A method for accelerating bio-orthogonal catalysis in situ in tumors by acid / reduction-sensitive polymersomes

Abstract

The application discloses a method for accelerating bio-orthogonal catalysis in situ of tumors by using acid / reduction-sensitive polymer vesicles, and the method comprises the following steps: introducing hydrophilic / hydrophobic precursors and Cu 2+ into the polymer vesicles, and then injecting the polymer vesicles into the body of a patient. The polymer vesicles are separated into an inner aqueous phase, a hydrophobic layer and a hydrophilic surface, so that side reactions and early reactions between the precursors and the divalent copper can be effectively avoided. The polymer vesicles are composed of linear copolymers of hyaluronic acid-bisulfur bond-poly-caprolactone with specific molecular weights, and the structure of the polymer vesicles is stable under normal physiological environment; under the acid / reduction environment in tumor cells, the membrane of the vesicles is degraded to cause perforation, so that the hydrophilic precursors are preferentially released from the inner aqueous phase to the outside of the membrane, chelate with the Cu + on the surface layer, and stabilize the structure of the Cu + and shorten the distance between the azide group and the catalytic center, so as to improve the catalytic efficiency. The co-delivery of the three substances (Azide, Alkyne and Cu 2+ ) and the improved catalytic efficiency after the chelation of the precursors and copper significantly reduce the dosage of intravenous injection, so that the nano-vesicle therapy has high efficiency and biological safety.

Description

TECHNICAL FIELD

[0001] The present technology relates to the co-delivery of polymer vesiculation, hydrophilic / hydrophobic precursors and copper ions and the accelerated bioorthogonal catalysis of controllable copper chelation of high molecular materials. BACKGROUND

[0002] The activation of traditional prodrugs depends on the human microenvironment of acid, enzyme, thiol, reactive oxygen, etc., which is often affected by enzyme activity, age-related physiological changes, drug interactions, etc. Bioorthogonal catalysis therapy is an emerging prodrug treatment technology. This therapy only relies on the efficient catalysis of transition metals, has more stable activation capacity, higher tumor specificity and lower off-target risk. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction frequently occurs in various bioorthogonal catalysis treatment programs, benefiting from the low cost and efficient catalysis of copper ions, simple precursor structure, good drugability of the product, and stable chemical structure and in vivo metabolism. Previous studies have shown that the co-delivery of nanomaterials and the precise control of the generation of catalytically active monovalent copper ions (Cu + ) are beneficial to improve the efficiency of bioorthogonal catalysis. However, the limitations of copper-catalyzed systems are copper toxicity (mainly caused by Cu + ) and the instability of copper ions (e.g., divalent copper (Cu 2+ ) can be reduced by alkyne; Cu + is prone to oxidation or disproportionation). The bottleneck of existing nanotechnology is that it cannot truly and safely co-deliver hydrophilic / hydrophobic precursors and Cu 2+ , and form stable Cu + structures in the complex physiological environment of tumor cells. The technical bottleneck brings low-efficiency in vivo delivery and in situ catalysis, followed by the toxic side effects of using high doses of copper ions.

[0003] The problems faced by copper-catalyzed bioorthogonal reactions are: Cu 2+ can be reduced to Cu + by alkyne; Cu + is prone to oxidation or disproportionation; the reaction requires a stable Cu + structure; and the systemic toxicity of copper ions. These bottlenecks leave room for improvement in bioorthogonal catalysis therapy to further improve the efficiency of in situ catalysis in tumors in a way to reduce the dose of exogenous copper ions and improve biological safety. SUMMARY

