PDA (at) P (NIPAAm-co-AM) nano hydrogel for resisting tumors through light-operated chemotherapy as well as preparation method and application of PDA (at) P (NIPAAm-co-AM) nano hydrogel

Abstract

The invention discloses PDA (at) P (NIPAAm-co-AM) nano hydrogel for resisting tumors through light-operated chemotherapy as well as a preparation method and application of the PDA (at) P (NIPAAm-co-AM) nano hydrogel. The preparation method comprises the following steps: adding NIPAAm and AM into water, stirring and vacuumizing to obtain an NIPAAm and AM aqueous solution; the preparation method comprises the following steps: sequentially adding C2H5OH and NH3.H2O into water, uniformly stirring, adding a DA.HCl aqueous solution, stirring in a dark place, centrifugally washing to obtain PDA nano-particles, and dispersing the PDA nano-particles in water to obtain a PDA solution; the preparation method comprises the following steps: mixing a PDA solution, an NIPAAm and AM aqueous solution, SBS, APS, BIS and SDS for reaction, dialyzing and freeze-drying after the reaction to obtain PDA (at) P (NIPAAm-co-AM) nanogel, and dissolving the PDA (at) P (NIPAAm-co-AM) nanogel in a tumor chemotherapy drug aqueous solution to obtain the light-operated chemotherapy anti-tumor PDA (at) P (NIPAAm-co-AM) nanohydrogel.

Description

Technical Field

[0001] The invention belongs to the field of biological hydrogel materials, and in particular relates to a light-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nano-hydrogel and a preparation method and application thereof. Background Art

[0002] Cancer treatment remains a challenge that needs to be solved. Currently, surgical resection, radiotherapy, chemotherapy and other methods are commonly used in clinical cancer treatment, but there are still problems such as poor targeting, tumor recurrence and metastasis. As a clinical adjuvant therapy, phototherapy has gradually attracted attention in cancer treatment. Near-infrared light has the characteristics of large tissue penetration depth and smaller attenuation during penetration. Taking advantage of the characteristics of high permeability and long retention of tumor tissue, nano-sized therapeutic drugs can be targeted to the tumor site and combined with locally irradiated near-infrared light to design a light-responsive treatment method. However, in the commonly used light-assisted treatment methods, near-infrared light irradiation produces a photothermal effect. High-temperature photothermal treatment above 50°C can kill cancer cells, but will cause thermal damage to normal tissues near the tumor tissue, reducing patient compliance; low-temperature photothermal treatment of 42-46°C will promote the synthesis of heat shock proteins in tumor cells, improve the heat resistance of tumor cells, and reduce the treatment effect. Therefore, the photothermal effect produced in light-assisted therapy is still one of the reasons that limit its large-scale clinical application.

[0003] Therefore, light-assisted tumor therapy faces photothermal problems in clinical practice, and it is necessary to develop new light-assisted targeted therapy systems to eliminate the photothermal effect with low controllability, achieve the anti-tumor properties of light-controlled chemotherapy, and improve the treatment effect. Summary of the invention

[0004] The purpose of the embodiments of the present application is to provide a light-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogel and a preparation method and application thereof in order to address the above-mentioned technical problems existing in the prior art.

[0005] According to a first aspect of an embodiment of the present application, a method for preparing a light-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogel is provided, comprising:

[0006] (1) Add NIPAAm (N-isopropylacrylamide) and AM (acrylamide) to water, stir and evacuate to obtain an aqueous solution of NIPAAm and AM, remove oxygen, and promote free radical polymerization;

[0007] (2) adding C2H5OH (ethanol) and NH3·H2O (ammonia water) to water in sequence and stirring evenly, adding DA·HCl (dopamine hydrochloride) aqueous solution and stirring in the dark, centrifuging and washing to obtain PDA nanoparticles, and dispersing the PDA nanoparticles in water to obtain a PDA solution;

[0008] (3) PDA (polydopamine) solution, NIPAAm and AM aqueous solution, SBS (sodium metabisulfite), APS (ammonium persulfate), BIS (methyl acrylamide), and SDS (sodium dodecyl sulfate) are mixed and reacted, and then dialyzed and freeze-dried to obtain PDA@P (NIPAAm-co-AM) nanogel, which is convenient for loading tumor chemotherapy drugs through swelling;

[0009] (4) The PDA@P (NIPAAm-co-AM) nanogel was dissolved in an aqueous solution of tumor chemotherapy drugs (such as doxorubicin DOX, paclitaxel PTX, cisplatin, 5-FU, gemcitabine, etc.) to obtain the light-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogel.

