CN110664753A

CN110664753A - Bone-targeting hypoxia-responsive nano micelle loaded with anticancer drug and preparation method thereof

Info

Publication number: CN110664753A
Application number: CN201911064781.XA
Authority: CN
Inventors: 陈忠平; 龙朦朦; 翁凌燕; 陆敏; 朱俐; 陈秋萍
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2019-11-04
Filing date: 2019-11-04
Publication date: 2020-01-10
Anticipated expiration: 2039-11-04
Also published as: CN110664753B

Abstract

The invention discloses a bone targeting hypoxia response nano micelle loaded with anticancer drugs and a preparation method thereof, wherein the preparation method comprises the following steps: carboxyl polyethylene glycol azobenzene is obtained through copolymerization reaction of polyethylene glycol and a low-oxygen response group (4, 4-azodiphenylamine); performing ring-opening polymerization reaction of carboxyl polyethylene glycol azobenzene and N-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride to obtain a polyethylene glycol-polylysine block copolymer with benzyloxycarbonyl on the surface, and removing the benzyloxycarbonyl to obtain the polyethylene glycol-polylysine block copolymer; obtaining a bone-targeted hypoxia-responsive polymer through copolymerization reaction of a polyethylene glycol-polylysine block copolymer and a bone-targeted group (alendronic acid); synthesizing the bone targeting hypoxia response nano micelle loaded with the anti-cancer drug by a dialysis method. The nano micelle prepared by the preparation method has stable structure, is not easy to decompose, has targeting effect on bone tissues, and can also play the effect of hypoxia controlled release drugs.

Description

Bone-targeting hypoxia-responsive nano micelle loaded with anticancer drug and preparation method thereof

Technical Field

The invention relates to the technical field of nano medicaments, in particular to a bone targeting hypoxia response nano micelle loaded with an anticancer medicament and a preparation method thereof.

Background

Bone is the third most preferred site of metastasis for cancer cell removal from the lung and liver, and some cancers show significant osteotrophy. Since bone metastases usually have multiple nodules, it is difficult to completely eliminate them by localized radiation therapy or surgical resection. For chemotherapy, the bone blood flow is low, so that the drug concentration in the bone part is not enough after administration, the treatment effect is poor under the condition of large dose, and the toxic and side effects are strong.

The nano-carrier drug delivery is one of important means for treating the tumor, the nano-carrier can not change the physical and chemical properties of the loaded drug, and the drug can be continuously released in a focus area, so that the drug can be maintained at a certain concentration. However, the nano-carrier of the non-bone targeting loaded drug can treat solid tumor, but has little effect on bone metastasis tumor as the traditional drug. Hypoxia is one of the basic characteristics of the microenvironment of bone metastasis, the oxygen concentration of normal tissues is generally about 30mmHg, while the oxygen concentration of tumor tissues at the bone part is lower, and sometimes is even close to 0, which is the main reason for the failure of many bone metastasis treatments. Therefore, it is urgent to design and prepare a nano-drug carrier with bone targeting function and hypoxia response function for treating hypoxic bone metastasis tumor.

Disclosure of Invention

The invention aims to provide an anti-cancer drug loaded bone targeting hypoxia response nano micelle and a preparation method thereof, and the prepared nano micelle has a stable structure, is not easy to decompose, has a targeting effect on bone tissues, and can also play a role in hypoxia controlled release of drugs.

In order to solve the above problems, the invention provides a preparation method of bone-targeted hypoxia-responsive nano-micelle loaded with an anticancer drug, which comprises the following steps:

1) dissolving polyethylene glycol in dichloromethane, adding 4, 4-azodiphenylamine, stirring at normal temperature and in dark place for reaction for 24 hours, and precipitating a product by using glacial ethyl ether to obtain carboxyl polyethylene glycol azobenzene shown in a formula (I):

wherein n is the degree of polymerization, and n is more than or equal to 14 and less than or equal to 70;

2) dissolving the carboxyl polyethylene glycol azobenzene obtained in the step 1) in tetrahydrofuran, adding N-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride, and reacting for 72 hours at normal temperature under stirring in the dark to obtain a polyethylene glycol-polylysine block copolymer with a benzyloxycarbonyl group on the surface as shown in a formula (II):

wherein n is the degree of polymerization, n is more than or equal to 14 and less than or equal to 70, m is the degree of polymerization, m is more than or equal to 9 and less than or equal to 39;

3) dissolving the polyethylene glycol-polylysine block copolymer obtained in the step 2) in trifluoroacetic acid, adding a hydrogen bromide acetic acid solution, stirring at 0 ℃ for reaction for 1h to remove carbobenzoxy, then melting into dichloromethane, and adding triethylamine to neutralize acid, so as to obtain the polyethylene glycol-polylysine block copolymer shown in the formula (III):

4) dissolving the polyethylene glycol-polylysine segmented copolymer obtained in the step 3) in N, N-dimethylformamide, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, stirring at normal temperature, adding alendronic acid, and stirring for reacting for 2 hours to obtain a bone targeting hypoxia response polymer shown in a formula (IV);

5) dissolving the bone-targeted hypoxia-responsive polymer and the anticancer drug obtained in the step 4) in N, N-dimethylformamide, dropwise adding water under magnetic stirring, stirring for 1.5h to obtain a mixed solution, transferring the mixed solution into a dialysis bag, dialyzing with pure water for 24h, and freeze-drying at-50 ℃ to obtain the bone-targeted hypoxia-responsive nano micelle loaded with the anticancer drug.

Preferably, the relative molecular mass of the polyethylene glycol in the step 1) is 1000-5000.

Preferably, the molar ratio of the polyethylene glycol to the 4, 4-azodiphenylamine in the step 1) is 1: 1.3.

preferably, the molar ratio of the carboxypolyethylene glycol azobenzene to the N-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride in step 2) is 1: 16.

preferably, the molar ratio of the polyethylene glycol-polylysine block copolymer to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, the N-hydroxysuccinimide and the alendronic acid in step 4) is 1:3:3.6: 5.

preferably, the mass ratio of the anticancer drug to the bone-targeting hypoxia-responsive polymer in the step 5) is 1: 4-10, wherein the anticancer drug is one of adriamycin, docetaxel and camptothecin.

