the sample obtained in Example 4;

[0036] Figure 6 This is a physical picture and extinction spectrum of the supernatant and substrate after the sample was centrifuged three times in comparative example 1;

[0037] Figure 7 It is the drug release curve diagram of Application Examples 1-4. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0039] Since Au@m-SiO2 has strong extinction at 200-250nm (232nm), and in order to reduce the influence of a small amount of mesoporous silica that may be separated during the processing step on the calculation, the present invention uses the extinction at 480nm to characterize the content of Dox. The linear equation is Y=0.01856X, where X is the concentration of Dox (in μg / mL) and Y is the extinction. The extinction spectra of Dox at different concentrations are shown in Figure 2. Figure 1 The standard curve of extinction at 480 nm is shown in Figure 2 .

[0040] Encapsulation efficiency (EE) refers to the ratio of the amount of drug encapsulated in the carrier to the total amount of drug input in the field of pharmaceutical preparations, usually expressed as a percentage. After the drug-loaded sample is treated according to the method of the present invention, the percentage of drug retained in the mesoporous silica is expressed as the encapsulation efficiency. Since the same gold / mesoporous silica core-shell structure model particles and the same concentration are used in the examples, comparative examples and application examples of this patent, drug loading = concentration of added Dox - sum of Dox concentrations in the centrifugal supernatant,

[0041] Preparation Example 1

[0042] This preparation example provides a gold / mesoporous silica core-shell structure nanoparticle, and the preparation method is as follows:

[0043] (1) In a 50 mL round-bottom flask, 8.2 mL of deionized water, 75 μL of 0.1 mol / L CTAB (cetyltrimethylammonium bromide) aqueous solution, 0.5 nmol / L gold particle (particle size approximately 45 nm) solution, and 10 μL of 1 mol / L NaOH aqueous solution were added in sequence.

[0044] (2) The above solution was placed in a 30°C water bath for 10 min and magnetically stirred at 450 rpm; then, 60 μL of ethyl silicate (dissolved in ethanol, with a volume fraction of 20%) was added dropwise and reacted for 24 h to obtain the Au@m-SiO2 sample.

[0045] (3) The Au@m-SiO2 sample was purified by centrifugation at 1000 rpm for 5 min three times and dispersed in ethanol.

[0046] (4) Add 0.25 mL of a 0.4 g / mL aqueous solution of NH₄NO₃ to the Au@m-SiO₂ sample purified by centrifugation. The mixture was magnetically stirred (450 rpm) at 50°C for 24 h to remove any CTAB remaining in the silica mesopores. Finally, the mixture was centrifuged twice at 1000 rpm for 5 min, dispersed in water, and set aside.

[0047] During the above preparation process, the particles were centrifuged several times, and the loss of particles was 10%. The final concentration of Au@m-SiO2 was 0.45nmol / L. The size of the gold core was 48.5±2.5nm and the thickness of the mesoporous silica shell was 34.6±1.9nm. The TEM images are shown in Figure 1. Figure 3 .

[0048] Example 1

[0049] This embodiment provides a method for increasing the doxorubicin loading capacity in a core-shell Au@m-SiO2 nanocarrier, as follows:

[0050] (1) Add 10 μL of 14.7 mg / mL doxorubicin hydrochloride aqueous solution to 1 mL of Au@m-SiO2 aqueous solution with a particle concentration of 0.45 nmol / L. Mix and incubate at 25°C for 30 min to allow Dox to reach adsorption equilibrium in the mesopores of silica (the color of the solution is a mixture of Au@m-SiO2 and doxorubicin, bright red).

[0051] (2) Add 12.5 μL of 1 mol / L Na2SO4 aqueous solution to the above solution, mix and incubate at 25°C for 30 min to allow the reaction to reach equilibrium;

[0052] (3) Add 5 μL of 0.2 mol / L NaOH aqueous solution to the above solution. After mixing, the solution turns purple-red.

[0053] (4) The above solution was placed on a tumbling mixer (MA9IC, manufactured by Aikemai Co., Ltd.) and tumbling mixed at 25°C for 96 h (tumbling speed 60 rpm);

[0054] (5) The above solution was centrifuged at 12000 rpm for 5 min, and the supernatant was collected. 1 mL of deionized water was added to the substrate and ultrasonicated (Kunshan Hechuang Ultrasonic Instrument Co., Ltd. KH3200B model, power 150 W) for 1 min to disperse it. This step was repeated 3 times (the solution turned bright red again) to remove the free drug.

