A carrier-free nano drug delivery system and its application

Abstract

The present invention discloses a carrier-free nano drug delivery system and its application. The carrier-free nano drug DHT NC@SF MPN constructed by the present invention does not contain a carrier material, so it has high safety. It also solves the problems of poor anti-tumor efficacy of metal-polyphenol networks and sudden release and leakage of nanocrystals in current research, and DHT NC@SF MPN has a high drug loading capacity and the characteristics of pH-responsive drug release. After intratumor injection, SF MPN decomposes in a weakly acidic tumor microenvironment, releasing DHT NC, Sil and Fe (III), DHT NC promotes tumor cell apoptosis, Sil inhibits tumor cell metastasis and proliferation, and Fe (III) triggers tumor cell ferroptosis. The three exert anti-tumor efficacy through the action of multiple pathways and multiple targets, realize chemotherapy / chemodynamic treatment of tumors, and have broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a carrier-free nano drug delivery system and its application. Background Art

[0002] Tumors have always been a major disease that has plagued mankind and seriously threatens human health. At present, the treatment strategies for tumors mainly include surgical treatment, chemotherapy (abbreviated as chemotherapy), radiotherapy, immunotherapy, cell therapy, and combination therapy that combines multiple treatment methods. Among them, chemotherapy occupies a dominant position among many therapies by directly killing tumor cells or inhibiting the proliferation of tumor cells. It is the most important treatment for tumors and the preferred treatment strategy for many tumor diseases. However, the poor water solubility, fast clearance rate, low bioavailability and narrow therapeutic window of most chemotherapy drugs have seriously hindered their clinical application.

[0003] In recent years, carrier-free nanomedicine has received extensive attention in the field of biomedicine, especially in anti-tumor, with broad application prospects and development potential. However, the development of carrier-free nanomedicine is currently limited by the structure and properties of chemotherapeutic drugs, and the types and number of reported carrier-free nanomedicines are very limited.

[0004] Therefore, constructing a safe and efficient carrier-free nanomedicine is crucial in the field of nanomedicine research. Summary of the invention

[0005] To make up for the deficiencies of the prior art, the present invention provides a carrier-free nano drug delivery system and its application.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a carrier-free nanomedicine, which comprises a metal-polyphenol network and a nanocrystalline drug.

[0008] Furthermore, the metal-polyphenol network includes metal ions and polyphenol compounds.

[0009] Furthermore, the metal ions include Fe(III), Ag(I), Au(I), Ba(II), Ca(II), Cd(II), Cu(II), Fe(II), Mn(II), Mg(II), Mo(II), Zn(II), Pt(II), Au(III), Al(III), Ce(III), Co(III), Cr(III), Eu(III), Gd(III), Ni(III), W(III), V(III) or Zr(IV).

[0010] Furthermore, the metal ion is selected from Fe(III).

[0011] Furthermore, the polyphenol compounds include flavonoid compounds, phenolic acid compounds, tannin compounds or anthocyanin compounds.

[0012] Furthermore, the polyphenol compounds are selected from flavonoid compounds.

[0013] Furthermore, the flavonoid compound is selected from silybin.

[0014] Furthermore, the nanocrystalline drug is tanshinone nanocrystal.

[0015] Furthermore, the tanshinone nanocrystals are dihydrotanshinone I nanocrystals.

[0016] Furthermore, the molar ratio of Fe(III) to silybin is 1:(3-7).

[0017] Furthermore, the molar ratio of Fe(III) to silybin is 1:(3-5).

[0018] Furthermore, the molar ratio of Fe(III) to silybin is 1:3.

[0019] Furthermore, the carrier-free nanomedicine also includes other functional small molecules.

[0020] Furthermore, the other functional small molecules include photothermal agents, photosensitizers, and contrast agents.

[0021] Furthermore, the other functional small molecules are selected from photosensitizers.

[0022] Furthermore, the photosensitizer includes one or more of IR808, ICG, Ce6, and TCPP.

[0023] Furthermore, the photosensitizer is selected from IR808.

[0024] Furthermore, the particle size of the dihydrotanshinone I nanocrystals is 227.8±7.9 nm.

[0025] Furthermore, the PDI of the dihydrotanshinone I nanocrystals is 0.297±0.007.

[0026] Furthermore, the particle size of the carrier-free nanomedicine is 262.12±5.61 nm.

[0027] Furthermore, the PDI of the carrier-free nanomedicine is 0.269±0.01.

[0028] The second aspect of the present invention provides a method for preparing a carrier-free nanomedicine, the method comprising:

[0029] 1) Preparation of nanocrystalline drugs;

[0030] 2) Adding polyphenol compounds and metal ions to the suspension of nanocrystalline drugs respectively, stirring, and obtaining carrier-free nanomedicine.

[0031] Furthermore, the nanocrystalline drug described in 1) is tanshinone nanocrystal.

[0032] Furthermore, the tanshinone nanocrystals are dihydrotanshinone I nanocrystals.

[0033] Furthermore, the metal ions described in 2) include Fe(III), Ag(I), Au(I), Ba(II), Ca(II), Cd(II), Cu(II), Fe(II), Mn(II), Mg(II), Mo(II), Zn(II), Pt(II), Au(III), Al(III), Ce(III), Co(III), Cr(III), Eu(III), Gd(III), Ni(III), W(III), V(III) or Zr(IV).

[0034] Furthermore, the metal ion is selected from Fe(III).

[0035] Furthermore, the polyphenol compounds described in 2) include flavonoid compounds, phenolic acid compounds, tannin compounds or anthocyanin compounds.

[0036] Furthermore, the polyphenol compounds are selected from flavonoid compounds.

[0037] Furthermore, the flavonoid compound is selected from silybin.

[0038] Furthermore, the molar ratio of Fe(III) to silybin is 1:(3-7).

[0039] Furthermore, the molar ratio of Fe(III) to silybin is 1:(3-5).

[0040] Furthermore, the molar ratio of Fe(III) to silybin is 1:3.

[0041] Further, 1) comprises the following steps:

[0042] a) dissolving dihydrotanshinone I in an organic solvent;

[0043] b) adding the solution obtained in a) to a dispersant to obtain a crude emulsion;

[0044] c) preparing a fine emulsion from the coarse emulsion in b) and solidifying the fine emulsion to obtain a nanocrystal suspension;

[0045] d) removing the organic solvent and drying to obtain dihydrotanshinone I nanocrystals.

[0046] Furthermore, the organic solvent described in a) is selected from acetone.

[0047] Furthermore, the specific step of b) includes adding the acetone solution of dihydrotanshinone I into water containing a dispersant under high-speed homogenization stirring.

[0048] Furthermore, the stirring speed in b) is 3000-5000 rpm.

[0049] Furthermore, the stirring speed in b) is 3000 rpm.

[0050] Furthermore, the stirring time in b) is 30 min.

[0051] Furthermore, the dispersant in b) is selected from PVP K30.

[0052] Furthermore, the ratio of PVP K30 to nanocrystalline drug is 1:(1-5).

[0053] Furthermore, the ratio of PVP K30 to nanocrystalline drug is 1:5.

[0054] Furthermore, in c), the coarse emulsion in b) is prepared into a fine emulsion by a high pressure homogenization method.

[0055] Furthermore, the homogenization pressure in c) is 10000-20000 psi.

[0056] Furthermore, the homogenization pressure in c) is 10,000 psi.

[0057] Furthermore, the number of cycles in c) is 20-60 times.

[0058] Furthermore, the number of cycles in c) is 60 times.

[0059] Furthermore, in d), the organic solvent is removed by rotary evaporation.

[0060] Further, step 2) comprises the following steps:

[0061] e) mixing the nanocrystalline drug suspension with the silybin solution;

[0062] f) mixing the suspension obtained in e) with a FeCl3 solution;

[0063] g) adjusting pH, stirring and centrifuging;

[0064] h) Pre-freeze and then dry.

[0065] Furthermore, the solution of silybin in e) is DMSO.

