WO2022253342A1 - 一种小胶束纳米药物及其制备方法与应用 - Google Patents

一种小胶束纳米药物及其制备方法与应用 Download PDF

Info

Publication number
WO2022253342A1
WO2022253342A1 PCT/CN2022/097029 CN2022097029W WO2022253342A1 WO 2022253342 A1 WO2022253342 A1 WO 2022253342A1 CN 2022097029 W CN2022097029 W CN 2022097029W WO 2022253342 A1 WO2022253342 A1 WO 2022253342A1
Authority
WO
WIPO (PCT)
Prior art keywords
small
mptx
ptx
medicine
micelles
Prior art date
Application number
PCT/CN2022/097029
Other languages
English (en)
French (fr)
Inventor
孟凤华
邱欣昀
郭贝贝
钟志远
Original Assignee
苏州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州大学 filed Critical 苏州大学
Publication of WO2022253342A1 publication Critical patent/WO2022253342A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the invention belongs to nano-medicine technology, in particular to a small micelle nano-medicine and its preparation method and application.
  • paclitaxel is a broad-spectrum anti-tumor taxane drug that acts on tubulin, and is used for the first-line or second-line treatment of various tumors.
  • PTX has extremely low water solubility and low bioavailability, so it cannot be used directly; its Taxol, which uses CremophorEL and absolute ethanol as solubilizers, is approved for the treatment of various tumors such as breast cancer, ovarian cancer, and non-small cell lung cancer.
  • CremophorEL can cause severe toxic side effects, and has no selectivity for normal cells and cancer cells.
  • nanoscale drug carriers such as liposomes, albumin, polymer micelles and nanoparticles, among which polymer micelles have better drug loading performance, circulation stability and release effect, but For PTX, the particle size of existing nanomicelle drugs is too large.
  • the invention discloses a small micelle nanomedicine and its preparation method and application, and prepares nanomedicine ATN1-MPTX, ATN2-MPTX and cRGD-MPTX coupled with PHSCNK (ATN1), PhScNK (ATN2) or cRGD polypeptide.
  • ATN2-MPTX can efficiently target and enrich the mouse 4T1 breast cancer tumors, increase the concentration of PTX in tumor cells, effectively delay the growth of mouse tumors, inhibit the lung metastasis of breast cancer, and prolong the survival time of small tumors. Rat lifespan.
  • the present invention adopts the following technical scheme: a small micellar nanomedicine, the preparation method of which is to add small molecular medicines and amphiphilic polymers into oligoethylene glycol to obtain a mixed solution, and then add the mixed solution to buffer In the solution, small micellar nanomedicines are obtained.
  • a targeted small micellar nanomedicine the preparation method of which is to add small molecule drugs, amphiphilic polymers, and targeted amphiphilic polymers into oligoethylene glycol to obtain a mixed solution, and then mix the The solution is added into the buffer solution to obtain the targeted small micellar nanomedicine.
  • the molecular weight ( M n ) of the oligoethylene glycol is 200-600, preferably 300-500; the molecular weight of the amphiphilic polymer is 2000-15000, and the molecular weight of the targeted amphiphilic polymer is 2000 ⁇ 15000.
  • the molecular weight of the polymer of the present invention is the number average molecular weight ( M n ) determined by NMR, and the unit is Da.
  • the small molecule drug includes paclitaxel PTX and the like.
  • a small micelle nano-medicine freezing solution the preparation method of which is to freeze the above-mentioned small micelle nano-medicine or targeted small micelle nano-medicine in liquid nitrogen and store it in a refrigerator to obtain a small micelle nano-medicine freezing solution;
  • the temperature of the refrigerator is -80°C to -10°C.
  • a small micelle nano drug freeze-dried powder the preparation method of which is to mix the above small micelle nano drug or targeting small micelle nano drug with a lyoprotectant, freeze in liquid nitrogen, and then use a lyophilizer to freeze dry to obtain the lyophilized powder of small micellar nano-medicine.
  • Lyoprotectants are preferably sucrose and mannitol.