[0010] Preferably, in step (1), the molar ratio of NIPAAm to AM is 80:20. The phase transition temperature range of the PDA@P (NIPAAm-co-AM) nanohydrogel prepared under this condition is 37°C-45°C, which is in line with the expected temperature range of the photothermal excitation nanohydrogel phase transition.

[0011] In a possible implementation, step (2) is specifically as follows:

[0012] Add 40 parts by volume of C2H5OH to 90 parts by volume of ultrapure water, stir evenly under heating conditions (35℃~40℃), immediately add 3 parts by volume of NH3·H2O and stir evenly, add DA·HCl aqueous solution, wherein the mass volume ratio of DA·HCl to C2H5OH is 50 mg:4 mL, stir under heating conditions (35℃~40℃) and protect from light to react, obtain PDA nanoparticles after centrifugation and washing, and disperse the PDA nanoparticles in water to obtain a PDA solution.

[0013] In a possible implementation, step (3) specifically includes:

[0014] (3.1) Add the PDA aqueous solution to the NIPAAm and AM aqueous solution, stir and evacuate to obtain a black solution, in which the mass of PDA is 1 / 30 of that of AM, exclude oxygen and promote free radical polymerization reaction;

[0015] (3.2) Add BIS and SDS to the black solution, stir and evacuate to form a black mixed solution, in which the mass ratio of PDA, BIS and SDS is 5:27:200;

[0016] (3.3) heating the black mixed solution, adding dropwise APS aqueous solution and SBS solution, and reacting under nitrogen protection to obtain a PDA@P(NIPAAm-co-AM) nanohydrogel precursor solution, wherein the molar ratios of APS and SBS added to the PDA@P(NIPAAm-co-AM) nanohydrogel precursor solution to the initially added AM are 50:21 and 25:21, respectively;

[0017] (3.4) The PDA@P(NIPAAm-co-AM) nanohydrogel precursor solution is dialyzed and centrifuged, and freeze-dried to obtain PDA@P(NIPAAm-co-AM) nanogel, which is convenient for loading tumor chemotherapy drugs through swelling.

[0018] Preferably, in step (3.4), the PDA@P(NIPAAm-co-AM) nanohydrogel precursor solution is placed in a 14 kDa dialysis bag, dialyzed with ultrapure water and centrifuged, and the precipitate is freeze-dried to obtain the PDA@P(NIPAAm-co-AM) nanogel, and unreacted monomers and by-products in the reaction process are removed.

[0019] Preferably, in step (4), the mass ratio of DOX to PDA@P (NIPAAm-co-AM) nanogel is 1:100, and the prepared light-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogel can achieve drug release by light control while eliminating the effect of photothermal on chemotherapy anti-tumor.

[0020] According to a second aspect of an embodiment of the present application, a light-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogel is provided, which is prepared by the method described in the first aspect.

[0021] According to a third aspect of an embodiment of the present application, there is provided an application of the light-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogel as described in the second aspect.

[0022] The technical solution provided by the embodiments of the present application may have the following beneficial effects:

[0023] It can be seen from the above embodiments that the present application forms PDA@P (NIPAAm-co-AM) nanohydrogel through free radical combination, and the nanohydrogel has good light response performance. The application of near-infrared light stimulates the contraction of the nanohydrogel and releases the loaded anti-tumor drug DOX, thereby overcoming the problem of photothermal effects during phototherapy, realizing light-controlled chemotherapy more accurately, and achieving a good tumor inhibition effect.

[0024] The preparation method of the invention is simple, the performance of the light-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nano-hydrogel is stable, and it has a relatively broad application prospect.