[0066] Furthermore, the FeCl3 solution in f) is DMSO.

[0067] Furthermore, in g), Tris buffer was used to adjust the pH.

[0068] Furthermore, the centrifugation condition in g) is 3000 rpm, 15 min.

[0069] The third aspect of the present invention provides the use of the carrier-free nanomedicine described in the first aspect of the present invention in the preparation of anti-tumor drugs.

[0070] Furthermore, the tumors include gastric cancer, breast cancer, liver cancer, and cervical cancer.

[0071] Furthermore, the tumor is selected from gastric cancer.

[0072] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0073] The fourth aspect of the present invention provides the use of the carrier-free nanomedicine described in the first aspect of the present invention in the preparation of a drug for regulating protein expression.

[0074] Furthermore, the protein includes one or more of apoptosis-related proteins, metastasis-related proteins or ferroptosis-related proteins.

[0075] Furthermore, the apoptosis-related proteins include one or more of caspase-3, caspase-8 or Bcl-2.

[0076] Furthermore, the metastasis-related proteins include one or more of MMP2, MMP9 or E-cadherin.

[0077] Furthermore, the ferroptosis-related proteins include TFRC and / or GPX4.

[0078] Advantages and beneficial effects of the present invention:

[0079] The carrier-free nano drug DHT NC@SF MPN constructed by the present invention does not contain carrier materials, so it has high safety. It also solves the problems of poor anti-tumor efficacy of metal-polyphenol network and sudden release and leakage of nanocrystals in current research, and DHT NC@SF MPN has a high drug loading capacity and the characteristics of pH-responsive drug release. After intratumor injection, SF MPN (silybin-iron ion network) decomposes in the weakly acidic tumor microenvironment, releasing DHT NC (dihydrotanshinone I nanocrystals), Sil (silybin) and Fe (III), DHT NC promotes tumor cell apoptosis, Sil inhibits tumor cell metastasis and proliferation, and Fe (III) triggers tumor cell ferroptosis. The three exert anti-tumor efficacy through multi-pathway and multi-target effects, realize chemotherapy / chemodynamic treatment of tumors, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 is the XRD spectra of DHT and DHT NCs prepared with different dispersants;

[0081] Figure 2 are SEM (left) (×35000) and TEM (right) (×40000) images of DHT NC;

[0082] Figure 3 is the drug loading and encapsulation efficiency diagram of DHT NC@SF MPN nanocomposites with different ratios;

[0083] Figure 4 are the infrared spectra of DHT, DHT NC, Sil, and DHT NC@SF MPN;

[0084] Figure 5 are SEM (left) (×20000) and TEM (right) (×40000) images of DHT NC@SF MPN;

[0085] Figure 6 6A is a graph showing the stability test results of DHT NC@SF MPN, wherein 6A is a graph showing the change in particle size, and 6B is a graph showing the change in PDI;

[0086] Figure 7 7A is the cumulative drug release curve of DHT and DHT NC@SF MPN, and 7B is the cumulative drug release curve of Sil and DHT NC@SF MPN;

[0087] Figure 8 8A is a graph showing the cell survival rate of human gastric cancer cell HGC 27 after 24 hours of drug administration, and 8B is a graph showing the cell survival rate of human gastric cancer cell HGC 27 after 48 hours of drug administration;

[0088] Fig. 9 This is the half-inhibitory concentration graph of human gastric cancer cell HGC 27 cells at 24h and 48h;

[0089] Fig.10 is a flow cytometry scatter plot of human gastric cancer cell HGC 27;

[0090] Fig.11 This is a statistical chart of apoptosis of human gastric cancer cell HGC 27;

[0091] Fig.12 This is the scratch image of human gastric cancer cell HGC 27 0h and 12h;

[0092] Fig.13 It is a statistical graph of the migration rate of human gastric cancer cell HGC 27;

[0093] Fig.14 This is a confocal laser image of human gastric cancer cell HGC 27 taking up DHT / IR808 NC@SF MPN nanocomplex;

[0094] Fig.15 It is the standard curve graph of protein quantification;

[0095] Fig.16 is a graph showing changes in DHT content in HGC 27 cells after treatment with different protein inhibitors;

[0096] Fig.17 This is the localization map of DHT / IR808 NC@SF MPN nanocomplex in the organelles of human gastric cancer cell HGC 27;

[0097] Fig.18 It is the curve diagram of intraperitoneal injection of drugs in different drug administration groups;

[0098] Fig.19 This is a statistical diagram of the tissue distribution of DHT in rats;

[0099] Fig. 20 is a graph of tumor inhibition rate, wherein 20A is a graph of tumor volume growth curve, and 20B is a graph of tumor growth inhibition rate curve;

[0100] Fig.21 is the tumor map of each group;

[0101] Fig. 22 It is a statistical graph of tumor weight;

[0102] Fig.23 is the biodistribution diagram of DHT / IR808 NC@SF MPN nanocomplex in nude mice;

[0103] Fig.24is the biodistribution diagram of DHT / IR808 NC@SF MPN nanocomplex in nude mouse tissues;

[0104] Fig.25 This is a statistical graph of the fluorescence intensity of DHT / IR808 NC@SF MPN nanocomplex in nude mouse tumor tissue;

[0105] Fig.26 are the H&E-stained sections of the tumor tissues of nude mice in each group;

[0106] Fig. 27 is the TUNEL staining section (×500) of the tumor tissue of nude mice in each group;

[0107] Fig.28 is the quantitative results of protein expression in tumor tissues of nude mice in each group;

[0108] Fig.29 29A is a graph showing the quantitative expression of proteins in tumor tissues of nude mice in each group, wherein 29B is a graph showing apoptosis-related proteins, 29C is a graph showing metastasis-related proteins, and 29A is a graph showing ferroptosis-related proteins. DETAILED DESCRIPTION

[0109] The following provides definitions of some terms used in this specification. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0110] The invention provides a carrier-free nano medicine, which comprises a metal-polyphenol network and a nano crystal medicine.

[0111] The metal-polyphenol network includes metal ions and polyphenol compounds.

[0112] The polyphenol compounds include flavonoid compounds, phenolic acid compounds, tannin compounds or anthocyanin compounds.

[0113] In one embodiment of the present invention, the carrier-free nanomedicine can also be referred to as a carrier-free nanocomplex.

[0114] In one embodiment of the present invention, flavonoids include but are not limited to silybin, quercetin, baicalin, apigenin, luteolin, green tea polyphenols, anthocyanins, kaempferol, chrysin, naringenin, and tea polyphenols.

[0115] In a specific embodiment of the present invention, the flavonoid compound is selected from silybin.

[0116] In one embodiment of the present invention, nanocrystal (NC) technology is a formulation technology that reduces the particle size of drug molecules to submicron level (100-1000nm) or even nanometer level (1-100nm) by crushing, dispersing or precipitating and crystallizing micron-sized drug particles, mainly including crushing technology and self-assembly technology.

[0117] In one embodiment of the present invention, nanocrystal drugs include but are not limited to tanshinone nanocrystals, paclitaxel nanocrystals, and camptothecin nanocrystals.

[0118] In a specific embodiment of the present invention, the nanocrystal drug is selected from tanshinone nanocrystals.

[0119] In one embodiment of the present invention, tanshinone includes dihydrotanshinone I, tanshinone IIA, cryptotanshinone, and tanshinone I.

[0120] In a specific embodiment of the present invention, the tanshinone is selected from dihydrotanshinone I.

[0121] The carrier-free nanomedicine also includes other functional small molecules.

[0122] The other functional small molecules include photothermal agents, photosensitizers, and contrast agents.

[0123] The photothermal agent includes but is not limited to IR820.

