A nano-carrier targeting anti-tumor cells and tumor stem cells and a preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a nano-carrier for targeting anti-tumor cells and tumor stem cells, and a preparation method and application thereof. The application provides an amphiphilic block copolymer, which is formed by connecting polycurcumin with a double sulfur bond and hyaluronic acid through an amide bond to form an amphiphilic block copolymer. The amphiphilic block copolymer has a higher curcumin loading capacity, and the content of curcumin can reach 25%. Meanwhile, the presence of the amide bond makes it more stable in a physiological environment when used as a carrier, and can reduce the shedding of the target head before reaching the tumor target. The amphiphilic block copolymer of the application can load chemotherapeutic drugs, can simultaneously target breast cancer cells and breast cancer stem cells, and can target breast cancer cells and breast cancer stem cells at the cell level and in the animal body after loading the drugs, and can play an anti-breast cancer effect, and can be used as a new nano-carrier for anti-tumor, and provides a new choice for anti-tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a nano-carrier targeting anti-tumor cells and tumor stem cells, and a preparation method and application thereof. BACKGROUND

[0002] Surgical treatment, radiotherapy, chemotherapy and the treatment of the combination of the three methods is the main means of clinical breast cancer treatment. The continuous updating and improvement of the clinical breast cancer treatment methods bring new hope to breast cancer patients, and also prolong the survival time of patients. However, the recurrence of breast cancer and the defects existing in the present methods (drug resistance, systemic toxicity, etc.) are still the problems that need to be solved in clinic. In recent years, a large number of studies have shown that breast cancer stem cells are closely related to the recurrence, drug resistance and metastasis of breast cancer. Breast cancer stem cells are a small part of cells in breast cancer, which maintain the growth and development of breast tumor through continuous self-renewal and differentiation (Zhang, Y., et al., The eradication of breast cancer and cancer stem cells using octreotide modified paclitaxel active targeting micelles and salinomycin passive targeting micelles. Biomaterials, 2012. 33(2): p. 679-91.). Most of the currently used chemotherapy drugs are ineffective on breast cancer stem cells, and the stem cells that survive after chemotherapy form new tumors through self-renewal and differentiation after a period of dormancy, leading to recurrence and metastasis of breast cancer. Therefore, it is crucial to kill breast cancer cells and breast cancer stem cells at the same time when treating breast cancer by drug therapy.

[0003] Nanomedicine prepared by nanocarriers has many advantages in the targeted therapy of tumors, such as improving the water solubility of hydrophobic drugs, improving the bioavailability of drugs, improving the tissue or cell targeting, etc., but also has problems such as being unable to effectively control the release of drugs from the carrier, incomplete drug release, unsatisfactory targeting effect, being unable to simultaneously target tumor cells and tumor stem cells, etc.; the carrier material also has problems such as being unable to degrade in vivo, low drug loading capacity, etc.; for example, a Chinese invention patent discloses an amphiphilic block copolymer with curcumin and hyaluronic acid embedded by ester bond as a nanocarrier, the copolymer carrier increases the water solubility of curcumin, improves the drug loading capacity and stability, but the drug loading capacity of the copolymer carrier curcumin is still limited, the copolymer carrier uses DCC / DMAP as a condensing agent, and the ester bond is formed by linking the phenolic hydroxyl group of curcumin and the branched carboxyl group on hyaluronic acid, however, the grafting rate of this connection method is generally not high, and the content of curcumin is only about 1.3% (Manju S, Sreenivasan K. Conjugation of curcumin onto hyaluronic acid enhances its aqueous solubility and stability [J]. Journal of colloid and interface science, 2011, 359 (1): 318-325.), and the loading capacity of curcumin is low. Therefore, it is necessary to research a new curcumin high-loading carrier that can simultaneously realize the targeted therapy of tumor cells and tumor stem cells. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the deficiencies in the prior art of tumor treatment, and to provide a nanocarrier for targeting anti-tumor cells and tumor stem cells, and a preparation method and application thereof.

[0005] The present application aims to provide an amphiphilic block copolymer which can be used as a nanocarrier for targeting anti-tumor cells and tumor stem cells.

[0006] The present application aims to provide a preparation method of the amphiphilic block copolymer.

[0007] The present application aims to provide the use of the amphiphilic block copolymer in the preparation of a drug for targeting anti-tumor cells and / or tumor stem cells.

[0008] The above object of the present application is achieved by the following technical means:

[0009] The application provides an amphiphilic block copolymer, which has a polycurcumin (PCDA) with double sulfur bonds as a hydrophobic end of a nano-carrier, has a hyaluronic acid (HA) capable of simultaneously specifically binding to CD44 receptors overexpressed on the surfaces of tumor cells and tumor stem cells as a hydrophilic end and a target head, and is formed by connecting the PCDA and the HA through an amide bond to form the amphiphilic block copolymer (HA-b-PCDA), the amphiphilic block copolymer as the nano-carrier can efficiently target tumor cells and tumor stem cells, and realize intelligent and rapid release of drugs in the target cells, so that the tumor cells and the tumor stem cells are killed. The amphiphilic block copolymer can efficiently graft curcumin, and improve the curcumin loading capacity; meanwhile, the existence of the amide bond in the amphiphilic block copolymer makes the amphiphilic block copolymer more stable in a physiological environment when the amphiphilic block copolymer is used as a carrier, and can reduce the shedding of the target head before the target head reaches a tumor target.