  • the amphiphilic polymer is PEG-P (CL-DTC), PEG-P (TMC-DTC), PEG-P (LA-DTC), etc.; the targeted amphiphilic polymer is the amphiphilic polymer
  • the targeting molecule is coupled to the affinity polymer.
  • the targeting molecule is a polypeptide, such as a PHSCNK polypeptide, a PhScNK polypeptide or a cRGD polypeptide.
  • the molecular weight ( M n ) of PEG is 1000-5000 Da.
  • the mixed solution under standing, the mixed solution is injected into the buffer solution, and then stirred, placed, vortexed, blown or inverted; injected is a prior art, such as using a syringe or a syringe pump; the stirring speed 100-1000rpm.
  • the concentration of the small molecule drug is 1 to 10 mg/mL
  • the concentration of the polymer is 1 to 100 mg/mL
  • the polymer is an amphiphilic polymer, or the polymer is an amphiphilic polymer and targets Amphiphilic polymers.
  • the dosage of the targeted amphiphilic polymer is 1-30% of the weight of the amphiphilic polymer, preferably 2.5-10%.
  • the amphiphilic polymer is not coupled with targeting molecules, and the targeting amphiphilic polymer is coupled with targeting molecules.
  • the volume ratio of oligoethylene glycol and buffer solution is 1: (5-40), preferably 1: (10-30).
  • the present invention has prepared micelles with high PTX drug loading, and the prepared small particle size micelles (20-40 nm) can penetrate deep into the tumor.
  • the present invention discloses the application of the above-mentioned small micelle nano-medicine or targeted small micelle nano-medicine in the preparation of anti-tumor drugs, preferably the tumor is triple-negative breast cancer (TNBC).
  • TNBC triple-negative breast cancer
  • the present invention designs and prepares high-efficiency PTX-loaded, disulfide-crosslinked micellar MPTX and three kinds of micellar nanomedicines actively targeting TNBC. Good stability, reduction-responsive drug release properties.
  • ATN2-MPTX has the strongest targeted endocytosis effect on 4T1 cells, the lowest IC 50 value, and the highest biodistribution in tumors.
  • ATN2-MPTX can induce ICD, promote the proliferation and maturation of BMDC, promote the polarization of BMDM to M1 macrophages, and create a favorable immune microenvironment. It can better inhibit the growth of TNBC in mice and the occurrence of lung metastasis, and prolong the survival period of mice, which reflects the advantages of treatment and brings hope for the treatment of highly metastatic tumors.
  • the main advantages of the present invention are as follows: 1)
  • the nano-carrier has a high surface area/volume ratio, which is beneficial to improve the solubility and loading efficiency of hydrophobic drugs such as PTX; 3) Nano-drugs at 20-40 nanometers can be enriched in tumor tissue through the enhanced permeability and retention (EPR) effect; 4) Nano-carriers are highly designable, and can be stimulated by designing the tumor microenvironment Responsive carriers and nanocarriers for active tumor targeting facilitate the controlled release of drugs and improve specificity.
  • Figure 1 shows the preparation and physicochemical properties of MPTX and ATN2-MPTX micelles (drug loading 4.8 wt. %).
  • A Schematic diagram of the preparation process of micelles;
  • B The particle size distribution of MPTX and ATN2-MPTX and the TEM schematic diagram of MPTX;
  • C MPTX with a drug loading of 23.1 wt.
  • Figure 2 shows the particle size distribution of MPTX and ATN2-MPTX after incubation in 10% FBS at 37 degrees for 24 h (A), and the particle size distribution after incubation in 10 mM DTT for 2 h (B); freezing solution-thawing and freezing Dry powder-reconstituted particle size distribution (C).
  • Figure 4 shows the endocytosis of Cy5-labeled MPTX, AT1-MPTX, ATN2-MPTX and cRGD-MPTX by 4T1 cells (incubated for 4 h) by flow cytometry (A) and CLSM (B&C).
  • B Semi-quantitative fluorescence intensity for Cy5.
  • PhScNK polypeptide ATN2 was used to incubate 4T1 cells for 2 h before adding Cy5/ATN2-MPTX.