[0124] Photosensitizers include, but are not limited to, IR808 (1-(5-carboxypentyl)-2-[2-[3-[[1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene]ethylidene]-2-chloro-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-3H-indole bromide, (1-(5-carboxypentyl)-2-[2-[3-[[1-(5-carboxypentyl)-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene]ethylidene]-2-chloro-1-cyclohexen-1-yl]ethenyl]-3,3-dimethyl-3H-indoleium bromide), indocyanine green (ICG), chlorin e6 (chlorin e6, Ce6) and Meso-Tetra(4-carboxyphenyl)porphine (TCPP).

[0125] In a preferred embodiment of the present invention, other functional small molecules are selected from photosensitizers.

[0126] In a specific embodiment of the present invention, the photosensitizer is selected from IR808.

[0127] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0128] Example 1 Preparation and Characterization of Dihydrotanshinone I Nanocrystals

[0129] 1 Experimental Materials

[0130] Dihydrotanshinone I (99%) (DHT, Chengdu Zhibiao Huachun Biotechnology Co., Ltd.), povidone K30 (PVPK30, Beijing Yitong Huamao Biotechnology Co., Ltd.), polyvinyl alcohol (PVA), hydroxypropyl methylcellulose (HPMC, Shanghai Yuanye Biotechnology Co., Ltd.), acetone (Tianjin Bailuns Biotechnology Co., Ltd.), Wahaha water (Wahaha Group Co., Ltd.).

[0131] 2 Experimental methods

[0132] 2.1 Preparation of dihydrotanshinone I nanocrystals

[0133] Accurately weigh 5 mg of DHT and dissolve it in acetone to form 1 mg mL -1 The acetone solution of DHT was added dropwise into the water containing the dispersant under high-speed homogenization stirring at 40,000 rpm, and the stirring was continued for 30 minutes to form a crude emulsion. The crude emulsion was circulated 40 times at 15,000 psi using a high-pressure homogenizer to obtain a fine emulsion, which was then dropped into 10 times the volume of pre-cooled Wahaha water for solidification to obtain a nanocrystal suspension, the organic solvent was removed by rotary evaporation, and the pre-frozen and vacuum dried for 48 hours to obtain dihydrotanshinone I nanocrystals (DHT NC).

[0134] 2.2 Investigation of the preparation process of dihydrotanshinone I nanocrystals

[0135] 2.2.1 Investigation of dispersant types

[0136] Free DHT is poorly soluble in water. In order to prepare it into nanocrystals, three dispersants, polyvinyl alcohol (PVA), povidone K30 (PVPK30), and hydroxypropyl methylcellulose (HPMC), were selected, and three types of DHT NCs were prepared by the above method. The three nanocrystals and DHT raw materials were analyzed by X-ray powder diffractometer to determine the crystal form of each group of samples. The specific operation is as follows: the samples were placed on a silicon substrate with zero background and measured in room temperature reflection mode. A 2D / max-2500Pc X-ray diffractometer was used, and the X-ray scanning mode was symmetrical coupling scanning mode to collect X-ray diffraction data. θ-2θ linkage, step scanning, step length 0.02 degrees; voltage 40 kV; current 100 mA; scanning speed 3 degrees per minute; Cu target; measurement range 3 to 60 degrees.

[0137] 2.2.2 Determination of the optimal preparation process

[0138] In order to obtain DHT NC with excellent properties, four key factors in the preparation process were selected: drug-excipient ratio, stirring speed, homogenization pressure, and number of cycles. Three levels were set for each factor. Particle size and PDI (polydispersity index) were used as evaluation indicators to design L9 (3 4 ) orthogonal experiment and determine the optimal preparation process of DHT NC, the specific parameters are shown in Table 1. Dynamic light scattering (DLS) was used to measure the particle size and PDI of 9 groups of DHT NC, the specific operation was as follows: 3 mL of DHT NC suspension was pipetted into a clean sample pool, and the DHT NC was measured using a Malvern Zetasizer Nano ZS laser particle size analyzer, with 3 parallel samples for each group.

[0139] Table 1L9(3 4 )Orthogonal experimental factor level table

[0140]

[0141] 2.3 Preparation of nanocomposites

[0142] The morphology of DHT NC was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The freeze-dried DHT NC powder was glued to the stage, dried and sprayed with gold, and then its morphology was observed by SEM. The freeze-dried DHT NC powder was dispersed in water to form a suspension, and the DHT NC suspension was dropped on a silicon wafer, and its morphology was observed by TEM after drying.

[0143] 3 Experimental results

[0144] 3.1 Investigation of the preparation process of dihydrotanshinone I nanocrystals

[0145] 3.1.1 Investigation of dispersant types

[0146] PVP K30, PVA and HPMC were used as dispersants to prepare DHT NCs. The XRD (X-ray diffraction) patterns of the three nanocrystals are shown in Figure 2. Figure 1 As shown in the figure, DHT NC prepared with PVP K30 as dispersant showed a flat baseline and sharp diffraction peaks, indicating that the crystal structure was good and the symmetry was good. When PVA or HPMC was used as a dispersant, the baseline was higher and drifted, and the diffraction peaks became broad and split, indicating that the nanocrystal lattice was unstable and there was lattice collapse. Therefore, PVP K30 was selected as a dispersant to prepare DHT NC.

[0147] 3.1.2 Determination of the optimal preparation process

[0148] The optimal preparation process of DHT NC was investigated. 4 ) The results of the orthogonal test are shown in Table 2. The results show that, with particle size and PDI as indicators, A3B1C1D3 is the best preparation process, that is, the ratio of dispersant to DHT is 1:5, the stirring speed is 3000rpm, the homogenization pressure is 10000psi, and the number of cycles is 60 times. The particle size of the DHT NC prepared under this condition is 227.8±7.9nm, and the PDI is 0.297±0.007, indicating that the particle size of the DHT NC is suitable and the stability is good. Therefore, the DHT NC used in this study was prepared under this condition.

[0149] Table 2L9(3 4 )Orthogonal test results (n=6)

[0150]

[0151]

[0152] 3.2 Characterization of dihydrotanshinone I nanocrystals

[0153] SEM and TEM images of DHT NCs are shown in Figure 2. Figure 2 As shown, DHT NC is spherical or ellipsoidal with clear outlines and a particle size of about 220nm to 230nm, which is consistent with the DLS measurement results.

[0154] Example 2 Preparation and characterization of multi-drug carrier-free nanocomplexes

[0155] 1 Experimental Materials

[0156] Experimental materials: silybin (98%) (Sil), anhydrous ferric chloride (Shanghai Dibai Biotechnology Co., Ltd.), Tris buffer (Beijing Bio-Toda Technology Co., Ltd.), 10% hydroxylamine hydrochloride solution, acetic acid-ammonium acetate buffer solution (Shanghai Yuanye Biotechnology Co., Ltd.), 0.1% o-phenanthroline solution (Beijing Bio-Ason Biotechnology Co., Ltd.), dimethyl sulfoxide (Tianjin Bailuns Biotechnology Co., Ltd.), chromatographic grade methanol, chromatographic grade formic acid (Modern Oriental (Beijing) Science and Technology Development Co., Ltd.), phosphate buffer, MD34-7000D dialysis bag (Beijing Solebold Technology Co., Ltd.).

[0157] 2 Experimental methods

[0158] 2.1 Preparation of nanocomposites

[0159] Accurately weigh 6.25 mg (0.0225 mmol) of DHT NC freeze-dried powder and disperse it in 8 mL of ultrapure water to form a DHTNC suspension. Add a certain amount of Sil DMSO solution (1.80 mmol mL -1 ), the suspension was stirred vigorously for 2 min, and then 1 mL of FeCl3 DMSO solution (0.18 mmol mL -1 ), the suspension was stirred vigorously for 2 min, and then 4 mL of Tris buffer (50 mM, pH 8.5) was added to increase the pH, and stirred vigorously. After the reaction was complete, the prepared silybin-iron ion-coated dihydrotanshinone I nanocrystal complex (DHT NC@SF MPN) was placed in a 50 mL 10 kD ultracentrifuge tube and centrifuged (3000 rpm, 15 min) until the concentration of DHT, Sil and FeCl3 in the lower layer of the centrifuge tube was less than 1 μg mL -1 To remove the unreacted drug, the pre-frozen solution was vacuum dried for 48 h to finally obtain the DHT NC@SF MPN nanocomposite.