[0025] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0027] Figure 1 The scanning electron micrographs of P(NIPAAm-co-AM) nanohydrogels with different NIPAAm and AM molar ratios in the present invention are shown;

[0028] Figure 2 The temperature-particle size curves of P(NIPAAm-co-AM) nanohydrogels with different NIPAAm and AM molar ratios in the present invention;

[0029] Figure 3 The DLS graph of the P(NIPAAm-co-AM) nanohydrogel with a molar ratio of NIPAAm to AM of 80:20 in the present invention;

[0030] Figure 4 It is the electron microscopy image of PDA and PDA@P(NIPAAm-co-AM) nanohydrogel in the present invention;

[0031] Figure 5 DLS images of PDA and PDA@P (NIPAAm-co-AM water) nanohydrogels in the present invention;

[0032] Figure 6 is the photothermal temperature rise curve of PDA@P(NIPAAm-co-AM) nanohydrogel in the present invention;

[0033] FIG7 is a drug release curve of different materials in the present invention;

[0034] Figure 8 This is a photo-induced drug release diagram of the PDA@P (NIPAAm-co-AM) nanohydrogel for light-controlled chemotherapy and anti-tumor in the present invention;

[0035] Fig. 9 This is the cytotoxicity graph of the PDA@P(NIPAAm-co-AM) nanohydrogel irradiated with light for 5 min in the present invention;

[0036] Fig.10 The cytotoxicity diagram of different materials in the present invention after 24 h of culture;

[0037] Fig.11 The cytotoxicity graphs of different groups. DETAILED DESCRIPTION

[0038] Here, exemplary embodiments are described in detail, and examples thereof are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application.

[0039] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0040] It should be understood that although the terms first, second, third, etc. may be used in the present application to describe various information, these information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "at the time of" or "when" or "in response to determining".

[0041] It should be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters of the following examples are also only an example within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0042] Example 1

[0043] This embodiment provides a method for preparing a P(NIPAAm-co-AM) nanohydrogel, which may include the following steps:

[0044] Step (1), weigh 2.15 g of NIPAAm and 0.15 g of AM, dissolve them in 100 mL of ultrapure water, and stir and evacuate for 30 min to obtain an aqueous solution of NIPAAm and AM, wherein the molar ratio of NIPAAM to AM is 90:10.

[0045] Step (2), weigh 27 mg of BIS and 200 mg of SDS and add them to the NIPAAm and AM aqueous solution, stir and evacuate for 30 min to form a uniform colorless solution.

[0046] Step (3), heat the precursor solution in an oil bath to 70°C, then dropwise add 8 mL of APS aqueous solution (5 μM) and 8 mL of SBS aqueous solution (2.5 μM). Stir in the oil bath and react for 5 h under nitrogen protection.

[0047] Step (4), placing the P(NIPAAm-co-AM) nanohydrogel precursor solution in a 14 kDa dialysis bag and dialyzing it with ultrapure water for 7 days. After dialysis, freeze-drying is performed to obtain P(NIPAAm-co-AM) nanogel, and the P(NIPAAm-co-AM) nanogel is dissolved in ultrapure water to obtain P(NIPAAm-co-AM) nanohydrogel.

[0048] Example 2

[0049] This embodiment provides a method for preparing a P(NIPAAm-co-AM) nanohydrogel, which is different from that of Embodiment 1 in that the molar ratio of NIPAAm to AM in this embodiment is 80:20, and the method may include the following steps:

[0050] Step (1), weigh 0.96 g of NIPAAm and 0.15 g of AM, dissolve them in 100 mL of ultrapure water, stir and evacuate for 30 min to obtain an aqueous solution of NIPAAm and AM, and the molar ratio of NIPAAm to AM is 80:20.

[0051] Step (2), weigh 27 mg of BIS and 200 mg of SDS and add them to the NIPAAm and AM aqueous solution, stir and evacuate for 30 min to form a uniform colorless solution.

[0052] Step (3), heat the uniform colorless solution in an oil bath to 70°C, then drop 8 mL of APS aqueous solution (5 μM) and 8 mL of SBS aqueous solution (2.5 μM). Stir in the oil bath and react under nitrogen protection for 5 h to obtain P(NIPAAm-co-AM) nanohydrogel precursor solution.