  • the Cy5 polymer concentration was 2 mg/mL.
  • the ruler is 25 mm.
  • Figure 5 shows the pharmacokinetic curves of cRGD-MPTX, ATN2-MPTX, MPTX and PTX in healthy Balb/c mice (7.5 mg PTX/kg).
  • Figure 6 shows the in vivo fluorescence imaging of Cy5/MPTX, Cy5/ATN2-MPTX and Cy5/cRGD-MPTX over time after intravenous administration in tumor-bearing mice (A), fluorescence quantitative analysis of tumor sites (B) and administration for 4 h
  • Ex vivo fluorescence imaging of posterior major organs and tumors C).
  • the enrichment of PTX in major organs and tumors after intravenous administration of MPTX, ATN2-MPTX, cRGD-MPTX and PTX for 4 h (D) and the ratio of PTX in tumors and viscera (T/N) E).
  • n 3.
  • Figure 7 is the ICD of 4T1 cells induced by MPTX, ATN2-MPTX and free PTX.
  • A CLSM observation of CRT on the cell surface, the scale bar is 25 mm;
  • Figure 8 is the proliferation of BMDCs in ATN2-MPTX, MPTX and free PTX after incubation for 24 h (A) and 48 h (B);
  • (C) and (D) are the curves of (A) and (B) respectively PTX low (0.04 mg/mL), moderate (1.25 mg/mL) and high (20 mg/mL) concentrations of BMDC proliferation.
  • n 5.
  • A Flow cytometry for the proportion of CD11c + total BMDCs and
  • B the proportion of CD11c + CD80 + CD86 + mature BMDCs. Concentrations of secreted (C) TNF- ⁇ and (D) IL-6 in the medium.
  • BMDM was first incubated with IL-4 (20 ng/mL) for 48 h, and then mixed with PBS, IL-4 (20 ng/mL), LPS (100 ng/mL), free PTX, MPTX and ATN2-MPTX (PTX concentration is 5 mg/mL) and incubated for 24 h.
  • TNF- ⁇ C and IL-6
  • D Concentrations of TNF- ⁇ (C) and IL-6 (D) in culture medium after stimulation with PTX preparations.
  • A Flow cytometry false-color plots of representative samples of BMDC (within the CD11c + total BMDC gate) and their statistical analysis (B). Concentrations of (C) IL-6 and (D) TNF- ⁇ in culture medium.
  • A Representative flow cytometry analysis and
  • B proportion of CD3 + CD4 + T cells (in CD45 + ring gate) in tumors.
  • C Typical flow cytometry analysis and
  • D proportion of CD3 + CD8 + T cells (in CD45 + ring gate) in tumors.
  • E Content of Tregs (CD3 + CD4 + FoxP3 + , CD45 + ) in CD4 + T cells and
  • F ratio of CD8 + T/Tregs.
  • Figure 14 is the flow cytometric analysis of the mouse spleen 24 hours after ATN2-MPTX administration.
  • A Representative flow cytometry false-color plots of CD3 + CD4 + , CD3 + CD8 + T cells (within the CD45 + total immune cell gate) and
  • B CD3 + CD4 + and
  • Figure 16 is the H&E staining images of heart, liver, kidney, and spleen sections of tumor-bearing mice treated with PBS, free PTX, MPTX, and ATN2-MPTX for 22 days (40 ⁇ , the bar is 50 mm).
  • WP white pulp
  • RP red pulp.
  • Figure 18 is the in vitro imaging (A), semi-quantitative fluorescence value (B) and mass (C) of the lungs of tumor-bearing mice after 22 days of administration.
  • ⁇ -caprolactone ( ⁇ -CL, 99%, Alfa Aesar) was dried over calcium hydride and distilled under reduced pressure for use.
  • 1,2-Dithiolane trimethylene carbonate (DTC) was synthesized and purified by our laboratory.