[0160] 2.2 Investigation of the dosage ratio of iron ion and silybin

[0161] In order to optimize the physicochemical properties of DHT NC@SF MPN nanocomposites, the molar ratio of Fe(III) to Sil was set to 1:3, 1:5 and 1:7 for encapsulation of DHT NC, and the effects of Fe(III) and Sil on the particle size, PDI, potential, drug loading and encapsulation efficiency of the nanocomposites were investigated.

[0162] 2.2.1 Determination of particle size, dispersion coefficient and potential

[0163] DLS was used to measure the particle size, PDI, and Zeta potential of DHT NC@SF MPN nanocomposites with different ratios. 3 mL of DHT NC suspension was pipetted into a clean sample pool, and the DHT NC@SF MPN nanocomposites with different ratios were measured using a Malvern Zetasizer Nano ZS laser particle size analyzer, with 3 parallel samples in each group.

[0164] 2.2.2 Determination of drug loading and encapsulation efficiency

[0165] The prepared DHT NC@SF MPNs with different ratios were placed in PBS at pH = 6.0 and stirred for 24 hours for acid hydrolysis. The reaction solution after acid hydrolysis was taken and the contents of DHT, Sil and Fe(III) were determined and calculated according to the above content determination method, and the drug loading (drug loading) and encapsulation efficiency (EE) were calculated according to formula (1) and formula (2), respectively. Three parallel samples were set for each group.

[0166]

[0167]

[0168] 2.3 Characterization of nanocomposites

[0169] 2.3.1 Structural characterization

[0170] Fourier transform infrared spectrometer (FT-IR) was used to characterize the structure of the nanocomposite. 2.00 mg of DHTNC@SF MPN nanocomposite freeze-dried powder, DHT NC freeze-dried powder, Sil powder and DHT powder were ground into fine powder in an agate mortar, mixed evenly with potassium bromide powder, and then loaded into molds, pressed into tablets on a tablet press, and tested by FTIR.

[0171] 2.3.2 Morphological characterization

[0172] The morphology of DHT NC@SF MPN nanocomposite was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The freeze-dried powder of the nanocomposite was glued to the stage, dried and sprayed with gold, and then its morphology was observed by SEM. The nanocomposite solution was prepared, and the solution was dropped on the silicon wafer, and its morphology was observed by TEM after drying.

[0173] 2.3.3 Stability study

[0174] The stability of the DHT NC@SF MPN nanocomposite was investigated. The prepared DHT NC@SF MPN nanocomposite was prepared into a suspension using PBS at pH = 7.4, with a concentration of 1 μg mL -1, respectively, at 25°C and 37°C for 7 days, and the particle size and PDI changes of the DHT NC@SF MPN nanocomposites were measured and recorded by DLS every day, with 3 parallel samples in each group.

[0175] 2.3.4 In vitro drug release study

[0176] The in vitro release behavior of free DHT, Sil and DHT NC@SF MPN was investigated by dialysis. 50 mL of PBS solution with pH values ​​of 6.0, 7.4 and 8.8 were prepared as release media. DHT, Sil and nanocomplex were accurately weighed and dissolved in the corresponding release media to obtain a concentration of 1 mg mL -1 The drug-containing solution (measured in DHT or Sil) was placed in a dialysis bag and oscillated in a constant temperature water bath oscillator at 37°C and 150 rpm. 1.0 mL of release medium was taken at 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h after placement, and the concentrations of DHT and Sil were determined by UPLC (ultra-high performance liquid chromatography). At the same time, 1.0 mL of fresh release medium was added to the original release medium to keep the total volume of the release medium unchanged. Three parallel samples were set for each group. The cumulative drug release amount was calculated according to formula (3) and formula (4).

[0177]

[0178]

[0179] 3 Experimental results

[0180] 3.1 Investigation of the dosage ratio of iron ion and silybin

[0181] 3.1.1 Determination of particle size, dispersion coefficient and potential

[0182] The particle size, PDI and potential of DHT NC@SF MPN nanocomposites prepared with different ratios of Sil and Fe(III) were measured, and the results are shown in Table 3. When the molar ratio was 1:3, the particle size of the DHT NC@SF MPN nanocomposites was significantly smaller than that of the other two groups (P<0.05), which was 262.12±5.61nm. When the molar ratio was 1:5, the PDI of the DHT NC@SF MPN nanocomposites was the smallest, and there was no significant difference with the nanocomposites prepared at a molar ratio of 1:3, indicating that the nanocomposites prepared at these two ratios had better dispersibility and stability. The potential gradually increased with the increase of the molar ratio, and there was no significant difference among the three groups.

[0183] Table 3 Particle size, PDI and potential of DHT NC@SF MPN nanocomposites with different ratios (n=3)

[0184]

[0185] Note: Compared with 1:3 group, *P<0.05

[0186] 3.1.2 Determination of drug loading and encapsulation efficiency

[0187] The contents of DHT, Sil and Fe(III) in the DHT NC@SF MPN nanocomposites with different ratios were determined after acid hydrolysis. Figure 3 As shown. As the molar ratio of Fe(III) to Sil increases from 1:3 to 1:7, the DHT loading and encapsulation efficiency gradually decrease, while the Sil loading gradually increases. This indicates that when the molar ratio of Fe(III) to Sil increases from 1:3, the encapsulation capacity of DHT NC formed by SF MPN has reached saturation. As the Sil dosage increases, Sil will be adsorbed on the surface of the nanocomposite, resulting in a decrease in the DHT loading. Considering that the DHT NC@SF MPN nanocomposite exerts its main antitumor effect with DHT, the DHT NC@SF MPN nanocomposite was prepared with a molar ratio of Fe(III) to Sil of 1:3. At this dosage ratio, the drug loadings of DHT, Sil and Fe(III) were 55.28±0.80%, 41.66±0.66% and 2.10±0.19%, respectively, and the DHT encapsulation efficiency was 90.46±2.27%.

[0188] 3.2 Characterization of nanocomposites

[0189] 3.2.1 Structural characterization

[0190] FT-IR was used to confirm the structure of DHT NC@SF MPN nanocomposites. Figure 4 As shown in Figure 2, compared with DHT NC, the DHTNC@SF MPN nanocomposite has a -1 The DHT NC@SF MPN nanocomposite showed a stronger hydroxide (-OH) peak at 680 cm -1 A new absorption peak obviously appeared at 2920cm, which is the characteristic peak of the hydroxyl complex of Fe(III), proving that the hydroxyl of Sil forms a complex with Fe(III). -1 The intensity of the hydrogen-carbon (-CH) peak at 240 nm decreased. The changes in these absorption peaks indicate that the DHTNC@SF MPN nanocomposite has been successfully constructed.

[0191] 3.2.2 Morphological characterization

[0192] SEM and TEM images of DHT NC@SF MPN nanocomposites are shown in Figure 2. Figure 5 As shown in the figure, the nanocomposite is also a spherical particle, but it shows a certain degree of adhesion, and the particle size is between 260nm and 270nm. Compared with the particle size of DHT NC, it has increased, which also shows that the SF MPN coating is successfully attached to the surface of DHT NC, and the DHT NC@SF MPN nanocomposite is successfully constructed.

[0193] 3.2.3 Stability study

[0194] The stability test results of DHT NC@SF MPN nanocomposites are shown in Figure 6 As shown, the particle size of the DHT NC@SF MPN nanocomposite was always between 260 and 270 nm within 7 days, and the PDI did not change significantly, indicating that the nanocomposite had good stability.