[0053] Step (4), placing the P(NIPAAm-co-AM) nanohydrogel precursor solution in a 14 kDa dialysis bag and dialyzing it with ultrapure water for 7 days. After dialysis, freeze-drying is performed to obtain P(NIPAAm-co-AM) nanogel, and the P(NIPAAm-co-AM) nanogel is dissolved in ultrapure water to obtain P(NIPAAm-co-AM) nanohydrogel.

[0054] Example 3

[0055] This embodiment provides a method for preparing a P(NIPAAm-co-AM) nanohydrogel, which is different from that of Embodiment 1 in that the molar ratio of NIPAAm to AM in this embodiment is 70:30, and the method may include the following steps:

[0056] Step (1), weigh 0.56 g of NIPAAm and 0.15 g of AM, dissolve them in 100 mL of ultrapure water, stir and evacuate for 30 min to obtain an aqueous solution of NIPAAm and AM, and the molar ratio of NIPAAm to AM is 70:30.

[0057] Step (2), weigh 27 mg of BIS and 200 mg of SDS and add them to the NIPAAm and AM aqueous solution, stir and evacuate for 30 min to form a uniform colorless solution.

[0058] Step (3), heat the uniform colorless solution in an oil bath to 70°C, then drop 8 mL of APS aqueous solution (5 μM) and 8 mL of SBS aqueous solution (2.5 μM). Stir in the oil bath and react under nitrogen protection for 5 h to obtain P(NIPAAm-co-AM) nanohydrogel precursor solution.

[0059] Step (4), placing the P(NIPAAm-co-AM) nanohydrogel precursor solution in a 14 kDa dialysis bag and dialyzing it with ultrapure water for 7 days. After dialysis, freeze-drying is performed to obtain P(NIPAAm-co-AM) nanogel, and the P(NIPAAm-co-AM) nanogel is dissolved in ultrapure water to obtain P(NIPAAm-co-AM) nanohydrogel.

[0060] Figure 1 The scanning electron micrographs of P(NIPAAm-co-AM) nanohydrogels with different NIPAAm to AM molar ratios are shown in Figures (a), (b), and (c) respectively. The scanning electron micrographs of nanohydrogels with NIPAAm to AM molar ratios of 90:10, 80:20, and 70:30 are shown in Figures (a), (b), and (c) respectively. It can be seen that the P(NIPAAm-co-AM) nanohydrogel has a network structure. As the NIPAAm to AM molar ratio decreases, the network of the P(NIPAAm-co-AM) nanohydrogel becomes denser.

[0061] Figure 2The temperature-particle size curves of P(NIPAAm-co-AM) nanohydrogels with different NIPAAm to AM molar ratios. The particle size of P(NIPAAm-co-AM) nanohydrogels is negatively correlated with temperature, and within a certain temperature range, the particle size of the nanohydrogel changes significantly, indicating that P(NIPAAm-co-AM) nanohydrogels have a low critical transition temperature. As the NIPAAm to AM molar ratio decreases, the particle size of the nanohydrogel increases at the same temperature. Among them, the transition temperatures of the nanohydrogels with NIPAAm to AM molar ratios of 90:10, 80:20, and 70:30 are in the range of 32℃-37℃, 37℃-45℃, and 37℃-50℃, respectively.

[0062] Figure 3 This is the DLS graph of the nanohydrogel with a molar ratio of NIPAAm to AM of 80:20. As shown in the figure, as the temperature increases, the particle size decreases, but the size of the nanohydrogel remains uniform.

[0063] Example 4

[0064] This embodiment provides a method for preparing a PDA@P (NIPAAm-co-AM) nanohydrogel, which may include the following steps:

[0065] Step (1), weigh 0.96 g of NIPAAm and 0.15 g of AM, dissolve them in 100 mL of ultrapure water, and stir and evacuate for 30 min to obtain an aqueous solution of NIPAAm and AM.

[0066] Step (2), add 40 mL of C2H5OH to 90 mL of ultrapure water, stir evenly, quickly add 3 mL of NH3·H2O, stir at 40°C for 30 min, then add 10 mL of DA·HCl aqueous solution (50 mg / mL) dropwise to the above solution, and stir at 40°C. After reacting in the dark for 24 h, collect the PDA nanoparticles by centrifugation at 12000 rpm for 10 min, wash three times with water, and finally disperse in water to obtain a PDA aqueous solution.