[0004] The purpose of the present application is to propose a method for accelerating bioorthogonal catalysis in situ in tumors by acid / reduction-sensitive polymer vesicles. The polymer vesicles formed by the hyaluronic acid-poly (caprolactone) copolymer (HA-SS-PCL) connected by disulfide bonds can spatially deliver azide, alkyne and Cu 2+The precursors / catalyst co-delivery system is separated in the inner water phase, the hydrophobic layer and the hydrophilic surface, and realizes high efficiency. By adjusting the molecular weight of the hydrophilic segment and the hydrophobic segment of the copolymer, the nano vesicle which is stable in normal physiological environment and quickly responds in acid / reduction environment is prepared. The surface membrane of the vesicle is perforated in the tumor cell internal environment, so that the hydrophilic precursor (azide) in the inner water phase is preferentially released from the inside to the outside, and is chelated with Cu + near the surface layer. Subsequently, the nano vesicle is further disintegrated in the acid / reduction environment, and the hydrophobic precursor (alkyne) is slowly released. The formation of the chelate helps to stabilize the structure of Cu + , and shortens the distance between the azide group and the Cu + catalytic center, thereby improving the efficiency of the Cu + catalyzed bio-orthogonal reaction. The chelation and catalysis behavior controlled by the programmed release of the nano vesicle realizes efficient bio-orthogonal catalytic therapy in situ in the tumor, so that the nano therapy has low demand for copper ions, and the biological safety risk brought by the exogenous copper is reduced.

[0005] Technical scheme: the acid / reduction sensitive linear copolymer formed by the nano vesicle material disclosed by the application is obtained by coupling two blocks of hyaluronic acid and polycaprolactone with specific molecular weight through a disulfide bond; the nano vesicle self-assembled by the copolymer can separate the hydrophilic precursor Azide, the hydrophobic precursor Alkyne and the divalent copper ion Cu 2+ physically in the inner water phase, the hydrophobic layer and the outer surface of the nano vesicle; the nano vesicle responds to the tumor microenvironment conditions and releases the two loaded precursors and the copper ion in a programmed manner. The copolymer is linearly copolymerized with hyaluronic acid and polycaprolactone with specific molecular weight through a disulfide bond, and the high stability of the nano vesicle assembled by the copolymer benefits from the close winding between the hydrophobic layers of polycaprolactone. Under the stimulation of acid / reduction conditions, the nano vesicle gradually undergoes polycaprolactone hydrolysis and disulfide bond rupture, resulting in membrane perforation, so as to trigger the preferential release of the hydrophilic precursor in the inner water phase from the inside to the outside; the hydrophobic precursor in the hydrophobic layer is slowly released as the nano vesicle is continuously disintegrated.

[0006] The precursors encapsulated by the nano vesicle material include a hydrophilic precursor and a hydrophobic precursor; the hydrophobic precursor is alkyne; the hydrophilic precursor is azide, and further includes a chelated cuprous ion structure for increasing the stability of Cu + and promoting the catalytic performance of Cu + , and the structure includes hydroxamic acid, imidazole group, pyridine, quinoline, phthalocyanine and porphyrin. In the acid / reduction environment, the hydrophilic precursor is preferentially released from the inner water phase of the nano vesicle, and is chelated with Cu 2+ near the surface layer which is reduced by glutathione (GSH). The chelate can stabilize Cu +The structure and the distance between the azide group and the copper catalytic center are shortened, which improves the rate of subsequent bio-orthogonal catalysis.

[0007] The blood circulation time (T 1 / 2 ) of the nanovesicle material in the living body is greater than 8 hours, and a Cu(I)-azide chelate is formed in situ at the tumor, promoting the synthesis of drugs by bio-orthogonal catalysis.

[0008] After intravenous injection of the nanomaterial into the living body (BALB / c mice), the half-life of the hydrophilic / hydrophobic precursor can be significantly prolonged, and the ability of hyaluronic acid to target tumors can efficiently co-deliver the precursor and copper ions to the tumor site; the special acid / reduction-sensitive structure of the nanovesicle can preferentially release the hydrophilic precursor and Cu + chelate in the tumor cell environment, thereby initiating chelate-promoted bio-orthogonal catalysis therapy in situ.