[0195] 3.2.4 In vitro drug release study

[0196] The drug release of DHT NC@SF MPN nanocomposites under different pH conditions was investigated. Figure 7 As shown. Although the release of DHT NC@SF MPN and free drugs are both related to pH, they show opposite drug release behaviors. Free DHT and Sil are released faster in alkaline conditions, i.e., pH = 8.8 medium. The release rate of DHT NC@SF MPN nanocomplexes is faster under pH = 6.0 than under pH = 7.4 or pH = 8.8. Under pH = 6.0, the nanocomplex releases more slowly than free DHT. In summary, the pH-responsive drug release exhibited by DHT NC@SF MPN nanocomplexes is conducive to the release and absorption of the nanocomplexes at the tumor site, allowing more DHT to be enriched at the tumor site. At the same time, its sustained release effect helps the nanocomplexes to continue to exert their efficacy.

[0197] Example 3 In vitro pharmacodynamic evaluation of multi-drug carrier-free nanocomplexes

[0198] 1 Experimental Materials

[0199] PBS (Corning, USA), fetal bovine serum, trypsin (Gibco, USA), Primocin antibiotics (Invivogen, USA), CCK-8 (Shanghai Bio-Tech Co., Ltd.), Annexin-V-FITC / PI kit (Suzhou Yuheng Biotechnology Co., Ltd., HY0821).

[0200] Human gastric cancer cell line HGC 27, Cell Bank of the Chinese Academy of Sciences.

[0201] 2 Experimental methods

[0202] 2.1 Cell culture

[0203] Human gastric cancer cell HGC 27 was cultured in RPMI-1640 medium containing 1% Primocin antibiotics and 10% fetal bovine serum at 37°C and 5% CO2 concentration.

[0204] 2.2 Cytotoxicity

[0205] Experimental groups: DHT group, DHT+Sil group, DHT+Sil+Fe(III) group, DHT NC group, DHT NC@SF MPN nanocomposite group. Human gastric cancer cells HGC 27 were cultured at 1×10 5 The cells were inoculated at a density of 10 cells / well in a 96-well plate and cultured for 24 h. Then, different concentrations of drug solutions (based on DHT, the final concentrations were 0.5, 1.0, 1.5, 2.5, 5.0, 10.0, 15.0, 20.0, 25.0 μmol·L -1 ) were co-incubated with cells for 24h and 48h, respectively. For the normal group, after the cells were co-incubated with the drug solution for 24h and 48h, the drug solution was carefully removed, the cells were washed 3 times with pre-cooled PBS, fresh culture medium was added, and culture was continued for 24h. After the culture was completed, the culture medium was removed, 10μL CCK-8 solution was added to each well, and the wells were placed in the incubator for further incubation for 2h, the 96-well plate was taken out, and the absorbance of each well at 490nm was measured by an ELISA instrument. The wells without drug addition were used as controls to calculate the cell survival rate.

[0206] 2.3 Apoptosis

[0207] Experimental groups: DHT group, DHT+Sil group, DHT+Sil+Fe(III) group, DHT NC group, DHT NC@SF MPN nanocomposite group. Annexin-V-FITC / PI double staining method was used to detect the effect of nanocomposite on apoptosis of human gastric cancer cell HGC 27. Human gastric cancer cell HGC 27 cells were cultured in 6-well plates, with 1×10 cells seeded in each well. 6 After 24 hours, 1 mL of 4 μmol·L -1(measured in DHT) drug solution incubated for 24h, carefully removed the drug solution, then washed the cells 3 times with pre-cooled PBS, added fresh culture medium, and continued incubation for 24h. After the incubation, digested with EDTA-free trypsin, centrifuged at 1800rpm for 5min, rinsed twice with pre-cooled PBS at room temperature, then added 100μL PBS to resuspend the cells, added 5μL YF488-AnnexinV working solution and 5μL PI working solution to each tube, mixed, incubated at room temperature in the dark for 15min, added 400μL PBS after the incubation, and detected by flow cytometry.

[0208] 2.4 Cell migration

[0209] Experimental groups: DHT group, DHT+Sil group, DHT+Sil+Fe(III) group, DHT NC group, DHT NC@SF MPN nanocomposite group. The effect of nanocomposite on the migration of human gastric cancer cells HGC 27 was investigated by cell scratch test. Five uniform straight lines were drawn on the back of a 6-well plate with a marker pen. Cells were plated at 1×10 6 The cells were inoculated at a density of 10 cells / well in a 6-well plate. After culturing for 24 h, the original serum-containing medium was removed and 3 mL of 4 μmol·L -1 (in terms of DHT) drug solution in serum-free medium, culture for 6 hours, then scratch the bottom of the 6-well plate with a 200 μL pipette tip vertically, wash the cells 3 times with pre-cooled PBS, wash off the scratched cells, add fresh medium, take pictures at 0h and 12h of culture, record the scratch area, and use the wells without drug solution as the blank control group. The blank control group and each drug group are set with 3 replicates. Calculate the cell migration rate according to formula (5).

[0210]

[0211] 3 Experimental results

[0212] 3.1 Cytotoxicity

[0213] The effect of DHT NC@SF MPN nanocomplex on cell viability was investigated in human gastric cancer cells HGC 27. The cell viability after 24h and 48h of administration was as follows Figure 8 As shown in the figure, each drug group showed a certain dose-effect relationship, and the cell survival rate in each drug group decreased with the increase of drug concentration. The cell half-inhibitory concentration (IC50) at 24h and 48h of drug administration is shown in the figure. Fig. 9 As shown, the IC50 of the nanocomplex is 7.60 μmol·L -1 / 6.43μmol·L -1 , compared with the original drug (14.47μmol·L -1 / 12.85μmol·L-1 ) and drug mixture (10.72 μmol·L -1 / 8.66μmol·L -1 ) was significantly reduced and had significant differences compared with the other groups (P<0.05), indicating that the cytotoxicity of the nanocomplex was stronger than that of the other groups.

[0214] 3.2 Apoptosis

[0215] Flow cytometry was used to detect the induced cell apoptosis. The flow cytometry scatter plot and apoptosis statistical graph are shown in Figure 2. Fig.10 and Fig.11 As shown. Fig.11 It can be seen that compared with the control group, the apoptosis rate of each drug-treated group increased to varying degrees (P<0.05). The apoptosis rate of the DHT NC@SF MPN group was 64.03%, significantly higher than that of the other four drug-treated groups (P<0.05). Compared with DHT and the drug mixture, the apoptosis rates increased by 46.98% and 6.07%, respectively, indicating that DHT NC@SF MPN has a stronger ability to induce apoptosis.

[0216] 3.3 Cell migration

[0217] The ability of DHT NC@SF MPN nanocomplexes to inhibit cell migration was evaluated by cell scratch assay. The cell scratch images and cell migration rate statistics at 0h and 12h are shown in Figure 2. Fig.12 and Fig.13 As shown, the scratch area of ​​each drug-treated group was significantly larger than that of the control group, and the scratch migration rate of the DHT NC@SF MPN group (19.75%) was significantly lower than that of the DHT group (22.85%) and the DHT NC group (23.27%) (P<0.05).

[0218] Example 4 Intracellular transport process of multi-drug carrier-free nanocomplex

[0219] 1 Experimental Materials

[0220] IR808 (99%) (Shanghai Kaiyulin Pharmaceutical Technology Co., Ltd.), RPMI-1640 culture medium, PBS (Corning Company, USA), Primocin antibiotics (Invivogen Company, USA), Hoechst33342 (Beijing Biotech Co., Ltd.), mitochondrial green fluorescent probe, lysosomal green fluorescent probe (Shanghai Biyuntian Biotechnology Co., Ltd.), endoplasmic reticulum green fluorescent probe, Golgi apparatus green fluorescent probe (Jiangsu KeyGen Biotechnology Co., Ltd.), radioimmunoprecipitation assay (RIPA) lysis buffer (Beijing Bairuiji Biotechnology Co., Ltd.), dichlorodiphenylamine (BCA) protein detection kit (Aladdin Co., Ltd. (Beijing, China)), methyl-β-cyclodextrin, amiloride hydrochloride, cytochalasin D, chlorpromazine hydrochloride, nystatin (Bosch Biotechnology (Shanghai, China)), protein-free cell freezing solution (Ecosai Biotechnology (Taicang) Co., Ltd.), chromatographic grade methanol, chromatographic grade formic acid (Modern Oriental (Beijing) Science and Technology Development Co., Ltd. Factory).