Preferably, the dialysis bag in step 5) has a molecular weight cut-off of 3000.

Preferably, the mass-to-volume ratio of the bone-targeting hypoxia-responsive polymer to water in step 5) is 2: 1.

the invention also provides the bone-targeting hypoxia-responsive nano micelle loaded with the anticancer drug, which is prepared by the preparation method.

Preferably, the hydration particle size of the bone targeting hypoxia response nano-micelle loaded with the anticancer drug is 200 nm.

Compared with the prior art, the invention has the following beneficial effects:

1. the nano micelle has a stable structure, is not easy to decompose in vivo under normal physiological conditions, and the loaded anticancer drug is not easy to leak, so that the toxic and side effects on normal organs and tissues of a human body are greatly reduced. And hydrophobic anticancer drugs, such as doxorubicin, docetaxel, camptothecin and the like, can be loaded in the nano-micelle.

2. The nano micelle contains bone targeting group alendronic acid, and can be combined with hydroxyapatite which is a main inorganic component in bone, so that the nano micelle has a targeting effect on bone tissues; meanwhile, the azobenzene containing the hypoxia response group can respond to a hypoxia environment under the hypoxia condition of the tumor, and the azo bond is reduced, so that the whole nano micelle system is damaged, the loaded anticancer drug is released, and the hypoxia controlled release effect is achieved.

3. The surface of the nano micelle is hydrophilic polyethylene glycol, so that the nano micelle can be prevented from being captured by an endothelial reticulum system, the blood circulation time of the nano micelle is effectively prolonged, and the nano micelle has sufficient time to be retained and enriched at a tumor part through an EPR effect.

4. The surface of the nano micelle provided by the invention is positively charged, so that the nano micelle is easier to enter tumor cells in vivo, and can release anticancer drugs in the tumor cells to inhibit the growth of the tumor cells.

5. The preparation method of the nano micelle is simple, is easy for scale-up production, and has higher encapsulation efficiency (up to 90%) and drug loading rate (up to 22%).

Drawings

FIG. 1 is a NMR chart of a bone-targeted hypoxia-responsive polymer (ALN-PEG-AZO-PLL) provided in example 1-1;

FIG. 2 is a graph of the in vitro hypoxia responsiveness-UV absorption characteristic peak changes of 4, 4-Azodiphenylamine (AZO), unloaded bone-targeting hypoxia-responsive nanomicelles provided in example 2-1 (ALN-PEG-AZO-PLLNPs), unloaded normal nanomicelles provided in example 2-2 (mPEG-PLL NPs), and unloaded bone-targeting nanomicelles provided in example 2-3 (ALN-PEG-PLL NPs); in FIG. 2, the A diagram is the ultraviolet absorption peak of AZO within the range of 200-700 nm, the B diagram is the ultraviolet absorption peak of mPEG-PLLNPs without AZO within the range of 200-700 nm, the C diagram is the ultraviolet absorption peak of ALN-PEG-PLL NPs within the range of 200-700 nm, and the D diagram is the ultraviolet absorption peak of ALN-PEG-AZO-PLL NPs within the range of 200-700 nm.

FIG. 3 is a transmission electron micrograph of unloaded bone-targeted hypoxia-responsive nanomicelles (ALN-PEG-AZO-PLL NPs) provided in example 2-1 before and after reduction;

FIG. 4 is a graph comparing the bone targeting effect of the adriamycin-loaded bone targeting hypoxia-responsive nanomicelles (ALN-PEF-AZO-PLL-DOX NPs) provided in example 4-1 and the adriamycin-loaded normal nanomicelles (mPEG-PLL-DOX NPs) provided in example 4-4; wherein, A is a graph comparing the effect of the combination of the adriamycin-loaded bone targeting hypoxia response nano-micelle (ALN-PEF-AZO-PLL-DOX NPs) provided in example 4-1 and the adriamycin-loaded normal nano-micelle (mPEG-PLL-DOXNPs) provided in example 4-4 with HA; panel B is a graph of the binding capacity of ALN-PEF-AZO-PLL-DOX NPs and mPEG-PLL-DOX NPs to HA at different time points.

FIG. 5 is the adriamycin release profiles under hypoxic and normoxic conditions of the adriamycin-loaded bone-targeting hypoxia-responsive nanomicelles (ALN-PEG-AZO-PLL-DOX NPs) provided in example 4-1, the adriamycin-loaded normal nanomicelles (mPEG-PLL-DOX NPs) provided in example 4-4, and the adriamycin-loaded bone-targeting nanomicelles (ALN-PEG-PLL-DOX NPs) provided in example 4-5;

FIG. 6 is the observation of the intracellular doxorubicin release behavior of the doxorubicin-loaded bone-targeted hypoxia-responsive nanomicelles (ALN-PEG-AZO-PLL-DOX NPs) provided in example 4-1 under hypoxic and normoxic conditions; FIG. 6, panel A shows the DOX release behavior observed in MDA-MB-321 cells, and panel B shows the DOX release behavior observed in RM-1 cells;

FIG. 7 shows the results of near-infrared in vivo imaging of bone-targeted hypoxia-responsive nanomicelles (ALN-PEG-AZO-PLL-Cy5.5NPs), common nanomicelles (mPEG-PLL-Cy5.5NPs) and bone-targeted nanomicelles (ALN-PEG-PLL-Cy5.5NPs) loaded with fluorescent dye Cy5.5 provided in example 8 on tumor-bearing mice; in FIG. 7, A is a fluorescence picture obtained by imaging the near-infrared living body of a tumor-bearing mouse by three kinds of nano-micelles loaded with a fluorescent dye Cy5.5; b, the fluorescence ratios of the tumor part and the normal tissue part of the three fluorescent dye Cy5.5-loaded nano-micelles at different time points are shown; c is a near infrared imaging fluorescence picture taken by a mouse which is sacrificed 24 hours later and then the mouse is taken out of the heart, liver, spleen, lung and kidney, and metastatic bones and normal bones (healthy bones);