[0009] The preparation method of the nanovesicle material comprises the following steps: dissolving the amphiphilic copolymer hyaluronic acid-disulfide-poly (caprolactone) and the hydrophobic precursor in a dimethyl sulfoxide solvent, dissolving the hydrophilic precursor in an aqueous solvent, and encapsulating the two precursors in the nanovesicle composed of hyaluronic acid-disulfide-poly (caprolactone) by a solvent exchange method; and the copper ion is coordinated with the chemical groups on the hyaluronic acid on the surface of the vesicle by non-covalent force.

[0010] The preparation method, the molecular weight of the copolymer hyaluronic acid-disulfide-poly (caprolactone) is in the range of 10-100 kDa, and the molecular weight distribution of hyaluronic acid and poly (caprolactone) is in the range of 5-50 kDa.

[0011] The preparation method, the specific operation of the solvent exchange method is: under room temperature conditions, the stirring condition is 600-1200 rpm, and the aqueous solvent is added dropwise into the dimethyl sulfoxide solvent.

[0012] The preparation method, after encapsulating the two precursors, the nanovesicle is transferred to a dialysis bag and dialyzed in a CuCl2 aqueous solution for 1-24 hours.

[0013] The preparation method, the concentration of the CuCl2 aqueous solution is in the range of 0.1-10 mg / mL.

[0014] The nanovesicle material is used in the preparation of drugs for bio-orthogonal catalysis therapy.

[0015] The nanovesicle material is used in the preparation of drugs for treating malignant tumors.

[0016] The application, the malignant tumor includes breast cancer.

[0017] The nanovesicle material physically separates the precursors and catalyst in space, which can avoid the side reaction between the precursors and Cu 2+ from being reduced by acetylene precursors into Cu + , and generates diacetylene byproducts and uncontrollable CuAAC reactions.

[0018] The two precursors in the nanovesicle material are two parts of the original drug, and after they complete the CuAAC reaction, they have comparable drug activity compared to the original drug.

[0019] The nanovesicle material has the ability to release drugs from the inside to the outside after being pore-forming, that is, the pH / GSH environment in tumor cells can perforate the membrane surface of the nanomaterial, so that the hydrophilic precursors in the nanomaterial are preferentially released.

[0020] The hydrophilic precursors preferentially released by the nanovesicle material can be chelated with Cu + reduced by GSH in the surface layer region of the nanomaterial, thereby stabilizing the structure of Cu + and shortening the distance between the azide group and Cu + , thereby improving the efficiency of copper catalysis.

[0021] The Cu + is only chelated by the precursors, without the participation of additional ligands.

[0022] The nanoreactor in the form of a vesicle self-assembled by the polymer of the present application initiates a chelation-promoted bio-orthogonal catalysis in tumor cells, as shown in Figure 1 .

[0023] Advantages: Compared with the prior art, the present application has the following significant advantages: The multi-component isolated co-delivery system constructed based on the polymer vesicle disclosed in the present application has the following effects: (1) The hydrophilic / hydrophobic precursors and Cu 2+ are physically separated in the inner water phase, the hydrophobic layer, and the hydrophilic surface by the vesicle-like nanostructure, which can effectively avoid side reactions and premature reactions between the precursors and divalent copper; (2) The pH / GSH-driven pore-forming behavior makes the hydrophilic precursors in the nanovesicle preferentially release, chelate with the external Cu + , stabilize the structure of Cu + , and shorten the distance between the azide group and Cu + , thereby improving the efficiency of copper catalysis; (3) The co-delivery of the three substances (Azide, Alkyne, and Cu 2+ ) and the improved catalytic efficiency after the chelation of the precursors and copper significantly reduces the demand for exogenous copper, improving the biosafety of the nanovesicle. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1Schematic diagram of chelation-promoted bio-orthogonal catalysis initiated by polymer vesicle-based nanoreactor in tumor cells;