[0221] 2 Experimental methods

[0222] 2.1 Cell culture

[0223] Human gastric cancer cell HGC 27 was cultured in RPMI-1640 medium containing 1% Primocin antibiotics and 10% fetal bovine serum at 37°C and 5% CO2 concentration.

[0224] 2.2 Cellular uptake

[0225] In a small petri dish, 5 × 10 4 Human gastric cancer cells HGC 27 were inoculated at a density of 10 cells / well. When the cells were about 80% confluent, they were washed with PBS for 3 times and 1 μM·L -1 , 2μM·L -1 , 4μM·L -1 DHT / IR808NC@SF MPN solution. After 4 hours of culture, slowly rinse with PBS for 3 times, and then add 1ml of serum-free culture medium. Place the small culture dish under a laser confocal microscope and take pictures to observe the drug uptake at different concentrations. 4 Human gastric cancer cells HGC 27 were inoculated at a density of 10 cells / well. When the cells were about 80% confluent, they were washed three times with PBS and 4 μM·L -1 DHT / IR808NC@SF MPN solution was cultured for 2, 4, and 8 hours, and then slowly rinsed with PBS for 3 times, and then 1 mL of serum-free culture medium was added. The small culture dish was placed under a laser confocal microscope and photos were taken to observe the uptake at different incubation times.

[0226] 2.3 Cellular uptake pathways

[0227] 2.3.1 Protein content determination

[0228] Use the BCA protein quantification kit to detect the protein concentration of cells. According to the instructions of the kit, mix reagent B and reagent A at a volume ratio of 1:50 to prepare an appropriate amount of BCA working solution. Use PBS buffer to gradually dilute the protein standard to a series of gradient standard solutions. Take 200μL of the prepared BCA working solution in a 96-well plate, add 10μL of a series of protein standard solutions or cell lysis samples, mix well, heat in a 37℃ water bath for 30min, measure the sample absorbance at a wavelength of 562nm with an enzyme reader, draw a standard curve and calculate the protein concentration of the cell lysate sample.

[0229] 2.3.2 Endocytosis protein inhibition experiment

[0230] The protein-mediated pathways of nanoparticle endocytosis are mainly clathrin- and caveolin-mediated endocytosis and macropinocytosis. The protein-mediated pathways of cell endocytosis can be analyzed by adding inhibitors of different endocytic pathways. 4 The cells were inoculated at a density of 100 / mL in a 24-well plate, and the medium was changed every day. When the cells formed a monolayer, they were pretreated with inhibitors of different endocytic pathways for 30 min. The types and concentrations of the inhibitors are shown in Table 4. Then 4 μmol·L -1 3 mL of DHT NC@SF MPN solution was added and incubated for 4 h. After the incubation, the cells were washed 3 times with ice-cold PBS buffer to terminate the reaction. Then, trypsin digestion solution was added to digest the HGC 27 cells and blown into a cell suspension. The suspension was centrifuged at 1000 rpm for 3 min, the supernatant was discarded, and the cells were washed with PBS. This was repeated 3 times to fully wash away the DHT NC@SF MPN nanocomplex adsorbed on the cell surface to obtain precipitated cells. The cells were collected, and 200 μL of 0.1% SDS solution was added to each well to lyse the cells. The cells were placed at room temperature for 10 min and then blown. The cell lysate was collected in an EP tube and centrifuged at 13000 rpm for 10 min. The supernatant was taken for analysis. 50 μL of cell lysate was taken to detect the content of DHT in the cell lysate. Another 10 μL of cell lysate was taken to determine the protein concentration, and the drug concentration was calculated according to formula (6).

[0231] Drug content per unit protein in cells = drug content per unit volume of cells / protein content per unit volume of cells (6)

[0232] Table 4 Types of cellular uptake inhibitors and their effective concentrations

[0233]

[0234] 2.4 Organelle colocalization

[0235] HGC 27 cells were cultured at 1×10 5 The cells were inoculated at a density of 100 / mL in a confocal dish, and the medium was changed every day until a cell monolayer was formed. A total of 12 dishes were cultured. The DHT / IR808NC@SF MPN nanocomplex was dispersed in the culture medium to a final concentration of 4 μmol·L -1 1 mL of drug-containing culture medium was added to each dish, and the cells were incubated for 4 h at 37 ° C in an incubator containing 5% CO2. After the incubation, the cells were washed 3 times with PBS buffer. Then 1 mL of Hoechst 33342 preheated at 37 ° C was added to each dish for staining, and the cells were cultured for 10 min. Then, the 12 dishes of cells were randomly divided into 4 groups, with 3 dishes in each group. 100 μL of Lyso-Tracker Green DND-99 dye solution, 100 μL of ER-TrackerGreen dye solution, 100 μL of Golgi-Tracker Green dye solution, and 100 μL of Mito TrackerGreen dye solution were added, respectively, and the cells were cultured for 10 min. After the staining, the cells were washed twice with culture medium, and fresh culture medium was added. The co-localization of DHT / IR808NC@SF MPN nanocomplexes with various organelles was observed using a laser confocal microscope.

[0236] 3 Experimental results

[0237] 3.1 Cellular uptake

[0238] The red fluorescent substance IR808 was doped into DHT NC and DHT NC@SF MPN to investigate the intracellular uptake. The laser confocal images taken at different drug concentrations at the same incubation time and at the same drug concentration at different incubation times are shown in Figure 2. Fig.14 As shown. The fluorescence intensity of DHT NC and DHT / IR808 NC@SF MPN is stronger than that of free IR808, indicating that the nanoparticles are conducive to cellular uptake. The fluorescence intensity of the DHT / IR808 NC@SF MPN group gradually increases with the increase of incubation time and incubation drug concentration, indicating that its cellular uptake is time / concentration dependent.

[0239] 3.2 Protein content determination

[0240] The protein was quantified using the BCA kit and a protein standard curve was established. Fig.15 shown.

[0241] 3.2.3 Endocytosis protein inhibition experiment

[0242] Different endocytic protein inhibitors were used to further explore the cellular uptake pathway of DHT NC@SF MPN nanocomplexes. Fig.16As shown in the figure, compared with the control group, the intracellular DHT content was significantly reduced after treatment with the five inhibitors (P<0.05), indicating that DHT, DHT NC, and DHT NC@SF MPN were taken up into HGC 27 cells through multiple pathways. After inhibiting the caveolin-mediated pathway with nystatin, the cellular uptake of DHT and DHT NC was only about 50% of that in the control group, indicating that the caveolin-mediated pathway is the main pathway for cellular uptake of DHT and DHT NC. For the DHT NC@SF MPN group, the intracellular DHT content was significantly reduced after treatment with methyl-β-cyclodextrin, mainly inhibiting the lipid raft / caveolin-mediated pathway. The different transport pathways of DHT, DHT NC, and DHT NC@SF MPN nanocomplexes may be related to their different particle sizes and surface properties.

[0243] 3.3 Organelle colocalization

[0244] Major organelles (nucleus, mitochondria, lysosomes, endoplasmic reticulum, and Golgi apparatus) were stained with specific fluorescent probes to locate the intracellular distribution of DHT / IR808 NC@SF MPN nanocomplexes. Fig.17 As shown in the figure, the red fluorescence of DHT / IR808 NC@SFMPN nanocomplex is widely distributed in mitochondria, lysosomes and endoplasmic reticulum. Since most nanoparticles enter the lysosomes for degradation and metabolism after being taken up into the cells, the nanocomplex appears in the lysosomes. The distribution of DHT / IR808 NC@SF MPN nanocomplexes in mitochondria and endoplasmic reticulum indicates that the nanocomplex can achieve lysosomal escape, which may be due to the acidity of SF MPN. Acidic substances have a high affinity with the lysosomal membrane, which will cause the lysosomal membrane line tension to decrease and form holes and maintain the stability of the holes, thereby causing the drug to escape from the lysosome. This shows that after entering the cell, the nanocomplex will not be completely degraded by the hydrolases present in the lysosome, and will be successfully released into the cell through lysosomal escape to exert its drug effect.