FIG. 8 shows the therapeutic effect of the adriamycin-loaded bone-targeting hypoxia-responsive nanomicelles (ALN-PEG-AZO-PLL-DOX NPs) provided in example 4-1, the adriamycin-loaded normal nanomicelles (mPEG-PLL-DOX NPs) provided in example 4-4, the adriamycin-loaded bone-targeting nanomicelles (ALN-PEG-PLL-DOX NPs), PBS and adriamycin (DOX) provided in example 4-5 on tumor-bearing mice; in FIG. 8, graph A shows the results of comparison of foot lifting times in 4 minutes for each group of mice on different modeling days, graph B shows the results of comparison of foot lifting times in 4 minutes for each group of mice on different modeling days, graph C shows the results of comparison of pain behavior scores for each group of mice on different modeling days, graph D shows the results of comparison of weight of each group of mice on different modeling days, and graph E shows the results of comparison of survival rates of each group of mice on different modeling days;

FIG. 9 is a graph of Micro-CT results of tumor-bearing femur after treatment of tumor-bearing mice with doxorubicin-loaded bone-targeted hypoxia-responsive nanomicelles (ALN-PEG-AZO-PLL-DOX NPs) provided in example 4-1, doxorubicin-loaded normal nanomicelles (mPEG-PLL-DOX NPs) provided in example 4-4, doxorubicin-loaded bone-targeted nanomicelles (ALN-PEG-PLL-DOX NPs) provided in example 4-5, PBS and DOX, respectively; in FIG. 9, graph A is a coronal plane and a sagittal plane of a femur of a tumor-bearing mouse photographed by Micro-CT, graph B is a reconstructed graph of Micro-CT3D of the front end of the femur, graph C is a comparison graph of bone density of mice of different administration groups, and graph D is a comparison graph of bone trabecular number of mice of different administration groups;

fig. 10 is a structural schematic diagram of an adriamycin-loaded bone-targeting hypoxia-responsive nano-micelle prepared by the invention, wherein-N ═ N-azo compounds represent 4, 4-azodiphenylamine.

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the present invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the present invention and is not intended to limit the scope of the claims which follow.

All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.

For further understanding of the present invention, the bone targeting hypoxia-responsive nanomicelle loaded with anticancer drug and the preparation method thereof provided by the present invention are described in detail below with reference to the following examples, and the scope of the present invention is not limited by the following examples.

Examples 1 to 1

Preparation of bone-targeting hypoxia-responsive polymer:

(1) dissolving 50mg (1 equivalent) of polyethylene glycol (HOOC-PEG-NHS, MW 1000) in 5mL of dichloromethane, adding 6.8mg (1.3 equivalents) of 4, 4-Azodiphenylamine (AZO), stirring at normal temperature and in the dark for 24 hours, and then precipitating a product by using 20mL of diethyl ether to obtain carboxyl polyethylene glycol azobenzene (HOOC-PEG-AZO);

(2) dissolving 50mg (1 equivalent) of HOOC-PEG-AZO in Tetrahydrofuran (THF), adding 124mg (16 equivalents) of N-benzyloxycarbonyl-L-lysine-N-carboxylic anhydride (zLL-NCA) and reacting for 72h under stirring at normal temperature and in the dark to obtain a polyethylene glycol-polylysine block copolymer (HOOC-PEG-AZO-PBLL) with benzyloxycarbonyl on the surface;

(3) dissolving 150mg (1 equivalent) of HOOC-PEG-AZO-PBLL in 5mL of trifluoroacetic acid (TFA), adding 1mL of 33% hydrogen bromide acetic acid solution (HBr/ACOH), stirring at 0 ℃ for reaction for 1h to remove carbobenzoxy, then melting into dichloromethane, adding 1mL of triethylamine to neutralize acid, and obtaining a polyethylene glycol-polylysine block copolymer (HOOC-PEG-AZO-PLL);

(4) dissolving 150mg (1 equivalent) of HOOC-PEG-AZO-PLL into 5mL of N, N-Dimethylformamide (DMF), adding 22mg (3 equivalents) of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 16mg (3.6 equivalents) of N-hydroxysuccinimide (NHS), stirring at normal temperature for reaction for 1h, adding 61mg (5 equivalents) of alendronic Acid (ALN), and stirring for reaction for 24h to obtain the bone targeting hypoxia-responsive polymer (ALN-PEG-AZO-PLL).

The structure of ALN-PEG-AZO-PLL was examined by hydrogen nuclear magnetic resonance spectroscopy (1H NMR), which is shown in FIG. 1.

Examples 1 to 2

Preparation of bone-targeting hypoxia-responsive polymer:

dissolving 50mg (1 equivalent) of polyethylene glycol (HOOC-PEG-NHS, MW 2000) in 5mL of dichloromethane, adding 6.8mg (1.3 equivalents) of 4, 4-Azodiphenylamine (AZO), stirring at normal temperature and in the dark for 24 hours, and then precipitating a product by using 20mL of diethyl ether to obtain carboxyl polyethylene glycol azobenzene (HOOC-PEG-AZO); the rest of the procedure was the same as in example 1-1.

Examples 1 to 3

Preparation of bone-targeting hypoxia-responsive polymer:

dissolving 50mg (1 equivalent) of polyethylene glycol (HOOC-PEG-NHS, MW 5000) in 5mL of dichloromethane, adding 6.8mg (1.3 equivalents) of 4, 4-Azodiphenylamine (AZO), stirring at normal temperature and in the dark for 24 hours, and then precipitating a product by using 20mL of diethyl ether to obtain carboxyl polyethylene glycol azobenzene (HOOC-PEG-AZO); the rest of the procedure was the same as in example 1-1.