[0055] The extinction spectrum of the supernatant and substrate after three centrifugations was tested respectively. The actual picture and extinction spectrum of the supernatant and substrate after centrifugation are shown in Figure 4As can be seen from the figure, Dox in the substrate has formed nanocrystals and is located within the mesoporous silica pores. The absorption peak (480 nm) of Dox molecules / ions has been significantly reduced. Using the Dox standard curve, the drug loading and encapsulation efficiency were calculated to be 0.134 mg / mL and 91.4%, respectively.

[0056] Example 2

[0057] This embodiment provides a method for increasing the doxorubicin loading capacity in a core-shell Au@m-SiO2 nanocarrier, as follows:

[0058] (1) Add 20 μL of 14.7 mg / mL doxorubicin hydrochloride aqueous solution to 1 mL of Au@m-SiO2 aqueous solution with a particle concentration of 0.45 nmol / L. Mix and incubate at 30°C for 20 min to allow Dox to reach adsorption equilibrium in the mesopores of silica (the color of the solution is a mixture of Au@m-SiO2 and doxorubicin, bright red).

[0059] (2) Add 12.5 μL of 1 mol / L Na2SO4 aqueous solution to the above solution, mix and incubate at 30°C for 20 min to allow the reaction to reach equilibrium;

[0060] (3) Add 5 μL of 0.2 mol / L NaOH aqueous solution to the above solution. After mixing, the solution turns purple-red.

[0061] (4) The above solution was placed on a tumbling mixer (MA9IC, manufactured by Aikemai Co., Ltd.) and tumbling mixed at 30°C for 72 h (tumbling speed 60 rpm);

[0062] (5) The above solution was centrifuged at 10,000 rpm for 8 min, and the supernatant was collected. 1 mL of deionized water was added to the substrate and ultrasonicated (Kunshan Hechuang Ultrasonic Instrument Co., Ltd. KH3200B model, power 150 W) for 1 min to disperse it. This step was repeated 3 times (the solution turned bright red again) to remove the free drug.

[0063] The drug loading capacity and encapsulation efficiency were calculated using the Dox standard curve to be 0.272 mg / mL and 92.4%, respectively.

[0064] Example 3

[0065] This embodiment provides a method for increasing the doxorubicin loading capacity in a core-shell Au@m-SiO2 nanocarrier, as follows:

[0066] (1) To 1 mL of an Au@m-SiO2 aqueous solution with a particle concentration of 0.45 nmol / L, 7 μL of a 14.7 mg / mL doxorubicin hydrochloride aqueous solution was added, and the mixture was mixed and incubated at 20°C for 40 min to allow Dox to reach adsorption equilibrium in the mesopores of the silica (the color of the solution was a mixture of Au@m-SiO2 and doxorubicin, bright red).

[0067] (2) Add 12.5 μL of 1 mol / L Na2SO4 aqueous solution to the above solution, mix and incubate at 20°C for 40 min to allow the reaction to reach equilibrium;

[0068] (3) Add 5 μL of 0.2 mol / L NaOH aqueous solution to the above solution. After mixing, the solution turns purple-red.

[0069] (4) The above solution was placed on a tumbling mixer (MA9IC, manufactured by Aikemai Co., Ltd.) and tumbling mixed at 20°C for 108 h (tumbling speed 60 rpm);

[0070] (5) The above solution was centrifuged at 15,000 rpm for 3 min, and the supernatant was collected. 1 mL of deionized water was added to the substrate and dispersed by ultrasound (Kunshan Hechuang Ultrasonic Instrument Co., Ltd. KH3200B model, power 150 W) for 1 min. This step was repeated 3 times (the solution turned bright red again) to remove free drugs.

[0071] The drug loading capacity and encapsulation efficiency were calculated using the Dox standard curve to be 0.094 mg / mL and 91.2%, respectively.

[0072] It can be seen from Examples 1-3 that when the amount of Dox added is within a certain range, the encapsulation efficiency remains basically unchanged, and the drug loading amount can be regulated by the amount of Dox added.