[0067] Step (3), add 5 mL of PDA aqueous solution (1 mg / mL) to NIPAAm and AM aqueous solution, stir and evacuate for 30 min to obtain a black solution; weigh 27 mg of BIS and 200 mg of SDS and add them to the black uniform solution in the three-necked flask, stir and evacuate for 30 min to form a uniform black mixed solution; heat the black mixed solution to 70°C in an oil bath, and then drop 8 mL of APS aqueous solution (5 μM) and 8 mL of SBS aqueous solution (2.5 μM). Stir in the oil bath and react under nitrogen protection for 5 h to obtain PDA@P (NIPAAm-co-AM) nanohydrogel precursor solution; place the PDA@P (NIPAAm-co-AM) nanohydrogel precursor solution in a 14 kDa dialysis bag, dialyze with ultrapure water for 7 days, centrifuge at 12000 rpm for 10 min after dialysis, and freeze-dry the precipitate to obtain PDA@P (NIPAAm-co-AM) nanogel.

[0068] Step (4), dissolving the PDA@P(NIPAAm-co-AM) nanogel in ultrapure water to obtain the PDA@P(NIPAAm-co-AM) nanohydrogel.

[0069] Figure 4 The electron micrographs of PDA nanoparticles and PDA@P(NIPAAm-co-AM) nanohydrogels, where (a) is a scanning electron micrograph of PDA nanoparticles and (b) is a transmission electron micrograph of PDA@P(NIPAAm-co-AM) nanohydrogels. The size of PDA nanoparticles is uniform, with an average particle size of about 200 nm. P(NIPAAm-co-AM) is coated on the surface of PDA nanoparticles, showing a layer with a smaller grayscale attached to the surface of PDA nanoparticles.

[0070] Figure 5 The DLS images of PDA nanoparticles and PDA@P(NIPAAm-co-AM) nanohydrogels. As shown in the figure, the particle sizes of PDA nanoparticles and PDA@P(NIPAAm-co-AM) nanohydrogels are evenly distributed. At the same temperature, the particle size of PDA@P(NIPAAm-co-AM) nanohydrogels is larger than that of PDA. At the same time, as the temperature increases, the particle size of PDA@P(NIPAAm-co-AM) nanohydrogels decreases. The DLS images and transmission electron microscopy images show that P(NIPAAm-co-AM) is coated on the surface of PDA nanoparticles to form PDA@P(NIPAAm-co-AM) nanohydrogels.

[0071] Example 5

[0072] In order to scientifically and accurately study the photothermal properties of PDA@P (NIPAAm-co-AM) nanohydrogel, this example selected PDA@P (NIPAAm-co-AM) nanohydrogels with multiple mass concentrations to measure the temperature rise of the material within 5 min of 808 nm near-infrared light irradiation.

[0073] Figure 6 The temperature rise curves of seven concentrations of PDA@P(NIPAAm-co-AM) nanohydrogel were recorded. The mass concentrations of PDA@P(NIPAAm-co-AM) nanogel in the PDA@P(NIPAAm-co-AM) nanohydrogel were 0μg / mL, 25μg / mL, 50 μg / mL, 75 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL, respectively. The near-infrared light irradiation power was 2 W. Among them, when the mass concentration was 50 μg / mL, the temperature of the material measured by FLIR after 5 minutes of near-infrared light irradiation was 39.7℃, which would not cause cell degeneration and had no killing effect on tumor cells and normal cells.

[0074] Example 6

[0075] This embodiment provides a method for preparing a PDA@P (NIPAAm-co-AM) nanohydrogel for light-controlled chemotherapy and anti-tumor, comprising:

[0076] Step (1), weigh 0.96 g of NIPAAm and 0.15 g of AM, dissolve them in 100 mL of ultrapure water, and stir and evacuate for 30 min to obtain an aqueous solution of NIPAAm and AM.