Example 2-1

Preparing unloaded bone targeting hypoxia response nano-micelle:

after 20mg of ALN-PEG-AZO-PLL obtained in example 1-2 was dissolved in 4mL of DMF, and fully dissolved, 10mL of water was added dropwise under magnetic stirring, and after stirring for 1.5h, the mixed solution was transferred to a dialysis bag (MW 3000) and dialyzed with pure water for 24h, and lyophilized at-50 ℃ to obtain unloaded bone-targeted low-oxygen responsive nanomicelles (ALN-PEG-AZO-PLL NPs).

Examples 2 to 2

Preparation of unloaded normal nano-micelles:

polyethylene glycol-polylysine block copolymers (mPEG-PLL) with methyl modification were prepared according to the method in the literature [ Lim C, Sim T, HonggNH, Oh KT. A stable nanoplatform for activity using PEG-PLL-PLA tribloc co-polyelectrolyte [ J ]. Colloidsurface B.2017,153:10-8 ].

20mg of mPEG-PLL is dissolved in 4mL of DMF, after the solution is fully dissolved, 10mL of water is added dropwise under magnetic stirring, after stirring is continued for 1.5h, the mixed solution is transferred into a dialysis bag (MW 3000), and the dialysis is carried out for 24h by pure water, and the empty nano-micelle (mPEG-PLL NPs) is obtained by freeze-drying at-50 ℃.

Examples 2 to 3

Preparing unloaded bone targeting nano-micelles:

dissolving 50mg of HOOC-PEG-NH2 in 5mL of THF, adding 123mg of zlL-NCA, stirring at normal temperature for reaction for 72h, carrying out rotary evaporation and concentration, adding excessive ethyl acetate for precipitation, centrifuging at a rotation speed of 8000r/min, dissolving the obtained product with dichloromethane, and carrying out rotary evaporation and evaporation to obtain HOOC-PEG-PBLL; dissolving HOOC-PEG-PBLL in 5mL of TFA, adding 1mL of 33% HBr/AcOH, stirring at 0 ℃ for reaction for 1h to remove carbobenzoxy, then melting into dichloromethane, adding 1mL of triethylamine to neutralize acid to obtain HOOC-PEG-PLL; 150mg of HOOC-PEG-PLL is dissolved in 5mL of DMF, 22mg of EDC (3 equiv.) and 16mg (3.6 equiv.) of NHS are added, the mixture is stirred at normal temperature for reaction for 1h, 61mg (5 equiv.) of ALN is added, and the mixture is stirred for reaction for 24h, so that the bone-targeting hypoxia-responsive polymer (ALN-PEG-PLL) is obtained.

Dissolving 20mg of ALN-PEG-PLL in 4mL of DMF, fully dissolving, dropwise adding 10mL of water under magnetic stirring, continuously stirring for 1.5h, transferring the mixed solution into a dialysis bag (MW & gt 3000), dialyzing with pure water for 24h, and freeze-drying at-50 ℃ to obtain the unloaded bone targeting nano-micelle (ALN-PEG-PLL NPs).

Example 3

In vitro hypoxia-responsive study of unloaded bone-targeted hypoxia-responsive nanomicelles:

AZO has a strong ultraviolet absorption peak at 388nm, so that an ultraviolet spectrophotometer (UV-2450, SHIMADZU, Japan) is used for detecting the reduction condition of the AZO after the nano micelle is added with reductase under the in vitro low oxygen condition, and the method specifically comprises the following steps: using 4, 4-Azodiphenylamine (AZO), the unloaded bone-targeting hypoxia-responsive nanomicelles (ALN-PEG-AZO-PLLNPs) provided in example 2-1, respectively, examples2-2 preparation of 1mg/mL concentrations of AZO solution, ALN-PEG-AZO-PLL NPs solution, mPEG-PLL NPs solution and ALN-PEG-PLL NPs solution from unloaded Normal Nano-micelles (mPEG-PLL NPs) and unloaded bone targeting Nano-micelles (ALN-PEG-PLL NPs) provided in examples 2-3; dividing the four solutions into two groups, each group is 2mL, and adding chemical reductant 2mg sodium hydrosulfite (Na)₂S₂O₄) And the other group is added with 20 mu L of liver microsome and 2mg of NADH (nicotinamide adenine dinucleotide), put in a low-oxygen environment for 12h (so as to simulate a reductase system under the low-oxygen condition in vivo), and then an ultraviolet spectrophotometer (UV-2450, SHIMADZU, Japan) is used for testing the ultraviolet absorption peak of each group of solution in the range of 200-700 nm respectively. As shown in FIG. 2, it can be found that AZO has a strong absorption peak around 388nm when Na is added₂S₂O₄Or liver microsomes and NADH, the strong absorption peak at 388 decreases, indicating that AZO is reduced to form amines and thus the UV absorption peak decreases or disappears. The same effect occurred in ALN-PEG-AZO-PLL NPs (FIG. 2D), while there was no absorption peak at 388nm in the mPEG-PLL NPs without AZO (FIG. 2B) and ALN-PEG-PLL NPs without AZO (FIG. 2C), indicating that no AZO was present, and Na was added₂S₂O₄Or liver microsomes and NADH did not change significantly at 388 nm. The no-load bone targeting hypoxia response nano micelle ALN-PEG-AZO-PLL NPs have hypoxia response in vitro because of containing AZO.

Adding chemical reducer with sodium sulfite (Na)₂S₂O₄) And respectively dripping the front and rear unloaded bone targeting hypoxia response nano-micelle solutions on a copper mesh, absorbing excessive liquid by using filter paper, dripping 1% phosphotungstic acid solution for dyeing, washing for 3 times by using clear water after dyeing for 1-2 min, absorbing excessive liquid by using filter paper, and observing the reduced form of the ALN-PEG-AZO-PLL NPs nano-micelle by using a transmission electron microscope (TEM, JEM-1230, Japan) after air drying, wherein the transmission electron microscope images of the unloaded bone targeting hypoxia response nano-micelle (ALN-PEG-AZO-PLL NPs) before and after reduction are shown in figure 3. Because the ALN-PEG-AZO-PLL contains AZO, the AZO is reduced under the condition that a chemical reducing agent is added to the formed nano micelle, so that the whole nano glue is formedThe bundle system is broken. From FIG. 3, it can be seen that ALN-PEG-AZO-PLL NPs have a uniform spherical appearance before being reduced, a particle size of about 200nm, and the entire nanoparticles are cleaved after reduction, further illustrating the low oxygen responsiveness of ALN-PEG-AZO-PLL NPs in vitro.