[0073] Example 4

[0074] This example provides a method for increasing the doxorubicin loading capacity of core-shell Au@m-SiO nanocarriers. The method differs from Example 2 only in that the 1-minute sonication in step (5) is replaced with vortexing (using a Vortex-Genie 2 vortex oscillator (Scientific Industries, USA, at gear 7) for 1 minute). All other conditions remain unchanged.

[0075] The product of Example 4 was subjected to elemental imaging test. The specific operation was as follows: 5 μL of the above-synthesized sample was dropped onto a TEM microgrid and allowed to dry; a single particle was randomly selected on the microgrid using a JEM-2100 TEM and line scanning elemental analysis was performed. The elemental scanning results are shown in FIG. Figure 5From the figure, we can see that the particles contain Au, Si, O, N and S elements, which means that the particles contain gold nanoparticles (Au), silicon dioxide (Si and O), Dox (chemical formula C 27 H 29 NO 11 , N and O) and SO4 2- (S and O).

[0076] Example 5

[0077] This example provides a method for increasing the doxorubicin loading capacity of core-shell Au@m-SiO2 nanocarriers. The only difference from Example 4 is that in step (3), 1 μL of a 0.2 mol / L NaOH aqueous solution is added to the above solution. Other conditions remain unchanged.

[0078] Example 6

[0079] This example provides a method for increasing the doxorubicin loading capacity of core-shell Au@m-SiO2 nanocarriers. The only difference from Example 4 is that in step (3), 2 μL of a 0.2 mol / L NaOH aqueous solution is added to the above solution. Other conditions remain unchanged.

[0080] Example 7

[0081] This example provides a method for increasing the doxorubicin loading capacity of core-shell Au@m-SiO2 nanocarriers. The only difference from Example 4 is that in step (3), 7.5 μL of a 0.2 mol / L NaOH aqueous solution is added to the above solution. Other conditions remain unchanged.

[0082] Example 8

[0083] This example provides a method for increasing the doxorubicin loading capacity of core-shell Au@m-SiO2 nanocarriers. The only difference from Example 4 is that in step (3), 10 μL of a 0.2 mol / L NaOH aqueous solution is added to the above solution. Other conditions remain unchanged.

[0084] Example 9

[0085] This example provides a method for increasing the doxorubicin loading capacity of core-shell Au@m-SiO2 nanocarriers. The only difference from Example 4 is that in step (3), 12.5 μL of a 0.2 mol / L NaOH aqueous solution is added to the above solution. All other conditions remain unchanged.

[0086] The supernatants of Examples 4-9 were centrifuged three times and subjected to extinction spectrum testing. The drug loading and encapsulation efficiency were calculated using the Dox standard curve. The results are shown in Table 1.

[0087] Table 1

[0088]

[0089] As shown in Table 1, a comparison of Examples 2 and 4 reveals that vortexing results in higher drug loading and encapsulation efficiency than ultrasonic re-dispersion, likely due to the dissolution and shedding of a small amount of Dox nanocrystals during ultrasound. Comparisons of Examples 4-9 reveal that the NaOH concentration in the mixed system affects the drug loading and encapsulation efficiency of doxorubicin hydrochloride, with an optimal concentration range of 1-2.5 mmol / L.

[0090] Example 10

[0091] This example provides a method for increasing the doxorubicin loading capacity of core-shell Au@m-SiO nanocarriers. The only difference from Example 4 is that in step (2), 25 μL of a 1 mol / L aqueous Na₂SO₄ solution is added to the above solution. Other conditions remain unchanged.

[0092] Example 11

[0093] This example provides a method for increasing the doxorubicin loading capacity of core-shell Au@m-SiO nanocarriers. The only difference from Example 4 is that in step (2), 1.25 μL of a 1 mol / L aqueous Na2SO4 solution is added to the above solution. Other conditions remain unchanged.

[0094] Example 12

[0095] This example provides a method for increasing the doxorubicin loading capacity of core-shell Au@m-SiO nanocarriers. The only difference between this method and Example 4 is that in step (2), 50 μL of a 1 mol / L Na2SO4 aqueous solution is added to the above solution. Other conditions remain unchanged.

[0096] The supernatants of Examples 10-12 were centrifuged three times and subjected to extinction spectrum testing. The drug loading and encapsulation efficiency were calculated using the Dox standard curve. The results are shown in Table 2.