[0077] Step (2), add 40 mL of C2H5OH to 90 mL of ultrapure water, stir evenly, quickly add 3 mL of NH3·H2O, stir at 40°C for 30 min, then add 10 mL of DA·HCl aqueous solution (50 mg / mL) dropwise to the above solution, and stir at 40°C. After reacting in the dark for 24 h, collect the PDA nanoparticles by centrifugation at 12000 rpm for 10 min, wash three times with water, and finally disperse in water to obtain a PDA aqueous solution.

[0078] Step (3), add 5 mL of PDA aqueous solution (1 mg / mL) to NIPAAm and AM aqueous solution, stir and evacuate for 30 min to obtain a black solution; weigh 27 mg of BIS and 200 mg of SDS and add them to the black uniform solution in the three-necked flask, stir and evacuate for 30 min to form a uniform black mixed solution; heat the black mixed solution to 70°C in an oil bath, and then drop 8 mL of APS aqueous solution (5 μM) and 8 mL of SBS aqueous solution (2.5 μM). Stir in the oil bath and react under nitrogen protection for 5 h to obtain PDA@P (NIPAAm-co-AM) nanohydrogel precursor solution; place the PDA@P (NIPAAm-co-AM) nanohydrogel precursor solution in a 14 kDa dialysis bag, dialyze with ultrapure water for 7 days, centrifuge at 12000 rpm for 10 min after dialysis, and freeze-dry the precipitate to obtain PDA@P (NIPAAm-co-AM) nanogel.

[0079] Step (4), weigh 40 μg of freeze-dried PDA@P(NIPAAm-co-AM) nanogel and dissolve it in 2 mL of DOX aqueous solution (20 μg / mL) (i.e., the mass ratio of DOX to PDA@P(NIPAAm-co-AM) nanogel is 1:1), stir evenly to obtain a light-controlled chemotherapeutic anti-tumor PDA@P(NIPAAm-co-AM) nanohydrogel (DOX-PDA@P(NIPAAm-co-AM) 1 / 1, abbreviated as DPN 1 / 1).

[0080] Example 7

[0081] This embodiment provides a method for preparing a PDA@P(NIPAAm-co-AM) nanohydrogel for light-controlled chemotherapy and anti-tumor, which is different from Example 6 in that the mass ratio of DOX to PDA@P(NIPAAm-co-AM) nanogel in Example 7 is 1:10, that is, 400 μg of freeze-dried PDA@P(NIPAAm-co-AM) nanogel is weighed. A PDA@P(NIPAAm-co-AM) nanohydrogel for light-controlled chemotherapy and anti-tumor (DOX-PDA@P(NIPAAm-co-AM) 1 / 10, abbreviated as DPN 1 / 10) is obtained.

[0082] Example 8

[0083] This embodiment provides a method for preparing a PDA@P(NIPAAm-co-AM) nanohydrogel for light-controlled chemotherapy and anti-tumor, which is different from Example 6 in that the mass ratio of DOX to PDA@P(NIPAAm-co-AM) nanogel in Example 8 is 1:50, that is, 2 mg of freeze-dried PDA@P(NIPAAm-co-AM) nanogel is weighed. A PDA@P(NIPAAm-co-AM) nanohydrogel for light-controlled chemotherapy and anti-tumor (DOX-PDA@P(NIPAAm-co-AM) 1 / 50, abbreviated as DPN 1 / 50) is obtained.

[0084] Example 9

[0085] This embodiment provides a method for preparing a PDA@P(NIPAAm-co-AM) nanohydrogel for light-controlled chemotherapy and anti-tumor, which is different from Example 6 in that the mass ratio of DOX to PDA@P(NIPAAm-co-AM) nanogel in Example 9 is 1:100, that is, 4 mg of freeze-dried PDA@P(NIPAAm-co-AM) nanogel is weighed. A PDA@P(NIPAAm-co-AM) nanohydrogel for light-controlled chemotherapy and anti-tumor (DOX-PDA@P(NIPAAm-co-AM) 1 / 100, abbreviated as DPN 1 / 100) is obtained.

[0086] The following examples characterize the performance of the light-controlled chemotherapeutic anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogels in Examples 6, 7, 8, and 9.