Example 4-1

Preparing the adriamycin-loaded bone-targeting hypoxia-responsive nano micelle:

dissolving 20mg of ALN-PEG-AZO-PLL obtained in example 1-2 in 4mL of DMF, adding 4mg of adriamycin (DOX), dropwise adding 10mL of water under magnetic stirring, stirring for 1.5h, transferring the mixed solution into a dialysis bag (MW 3000), dialyzing with pure water for 24h, freeze-drying to obtain bone targeting low-oxygen response nano-micelles (ALN-PEF-AZO-PLL-DOX NPs) loaded with the adriamycin, wherein the entrapment rate of ALN-PEF-AZO-PLL-DOXNPs is 90.5 +/-0.29 (%), the drug loading rate is 22.3 +/-0.23%, and the hydrated particle size is 200nm as measured by a particle size potential tester.

Example 4 to 2

Preparing bone targeting hypoxia response nano micelle loaded with docetaxel:

20mg of ALN-PEG-AZO-PLL obtained in example 1-2 is dissolved in 4mL of DMF, 5mg of docetaxel is added, 10mL of water is added dropwise under vigorous magnetic stirring, then stirring is carried out for 1.5h, the mixed solution is transferred into a dialysis bag (MW & gt 3000) and dialyzed for 24h by pure water, and bone-targeted hypoxia-responsive nano-micelle loaded with docetaxel is obtained after freeze-drying.

Examples 4 to 3

Preparing the camptothecin-loaded bone-targeted hypoxia-responsive nano micelle:

20mg of ALN-PEG-AZO-PLL obtained in example 1-2 is dissolved in 4mL of DMF, 2mg of camptothecin is added, 10mL of water is added dropwise under vigorous magnetic stirring, then stirring is carried out for 1.5h, the mixed solution is transferred into a dialysis bag (MW & gt 3000) and dialyzed for 24h by pure water, and the bone-targeted hypoxia-responsive nano-micelle loaded with the camptothecin is obtained after freeze-drying.

Examples 4 to 4

Preparation of adriamycin-loaded common nano-micelle:

20mg of mPEG-PLL obtained in example 2-2 was dissolved in 4mL of DMF, 4mg of doxorubicin was added, 10mL of water was added dropwise under vigorous magnetic stirring, followed by stirring for 1.5h, the mixed solution was transferred to a dialysis bag (MW. 3000) and dialyzed with pure water for 24h, and ordinary nanomicelles (mPEG-PLL-DOX NPs) loaded with doxorubicin were obtained after lyophilization.

Examples 4 to 5

Preparing the adriamycin-loaded bone targeting nano micelle:

20mg of ALN-PEG-PLL obtained in example 2-3 was dissolved in 4mL of DMF, 4mg of doxorubicin was added, 10mL of water was added dropwise under vigorous magnetic stirring, followed by stirring for 1.5h, the mixed solution was transferred to a dialysis bag (MW 3000) and dialyzed for 24h with pure water, and bone-targeted nanomicelles (ALN-PEG-PLL-DOX NPs) loaded with anticancer drugs were obtained after lyophilization.

Example 5

The binding capacity of the adriamycin (DOX) -loaded bone-targeted hypoxia-responsive nano-micelle to Hydroxyapatite (HA) in vitro is investigated:

ALN can be combined with Hydroxyapatite (HA) which is the main inorganic component in bone, so the bone targeting effect of ALN is examined in vitro by using ALN-PEG-AZO-PLL-DOX NPs which are the bone targeting hypoxia response nano-micelles obtained in example 4-1 and mPEG-PLL-DOX NPs which are the common nano-micelles obtained in example 4-4 and are loaded with adriamycin, mixed with HA, as shown in FIG. 4. Respectively diluting ALN-PEG-AZO-PLL-DOX NPs and mPEG-PLL-DOX NPs by pure water by 1 time, respectively taking 5mL, respectively adding 5mg of HA, magnetically stirring, respectively taking out 200 mu L of mixed solution for centrifugation at 30min, 1h, 2h, 3h and 4h, taking supernatant, and breaking a membrane by using methanol to measure the content of DOX, so that the content of ALN-PEG-AZO-PLL-DOX NPs or mPEG-PLL-DOX NPs which are not combined with HA can be measured, and further the content of ALN-PEG-AZO-PLL-DOXNPs or mPEG-PLL-DOX NPs which are combined with each gram of HA can be converted.

As HA is insoluble in water and the DOX-loaded nano micelle is not clear, HA can be added into mPEG-PLL-DOX NPs and ALN-PEG-AZO-PLL-DOX NPs solutions, whether NPs are combined with HA or not is judged by observing whether the solutions are clear or not, once the solutions are combined, precipitation occurs, and the solutions become clear. From FIG. 4A, it can be seen that the solution of ALN-PEG-AZO-PLL-DOX NPs becomes clear after mixing with water-insoluble HA for 12h (right in FIG. 4A), indicating that the ALN-PEG-AZO-PLL-DOX NPs are combined with HA and precipitated at the bottom of the centrifuge tube, while under the same conditions, the solution of mPEG-PLL-DOX NPs without ALN is still not clear (left in FIG. 4A), indicating that the solution is not combined with HA. By measuring the binding capacity of two drug-loaded nano-micelles and HA at different time points, as shown in FIG. 4B, FIG. 4B shows that ALN-PEG-AZO-PLL-DOXNPs and HA are really strongly bound, about 60mg of ALN-PEG-AZO-PLL-DOX NPs are bound to HA per gram within 4h, which is more than 3 times of the binding capacity of mPEG-PLL-DOX NPs, and the drug-loaded nano-micelles containing ALN can be strongly bound to HA, so that the bone targeting effect is achieved.