[0025] Figure 2 Physicochemical property characterization of nanovesicle PCHP under different pH, GSH or buffer environment;

[0026] Figure 3 pH / GSH-driven PCHP1 membrane perforation and inside-out programmed precursor / copper release;

[0027] Figure 4 Hydroxamic acid-accelerated CuAAC reaction and uncontrolled CuAAC reaction initiated by alkyne group;

[0028] Figure 5 In vitro cell uptake, GSH response and anti-tumor effect characterization;

[0029] Figure 6 In vivo pharmacokinetics, drug distribution and anti-tumor experiments. DETAILED DESCRIPTION

[0030] The application is further described below in conjunction with the accompanying drawings and examples.

[0031] Step 1: Synthesis of hyaluronic acid-mercapto pyridine (HA-SS-Py), taking 10 kDa hyaluronic acid as an example: under nitrogen protection, HA (10 kDa, 1.000 g, 0.10 mmol) and cysteamine (0.812 g, 10.00 mmol) were dissolved in 80 mL of deionized water containing sodium cyanoborohydride (0.772 g, 10.00 mmol), and then reacted at a temperature of 50 °C and a rotation speed of 500 rpm for 5 days. After the reaction was completed, the mixture (HA 10000 -SH and HA 10000 -SS-HA 10000 ) was purified with an ultrafiltration tube (MWCO = 1000 Da) for 3 times, and then freeze-dried (yield: 81%). Under nitrogen protection, the mixture was dissolved in a 20 mM aqueous solution of dithiothreitol (DTT), and then stirred at a temperature of 25 °C and a rotation speed of 400 rpm for 2 days. After the reaction was completed, it was ultrafiltrated (MWCO = 1000 Da) at 4 °C for 3 times, and then freeze-dried to obtain HA 10000 -SH (yield 61%). Under nitrogen protection, HA 10000-SH (0.600 g, 0.06 mmol) and 2,2′-disulfide dipyridine (0.027 g, 0.12 mmol) were added to 50 mL of deionized water. The solution was adjusted to pH 2.0 with 1.0 M HCl and stirred at 25°C and 500 rpm for 24 h. After the reaction was completed, the pH was adjusted to 7.0 with 1.0 M NaOH solution and then filtered using a 0.22 μm MCE filter membrane. The product HA 10000 -SS-Py was purified by ultrafiltration (MWCO=1000Da) three times and then lyophilized to obtain (yield: 73%).

[0032] Step 2: Synthesis of hyaluronic acid-disulfide bond-polycaprolactone (HA-SS-PCL): Under nitrogen atmosphere, HA 10000 -SS-Py (0.440 g, 0.04 mmol) and PCL 6522 -SH (0.257 g, 0.04 mmol) was dissolved in 8 mL of deionized water (containing a catalytic amount of glacial acetic acid) and 15 mL of DMF solvent, and then the deionized water solution was dripped into the DMF solvent under anaerobic conditions at a temperature of 25°C and a rotation speed of 500 rpm for 24 hours. After the reaction, the product (HA 10000 -SS-PCL 6522 ) was precipitated with ice ethanol, washed with deionized water and acetone, and dried in vacuum at 42°C overnight (yield 47%). 4000 -SS-PCL 5422 Made from hyaluronic acid with a molecular weight of 4000Da.

[0033] Step 3: Synthesis of the precursor 6-azidohexyl hydroxamic acid (Azide): Hydroxylamine hydrochloride (1.623 g, 23.36 mmol) and sodium hydroxide (1.445 g, 35.04 mmol) were added to 20 mL of methanol and reacted at 25°C for 30 minutes. Methyl 6-azidohexanoate (0.530 g, 2.94 mmol) was then added dropwise to 10 mL of the methanol solution and reacted at 25°C and 500 rpm for 30 minutes. After the reaction, the methanol was removed by rotary evaporation and the product was redissolved in 20 mL of deionized water. The pH was adjusted to 6.0, and the product was quickly extracted with ethyl acetate and then rotary evaporated. The yield was 88%.