[0087] Example 10

[0088] The light-controlled chemotherapy anti-tumor PDA@P(NIPAAm-co-AM) nanohydrogels prepared in Examples 6-9 were placed in dialysis bags respectively, and then placed in a release medium for drug release experiments to determine the cumulative release amount of the drug;

[0089] Specifically, the dialysis bag containing the drug-loaded nanogel aqueous solution was placed in 2 mL of the release medium PBS, and the drug release experiment was carried out at 37°C and 45°C. At a specific time point, 0.5 mL of the release medium PBS solution was taken and supplemented with an equal volume of blank medium. The drug concentration released at each time point was measured, and the drug release amount was calculated.

[0090] Figure 7 shows the cumulative drug release curves of PDA@P(NIPAAm-co-AM) nanohydrogels for light-controlled chemotherapy and anti-tumor at 37°C and 45°C, respectively, with the mass ratios of DOX and PDA@P(NIPAAm-co-AM) nanogels being 1:1, 1:10, 1:50, and 1:100, respectively. Each group was run three times in parallel. It can be seen from the figure that at the same temperature, as the mass ratio of DOX and PDA@P(NIPAAm-co-AM) nanogels decreases, the amount of drug released gradually increases; at the same mass ratio, as the temperature increases, the amount of drug released increases.

[0091] Embodiment 11

[0092] The light-controlled chemotherapy anti-tumor PDA@P(NIPAAm-co-AM) nanohydrogel (DPN 1 / 100) prepared in Example 9 was selected to measure the relative release amount of the drug before and after illumination;

[0093] Specifically, 900 mg of PDA@P(NIPAAm-co-AM) was weighed and dissolved in 18 mL of DOX aqueous solution (500 μg / mL), and then divided equally into a non-irradiated group and an illuminated group, with three parallel experiments set up in each group. The equally divided light-controlled chemotherapy anti-tumor PDA@P(NIPAAm-co-AM) nanohydrogels were placed in dialysis bags, and then the dialysis bags containing the drug-loaded nanogel aqueous solution were placed in 3 mL of release medium PBS. The drug release experiment was carried out at 37°C. The irradiated group was applied with near-infrared light with a power of 2 W for 5 min, and each irradiation was a cycle. Before and after irradiation, 2 mL of the release medium PBS solution was taken and supplemented with an equal volume of blank medium. The non-irradiated group took an equal volume of release medium and supplemented with blank medium at the same time. The relative release amount of the drug in each cycle was determined.

[0094] Figure 8 The relative release graph of the drug in each cycle. The relative release of the drug in each cycle of the irradiated group was higher than that of the non-irradiated group.

[0095] Example 12

[0096] This example verifies the application of the material in tumor treatment by its killing effect at the cellular level. The cells used in the experiment are mouse breast cancer cells (4T1). 4T1 cells were co-cultured with the photo-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogel for 24 hours. During the culture process, 5 minutes of light was applied, and the concentration of PDA@P (NIPAAm-co-AM) nanohydrogel that produced photothermal killing effect on cells under the same lighting conditions was measured. Fig. 9As shown, when the concentration of PDA@P(NIPAAm-co-AM) nanohydrogel is greater than 50 μg / mL, the photothermal effect generated by the material significantly affects the survival rate of 4T1 cells.

[0097] 4T1 cells were co-cultured with DPN 1 / 1, DPN 1 / 10, DPN 1 / 50, and DPN 1 / 100 light-controlled chemotherapeutic anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogels for 24 h, and the cell survival rate was measured with or without 5 min of light irradiation. Fig.10 As shown in the figure, when the material concentrations of DPN 1 / 1, DPN 1 / 10, DPN 1 / 50, and DPN 1 / 100 were 0 μg / mL, simple illumination had no killing effect on cells. When the material and illumination acted together, the survival rate of 4T1 cells gradually decreased with the increase of material concentration. The figure shows that the cell killing effect was more obvious when illumination was applied than when no illumination was applied. Among them, DPN 1 / 100 had a significant killing effect under illumination. When the DOX concentration was 0.25 μg / mL, illumination simply promoted drug release to produce obvious cell killing. When the DOX concentration was 0.5 μg / mL, the photothermal generated by illumination synergistically killed 4T1 cells with the drug.