Example 6

In vitro release profiles of mPEG-PLL-DOXNPs, ALN-PEG-PLL-DOX NPs and ALN-PEG-AZO-PLL-DOX NPs under normoxic and hypoxic conditions, respectively, were determined:

ALN-PEG-AZO-PLL-DOX NPs solution, mPEG-PLL-DOX NPs solution and ALN-PEG-PLL-DOX NPs solution with the concentration of 200 mu g/mL are respectively prepared by using the adriamycin-loaded bone targeting hypoxia-responsive nano micelle ALN-PEG-AZO-PLL-DOX NPs obtained in the embodiment 4-1, the adriamycin-loaded common nano micelle mPEG-PLL-DOX NPs obtained in the embodiment 4-4 and the adriamycin-loaded bone targeting nano micelle ALN-PEG-PLL-DOX NPs obtained in the embodiment 4-5, 10mL of each of the solutions was added with 20mg of NADH and 100. mu.L of liver microsomes, the mixed solution was transferred to a dialysis bag (MW 3000), the dialysis bag was placed in 30mL of PBS solution under hypoxic conditions (37 ℃, 5% CO).₂，1％O₂) And (5) performing dialysis. At dialysis times of 0.5h, 1h, 2h, 4h, 6h, 12h and 24h, 0.5mL of PBS dialysate was removed at each time node, centrifuged at 8000r/s, and the supernatant was subjected to HPLC to determine DOX content, and the cumulative percentage of DOX release was calculated. While setting the normoxic condition (37 ℃, 5 percent CO)₂，20％O₂) Is a control group. The same volume of fresh PBS solution was replenished while removing 0.5mL of PBS dialysate each time, so that the volume of the entire system remained unchanged. As shown in FIG. 5, it can be seen from FIG. 5 that the drug release amount of ALN-PEG-AZO-PLL-DOX NPs containing AZO was significantly higher than that of normoxic (hypoxia: 74%, normoxic: 24%) after liver microsomes and NADH were added under hypoxia, indicating that AZO was reduced under hypoxia and the whole load was loadedThe drug nanoparticles are destroyed thereby releasing the loaded DOX. However, the cumulative release of DOX did not change significantly for the AZO-free mPEG-PLL-DOX NPs and ALN-PEG-PLL-DOXNPs, both normoxic and hypoxic.

Example 7

The drug release behavior of the adriamycin-loaded bone targeting hypoxia response nano micelle ALN-PEG-AZO-PLL-DOX NPs in hypoxic and normoxic cells is observed:

according to previous studies, a cover slip is applied to the surface of a layer of cells to produce a decreasing oxygen concentration from the edge of the slide to the center of the slide, so different oxygen gradients are produced by applying a cover slip to a glass bottom dish containing a layer of cells, and after co-incubation of the cells with the loaded nanomicelles, the DOX fluorescence intensity from the center to the edge of the slide is observed using a confocal laser microscope (TCS SP8, Leica). On a glass bottom capsule (

Surface treated, JET BIOFIL, Canada) into MDA-MB-231 cells and RM-1 cells, each dish has 20000 cells, after 24h of culture, the liquid in the dish is sucked off, washed three times with PBS, then 2mL of complete culture solution containing ALN-PEG-AZO-PLL-DOX NPs is added, the drug solubility is 10 mug/mL, meanwhile, a cover glass is added on the glass bottom, and the mixture is placed into a cell culture box for 3h of culture and observed by a laser confocal microscope. The results are shown in FIG. 6, in which A is the DOX release behavior observed in MDA-MB-321 cells, and B is the DOX release behavior observed in RM-1 cells. After coverslipping on MDA-MB-321 cells (FIG. 6A) and RM-1 cells (FIG. 6B), the central DOX fluorescence intensity was much stronger than the edge, indicating that the drug release was much greater in cells in the center of the slide, indicating that the drug release of ALN-PEG-AZO-PLL-DOX NPs is hypoxia-dependent, further indicating that it has hypoxia-responsiveness.

Example 8

And (3) investigating the bone targeting effect of the bone targeting hypoxia response nano-micelle in a C57BL/6 tumor-bearing mouse:

and (3) dissolving 20mg of the bone-targeting hypoxia-responsive polymer ALN-PEG-AZO-PLL obtained in example 1-2 in 4mL of DMF, adding 4mg of fluorescent dye Cy5.5, dropwise adding 10mL of water under vigorous magnetic stirring, stirring for 1.5h, transferring the mixed solution into a dialysis bag (MW & gt 3000), dialyzing for 24h with pure water, and freeze-drying to obtain the bone-targeting hypoxia-responsive nano-micelle (ALN-PEG-AZO-PLL-Cy5.5NPs) loaded with the fluorescent dye Cy5.5.

20mg of mPEG-PLL obtained in example 2-2 was dissolved in 4mL of DMF, 4mg of fluorescent dye Cy5.5 was added thereto, 10mL of water was added dropwise under vigorous magnetic stirring, followed by stirring for 1.5h, the mixed solution was transferred to a dialysis bag (MW. 3000) and dialyzed with pure water for 24h, and then lyophilized to obtain fluorescent dye Cy5.5-loaded nanomicelles (mPEG-PLL-Cy5.5NPs).

20mg of ALN-PEG-PLL obtained in example 2-3 was dissolved in 4mL of DMF, 4mg of the fluorescent dye Cy5.5 was added thereto, 10mL of water was added dropwise under vigorous magnetic stirring, followed by stirring for 1.5h, the mixed solution was transferred to a dialysis bag (MW. about.3000) and dialyzed with pure water for 24h, and then lyophilized to obtain bone-targeted nanomicelles (ALN-PEG-PLL-Cy5.5NPs) carrying the fluorescent dye Cy5.5.