[0010] An amphiphilic block copolymer (HA-b-PCDA) has a polycurcumin (PCDA) with double sulfur bonds as a hydrophobic end, has a hyaluronic acid (HA) as a hydrophilic end and a target head, and the polycurcumin with double sulfur bonds is connected to the hyaluronic acid through an amide bond to form the amphiphilic block copolymer (HA-b-PCDA), and a structural formula of the polycurcumin with double sulfur bonds (PCDA) is shown as formula (I):

[0011]

[0012] A structural formula of the amphiphilic block copolymer is shown as formula (II):

[0013]

[0014] A preparation method of an amphiphilic block copolymer, comprising the following steps:

[0015] S1. Preparation of polycurcumin (PCDA) with double sulfur bonds

[0016] Curcumin, 3,3'-dithiodipropionic acid, N-N-dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) are mixed with anhydrous dichloromethane, and the mixture is fully reacted at room temperature; filtration is performed, the filtrate is crystallized with anhydrous diethyl ether, and the crystals are purified and dried to obtain the polycurcumin (PCDA) with double sulfur bonds; the polycurcumin with double sulfur bonds is an orange yellow powder;

[0017] Preferably, the mass ratio of the curcumin to the 3,3'-dithiodipropionic acid in step S1 is 1-1.2:0.5-0.6.

[0018] Further preferably, the mass ratio of the curcumin to the 3,3'-dithiodipropionic acid in step S1 is 1.008:0.572.

[0019] S2. Preparation of amino-functionalized hyaluronic acid

[0020] The hyaluronic acid (HA) is mixed with a buffer solution at pH 5-6, and then mixed with 1,4-butanediamine, and reacted at 50-55°C; then sodium cyanoborohydride is added, and reacted at 50-55°C, after the reaction is completed, the reaction product is dialyzed, and dried to obtain the amino-functionalized hyaluronic acid; the amino-functionalized hyaluronic acid is a milky white solid.

[0021] Preferably, the buffer solution in step S2 is a 2wt% acetic acid buffer solution at pH 5.6.

[0022] Preferably, the sufficient reaction in step S2 is stirring at 50-55°C for 20-24h.

[0023] Further preferably, the sufficient reaction in step S2 is magnetic stirring at 50°C for 24h.

[0024] Preferably, the step of adding sodium cyanoborohydride and sufficient reaction in step S2 is performed in three times.

[0025] Preferably, the dialysis in step S2 is dialysis in deionized water for 70-72h.

[0026] Preferably, the dialysis in step S2 is dialysis in deionized water for 72h.

[0027] S3. Preparation of polycurcumin succinimidyl ester (PCDA-NHS)

[0028] The dichloromethane solution of polycurcumin (PCDA) with disulfide bond prepared in step S1, N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) are mixed, and reacted at room temperature, then the reaction product is crystallized with anhydrous ether at 0-10°C, and the crystallized solid is dried to obtain the polycurcumin succinimidyl ester (PCDA-NHS); the PCDA-NHS is an orange yellow gummy solid.

[0029] Preferably, the dichloromethane in step S3 is anhydrous dichloromethane.

[0030] Preferably, the sufficient reaction in step S3 is stirring at room temperature for 20-22h.

[0031] Further preferably, the sufficient reaction in step S3 is stirring at room temperature for 20h.

[0032] Preferably, the mass ratio of the polycurcumin with disulfide bond (PCDA) : N-hydroxysuccinimide (NHS) : 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) in step S3 is 0.5-0.6: 0.1-0.2: 0.2-0.3.

[0033] Further preferably, the mass ratio of the polycurcumin with disulfide bond (PCDA) : N-hydroxysuccinimide (NHS) : 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) in step S3 is 0.506: 0.134: 0.234.

[0034] Preferably, the temperature of the crystallization in step S3 is 3-6℃.

[0035] Further preferably, the temperature of the crystallization in step S3 is 4℃.

[0036] Preferably, the time of the crystallization in step S3 is 10-12h.

[0037] Further preferably, the time of the crystallization in step S3 is 12h.

[0038] S4. Preparation of the amphiphilic block nanocarrier (HA-b-PCDA)

[0039] The amino-functionalized hyaluronic acid prepared in step S2 and the polycurcumin succinimidyl ester (PCDA-NHS) prepared in step S3 are mixed with dimethyl sulfoxide (DMSO), and then mixed with N,N-diisopropylethylamine, and reacted sufficiently at 45-55℃. The reaction solution is dialyzed and dried to obtain the amphiphilic block copolymer (HA-b-PCDA); the HA-b-PCDA is an orange powder.

[0040] Preferably, the sufficient reaction in step S4 is stirring at 45-55℃ for 46-48h.

[0041] Preferably, the sufficient reaction in step S4 is stirring at 50℃ oil bath for 48h.

[0042] Preferably, the mass-volume ratio of the amino-functionalized hyaluronic acid : polycurcumin succinimidyl ester : N,N-diisopropylethylamine in step S4 is 0.2-0.8g : 0.3-0.4g : 15-20μL.

[0043] Further preferably, the mass-volume ratio of the amino-functionalized hyaluronic acid : polycurcumin succinimidyl ester : N,N-diisopropylethylamine in step S4 is 0.280g : 0.352g : 20μL.

[0044] Preferably, the dialysis is dialyzed with deionized water for 70-72h.

[0045] Further preferably, the dialysis is dialysis with deionized water for 72 hours, and the deionized water is replaced every 12 hours.

[0046] The amphiphilic block copolymer prepared by the above preparation method is also within the protection scope of the present application.

[0047] The use of the amphiphilic block copolymer in the preparation of a drug for targeting anti-tumor cells and / or tumor stem cells is also within the protection scope of the present application.

[0048] Preferably, the use of the amphiphilic block copolymer as a nanocarrier in the preparation of a nanodrug for targeting anti-tumor cells and / or tumor stem cells.

[0049] Preferably, the tumor cells and / or tumor stem cells are tumor cells and / or tumor stem cells expressing CD44 receptors on the surface.

[0050] Further preferably, the tumor is one or more of breast cancer, pancreatic cancer, salivary gland cancer, gastric cancer, colon cancer, lung cancer, ovarian cancer or leukemia.

[0051] Further preferably, the tumor is breast cancer.

[0052] A drug for targeting anti-tumor cells and / or tumor stem cells is prepared from the amphiphilic block copolymer.