[0245] Example 5 Pharmacokinetics and tissue distribution study of multi-drug carrier-free nanocomplexes

[0246] 1 Experimental Materials

[0247] Healthy male Sprague-Dawley (SD) rats, weighing 200±20 g, were provided by Beijing Biotechnology Co., Ltd., license number: SCXK(Beijing)2019-0010.

[0248] 2 Experimental methods

[0249] 2.1 Dosage regimen

[0250] SD rats were randomly divided into blank group, DHT original drug group, DHT NC group, DHT+Sil+Fe(III) group (DHT NC@SFMPN nanocomposite group, 6 rats in each group, fasted for 12h before the experiment, and had free access to water. Rats in the drug group were given 15mg·kg -1 The dose was intraperitoneally injected (in terms of DHT), and the blank group was given normal saline at the same dose. 0.75 mL of blood was collected from the rat orbital venous plexus at 0min, 5min, 10min, 15min, 0.5h, 1h, 2h, 4h, 8h, 12h, and 24h after administration, and placed in a centrifuge tube pretreated with sodium heparin. After centrifugation at 13000rpm for 10min, the supernatant was taken, and the plasma was combined and stored in a -80℃ refrigerator for future use. 24h after administration, the heart, liver, spleen, lung, and kidney tissues of each group of rats were weighed and stored in a -80℃ refrigerator for future use.

[0251] 2.2 Plasma pretreatment

[0252] The plasma sample was processed by taking 200 μL of blank rat plasma, adding 800 μL of methanol to precipitate plasma protein, vortexing at room temperature for 3 min to mix it evenly (the purpose is to fully precipitate the plasma protein), then centrifuging at 12000 rpm for 10 min, blowing dry with nitrogen in a 40°C water bath, adding 600 μL of methanol to the residue to re-dissolve it, vortexing for 1 min, centrifuging at 12000 rpm for 10 min, and taking the supernatant for UPLC analysis.

[0253] 2.3 Tissue sample pretreatment

[0254] Each tissue sample (heart, liver, spleen, lung, kidney) was drained and weighed, and physiological saline was used as the homogenate, and the tissue homogenate was prepared at a ratio of tissue mass (g): physiological saline volume (mL) of 1:2. Take 200μL of tissue homogenate, add 800μL of methanol, vortex and oscillate evenly, centrifuge at 12000rpm for 10min under low temperature conditions, take the supernatant, blow dry with nitrogen in a 40℃ water bath, add 600μL of methanol to the residue, vortex for 1min, centrifuge at 12000rpm for 10min, and take the supernatant for analysis by UPLC.

[0255] 3 Experimental results

[0256] 3.1 Pharmacokinetics

[0257] DHT medication time curve Fig.18As shown in the figure, it can be seen from the drug-time curve that the blood drug concentration first increases and then decreases, which is consistent with the pharmacokinetic characteristics of intraperitoneal administration. The time for the blood drug concentration to reach the maximum value is about 1h. Other pharmacokinetic parameters are shown in Table 5. The area under the curve (AUC) of the DHT NC@SF MPN nanocomposite group is the highest (14.11±0.11mg / L*h), which is 1.60 times and 1.86 times that of the free DHT and DHT NC groups, respectively. At the same time, the mean residence time (MRT) and half-life (t1 / 2) are significantly prolonged, which are 1.68 times and 2.06 times that of the free drug, respectively, indicating that the drug elimination function is impaired. Therefore, the improvement of DHT bioavailability may be related to the sustained drug release and weakened drug elimination of DHT NC@SF MPN.

[0258] Table 5 Pharmacokinetic parameters of intraperitoneal injection in different dosing groups

[0259]

[0260] 3.2 Organizational Distribution

[0261] The distribution of DHT in tissues is as follows Fig.19 As shown in the figure, from the perspective of drug tissue distribution, DHT is mainly concentrated in liver and kidney tissues. The DHT content in kidney tissue of the DHT NC@SF MPN nanocomposite group was significantly lower than that of the other groups, confirming its low drug elimination ability.

[0262] Example 6 In vivo pharmacodynamic evaluation and mechanism of action study of multi-drug carrier-free nanocomplexes

[0263] 1 Experimental Materials

[0264] Healthy male Balb / c nude mice, weighing 20 ± 2 g, were provided by Beijing Weitonglihua Biotechnology Co., Ltd., license number: SCXK (Beijing) 2021-0006. This study was approved by the Medical and Laboratory Animal Ethics Committee of Beijing University of Chinese Medicine, number: BUCM-4-20220627-2126.

[0265] 2 Experimental methods

[0266] 2.1 Establishment of HGC 27-CDX nude mouse tumor model

[0267] The HGC 27-CDX nude mouse tumor model was established by subcutaneous injection of tumor cell lines. The cultured human gastric cancer cells HGC 27 were digested with trypsin and collected by centrifugation. The cells were resuspended in PBS to a final concentration of 2×10 8 / mL human gastric cancer cell HGC 27 cell suspension. 0.2mL of human gastric cancer cell HGC 27 cell suspension was injected into the right armpit of nude mice, and the modeling time was 2 weeks. HGC 27-CDX tumor nude mice were placed in the SPF animal laboratory of Beijing University of Chinese Medicine [SYXK (Beijing) 2016-0038] and were free to eat and drink during the feeding period.

[0268] 2.2 Tumor size monitoring

[0269] When the tumor volume reaches 100mm 3 At the same time, 48 nude mice were randomly divided into 8 groups: Model group, Saline group, DHT group, DHT NC group, DHT+Sil group, DHT+Sil+Fe(III) group, DHT NC@SF MPN group and Lps-P (paclitaxel liposome for injection), with 6 mice in each group. Each drug-loaded conjugate administration group was intratumorally injected with a DHT dose of 10 mg / (kg·d), and the positive drug group was administered with a dose of 10 mg / (kg·d), once every other day, and the administration cycle was 14 days. During the entire experiment, the length and width of the tumor were manually measured with a digital vernier caliper, and the tumor volume was calculated according to formula (7). After the experiment, all nude mice were killed by cervical dislocation, and the tumor tissues were taken and weighed. In addition, the tumor tissues and organs were fixed with 4% paraformaldehyde.

[0270] Tumor volume (mm 3 ) = length × width 2 ×0.5 (7)

[0271] 2.3 Biodistribution

[0272] When the tumor volume reaches 100mm 3 At the time of intravenous injection, 18 nude mice were randomly divided into 3 groups: IR808 group, DHT / IR808 NC group, and DHT / IR808 NC@Sil-Fe(III) group, with 6 mice in each group. Each drug-loaded conjugate administration group was intratumorally injected with 10 mg / (kg·d) DHT. After administration, the nude mice were anesthetized with isoflurane. When the nude mice were in a deep anesthetized state, they were placed in a small animal in vivo imager for observation. The excitation wavelength was set to 660 nm and the emission wavelength was set to 780 nm. The fluorescence distribution in the nude mice was recorded at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h after intratumoral injection. After 24 h of administration, the nude mice were anesthetized with sodium pentobarbital, and the tumors, heart, liver, spleen, lung, kidney and other tissues of the nude mice were removed. The blood in the tissues was flushed with saline, and after being dried with filter paper, they were immediately placed in the in vivo imager for imaging. The fluorescence intensity of nude mouse images taken at different time periods was analyzed using Image J software.