[0034] Step 4: Preparation of polymer vesicles co-loaded with precursor and copper: 10000 -SS-PCL 6522 For example: 20mg of HA 10000 -SS-PCL 6522and 5.0 mg of 2-ethynylquinoline (Alkyne) were dissolved in 5 mL of dimethyl sulfoxide (DMSO). Then, 5 mL of deionized water containing 5.0 mg of Azide was added into the DMSO phase with vigorous stirring at 800 rpm for 20 min. Subsequently, the mixed solvents were moved into a dialysis bag (MWCO 8-14 kDa) and dialyzed in deionized water for 24 h, after which the polymerized vesicles (PHP1) loaded with precursors (Azide and Alkyne) were collected. PCHP1 was obtained after dialyzing the dialysis bag containing PHP1 in 1 mg / mL CuCl2solution for 3 h, followed by dialysis in deionized water for 48 h to remove excess CuCl2. The preparation process is shown in Figure 2 A.

[0035] PHP2 and PCHP2 were prepared according to the above steps, except that HA 10000 -SS-PCL 6522 was changed to HA 4000 -SS-PCL 6522 .

[0036] Physicochemical properties of PCHP1 and PCHP2. PCHP1 and PCHP2 were characterized physically and chemically, including particle size, polydispersity index (PDI), zeta potential, storage and recycling stability, and pH / GSH response of the polymer. The polymers were dispersed in 0.01 M phosphate buffer (PB) under different conditions (pH 7.4, pH 6.5, pH 5.0, and pH 5.0 / GSH) and 10% fetal bovine serum (FBS), respectively. The particle size and PDI Figure 2 B, 2C), zeta potential Figure 2 D) were recorded at predetermined time intervals by DLS. The morphology, type, and distribution of the micro-area elements were observed by transmission electron microscopy (TEM). The results are shown in Figure 2 E, which show that PCHP1 has smaller particle size changes in PBS and FBS, exhibiting higher storage stability. In addition, the results are shown in Figure 2 F and 2G, which show that PCHP1 can respond to the stimulation of GSH and has a more sensitive acid response ability. Figure 2 H, which show that copper is loaded on the outer layer of PCHP1, achieving spatial separation of the catalyst copper and the precursors.

[0037] In vitro copper and precursor release from PCHP1. Drug release from PCHP1 was determined by dialysis. Briefly, 1 mL of a PCHP1 solution containing 6.54 μmol Azide, 6.00 μmol Alkyne, and 0.31 μmol Cu(II) was placed in a dialysis bag (MWCO 8-14 kDa) and then placed in four PB conditions (5 mL, pH 7.4, pH 6.5, pH 5.0, and pH 5.0 / GSH) in a thermostatic shaker (37°C, 100 rpm). 5 mL of dialysate was collected and replaced with 5 mL of fresh PB at predetermined time intervals. The degradation of PCHP1 was followed by transmission electron microscopy (TEM), and the release of precursor and Cu(II) was measured by HPLC and ICP-OES.

[0038] like Figure 3 As shown, TEM images demonstrate that at pH 5.0, the PCHP1 membrane undergoes perforation. The addition of GSH to pH 5.0 accelerates this membrane perforation process, leading to complete disaggregation of PCHP1. Drug release experiments revealed that the hydrophilic precursor, Azide, exhibited a significantly higher release rate within 6 hours than the surface copper ions and the hydrophobic precursor, Alkyne. Therefore, PCHP1 membrane perforation in the tumor cell microenvironment preferentially releases Azide from the inner aqueous phase through concentration diffusion. Subsequently, driven by GSH, disaggregated polymersomes gradually release copper ions and Alkyne. These data demonstrate that PCHP1 possesses programmed drug release capabilities.