[0053] A preparation method of a drug for targeting anti-tumor cells and / or tumor stem cells, wherein a dimethyl sulfoxide (DMSO) solution of the amphiphilic block copolymer (HA-b-PCDA) is mixed with water, or a dimethyl sulfoxide (DMSO) solution of the amphiphilic block copolymer (HA-b-PCDA) and an anti-tumor drug is mixed with water, and the mixture is dialyzed to obtain the drug.

[0054] Preferably, the drug uses low-toxic dimethyl sulfoxide as an organic solvent, is safer, and can be prepared into a nanodrug through simple mixing and dialysis, and the preparation process is simple.

[0055] Preferably, the water is ultrapure water.

[0056] Preferably, the mass ratio of the amphiphilic block copolymer to the anti-tumor drug is 1-2:0.1-0.2.

[0057] Further preferably, the mass ratio of the amphiphilic block copolymer to the anti-tumor drug is 1:0.1.

[0058] Preferably, the anti-tumor drug is doxorubicin or docetaxel.

[0059] Compared with the prior art, the present application has the following beneficial effects:

[0060] The present application provides an amphiphilic block copolymer, which connects polycurcumin and hyaluronic acid through an amide bond, can load chemotherapeutic drugs as a carrier, can target breast cancer cells and breast cancer stem cells, can target breast cancer cells and breast cancer stem cells at the cellular level and in vivo after loading drugs, and can play an anti-breast cancer effect, and can be used as a new carrier of anti-tumor drugs; meanwhile, the amphiphilic block copolymer has a higher curcumin loading capacity, and the content of curcumin can reach 25%; meanwhile, the presence of the amide bond makes the amphiphilic block copolymer more stable in a physiological environment when used as a carrier, can reduce the shedding of the targeting head before reaching the tumor target, and provides a new drug selection for anti-tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 The synthesis route of HA-b-PCDA in the present application is shown in the figure.

[0062] Figure 2 The nuclear magnetic resonance hydrogen spectrum of HA-b-PCDA synthesized in the present application is shown in the figure.

[0063] Figure 3 The results of the nanoparticles targeting breast cancer cells are shown in the figure; A is the uptake of DOX / HA-b-PCDA NPs and free DOX by 4T1 cells under a fluorescence microscope, and the scale is 100 μm; B is the uptake of DOX / HA-b-PCDA NPs and free DOX by 293T cells (B) under a fluorescence microscope, and the scale is 100 μm; C is the intracellular distribution of DOX / HA-b-PCDA NPs in 4T1 cells under a confocal microscope, red represents the fluorescence of doxorubicin, blue is DAPI staining of the cell nucleus, green is Lysosome (lysosome) staining green, Overlay is the superposition of the three channels, indicating the co-localization of the cell nucleus, lysosome and doxorubicin, and the scale is 20 μm; D is the average fluorescence intensity of DOX in 4T1 cells and 293T cells after incubation with a DOX solution or a DOX / HA-b-PCDA NPs solution, and "*" group, p<0.01 compared with the 4T1 group; E is the average fluorescence intensity of DOX in 4T1 cells after incubation with a DOX solution or a DOX / HA-b-PCDA NPs solution, and "*", p<0.01 compared with the HA solution pre-incubation group; DOX represents free DOX treatment; NPs represents DOX / HA-b-PCDA NPs treatment; HA+NPs represents NPs treated with free HA pre-incubation; HA+DOX represents free DOX treated with free HA pre-incubation; Overlay is the superposition of the pictures of DAPI (the cell nucleus is dyed blue) and DOX (doxorubicin is red), indicating the co-localization of DAPI and DOX.

[0064] Figure 4 Results of the nanoparticles of the present application targeting breast cancer stem cells; Wherein A is the fluorescence microscope observation results of DOX / HA-b-PCDA NPs and free DOX uptake by 4T1 mammosphere cells rich in breast cancer stem cells, the scale is 100 μm; B is the confocal microscope observation results of intracellular distribution of DOX / HA-b-PCDA NPs in 4T1 mammosphere cells rich in breast cancer stem cells, red represents doxorubicin fluorescence, blue is DAPI staining of cell nucleus, green is Lysosome (lysosome) staining green, Overlay is the superposition of the three channels, indicating the co-localization of cell nucleus, lysosome and doxorubicin, the scale is 20 μm; DOX represents free DOX treatment, NPs represents DOX / HA-b-PCDA NPs treatment, HA+NPs represents NPs treated with free HA pre-incubation, HA+DOX represents free DOX treated with free HA pre-incubation, Overlay is the superposition of DAPI (cell nucleus stained blue) and DOX (doxorubicin red) pictures, indicating the co-localization of DAPI and DOX.

[0065] Figure 5 Results of the effect of the nanoparticles of the present application on cell viability; Wherein A is breast cancer cell line 4T1 cells, B is normal cell line 293T cells; C is 4T1 breast cancer stem cells.

[0066] Figure 6 Results of the effect of the nanoparticles of the present application on the destruction of existing 4T1 mammospheres rich in breast cancer stem cells; a is the blank control group (without any drug treatment), b is the doxorubicin treatment group, c is the DOX / HA-b-PCDA NPs treatment group.

[0067] Figure 7 Results of the effect of the nanoparticles of the present application on the reformation of 4T1 mammospheres rich in breast cancer stem cells; a is the blank control group (without any drug treatment), b is the doxorubicin treatment group, c is the DOX / HA-b-PCDA NPs treatment group.

[0068] Figure 8 Results of the effect of the nanoparticles of the present application on ALDH1+ cells in 4T1 mammospheres rich in breast cancer stem cells, a is the blank control group (without any drug treatment), b is the doxorubicin treatment group, c is the HA-b-PCDA NPs treatment group, d is the DOX / HA-b-PCDA NPs treatment group; TEST represents the test sample, DEAB represents the negative control sample. DETAILED DESCRIPTION

[0069] The application will be further described in conjunction with specific examples, but the examples do not limit the application in any form. Unless otherwise specified, the reagents, methods and devices used in the application are conventional reagents, methods and devices in the art.