[0098] Fig.11 To select DPN 1 / 100, the control group was set at a DOX concentration of 0.25 μg / mL, the illumination time was 5 min, and the near-infrared light irradiation power was 2 W. The DN group was a drug-loaded nanohydrogel obtained by dissolving P(NIPAAm-co-AM) nanogel in a DOX aqueous solution; NIR represents applied light. Fig.11 The results show that the irradiated group had a significant cell killing effect compared with the non-irradiated group and the no-PDA group, indicating that light irradiation of PDA nanoparticles generated photothermal energy, which in turn stimulated the P(NIPAAm-co-AM) nanohydrogel to reduce its particle size, shrink and discharge the DOX-containing aqueous solution, thereby killing 4T1 cells. At the same time, the photothermal energy itself had no effect on the cell survival rate.

Claims

1. A method for preparing a PDA@P (NIPAAm-co-AM) nanohydrogel for light-controlled chemotherapy and anti-tumor, characterized in that: include: (1) Add NIPAAm and AM to water, stir and evacuate to obtain an aqueous solution of NIPAAm and AM; (2) adding C2H5OH and NH3·H2O to water in sequence and stirring evenly, adding DA·HCl aqueous solution and stirring in the dark, centrifuging and washing to obtain PDA nanoparticles, and dispersing the PDA nanoparticles in water to obtain a PDA solution; (3) PDA solution, NIPAAm and AM aqueous solution, SBS, APS, BIS, and SDS are mixed for reaction, and after the reaction, dialyzed and freeze-dried to obtain PDA@P(NIPAAm-co-AM) nanogel; (4) The PDA@P (NIPAAm-co-AM) nanogel was dissolved in an aqueous solution of tumor chemotherapy drugs to obtain a light-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogel.

2. The method according to claim 1, characterized in that In step (1), the molar ratio of NIPAAm to AM is 80:

20.

3. The method according to claim 1, characterized in that: Step (2) is as follows: 40 parts by volume of C2H5OH were added to 90 parts by volume of ultrapure water. After stirring evenly under heating conditions, 3 parts by volume of NH3·H2O were immediately added and stirred evenly. A DA·HCl aqueous solution was added, wherein the mass volume ratio of DA·HCl to C2H5OH was 50 mg:4 ml. The mixture was stirred under heating conditions and protected from light for reaction. After centrifugation and washing, PDA nanoparticles were obtained. The PDA nanoparticles were dispersed in water to obtain a PDA solution.

4. The method according to claim 1, characterized in that: Step (3) specifically includes: (3.1) Add the PDA aqueous solution to the NIPAAm and AM aqueous solutions, stir and evacuate to obtain a black solution, in which the mass of PDA is 1 / 30 of that of AM; (3.2) Add BIS and SDS to the black solution, stir and evacuate to form a black mixed solution, in which the mass ratio of PDA, BIS and SDS is 5:27:200; (3.3) heating the black mixed solution, adding dropwise APS aqueous solution and SBS solution, and reacting under nitrogen protection to obtain a PDA@P(NIPAAm-co-AM) nanohydrogel precursor solution, wherein the molar ratios of APS and SBS added to the PDA@P(NIPAAm-co-AM) nanohydrogel precursor solution to the initially added AM are 50:21 and 25:21, respectively; (3.4) The PDA@P(NIPAAm-co-AM) nanohydrogel precursor solution was dialyzed and centrifuged, and then freeze-dried to obtain the PDA@P(NIPAAm-co-AM) nanogel.

5. The method according to claim 4, characterized in that In step (3.4), the PDA@P(NIPAAm-co-AM) nanohydrogel precursor solution was placed in a 14 kDa dialysis bag, dialyzed with ultrapure water and centrifuged, and the precipitate was freeze-dried to obtain the PDA@P(NIPAAm-co-AM) nanogel.

6. The method according to claim 1, characterized in that In step (4), the mass ratio of DOX to PDA@P(NIPAAm-co-AM) nanogel was 1:

100.

7. A PDA@P (NIPAAm-co-AM) nanohydrogel for light-controlled chemotherapy and anti-tumor, characterized in that: Prepared by the method according to any one of claims 1 to 6.

8. An application of the light-controlled chemotherapy anti-tumor PDA@P (NIPAAm-co-AM) nanohydrogel as claimed in claim 7.

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