[0273] 3 Experimental results

[0274] 3.1 Tumor size monitoring

[0275] The in vivo pharmacodynamics of HGC 27 tumor-bearing nude mice were evaluated. The tumor volume growth curves and tumor growth inhibition rates of each group during the administration period were as follows: Fig. 20 As shown, after the administration, the tumors and tumor weights of each group were Fig.21 and Fig. 22 As shown. Compared with the model group and the saline group, the tumor volume and tumor weight of each drug-treated group were significantly reduced (P<0.05). The tumor inhibition rate of the DHT NC@SFMPN group was the highest, at 39.74%, which was significantly enhanced compared with the inhibition rate of DHT (11.35%), and there was no significant difference in inhibition rate compared with the positive control group Lps-P (P<0.05).

[0276] 3.2 Biodistribution

[0277] The biodistribution of IR808-doped DHT NC@SF MPNs after intratumoral or intraperitoneal injection is shown in Figure 2. Fig.23 and Fig.24 As shown, the fluorescence intensity analysis was Fig.25 As shown. It can be seen that the fluorescence intensity gradually increased within 0-8h and was not eliminated until 24h. For the intratumoral injection group, the fluorescence was mainly located in the tumor tissue and was almost undetectable in other tissues. In the intraperitoneal injection group, although the fluorescence accumulated in the tumor was stronger than that in other tissues, its widespread distribution could not be avoided. At the same time, the fluorescence intensity of DHT / IR808 NC@SF MPN was significantly lower than that of the intratumoral injection group 24h after intraperitoneal injection (P<0.05). The biodistribution characteristics indicate that the DHT NC@SF MPN nanocomplex can selectively accumulate in the tumor and remain for a long time, which is conducive to its good anti-tumor effect.

[0278] 3.3 Study on anti-tumor mechanism

[0279] After the administration, the H&E stained sections of the tumors in each group were as follows: Fig.26 As shown in the figure, a small amount of necrotic tumor tissue appeared in the center of the tumor in the model group and the saline group. This phenomenon may be due to the rapid growth of the tumor and the delayed construction of blood vessels, which leads to the lack of nutrition and oxygen in the central tumor, and then leads to necrosis in the center of the tumor tissue. The necrotic area of ​​the DHT NC@SFMPN and Lps-P groups was significantly larger than that of the other drug-treated groups, indicating that their anti-tumor effects were better. No obvious tumor metastasis was found in the H&E stained sections of several groups.

[0280] In order to clarify its anti-tumor mechanism in vivo, apoptosis analysis was performed using apoptosis staining (TUNEL staining). Fig. 27As shown in the figure, almost no green fluorescence was observed in the model group and the saline group, indicating that tumor cells did not undergo apoptosis. Among all the drug-treated groups, the green fluorescence area of ​​the DHT NC@SF MPN and Lps-P groups was the largest, indicating that they could effectively induce tumor cell apoptosis. When the drugs were used in combination, the apoptosis rate was higher than that of a single drug.

[0281] The expression of proteins related to tumor cell apoptosis, metastasis and ferroptosis was studied. Fig.28 and Fig.29 As shown in the figure, both DHT NC@SF MPN nanocomplexes and Lps-P can significantly upregulate the expression of caspase-3 and caspase-8, downregulate the expression of Bcl-2, and promote tumor cell apoptosis. At the same time, the nanocomplex can reduce the expression of MMP2 and MMP9, indicating that they can inhibit the decomposition of the extracellular matrix and prevent tumor metastasis. At the same time, the increased expression of E-cadherin also helps to prevent the spread of tumor cells. The expression of ferroptosis-related protein TFRC increased and the expression of GPX4 decreased, indicating that ferroptosis was activated. These results indicate that the anti-tumor effect of DHT NC@SF MPN is mediated by multiple pathways.

[0282] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A carrier-free nanomedicine, characterized in that: The carrier-free nanomedicine includes a metal-polyphenol network and a nanocrystalline drug; The metal-polyphenol network includes metal ions and polyphenol compounds; The metal ion is selected from Fe(III); The polyphenolic compounds are selected from flavonoid compounds; The flavonoid compound is selected from silybin; The nanocrystal drug is tanshinone nanocrystal; The tanshinone nanocrystals are dihydrotanshinone I nanocrystals; The molar ratio of Fe(III) to silybin is 1:3; The carrier-free nanomedicine also includes other functional small molecules; The other functional small molecules are selected from photosensitizers; The photosensitizer is selected from IR808.

2. The carrier-free nanomedicine according to claim 1, characterized in that: The particle size of the dihydrotanshinone I nanocrystals is 227.8±7.9 nm.

3. The carrier-free nano drug according to claim 1, characterized in that The PDI of the dihydrotanshinone I nanocrystals is 0.297±0.

007.

4. The carrier-free nanomedicine according to claim 1, characterized in that: The particle size of the carrier-free nanomedicine is 262.12±5.61 nm.

5. The carrier-free nanomedicine according to claim 1, characterized in that: The PDI of the carrier-free nanomedicine is 0.269±0.

01.

6. A method for preparing a carrier-free nanomedicine, characterized in that: The method comprises: 1) Preparation of nanocrystalline drugs; 2) adding polyphenolic compounds and metal ions to the suspension of nanocrystalline drugs respectively, stirring, and obtaining carrier-free nanomedicines; The nanocrystal drug described in step 1) is tanshinone nanocrystal; The tanshinone nanocrystals are dihydrotanshinone I nanocrystals; In step 2), the metal ion is selected from Fe(III); The polyphenolic compounds are selected from flavonoid compounds; The flavonoid compound is selected from silybin; The molar ratio of Fe(III) to silybin is 1:3; Step 1) includes the following steps: a) dissolving dihydrotanshinone I in an organic solvent; b) adding the solution obtained in a) to a dispersant to obtain a crude emulsion; c) preparing the coarse emulsion in b) into a fine emulsion, and solidifying it to obtain a nanocrystal suspension; d) removing the organic solvent and drying to obtain dihydrotanshinone I nanocrystals; Step 2) includes the following steps: e) mixing the nanocrystalline drug suspension with the silybin solution; f) mixing the suspension obtained in e) with a FeCl3 solution; g) adjusting pH, stirring and centrifuging; The dispersant is selected from PVP K30.

7. The method according to claim 6, characterized in that The organic solvent described in a) is selected from acetone.

8. The method according to claim 6, characterized in that b) The specific step comprises adding the acetone solution of dihydrotanshinone I into water containing a dispersant under high-speed homogenization stirring.

9. The method according to claim 8, characterized in that The stirring speed in b) is 3000-5000 rpm.

10. The method according to claim 9, characterized in that The stirring speed in b) was 3000 rpm.

11. The method according to claim 8, characterized in that b) The stirring time is 30 min.

12. The method according to claim 6, characterized in that The ratio of PVP K30 to nanocrystalline drug is 1:(1-5).

13. The method according to claim 12, characterized in that The ratio of PVP K30 to nanocrystalline drug is 1:

5.

14. The method according to claim 6, characterized in that In c), the coarse emulsion in b) is prepared into a fine emulsion by high pressure homogenization.

15. The method according to claim 14, characterized in that The homogenization pressure in c) is 10,000-20,000 psi.

16. The method according to claim 15, characterized in that The homogenizing pressure in c) is 10,000 psi.

17. The method according to claim 14, characterized in that The number of cycles in c) is 20-60 times.

18. The method according to claim 17, characterized in that The number of cycles in c) is 60.

19. The method according to claim 6, characterized in that d) The organic solvent is removed by rotary evaporation.

20. The method according to claim 6, characterized in that e) The solution of silybin is DMSO.

21. The method according to claim 6, characterized in that f) The FeCl3 solution is DMSO.

22. The method according to claim 6, characterized in that g) Tris buffer was used to adjust the pH.

23. The method according to claim 6, characterized in that g) The centrifugation condition is 3000 rpm for 15 min.

24. The method according to claim 6, step 2) further comprising step h) pre-freezing and then drying.

25. Use of the carrier-free nanomedicine according to any one of claims 1 to 5 in the preparation of a drug for treating gastric cancer.

26. The use according to claim 25, characterized in that The medicament further includes a pharmaceutically acceptable carrier.

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