[0070] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0071] RPMI 1640 medium (Cat. No. 11835-030, Lot No. 1951096, Life Technologies Corporation, 500 mL);

[0072] DMEM / F12 + GlutaMAX TM Medium (Cat. No. 10565-018, Lot No. 2277125, Life Technologies Corporation, 500 mL);

[0073] Aldefluor TM fluorescent reagent (Cat. No. 01700, Lot No. 18E91436, STEMCELL Technologies);

[0074] PBS buffer: containing 135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4, pH 7.3 ± 0.1;

[0075] Stem cell medium: 10 mL B27 (1:50, Invitrogen) + 10 μg EGF (20 ng / mL) + 5 μg bFGF (10 ng / mL) + 2 g 0.4% BSA (bovine serum albumin) + 2.5 mg insulin (5 μg / mL) + 500 mL DMEM / F12 (1:1).

[0076] The nanoparticles of the application are nanoparticles of a drug prepared using HA-b-PCDA, or nanoparticles of a drug prepared using HA-b-PCDA and an anti-tumor drug.

[0077] Example 1 Preparation and identification of HA-b-PCDA

[0078] 1. Preparation of polycurcumin (PCDA) with disulfide bond

[0079] Curcumin (Cur) 1.008 g (2.736 mmol), 3,3'-dithiodipropionic acid 0.572 g (4.827 mmol), N-N-dicyclohexyl carbodiimide (DCC) 1.008 g (4.893 mmol) and 4-dimethylamino pyridine (DMAP) 0.121 g (0.9904 mmol) were weighed into 40 mL of anhydrous dichloromethane and stirred at room temperature for 24 h. The reaction was filtered under vacuum to obtain an orange-yellow turbid solution. 600 mL of anhydrous ether was added to the filtrate to precipitate the product. The precipitate was washed with a small amount of anhydrous ether and dried under vacuum to obtain 0.603 g (0.0754 mmol) of an orange-yellow powder, which is PCDA.

[0080] 2. Preparation of Amino-functionalized Hyaluronic Acid

[0081] Hyaluronic acid (HA) was end-reduced and aminated with 1,4-diaminobutane using sodium cyanoborohydride (NaCNBH3) as a reducing agent.

[0082] One gram of HA (5.6 kDa, 0.18 mmol) was dissolved in 30 mL of an acetic acid buffer (pH = 5.6, 2 wt%). Then, 1 mL of 1,4-diaminobutane (11.4 mmol) was added to the HA solution under magnetic stirring. The HA reacted with 1,4-diaminobutane to form an imine mixture under stirring at 50 °C for 24 h. Subsequently, 0.2 g of sodium cyanoborohydride (3.2 mmol) was added to the mixture under stirring every day for three consecutive days. The mixture was purified by dialysis against deionized water through a dialysis bag (Spectra / Por, MWCO 3500) for 72 h to remove the excess 1,4-diaminobutane and sodium cyanoborohydride. The final product was collected and lyophilized to obtain 1.569 g (0.0785 mmol) of a milky white solid, which is amino-functionalized HA.

[0083] 3. Polycurcumin succinimidyl ester (PCDA-NHS)

[0084] PCDA prepared in 1 was weighed into 40 mL of anhydrous dichloromethane, and 0.234 g (1.22 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.134 g (1.164 mmol) of N-hydroxysuccinimide (NHS) were added. The mixture was stirred at room temperature for 20 h to obtain an orange-red solution. 40 mL of anhydrous ether was added to the reaction solution. The product was precipitated in the refrigerator at 4 °C for 12 h. The precipitate was filtered under suction and dried under vacuum to constant weight to obtain 0.301 g (0.01501 mmol) of an orange-yellow gummy solid, which is PCDA-NHS.

[0085] 4. Preparation of HA-b-PCDA

[0086] Amino-functionalized hyaluronic acid 0.352 g (0.0176 mmol) prepared in 2 and PCDA-NHS 0.280 g (0.0187 mmol) prepared in 3 were dissolved in 20 mL DMSO, 20 μl of N-N-diisopropyl ethylamine was added, and the solution was stirred at 50 °C oil bath for 48 h. After the reaction was completed, the solution was loaded into a dialysis bag (MWCO = 8000-14000) with deionized water as the dialysate, and dialyzed for 72 h, with deionized water changed every 12 h. The solution in the dialysis bag was collected, and after freeze-drying, an orange powder was obtained, which was HA-b-PCDA.

[0087] The synthetic route diagram of HA-b-PCDA is shown in Figure 1 .

[0088] 5. Identification of HA-b-PCDA

[0089] The prepared HA-b-PCDA was identified, and the nuclear magnetic resonance hydrogen spectrum of HA-b-PCDA is shown in Figure 2 . From Figure 2 , the nuclear magnetic result of HA-b-PCDA is as follows:

[0090] 1 H NMR (600 MHz, DMSO-d6) δ 9.85 (d, J = 10.6 Hz, 1H, -OHC=CH), 8.06 (s, 1H, -NHCO-), 8.00 (s, 1H, -NHCO-), 7.59-7.51 (m, 2H, -CHCHC-), 7.31 (s, 4H, -OCCHC-), 7.16 (s, H, -OCCHC-), 6.83 (s, H, -CCHCH-), 6.78 (s, 3H, -C=CHCH-), 6.16 (d, J = 7.0 Hz, 2H, 2nH, -CH=CH-), 6.10 (s, 2H, -CH=CH-), 4.31-4.34 (d, 4H, -OCH2CH2-, OHCH2CH2-), 3.83 (t, 6H, -OCH3), 3.85-3.82 (m, 16H, HOCHCHHO,), 3.19 (s, 2H, SSCH2CO), 3.10-3.00 (m, 4H, -CH2CH2S-), 2.89-2.91 (d, 2H, -CH2CH2S-), 2.92 (s, 2H, -CH2CH2S-), 2.64 (s, 2H, -CH2CH2S-), 1.39-1.23 (s, 6H, -NOCCH3-), 1.07 (s, 2H, OH), 0.99 (s, 1H, OH).