  • c(RGDfC) cyclic peptide (Arg-Gly-Asp-D-Phe-Cys)
  • Ac-PHSCNK-NH 2 ATN-1
  • Ac-PhScNK-NH 2 ATN-2
  • HPLC high performance liquid chromatography
  • Micro BCA Kit (Pierce, Thermo Scientific), Enhanced ATP Detection Kit (S0027, Beyond), mouse high mobility group box B1 (HMGB-1) (E-EL-M0676c, Elabscience), mouse white blood cells Interferon-6 (IL-6) ELISA kit (JN-029562-S, Shanghai Research Field), mouse interferon- , Interferon- (IFN- , IFN- ) and cell necrosis factor- (TNF- ) ELISA detection kit (Invivogen), calreticulin (CRT) antibody (Ab2907, Abcam), and mouse fluorescently labeled various antibodies (Biolegend) CD45-PerCP/Cyanine5.5, CD80-APC, CD86-PE, Kits and antibodies such as CD11c-FITC, CD11b-FITC, CD206-Alexa Fluor 647, F4/80-PE, CD4-PE, CD8-FITC, FoxP3-Alexa Fluor 647 and goat anti-rabbit-Alexa
  • the raw materials of the present invention are all existing products, and the specific operation method and test method are conventional methods in this field. All data of the present invention are all presented with average value, and intergroup difference is assessed by ANOVA single-factor analysis of variance, and *p ⁇ 0.05 is considered to have significant difference, **p ⁇ 0.01 and ***p ⁇ 0.001 were considered highly significant differences.
  • MeO-PEG-OH 800 mg, 0.4 mmol
  • DPP 1.0 g, 4 mmol
  • DTC 400 mg, 2.1 mmol
  • DTC 400 mg, 2.1 mmol
  • CL 400 mg, 3.5 mmol
  • anhydrous DCM 3.2 mL
  • the PEG-P (CL-DTC) is calculated according to the integral ratio of the characteristic peaks of PEG and DTC ( ⁇ 3.00), CL ( ⁇ 1.04 and 2.31)
  • the molecular weight is 2k-0.9k-1.1k
  • the molecular weight measured by GPC is 7.1 kg/mol
  • the molecular weight distribution is 1.2 (Table 1).
  • ATN-PEG-P(CL-DTC) is obtained by amidation reaction between PHSCNK or PhScNK polypeptide and NHS-PEG-P(CL-DTC). Briefly, NHS-PEG-P(CL-DTC) (132 mg, 0.024 mmol) was dissolved in 1 mL of anhydrous DMSO, and PHSCNK peptide or PhScNK peptide (20 mg, 0.028 mmol) was dissolved in 0.5 mL of anhydrous DMSO.
  • the polymer solution was added dropwise to the polypeptide solution at 30 °C, and after 30 min of dropping, 7 mL of triethylamine was added to adjust the pH of the system to 8.0, and the reactor was sealed and reacted at 30 °C for 48 h.
  • the reaction solution was put into a dialysis bag (MWCO 3500 Da) and dialyzed in DMSO for 4 h, DCM for 2 h, and then precipitated twice in 30 times volume of ice ether/absolute ethanol (9/1, v/v) , centrifuged and vacuum-dried for 24 h to obtain ATN-PEG-P(CL-DTC).
  • the spectra obtained by 1 H NMR (600 MHz, DMSO- d 6 ) can be used to analyze the polymer structure and molecular weight, and the functionalization degree of the polypeptide can be calculated by using the Micro BCA protein kit.
  • the response is as follows: .
  • the NHS characteristic peak ( ⁇ 2.59) appeared in its NMR image, and the ratio of its integral value to the main peak of PEG was close to the theoretical value.
  • two different configurations of polypeptides PHSCNK (ATN1) and PhScNK (ATN2) were reacted through the amidation reaction of the primary amine of the C-terminal lysine and the NHS end group of the polymer to obtain ATN1-PEG-P(CL- DTC) and ATN2-PEG-P (CL-DTC).
  • PhScN is the D-type (D-His and D-Cys) isomers of histidine and cysteine in PHSCN.
  • a lysine K is introduced at the C-terminus, which can efficiently bond the polypeptide to the end of the polymer through its primary amine.