[0097] Table 2

[0098] Example 4 Example 10 Example 11 Example 12 <![CDATA[Volume of Na2SO4 solution added]]> 12.5μL 25 μL 1.25 μL 50 μL Drug loading (mg / mL) 0.283 0.281 0.119 0.248 Encapsulation efficiency 96.3％ 95.6％ 40.4％ 84.3％

[0099] As shown in Table 2, and by comparing Examples 4, 10-12, the Na2SO4 concentration in the mixed system affects the drug loading and encapsulation efficiency of doxorubicin hydrochloride. Both excessively high and low Na2SO4 concentrations are detrimental to drug loading and encapsulation efficiency. When the Na2SO4 concentration is too low, Dox cannot form nanocrystals within the silica mesopores. When the Na2SO4 concentration is too high, the excessively high salt concentration can reduce the surface charge of Au@m-SiO2 particles with a low zeta potential (~30 mV), hindering particle stability. The optimal concentration range is 5-25 mmol / L.

[0100] Example 13

[0101] This example provides a method for increasing the doxorubicin loading capacity of core-shell Au@m-SiO2 nanocarriers. The only difference from Example 1 is that the tumbling mixing time in step (4) is changed from 96 h to 24 h. Other conditions remain unchanged.

[0102] The extinction spectrum of the supernatant after three centrifugations was tested, and the drug loading capacity and encapsulation efficiency were calculated using the Dox standard curve to be 0.131 mg / mL and 44.6%, respectively.

[0103] Comparative Example 1

[0104] The only difference between this comparative example and Example 1 is that steps (2) and (3) are omitted. Other conditions remain unchanged.

[0105] The extinction spectrum of the supernatant and substrate after three centrifugations was tested. The actual picture and extinction spectrum of the supernatant and substrate after centrifugation are shown in Figure 6 As can be seen from the figure, a large amount of Dox is lost after one centrifugation, and Dox exists in the form of molecules / ions. The drug loading and encapsulation efficiency were calculated using the Dox standard curve to be 0.015 mg / mL and 10.4%, respectively.

[0106] Comparative Example 2

[0107] The only difference between this comparative example and Example 1 is that step (2) is omitted. Other conditions remain unchanged.

[0108] The extinction spectrum of the supernatant after three centrifugations was tested, and the drug loading capacity and encapsulation efficiency were calculated to be 0.029 mg / mL and 19.4%, respectively, using the Dox standard curve.

[0109] Comparative Example 3

[0110] The only difference between this comparative example and Example 1 is that step (3) is omitted. Other conditions remain unchanged.

[0111] The extinction spectrum of the supernatant after three centrifugations was tested, and the drug loading capacity and encapsulation efficiency were calculated to be 0.054 mg / mL and 36.5%, respectively, using the Dox standard curve.

[0112] By comparing the test results of Example 1 with Comparative Examples 1-3, it can be seen that for the method involved in the present invention, the addition of sodium sulfate and sodium hydroxide are both essential steps to improve the drug encapsulation efficiency. Among them, Comparative Example 1 is a method commonly used in current literature, and the encapsulation efficiency is very low.

[0113] Application Example 1

[0114] The substrate obtained from Example 2 was centrifuged three times and subjected to drug release test:

[0115] (1) Add 3 mL of PBS solution (10 mM, pH = 4.5) to the substrate and disperse it by ultrasonication for 1 min to obtain the sample solution to be tested.

[0116] (2) Add 100 mL of PBS solution (10 mM, pH = 4.5) as the external medium to a 200 mL beaker and adjust the temperature to 37°C;

[0117] (3) Place 3 mL of the sample solution to be tested into a dialysis bag (molecular weight cut-off 3500-5000 Da, diameter 15 mm) and clamp the two ends;

[0118] (4) Place the dialysis bag in the external medium without slow stirring, test the extinction spectrum of the external medium at different release times, and calculate the amount of Dox released into the external medium and the released amount.

[0119] Application Example 2

[0120] The substrate obtained by centrifuging the sample obtained in Example 2 three times was subjected to a drug release test. The only difference between the test and Application Example 1 was that the pH of the PBS solution used in step (1) was 7.4.