[0091] The structural formula of HA-b-PCDA is shown in formula (II):

[0092]

[0093] Determination of the content of curcumin in HA-b-PCDA of Example 2

[0094] 10 mg of HA-b-PCDA prepared in Example 1 was dissolved in 10 mL of ultrapure water. After complete dissolution, 1 mL of the aqueous solution of HA-b-PCDA was taken and diluted to 10 mL with DMSO as a sample solution. At this time, the sample concentration was represented by C 样品 . 5 mg of curcumin was dissolved in DMSO and diluted to 10 mL as a control solution. At this time, the concentration of the control solution was represented by C 对照 . The absorbance values A 样品 and A 对照 of the sample solution and the control solution at 420 nm were measured by a UV spectrophotometer (U-2910, HITACHI, Japan), respectively. The concentration of curcumin in the sample solution was calculated according to the following formula:

[0095] C 样品 = C 对照 × A 对照 / A 样品

[0096] The content of curcumin in HA-b-PCDA (%) = C 样品 × 100

[0097] The content of curcumin in HA-b-PCDA prepared in Example 1 was measured by the method of this example to be 25%.

[0098] Comparative Example

[0099] The amphiphilic block copolymer PCDA-HA of curcumin and hyaluronic acid (HA) grafted by ester bond was synthesized according to the method of the patent CN105646861A.

[0100] 0.9 g of curcumin, 9 g of hyaluronic acid, 400 mg of condensing agent DCC and 36 mg of catalyst DMAP were dissolved in 40 mL of anhydrous DMSO, and reacted at room temperature for 1 day. The product was precipitated by adding an appropriate amount of cold ethyl acetate, and repeated precipitation for 3 times. After filtration, vacuum drying was performed to obtain PCDA-HA. The content of curcumin in PCDA-HA was measured by the method of Example 2 to be 5%.

[0101] Example 3 Preparation of nanoparticles and characterization of nanoparticles

[0102] 1. Preparation of nanoparticles

[0103] 1.1 HA-b-PCDA Nanoparticles (HA-b-PCDA NPs)

[0104] Take 2.5 mg of HA-b-PCDA prepared in Example 1 dissolved in 0.5 mL of dimethyl sulfoxide (DMSO), and dropwise add to 5 mL of ultrapure water while stirring, after the dropwise addition is completed, dialysis for 12 h (molecular weight cut-off is 3500), to obtain a nanoparticle solution, freeze-drying to obtain a nanoparticle freeze-dried powder.

[0105] 1.2 DOX / HA-b-PCDA Nanoparticles (DOX / HA-b-PCDA NPs)

[0106] Take 2.5 mg of HA-b-PCDA and 0.25 mg of free doxorubicin (DOX) dissolved in 0.5 mL of DMSO, and dropwise add to 5 mL of ultrapure water while stirring, after the dropwise addition is completed, dialysis for 12 h (molecular weight cut-off is 3500), to obtain a nanoparticle solution, freeze-drying to obtain a nanoparticle freeze-dried powder.

[0107] 1.3 DTX / HA-b-PCDA Nanoparticles (DTX / HA-b-PCDA NPs)

[0108] Take 2.5 mg of HA-b-PCDA and 0.25 mg of docetaxel (DTX) dissolved in 0.5 mL of DMSO, and dropwise add to 5 mL of ultrapure water while stirring, after the dropwise addition is completed, dialysis for 12 h (molecular weight cut-off is 3500), to obtain a nanoparticle solution, freeze-drying to obtain a nanoparticle freeze-dried powder.

[0109] 2. Characterization of Nanoparticles

[0110] The particle size and surface charge of each nanoparticle were determined by dynamic laser light scattering (DLS).

[0111] The experimental results show that the particle size of HA-b-PCDA NPs is 170.8 nm, and the potential is -43.5 mv; the particle size of DOX / HA-b-PCDA NPs is 175.1 nm, and the potential is -36.1 mv; the particle size of DTX / HA-b-PCDA NPs is 125.8 nm, and the potential is -38.9 mv; indicating that the HA-b-PCDA carrier can load various clinically used chemotherapeutic drugs to form nanoparticle drugs.

[0112] Example 4 Evaluation of the Effect of Nanoparticles Targeting Breast Cancer Cells

[0113] 1. Experiment

[0114] 4T1 breast cancer cell line and 293T cell line were used as tumor cell line and normal cell line model respectively, and the effect of nanoparticles targeting breast cancer cells was evaluated by microscope observation and flow cytometry.

[0115] Microscopic observation: Cells at 5×10 5 Cells were seeded at a density of 15 mm in RPMI 1640 medium in confocal culture dishes and incubated overnight. Then, DOX / HA-b-PCDA NPs or free DOX were added to the culture dishes and incubated with the cells for 4 h. After incubation, the RPMI 1640 medium containing the drug DOX was removed, the cells were washed three times with PBS buffer, fixed with 4% paraformaldehyde, stained with DAPI, and observed with a fluorescence microscope or confocal microscope.

[0116] Flow cytometry assay: Cells were analyzed at 5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of cells / well in 24-well plates and incubated overnight in RPMI 1640 medium. Then, DOX / HA-b-PCDA NPs or free DOX were added to the culture dishes and incubated with the cells for 4 hours. After incubation, the RPMI 1640 medium containing the drug-treated DOX was removed, the cells were washed three times with PBS buffer, digested with 0.25% (wt / v%) trypsin, and resuspended in 0.5 mL of PBS buffer. The cells were then analyzed by flow cytometry.