  • the present invention studies the effects of two polypeptides on the endocytosis of micelles. Taking the H NMR image of ATN2-PEG-P (CL-DTC) as an example, it was found that the characteristic peaks of NHS became significantly smaller, and the characteristic peaks of PhScNK ( ⁇ 1.7-2.1 and ⁇ 7.0-7.1) appeared. The content of polymer polypeptide was determined by Micro BCA microprotein kit, and the functionalization degrees of PhScNK and PHSCNK were calculated to be 71.2% and 79.6%, respectively.
  • cRGD-PEG-P(CL-DTC) is prepared from the sulfhydryl group of c(RGDfC) polypeptide and Mal-PEG-P(CL-DTC) through Michael addition reaction. The steps, purification and characterization of the polymer are the same as above. The response is as follows: .
  • the functionalization degree of cRGD was calculated to be about 89.2%; the amount of polypeptide contained in the polymer was tested with Micro BCA reagent, and the functionalization degree of cRGD was about 86.6% %.
  • PTX and PEG 2k -P(CL 1k -DTC 1k ) were dissolved in PEG 350 according to the mass ratio (1/20), and the polymer concentration was kept at 1 mg/mL.
  • 100 mL of the mixed solution was poured into the bottom of 900 mL of PB solution (pH 7.4, 10 mM) at 37 °C, and stood without stirring during the process.
  • PB solution pH 7.4, 10 mM
  • PTX drug-loaded micelles which are non-targeting small micellar nanomedicines.
  • the particle size is 27.8nm and the particle size distribution (PDI) is 0.18 as determined by DLS.
  • Example 1 Preparation and characterization of PTX micelles: PTX and PEG 2k -P(CL 1k -DTC 1k ) were dissolved in PEG 350 according to different mass ratios (5/100, 10/100, 20/100, 30/100) , where the polymer concentration was 50 mg/mL. After conventional ultrasonication for 5 min, 100 mL of the mixed solution was poured into the bottom of 900 mL of PB solution (pH 7.4, 10 mM) at 37 °C, and stood without stirring during the process. After injection, use a pipette gun to stick to the liquid surface and pipette 5 times to obtain micellar MPTX with a theoretical PTX drug loading of 4.8 wt.
  • PB solution pH 7.4, 10 mM
  • % to 23.1 wt. % which is a non-targeting small micellar nano drug.
  • the particle size and particle size distribution (PDI) were determined by DLS, and the micelle morphology was determined by TEM. MPTX was dissolved in acetonitrile containing 20 mM DTT (the polymer concentration was 0.1 mg/mL), and then the PTX concentration was tested by HPLC, and the drug loading and drug loading efficiency were calculated. DLS was used to monitor the particle size and particle size distribution of PTX micelles stored at 25 and 37°C, diluted to a low concentration (20 mg/mL) and in a solution containing 10% FBS at different time points, see Table 2.
  • PEG-PCL-based PTX-loaded micelles were also prepared by the same method.
  • the drug loading capacity of PTX nanocarriers reported in most literatures in the prior art is relatively low.
  • the present invention utilizes a small amount of PEG350 to dissolve the polymer PEG-P (CL-DTC) and PTX, and the mixture is added to the water phase (the final volume content of PEG350 is 10%), and the polymer forms the micelles of the core-shell structure through the hydrophobic interaction.
  • PTX is encapsulated in the hydrophobic core, and DTC in the core can quickly self-crosslink, which can fix PTX in the micelle core more stably, and obtain the PTX micelle nano drug MPTX (Fig. 1 A).
  • the PEG350 used here is non-toxic and FDA-approved for use.
  • the present invention prepared micellar MPTX with drug loading from 4.8 wt. % to 23.1 wt. % by this method, with a particle size range of 30-38 nm and a particle size distribution of 0.13-0.17 (Table 2).
  • DLS test results showed that MPTX loaded with 4.8 wt. % had a particle size of 32.7 nm and a PDI of 0.16, and the TEM image showed that it had a more regular shape of solid spheres (Fig. 1B).