[0039] Determination of Copper(I) Chelates. Mass spectrometry confirmed the presence of Cu(I)-azide and Cu(I)-SAHA-D complexes. Methyl 6-azidohexanoate (without the hydroxamic acid moiety) and Azide (with the hydroxamic acid moiety) were added at equimolar concentrations (20 mM) to 1 mL of a mixed solvent (CH3CN:PBS = 1:5, v / v) containing 5 mol% Cu(II) and 10 mM GSH. The mixture was stirred at 500 rpm for 1 hour at 25°C and used as the sample. Additionally, Azide (6.6 mM), Alkyne (6.0 mM), Cu(II) (0.3 mM), and glutathione (10.0 mM) were dissolved in 1 mL of a mixed solvent (CH3CN:PBS = 1:5, v / v) and stirred at 500 rpm for 1 hour at 25°C and used as the sample. All samples were analyzed by MODI-TOF mass spectrometry.

[0040] In vitro kinetic analysis of CuAAC reaction with or without hydroxamic acid fragment. 22 mM of Azide, 20 mM of Alkyne, 100 mM of NaAsc and 1 mM of CuCl2 were reacted in 0.5 mL of mixed solvent (9D2O:DMSO-d6=3:2, v / v) in 2 mL centrifuge tube at 37 °C with 900 rpm rotation. Each mixture was detected at pre-set time period (0, 10, 30, 60, 360 min) 1 H NMR spectra. The yield of CuAAC reaction was calculated by the ratio of integral area of characteristic peaks.

[0041] CuAAC reaction with or without glutathione in vitro. GSH group was incubated PCHP1 in 0.01 M phosphate buffered saline (PBS) with 10 mM GSH at 25 °C with 300 rpm stirring for pre-set time. In GSH-free group, precursors (Azide+Alkyne), precursors / Cu(II) and PCHP1 were incubated in mixed solvent (CH3CN:PBS=1:5, v / v) at 25 °C with 300 rpm stirring for pre-set time. The molar ratio of Azide, Alkyne, CuCl2 was 1.1:1:0.05. All samples were detected by MODI-TOF MS with DHB as matrix and analyzed by HPLC (C18 column (25 cm x 0.46 cm, Syncronis TM ), eluted with 60% CH3CN and 40% water at a flow rate of 0.8 mL / min).

[0042] As shown in Figure 4 A-C, the fragment peaks of Cu(I)-Azide complex and Cu(I)-SAHA-D complex were found by mass spectrometry, which indicated that the precursors and their products could stabilize copper ions as chelate structure. The effect of hydroxamic acid on catalytic rate was tracked by hydrogen nuclear magnetic resonance spectroscopy. As shown in Figure 4 D-F, when the reaction group contained precursors with hydroxamic acid fragment, higher yield was maintained at different time periods. The results showed that the catalytic efficiency of Cu + was improved by about two times after chelation with hydroxamic acid. The data of mass spectrometry and hydrogen spectrum showed the possibility that the chelation of precursors with copper ions could accelerate the CuAAC reaction rate. In addition, as shown in G and H, the product SAHA-D was detected by mass spectrometry after mixing of precursors and Cu 2+ , which was attributed to the redox side reaction between Cu 2+ and Alkyne, and the CuAAC reaction was induced by Cu ion reduced by alkyne group. PCHP1 prevented the occurrence of side reaction because it spatially separated the precursors and copper ions.