[0117] 2. Results

[0118] Results of nanoparticle targeting breast cancer cells observed by microscopy and measured by flow cytometry are as follows: Figure 3As shown. A shows the uptake of DOX / HA-b-PCDA NPs and free DOX by 4T1 cells under fluorescence microscopy (scale bar: 100 μm); B shows the uptake of DOX / HA-b-PCDA NPs and free DOX by 293T cells under fluorescence microscopy (scale bar: 100 μm); C shows the intracellular distribution of DOX / HA-b-PCDA NPs in 4T1 cells under confocal microscopy, with DAPI staining (blue) for the nucleus and lysosome staining (green). Red indicates doxorubicin fluorescence, blue indicates DAPI staining of the nucleus, and green indicates lysosome staining. The overlay shows the superposition of the three channels, indicating the co-localization of the nucleus, lysosomes, and doxorubicin (scale bar: 20 μm); D shows the uptake of DOX / HA-b-PCDA NPs by 4T1 cells with DOX solution or DOX / HA-b-PCDA NPs. After incubation with NPs solution, the mean fluorescence intensity of DOX in 4T1 and 293T cells was shown in the "*" group, with p < 0.01 compared to the 4T1 group. E represents the mean fluorescence intensity of DOX in 4T1 cells after incubation with DOX solution or DOX / HA-b-PCDA NPs solution, with "*" compared to the HA solution pre-incubation group, with p < 0.01. DOX represents free DOX treatment, NPs represents DOX / HA-b-PCDA NPs treatment, HA+NPs represents NPs pre-incubated with free HA, HA+DOX represents free DOX pre-incubated with free HA, and Overlay is the superposition of DAPI (nuclei stained blue) and DOX (doxorubicin red) images, indicating the co-localization of DAPI and DOX.

[0119] from Figure 3 Figures A and B show that after 4 hours of treatment with DOX / HA-b-PCDA NPs, the intracellular red fluorescence of 4T1 cells (tumor cell line) was significantly stronger than that of 293T cells (normal cells), while in the free DOX treatment group, the intracellular red fluorescence of 4T1 cells was weaker than that of 293T cells. Figure D shows that in the DOX / HA-b-PCDA NPs treatment group, the average fluorescence intensity of 4T1 cells (tumor cell line) was significantly stronger than that of 293T cells (normal cells), while in the free DOX treatment group, the average intracellular fluorescence intensity of 4T1 cells was weaker than that of 293T cells. These results indicate that compared with free DOX, DOX / HA-b-PCDA NPs have higher cell uptake selectivity in 4T1 cancer cells than in non-cancerous 293T cells, and DOX / HA-b-PCDA NPs can significantly improve the effect of DOX targeting tumor cells.

[0120] from Figure 3 The C-axis shows that DOX / HA-b-PCDA NPs are specifically localized to lysosomes, indicating that DOX / HA-b-PCDA NPs enter cells via endocytosis. Figure 3As can be seen from Figures 3A, 3B and 3E, for the free DOX treatment group, there is no significant difference in fluorescence intensity between the cells treated with excess free HA and the cells not treated with excess free HA; and for the DOX / HA-b-PCDA NPs treatment group, the fluorescence intensity of the 4T1 cells pretreated with excess free HA is significantly lower than that of the cells not pretreated with excess free HA, which indicates that the DOX / HA-b-PCDA NPs have high selectivity for the breast cancer cells overexpressing CD44, and the DOX / HA-b-PCDA NPs can enter the cells through CD44 receptor-mediated endocytosis.

[0121] In summary, the DOX / HA-b-PCDA NPs can enter the cells through CD44 receptor-mediated endocytosis, and can significantly improve the effect of DOX on targeting tumor cells.

[0122] Example 5 Evaluation of the effect of the nanoparticles on targeting breast cancer stem cells

[0123] 1. Experiment

[0124] The 4T1 breast cancer stem cells obtained by the suspension culture method were used as a breast cancer stem cell model, and the effect of the nanoparticles on targeting breast cancer stem cells was evaluated by using a microscope and a flow cytometer.

[0125] Microscope observation: The cells were inoculated in a 15-mm confocal culture dish containing stem cell culture medium at a density of 5 x 105cells / dish, and incubated overnight. Then, the DOX / HA-b-PCDA NPs or free DOX were added to the culture dish and incubated with the cells for 4 hours. After the incubation, the DOX-containing stem cell culture medium was removed, the cells were washed with PBS buffer for three times, fixed with 4% paraformaldehyde, and stained with DAPI. Then, the fluorescence microscope or confocal microscope was used for observation. 5

[0126] Flow cytometer determination: The cells were inoculated in a 24-well plate at a density of 5 x 105cells / well, and incubated overnight with stem cell culture medium. Then, the DOX / HA-b-PCDA NPs or free DOX were added to the culture dish and incubated with the cells for 4 hours. After the incubation, the DOX-containing stem cell culture medium was removed, the cells were washed with PBS buffer for three times, and resuspended in 0.5 mL of PBS buffer after being digested with 0.25% (wt / v%) trypsin. Then, the flow cytometer was used for determination. 4

[0127] 2. Results

[0128] The results of the microscope observation and flow cytometer determination of the nanoparticles targeting breast cancer stem cells are shown in Table 1. Figure 4 Figure 4 ​​​A shows the fluorescence microscopy results of DOX / HA-b-PCDA NPs uptake and free DOX in 4T1 mammary glomeruli rich in breast cancer stem cells, with a scale bar of 100 μm; B shows the confocal microscopy results of the intracellular distribution of DOX / HA-b-PCDA NPs in 4T1 mammary glomeruli rich in breast cancer stem cells, with red indicating doxorubicin fluorescence, blue indicating DAPI staining of the cell nucleus, and green indicating lysosome staining. The overlay shows the superposition of the three channels, indicating the co-localization of the cell nucleus, lysosomes, and doxorubicin, with a scale bar of 20 μm; DOX represents free DOX treatment, NPs represent DOX / HA-b-PCDA NPs treatment, HA+NPs represent NPs pre-incubated with free HA treatment, and HA+DOX represents free DOX treatment pre-incubated with free HA treatment.