Construction of bone metastasis models using C57BL/6 mice: after C57BL/6 mice were anesthetized with chloral hydrate, the femurs of the right legs were carefully exposed, the femurs were punched from the ends of the femurs into the femurs using a 29gauge needle, then 20. mu.L or more of a 5X 107cells/mL cell suspension was injected, the needle holes were sealed with bone wax and sutured. Sufficient grain and water were given to the molded mice, and the mice were randomly divided into 3 groups on the 10 th day of molding, each group of 3 mice was anesthetized by intraperitoneal injection of chloral hydrate, then mPEG-PLL-cy5.5nps, ALN-PEG-PLL-cy5.5nps, or ALN-PEG-AZO-PLL-cy5.5nps were injected into the tail vein, and fluorescence pictures were taken with a small animal living body imager (luminea II, calipers life Sciences) at 0h, 0.5h, 1h, 2h, 4h, 6h, 12h, and 24h after intravenous injection, as shown in a of fig. 7. Then, the fluorescence intensity of the fluorescence image shown in a in fig. 7 was analyzed using IVIS imaging software, and the fluorescence ratio of the tumor site to the normal tissue site at different time points of different groups was compared, and the result is shown in B in fig. 7. After the photos are collected at 24h, the mice are euthanized, and the heart, liver, spleen, lung, kidney, normal bone and bone metastasis bone of each group of mice are taken out and imaged, wherein the imaging picture is shown as a C picture in figure 7. The image acquisition parameters were as follows: the Exposure time is auto, Pixel width is 1cm, Pixel Height is 1cm, Binning Factor is 8, fNumber is 2, and Field of view (FOV) is 10 cm.

According to the graph A in the graph in the figure 7, the right ring at 0h is the femur of the right leg of the mouse with the bone metastasis tumor, the left ring is the normal femur of the left leg of the mouse, mPEG-PLL-Cy5.5NPs slightly reach the bone tumor due to the EPR effect of the nanoparticles, and the mPEG-PLL-Cy5.5NPs in the bone tumor are metabolized with the time; due to the bone targeting effect of ALN, ALN-PEG-PLL-Cy5.5NPs are more gathered in bone tumors than mPEG-PLL-Cy5.5NPs, but most of ALN-PEG-PLL-Cy5.5NPs are metabolized by 24 hours due to the lack of good controlled release effect; the ALN-PEG-AZO-PLL-Cy5.5NPs not only have the bone targeting effect of ALN, but also have the hypoxia response effect of AZO, so that the ALN-PEG-AZO-PLL-Cy5.5NPs can be greatly gathered in bone tumors, AZO is reduced in the hypoxia environment of the bone tumors, Cy5.5 is released from the ALN-PEG-AZO-PLL-Cy5.5NPs, so that the fluorescence intensity at the bone tumor sites is higher, and Cy5.5 still exists in the bone tumors by 24h (FIG. 7A). By 24h, mice from different groups were sacrificed and their vital organs (heart, liver, spleen, lung and kidney) were removed and the metastatic and normal bones were imaged near infrared with the same results, and compared to the other two groups, the fluorescence was strongest in the right leg with bone metastases after tail vein injection of ALN-PEG-AZO-PLL-cy5.5nps (fig. 7C), further indicating that it was more enriched in bone tumor sites due to the bone targeting effect of ALN and the controlled release effect of AZO hypoxia. The fluorescence ratios of tumor sites to normal tissue sites at different time points of different groups were compared by analyzing the fluorescence intensity of the circled sites of the left and right legs using IVIS imaging software, with the ratio of ALN-PEG-AZO-PLL-cy5.5nps being the largest at the different time points (fig. 7B).

Example 9

Mice with bone metastases were treated with ALN-PEG-AZO-PLL-DOX NPs, mPEG-PLL-DOX NPs, and ALN-PEG-PLL-DOXNPs obtained in examples 4-1, 4-4, and 4-5, according to the following specific embodiments:

tumor-bearing mice were randomly divided into 5 groups of 6 mice each, and on day 7 of molding, the groups were administered with 5mg DOX/kg body weight of the drug in tail vein injection of the following drugs: PBS, DOX, mPEG-PLL-DOX NPs, ALN-PEG-PLL-DOX NPs or ALN-PEG-AZO-PLL-DOX NPs, administered once every 4 days. Starting from the first day of molding, the number of foot lifts and the time of foot lifts of the right leg of each group of mice within 4min were measured every other day and the pain behavior of each group of mice was scored according to the following criteria: and 4, dividing: normal use; and 3, dividing: slight lameness; and 2, dividing: the lameness is obvious, and the affected limb is lifted after stopping; 1 minute: the affected limb can not be used partially; 0 minute: the affected limb can not be used at all. The times and time of raising the feet of the mice are measured by a camera observation method, the mice are put into a transparent glass box (10cm multiplied by 10cm) to be pre-adapted for 2 to 3 days, one mouse is put into one box, and the measurement is started after the mice are adapted to the environment of the glass box. Before measurement, each mouse is adapted in a glass box for 10min, then a digital camera is used for shooting a moving video of the mouse in the glass box within 10min, the foot lifting times and the foot lifting time of the mouse within 4min are observed according to the 4 th min-8 th min of the shot video, the moving conditions of the mouse are scored according to the whole video, and a treatment effect graph is obtained and shown in figure 8, wherein A in figure 8 is different modeling days, the foot lifting times of each group of mice within 4min are compared, B is different modeling days, the foot lifting time of each group of mice within 4min is compared, C is different modeling days, the pain behavior of each group of mice is scored and compared, D is different modeling days, the weight of each group of mice is compared, E is different modeling days, and the survival rate of each group of mice is compared. As can be seen from FIGS. 8(A-C), after the tumor-bearing mice were treated with PBS, DOX, mPEG-PLL-DOX NPs or ALN-PEG-PLL-DOX NPs, the number of times of lifting the feet and the time of lifting the feet reached the peak values on day 17, the number of times of lifting the feet was 20,19,18,17 (FIG. 8A), the time of lifting the feet was 4.6s,3.4s,4.2s,3.6s (FIG. 8B), while the number of times of lifting the feet and the time of the ALN-PEG-AZO-PLL-DOXNPs treatment group on day 17 were 4 and 1.9s, respectively, which is significantly different from the other groups, indicating that the tumor-bearing mice could significantly reduce bone pain after being treated with ALN-PEG-AZO-PLL-DOX NPs. After day 17, the number of times and time of raising the feet of mice in different administration groups tended to decrease, because some tumor-bearing mice were paralyzed and could not use the right leg. It can also be seen from the scoring of the right leg usage of tumor-bearing mice (fig. 8C) that ALN-PEG-AZO-PLL-DOX NPs significantly reduced pain and improved therapeutic effect compared to the other groups.