[0121] Application Example 3

[0122] The substrate obtained by centrifuging the sample three times in Example 2 was subjected to a drug release test. The only difference from Application Example 1 was that the temperature was controlled to 48 degrees in step (2).

[0123] Application Example 4

[0124] The substrate obtained by centrifuging the sample obtained in Example 2 three times was subjected to a drug release test. The only difference between the test and Application Example 1 was that the pH of the PBS solution used in step (1) was 7.4, and the temperature was controlled to 48 degrees in step (2).

[0125] The drug release curves of application examples 1-4 are shown in Figure 7 .

[0126] As can be seen from Application Examples 1-2 in the figure, under acidic conditions, the Dox aggregates in the silica mesopores will disaggregate after the amine groups are protonated, resulting in the release of Dox. Therefore, the drug release rate under acidic conditions (pH = 4.5) is significantly higher than that under neutral conditions (pH = 7.4).

[0127] As shown in Examples 1 and 3, and 2 and 4, the drug release rate at 48°C is significantly higher than at 37°C. This demonstrates that laser irradiation of the nanoparticle drug-carrying platform, with the gold nanoparticle core absorbing the heat generated by the laser, can regulate the drug release rate. The drug delivery system prepared by the present method can regulate drug release rate by controlling pH and temperature.

[0128] The applicant declares that the present invention is illustrated by the above-described embodiments, but the present invention is not limited to the above-described embodiments. This does not mean that the present invention must rely on the above-described embodiments in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent replacements for raw materials in the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0129] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0130] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for increasing the doxorubicin loading capacity in a core-shell Au@m-SiO2 nanocarrier, characterized in that: The method comprises the following steps: (1) mixing and incubating the core-shell structured nanoparticles of mesoporous silica-coated gold nanoparticles and the drug doxorubicin hydrochloride in an aqueous solution; (2) mixing the product of step (1) with Na2SO4 in an aqueous solution and incubating; (3) mixing the product of step (2) with NaOH in an aqueous solution; (4) The product of step (3) is aged and then centrifuged to remove free drugs, thereby ultimately increasing the loading amount of doxorubicin in the core-shell structure Au@m-SiO2 nanocarrier.

2. The method according to claim 1, characterized in that The size of the gold nanoparticles is 5-100 nm.

3. The method according to claim 1 or 2, characterized in that The shell thickness of the mesoporous silica is 1-100 nm.

4. The method according to any one of claims 1 to 3, characterized in that The concentration of the core-shell structured nanoparticles in the aqueous solution in step (1) is 0.01-10 nmol / L; Preferably, the concentration of doxorubicin hydrochloride in the solution in step (1) is 0.01-3 mg / mL.

5. The method according to any one of claims 1 to 4, characterized in that The temperature of the mixed incubation in step (1) is 10-35° C. and the time is not less than 10 minutes; Preferably, during the mixed incubation in step (1), any one or at least two of stirring treatment, ultrasonic treatment, oscillation treatment or mixer treatment are also performed.

6. The method according to any one of claims 1 to 5, characterized in that The concentration of Na2SO4 in the aqueous solution in step (2) is 0.1-50 mmol / L; more preferably 5-25 mmol / L; Preferably, the temperature of the mixed incubation in step (2) is 10-35°C and the time is not less than 10 minutes; Preferably, during the mixed incubation in step (2), any one or at least two of stirring treatment, ultrasonic treatment, oscillation treatment or mixer treatment are also performed.

7. The method according to any one of claims 1 to 6, characterized in that The concentration of NaOH in the aqueous solution in step (3) is 0.1-3 mmol / L; more preferably 1-2.5 mmol / L.

8. The method according to any one of claims 1 to 7, characterized in that The aging temperature in step (4) is 10-35° C. and the aging time is not less than 48 hours; Preferably, during the aging process of step (4), any one or at least two of stirring treatment, ultrasonic treatment, oscillation treatment or mixer treatment are performed.

9. The method according to any one of claims 1 to 8, characterized in that The number of centrifugation in step (4) is 1-5 times, each time independently performed at 8000-15000 rpm for 1-10 min.

10. Use of the method according to any one of claims 1 to 9 in preparing a core-shell structured Au@m-SiO2 nano drug delivery system loaded with doxorubicin hydrochloride.

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