[0129] from Figure 4 As can be seen from A and B, in the DOX / HA-b-PCDA NPs treatment group, 4T1 breast cancer stem cells exhibited a very strong red fluorescence signal (A), and some of the red fluorescence signal co-localized with the green signal of lysosomes (B). Figure 4 (Figure B) indicates that DOX / HA-b-PCDA NPs enter 4T1 breast cancer stem cells via endocytosis. In the free DOX treatment group, a slight red fluorescence signal was observed in 4T1 breast cancer stem cells, suggesting that DOX / HA-b-PCDA NPs can enhance the effect of DOX targeting 4T1 breast cancer stem cells. In the DOX / HA-b-PCDA NPs treatment group, when cells were pre-incubated with free HA to block the CD44 receptor, the red fluorescence signal significantly decreased (compared to NPs with HA+NPs). However, in the free DOX treatment group, there was no significant difference in red fluorescence signal between cells pre-incubated with free HA and those not pre-incubated (compared to DOX with HA+DOX).

[0130] In summary, DOX / HA-b-PCDA NPs can significantly improve the effect of DOX targeting 4T1 breast cancer stem cells.

[0131] Example 6: Evaluation of the efficacy of nanoparticles against breast cancer cells and breast cancer stem cells

[0132] 1. Experiment

[0133] 1.1 Evaluation of the effect of nanoparticles on breast cancer cells using the MTT assay

[0134] 4T1 breast cancer cells, 293T normal cells, were seeded in 96-well plates at a density of 5000 cells per well, and incubated with RPMI1640 medium overnight. Then different concentrations of DOX / HA-b-PCDA NPs or free DOX were added to the well plates, respectively, and then incubated for 72 h. After incubation, the cell viability of each group was determined by MTT method.

[0135] 1.2 Evaluation of the effect of nanoparticles on breast cancer stem cells by CCK-8 method

[0136] 4T1 breast cancer stem cells obtained by suspension culture method were seeded in 96-well plates at a density of 5000 cells per well, and incubated with stem cell medium overnight. Then different concentrations of DOX / HA-b-PCDA NPs or free DOX were added to the well plates, respectively, and then incubated for 72 h. After incubation, the cell viability of each group was determined by CCK-8 method.

[0137] 1.3 Evaluation of the effect of nanoparticles on breast cancer stem cells by nanoparticle destruction experiment of breast cancer stem cell spheres

[0138] 4T1 breast cancer stem cell spheres were incubated with 5 μg / mL DOX / HA-b-PCDA NPs and free DOX in stem cell medium for 10 days, and the morphology of 4T1 breast cancer stem cell spheres was photographed using an inverted microscope (IX81, Olympus, Japan).

[0139] 1.4 Evaluation of the effect of nanoparticles on breast cancer stem cells by nanoparticle prevention of 4T1 breast cancer stem cell sphere reformation experiment

[0140] After the 4T1 breast cancer stem cell spheres were dispersed into single cells, they were seeded into ultra-low attachment 6-well plates at a density of 2 x 10 5 cells per well, and cultured in stem cell medium; then DOX / HA-b-PCDA NPs or free DOX were added to the well plates at a DOX concentration of 5 μg / mL per well, and incubated with 4T1 breast cancer stem cells for 5 days. After incubation, the formed breast spheres were photographed and observed using a microscope.

[0141] 1.5 Evaluation of the effect of nanoparticles on breast cancer stem cells by flow cytometry determination of the proportion of ALDH1+ cells

[0142] 4T1 breast cancer stem cells were seeded into ultra-low attachment 24-well plates in stem cell medium, and after incubation for 24 h, the stem cell medium containing DOX / HA-b-PCDA NPs or free DOX was replaced, and incubation was continued for 48 h. After incubation, the cells were stained with Aldefluor TM fluorescent reagent, and the proportion of ALDH1+ cells in each group of cells was analyzed by flow cytometry.

[0143] 2. Results

[0144] The effects of the nanoparticles on the cell viability of 4T1 breast cancer cell line, 293T normal cell line and 4T1 breast cancer stem cells are shown in A, B and C of FIG. 6, respectively; the effects of the nanoparticles on the destruction of the existing 4T1 mammospheres enriched with breast cancer stem cells are shown in FIG. 7, where a is a blank control group (without any drug treatment), b is an Adriamycin treatment group, and c is a DOX / HA-b-PCDA NPs treatment group; the effects of the nanoparticles on the reformation of 4T1 mammospheres enriched with breast cancer stem cells are shown in FIG. 8, where a is a blank control group (without any drug treatment), b is an Adriamycin treatment group, and c is a DOX / HA-b-PCDA NPs treatment group; the effects of the nanoparticles on the ALDH1+ cells in 4T1 mammospheres enriched with breast cancer stem cells are shown in FIG. 9, where a is a blank control group (without any drug treatment), b is an Adriamycin treatment group, c is a HA-b-PCDA NPs treatment group, and d is a DOX / HA-b-PCDA NPs treatment group; TEST represents a test sample, and DEAB represents a negative control sample. Figure 5 Figure 6 Figure 7 Figure 8

[0145] From A and B of FIG. 6, it can be seen that the inhibitory effect of DOX / HA-b-PCDA NPs on 4T1 cells (tumor cells) is greater than that on 293T cells (normal cells), while the free DOX is just the opposite, which indicates that DOX / HA-b-PCDA NPs can significantly increase the effect of DOX on tumor cells, while reducing the toxicity of DOX on normal cells. Figure 5 From C of FIG. 6, it can be seen that the IC50value of free DOX on 4T1 breast cancer stem cells is significantly greater than the IC50value on 4T1 breast cancer cells, which indicates that 4T1 breast cancer stem cells have developed obvious drug resistance to DOX; in addition, the effect of DOX / HA-b-PCDA NPs on 4T1 breast cancer stem cells is significantly stronger than that of free DOX. 50 50

[0146] Figure 5 From FIG. 7, it can be seen that after 10 days of treatment, the 4T1 breast cancer stem cell spheres in the PBS treatment group and the free DOX treatment group are not significantly destroyed, but are significantly destroyed in the DOX / HA-b-PCDA NPs group, which indicates that DOX / HA-b-PCDA NPs have the ability to eliminate breast cancer stem cell spheres.