[0043] Coumarin 6 / Rhodamine B@HA 10000 -SS-PCL 6522 (CRHP) and GSH tracer Cu@HA 10000 -SS-PCL 6522 Preparation of (GCHP). CRHP and GCHP were prepared by solvent exchange method. Briefly, 20 mg of HA 10000 -SS-PCL 6522 and 5.0 mg of coumarin 6 were dissolved in 5 mL of DMSO. Then, 5 mL of Milli Q water containing 5.0 mg of rhodamine B was added dropwise to the DMSO phase under vigorous stirring at 800 rpm for 20 min. The solution was transferred to a dialysis bag (MWCO 8-14 kDa) and dialyzed against deionized water for 24 h, and the CRHP was collected. GCHP was prepared according to the PCHP1 preparation protocol using 20 mg of HA 10000 -SS-PCL 6522 , 5.0 mg of GSH tracer, CuCl2solution (1 mg / mL). The content of coumarin 6 (λexc= 450 nm, λem= 505 nm), rhodamine B (λexc= 553 nm, λem= 610 nm) and gsh tracer (λexc= 520 nm, λem= 580 nm) in CRHP and GCHP was determined by fluorescence spectrometer.

[0044] Cellular uptake of CRHP and GCHP. MCF-7, 4T1 and 293T cells were seeded in 35 mm confocal dishes (1 x 10 5 MCF-7 and 4T1 cells were treated with CRHP for 1 h and 4 h, respectively, washed twice with cold PBS, fixed with paraformaldehyde (4%, v:v) for 10 min, stained with DAPI for 6 min, washed three times with cold PBS, and then observed by confocal laser scanning microscopy (CLSM). 293T, MCF-7 and 4T1 cells were treated with GCHP for 2 h, and the same pre-staining procedure as described above was performed before CLSM imaging.

[0045] Cytotoxicity test. Cell relative viability was determined by cell titer-fluorescence (CTG) assay. MCF-7, 4T1 and 293T cells were seeded in 96-well cell culture plates (5 x 10 3The cells were cultured in a 400 μM flask (100 μL / well) for 24 hours. The old culture medium was then removed and fresh culture medium containing various concentrations (0-25 μM) of drugs (Azide, Alkyne, SAHA, SAHA-D, Precursors (Azide + Alkyne), Precursors + Cu(II), and PCHP1) was added. After 48 hours of incubation, 20 μL of CTG solution was added to each well and the cells were allowed to equilibrate at room temperature for 10 minutes. The luminescence intensity was measured using a microplate reader. The relative cell viability was calculated as follows:

[0046] Relative activity (%) = (Lum treat -Lum blank ) / (Lum control -Lum blank )×100%

[0047] like Figure 5 As shown, laser confocal imaging demonstrates that CRHP polymersomes loaded with both hydrophobic coumarin 6 and hydrophilic rhodamine B can rapidly and time-dependently accumulate in tumor cells, owing to the targeting of HA to CD44 overexpressed on the tumor cell membrane. GCHP loaded with a GSH tracer is not activated in normal cells but is abundantly activated in tumor cells, indicating that polymersomes tend to aggregate, degrade, and release drugs in a high-GSH environment. Cytotoxicity experiments revealed that PCHP1 has a significant inhibitory effect on tumor cells while showing no significant toxicity to normal cells.

[0048] Pharmacokinetics and Biodistribution. 4T1 tumor-bearing mice were randomly divided into two groups (n = 3) and intravenously injected with the precursor plus Cu(II) and PCHP1 (Azide = 190 μg, Alkyne = 150 μg, Cu(II) = 3 μg). Blood samples were collected at 0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h, and tumor samples were collected at 3 h, 12 h, and 24 h. Tumor tissues were lysed by RIPA, homogenized, extracted with acetonitrile, and filtered. The contents of Azide, Alkyne, and SAHA-D in each sample were analyzed by high-performance liquid chromatography.

[0049] In vivo tumor inhibition. 4T1 tumor-bearing mice were randomly divided into 5 groups (n=6) and intravenously injected with normal saline (control group), precursor (Azide+Alkyne), SAHA-D, precursor+Cu(II) and PCHP1 (Azide=1.9mg / kg, Alkyne=1.5mg / kg, SAHA-D=3.1mg / kg, Cu(II)=30μg / kg) on ​​days 0, 3, 6 and 9, respectively. Tumor volume and body weight were monitored during treatment. When the average tumor volume of the control group reached ~1500mm3 (day 12), the mice were sacrificed and the tumors were recorded. Afterwards, the tumors were weighed and photographed.Figure 6 .