After tumor-bearing mice were administered PBS, DOX, mPEG-PLL-DOX NPs, ALN-PEG-PLL-DOX NPs or ALN-PEG-AZO-PLL-DOX NPs, respectively, systemic toxicity of the different formulations was evaluated by monitoring the body weight of the mice every two days, and the treatment effect was evaluated by observing the survival of the mice in the different treatment groups. As can be seen in fig. 8D, the body weight did not change much in the tumor-bearing mice after the administration of the different dosage forms, indicating that there was no significant systemic toxicity for the different drug dosage forms. As can be seen from the survival plots in FIG. 8E, the ALN-PEG-AZO-PLL-DOX NPs treatment group was able to prolong the survival time of tumor-bearing mice compared to the other treatment groups.

Mice from different dosing groups (PBS, DOX, mPEG-PLL-DOX NPs, ALN-PEG-PLL-DOX NPs and ALN-PEG-AZO-PLL-DOX NPs) were euthanized on day 21 of modeling, tumor-bearing femurs were removed, blood was washed with physiological saline, tomographic scanning was performed using Micro-CT (SKYSCAN 1176, Belgium), and 3D reconstruction was performed using corresponding bone analysis software to evaluate bone density (BMD) and trabecular bone number (Tb.N), respectively. Micro-CT further evaluates the treatment effect of different drug-loaded nanoparticles on bone metastasis. The therapeutic effect is shown in FIG. 9, wherein graph A in FIG. 9 is the coronal plane and the sagittal plane of the femur of a tumor-bearing mouse photographed by Micro-CT, graph B is the Micro-CT3D reconstructed image of the front end of the femur, graph C is the bone density comparison graph of mice of different administration groups, and graph D is the bone trabecular number comparison graph of mice of different administration groups. After tumor-bearing mice are treated by different drug formulations, the thighbone of the mice of the PBS group, the DOX group and the mPEG-PLL-DOX NPs group is seriously damaged and is shown as eroded and comminuted fracture of bone, while the ALN-PEG-PLL-DOX NPs treatment group has smaller damage to the bone, probably because ALN has a targeting effect on diseased bone, the bone is protected to a certain extent, and the dissolution of the bone can still be seen. The protection of bone mass was most effective only in the group treated with ALN-PEG-AZO-PLL-DOX NPs. The same results are shown in the 3D reconstructed Micr-CT image, and the Micro-CT3D reconstructed image of the front end of the femur shows that the surface of the normal front end of the femur is smooth and flat, and the bone is firm, but after PBS, DOX or mPEG-PLL-DOX NPs treatment, the bone structure of the front end of the femur becomes incomplete, the surface is corroded and becomes rough, and the bone also becomes loose; the ALN-PEG-PLL-DOX NPs treatment group protected the bone better than the first three groups, but the bone was still damaged, and only the ALN-EPG-AZO-PLL-DOX NPs treatment group could substantially preserve the intact bone, indicating that the treatment effect on bone metastasis was optimal (FIG. 9B). As can be seen from FIGS. 9C and 9D, the bone density and trabecular number of ALN-PEG-AZO-PLL-DOX NPs were higher than those of the other groups after the treatment, indicating that ALN-PEG-AZO-PLL-DOX NPs have superior therapeutic effects.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing exemplary embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims

1. A preparation method of bone-targeted hypoxia-responsive nano-micelle loaded with anticancer drugs is characterized by comprising the following steps:

1) dissolving polyethylene glycol in dichloromethane, adding 4, 4-azodiphenylamine, stirring at normal temperature and in dark place for reaction for 24 hours, and precipitating by using ethyl glacial ether to obtain carboxyl polyethylene glycol azobenzene shown in a formula (I):

4) dissolving the polyethylene glycol-polylysine segmented copolymer obtained in the step 3) in N, N-dimethylformamide, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, stirring at normal temperature, adding alendronic acid, and stirring for reacting for 2h to obtain the bone targeting hypoxia response polymer shown in the formula (IV):

2. The method according to claim 1, wherein the polyethylene glycol of step 1) has a relative molecular mass of 1000 to 5000.

3. The method according to claim 1, wherein the molar ratio of the polyethylene glycol to the 4, 4-azodiphenylamine in the step 1) is 1: 1.3.

4. the method according to claim 1, wherein the molar ratio of carboxypolyethyleneglycol azobenzene to N-benzyloxycarbonyl-L-lysine-N-carboxylic acid anhydride in step 2) is 1: 16.

5. The method according to claim 1, wherein the molar ratio of the polyethylene glycol-polylysine block copolymer to 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, N-hydroxysuccinimide and alendronic acid in step 4) is 1:3:3.6: 5.

6. The preparation method of claim 1, wherein the mass ratio of the anticancer drug to the bone-targeted hypoxia-responsive polymer in the step 5) is 1: 4-10, and the anticancer drug is one of doxorubicin, docetaxel and camptothecin.

7. A method of manufacturing as claimed in claim 1 wherein the dialysis bag of step 5) has a molecular weight cut-off of 3000.

8. The method of claim 1, wherein the mass to volume ratio of the bone-targeting hypoxia-responsive polymer to water in step 5) is 2:1 in g/L.

9. The bone-targeting hypoxia-responsive nano-micelle loaded with the anticancer drug, prepared by the preparation method according to any one of claims 1 to 8.

10. The anticancer drug-loaded bone-targeting hypoxia-responsive nanomicelle according to claim 9, wherein the hydrated particle size of the anticancer drug-loaded bone-targeting hypoxia-responsive nanomicelle is 200 nm.