[0147] From FIG. 8, it can be seen that after 10 days of treatment, the 4T1 breast cancer stem cell spheres in the PBS treatment group and the free DOX treatment group are not significantly destroyed, but are significantly destroyed in the DOX / HA-b-PCDA NPs group, which indicates that DOX / HA-b-PCDA NPs have the ability to eliminate breast cancer stem cell spheres. Figure 6 From FIG. 9, it can be seen that after 10 days of treatment, the 4T1 breast cancer stem cell spheres in the PBS treatment group and the free DOX treatment group are not significantly destroyed, but are significantly destroyed in the DOX / HA-b-PCDA NPs group, which indicates that DOX / HA-b-PCDA NPs have the ability to eliminate breast cancer stem cell spheres.

[0148] Figure 7 ​​​​​​​​It can be seen that PBS or free DOX treatment group, breast cancer stem cell spheres were significantly larger, while DOX / HA-b-PCDA NPs treatment group, no obvious breast cancer stem cell spheres were formed; these results show that DOX / HA-b-PCDA NPs can significantly inhibit breast cancer stem cell re-sphere formation.

[0149] From Figure 8 It can be seen that the proportion of ALDH1+ cells in HA-b-PCDA and DOX / HA-b-PCDA NPs treatment groups were significantly reduced from 31.9% (negative control group) to 9.98% and 12.30%, respectively. In contrast, the proportion of ALDH1+ cells in free DOX treatment group was significantly increased; these results show that HA-b-PCDA is a carrier with the ability to eliminate breast cancer stem cells.

[0150] In summary, HA-b-PCDA can significantly increase the anti-tumor cell and anti-4T1 breast cancer stem cell effects of DOX; at the same time, it has the ability to eliminate breast cancer stem cell spheres and significantly inhibit breast cancer stem cell re-sphere formation; HA-b-PCDA is a carrier with the ability to eliminate breast cancer stem cells.

[0151] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. The use of an amphiphilic block copolymer loaded with doxorubicin in the preparation of a drug targeting anti-tumor cells and / or tumor stem cells, characterized in that, The amphiphilic block copolymer is a polycurcumin with disulfide bonds linked to hyaluronic acid via amide bonds, and the structural formula of the polycurcumin with disulfide bonds is shown in formula (I): Formula (I), The structural formula of the amphiphilic block copolymer is shown in formula (II): Equation (II).

2. The application according to claim 1, characterized in that, The preparation method of the amphiphilic block copolymer includes the following steps: S1. Preparation of polycurcumin with disulfide bonds Curcumin, 3,3'-dithiodipropionic acid, NN-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were mixed with anhydrous dichloromethane and reacted fully at room temperature. The mixture was filtered, and the filtrate was crystallized with anhydrous diethyl ether. After purification and drying of the crystals, polycurcumin with disulfide bonds was obtained. S2. Preparation of amino-functionalized hyaluronic acid Hyaluronic acid was mixed with a buffer solution of pH 5-6 and then mixed with 1,4-butanediamine. The mixture was reacted thoroughly at 50-55°C. Sodium cyanoborohydride was then added and the mixture was reacted thoroughly at 50-55°C. After the reaction was completed, the reactants were dialyzed and dried to obtain amino-functionalized hyaluronic acid. S3. Preparation of polycurcumin succinimide ester The dichloromethane solution of polycurcumin with disulfide bonds prepared in step S1, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed and reacted fully at room temperature. The reaction product was then crystallized with anhydrous diethyl ether at 0-10°C. The crystallized solid was dried to obtain polycurcumin succinimide ester. S4. Preparation of amphiphilic block copolymers The amino-functionalized hyaluronic acid prepared in step S2 and the polycurcumin succinimide ester prepared in step S3 were mixed with dimethyl sulfoxide, and then mixed with N,N-diisopropylethylamine. The mixture was reacted thoroughly at 45-55°C. The reaction solution was dialyzed and dried to obtain the amphiphilic block copolymer.

3. The application according to claim 2, characterized in that, In step S2, the buffer solution is a 2wt% acetic acid buffer solution with a pH of 5.

6.

4. The application according to claim 2, characterized in that, In step S3, the mass ratio of polycurcumin with disulfide bonds: N-hydroxysuccinimide: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 0.5-0.6: 0.1-0.2: 0.2-0.3; in step S4, the mass-volume ratio of amino-functionalized hyaluronic acid: polycurcumin succinimide ester: N,N-diisopropylethylamine is 0.2-0.8 g: 0.3-0.4 g: 15-20 μL.

5. The application according to claim 1, characterized in that, The tumor cells and / or tumor stem cells are tumor cells and / or tumor stem cells that express the CD44 receptor on their surface.

6. A drug targeting anti-tumor cells and / or tumor stem cells, characterized in that, It was prepared by loading doxorubicin onto the amphiphilic block copolymer described in claim 1.

7. A method for preparing a drug targeting anti-tumor cells and / or tumor stem cells, characterized in that, The amphiphilic block copolymer described in claim 1 and a dimethyl sulfoxide solution of doxorubicin are mixed with water, and the mixture is dialyzed to obtain the drug.

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