[0050] As shown in Figure 6 pharmacokinetics and biodistribution experiments, the co-delivery ability of PCHP1 makes the drug half-lives of Azide and Alkyne similar and prolonged, and the tumor specificity of PCHP1 makes the precursors highly enriched at tumor sites. In addition, due to the chelation-promoting effect of the precursors, the Cu + catalyzed reaction proceeds rapidly, and the generation rate of SAHA-D increases from 0.74 μg / h to 1.46 μg / h. The efficient co-delivery of precursors and copper ions, as well as the chelation-first and catalysis-second behavior controlled by the programmed release of PCHP1, realizes efficient bio-orthogonal catalytic therapy in situ at the tumor, making this nanotherapy have a low demand for copper ions, reducing the biological safety risk brought by exogenous copper. At this time, the catalytic efficiency after chelation promotion is normalized by the total dosage of copper ions (0.1 mg / kg), and the results show that the catalytic efficiency per milligram of copper ions in a kilogram of mice is increased by 2800%. These data show that the excellent therapeutic effect of PCHP1 benefits from reliable co-delivery of precursor / copper separation, tumor-specific distribution, and accelerated bio-orthogonal catalysis in tumor cells.

Claims

1. A nanovesicle material formed by an acid / reduction-sensitive linear copolymer, characterized in that: The linear block copolymer is obtained by coupling two blocks of hyaluronic acid and polycaprolactone via a disulfide bond; The nanovesicles formed by the self-assembly of the copolymer can transport 6-azidohexylhydroxamic acid, 2-ethynylquinoline and divalent copper ion Cu 2+ Physically separated into an inner aqueous phase, a hydrophobic layer, and an outer surface of the nanovesicle; the nanovesicle responds to tumor microenvironment conditions and programmedly releases the two loaded precursors and copper ions; the molecular weight distribution of hyaluronic acid and polycaprolactone ranges from 5-50 kDa.

2. The nanovesicle material according to claim 1, characterized in that The in vivo blood circulation time T of the nanovesicle material 1 / 2 For more than 8 hours, a Cu(I)-Azide chelate complex is formed in situ in the tumor, promoting bioorthogonal catalytic drug synthesis.

3. A method for preparing the nanovesicle material according to claim 1, characterized in that: The following steps are involved: The amphiphilic copolymer hyaluronic acid-disulfide bond-polycaprolactone and the hydrophobic precursor are dissolved in dimethyl sulfoxide solvent, and the hydrophilic precursor is dissolved in water solvent. The two precursors are encapsulated in polymer vesicles composed of hyaluronic acid-disulfide bond-polycaprolactone through a solvent conversion method; copper ions are coordinated with chemical groups on the hyaluronic acid on the surface of the vesicles through non-covalent forces.

4. The preparation method according to claim 3, characterized in that The molecular weight of the copolymer hyaluronic acid-disulfide bond-polycaprolactone is in the range of 10-100 kDa.

5. The preparation method according to claim 3, characterized in that The specific operation of the solvent conversion method is: under room temperature conditions, stirring conditions are 600-1200 rpm, and water solvent is added dropwise to dimethyl sulfoxide solvent.

6. The preparation method according to claim 3, characterized in that The polymer vesicles after encapsulating the two precursors were transferred to a dialysis bag and dialyzed in a CuCl2 aqueous solution for 1-24 hours.

7. The preparation method according to claim 6, characterized in that The concentration range of CuCl2 aqueous solution is 0.1-10 mg / mL.

8. Use of the nanovesicle material according to claim 1 in preparing drugs in the field of bioorthogonal catalytic therapy.

9. Use of the nanovesicle material according to claim 1 in preparing drugs for treating malignant tumors.

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