  • MPTX micelles remained clear and transparent, with a particle size of 38.1 nm and a PDI of 0.13 (Table 2).
  • micellar nanomedicine Due to the low water solubility of PTX (water solubility is 5.56 mg/L), the same amount of PTX was dissolved in PEG350 and directly added to water, PTX precipitated rapidly, while MPTX loaded with 23.1 wt. % PTX remained clear, indicating that PTX was completely Loaded inside the micelles (Fig. 1C).
  • Using this method to prepare micellar nanomedicine is simple, reproducible, and the stability of the micelles is good. After three weeks of storage at room temperature, the particle size and particle size distribution of the micelles are basically unchanged ( Figure 1 D), and there is no precipitation.
  • MPTX prepared by the present invention has a higher drug loading than liposomes, most nanocarriers and Genexol-PM (PTX drug loading 10 wt. %) reported in the literature.
  • the effect of DTC in the polymer on the drug loading and stability of the formed PTX micelles was studied.
  • the PEG-PCL-loaded PTX uncrosslinked micelles ncMPTX and PEG-P(CL -DTC) (MPTX), half of which was stored at room temperature and half of which was dialyzed.
  • the particle size and PDI changes before and after dialysis and the changes of PTX drug loading in the two groups were measured.
  • the 330 nm ultraviolet absorption value of the dithiolane of DTC in the nanoparticles decreased a lot, indicating that the sulfur-sulfur cross-linking reaction occurred to obtain cross-linked nanoparticles.
  • the present invention firstly measures the ultraviolet absorption of two kinds of micelles and corresponding polymer solution (5 mg/mL) with the same concentration, and finds that the PEG-PCL polymer solution and its micellar ncMs have no obvious absorption peak.
  • the DTC in Ms showed the ultraviolet absorption peak of dithiolane at 326 nm, but its intensity was lower than that of DTC in the corresponding polymer solution (Fig. 1 E), which decreased by 40.7%.
  • micellar MPTX based on PEG-P(CL-DTC) of the present invention is much more stable than Genexol-PM based on PEG-PDLLA with much higher Tg .
  • Table 2 The particle size of MPTX with different drug loadings and the particle size after reconstitution of frozen liquid/lyophilized powder.
  • Table 3 Changes in particle size and drug loading of MPTX before and after dialysis.
  • MPTX 50 mg/mL, 1 mL
  • GSH reduced glutathione
  • DLS DLS monitors changes in micelle size and PDI.
  • ATN2-MPTX 50 mg/mL, 1 mL was packed into dialysis bags (MWCO 14 kDa) respectively, soaked in 25 mL of PB with and without 10 mM GSH solution (pH 7.4, 10 mM, containing 0.1% Tween 80) and incubated at 37oC, 200 rpm on a shaker.
  • micellar frozen liquid and freeze-dried powder were prepared.
  • the freezing liquid is the PTX micelles (1 mg/mL, 1 mL) glass vials were frozen in liquid nitrogen for 10 minutes, transferred to a -20°C refrigerator for storage, and thawed at room temperature before use.
  • the lyophilized powder is to add 110 mL of lyoprotectant mixed with sucrose and mannitol (1/1, 80 mg/mL) into PTX micelles (4 mg/mL, 200 mL), mix well and immerse in Freeze in liquid nitrogen for 10 minutes, then freeze-dry for 24 hours with a lyophilizer, and reconstitute with 800 mL of secondary water before use to obtain 1 mg/mL PTX micelles (the protective agent accounts for 1.1% w/v).
  • the changes in particle size and PDI of PTX micelles before freezing/lyophilization and after thawing/reconstitution were measured, and the changes in drug loading were measured by HPLC.
  • the micelles were thawed/reconstituted and tested with DLS.
  • PTX-loaded targeting micelles are composed of amphiphilic block polymer PEG 2k -P(CL 1k -DTC 1k ) and polymers coupled with targeting molecules Ta-PEG-P(CL-DTC) (Ta is the polypeptide cRGD, ATN1 or ATN2) self-assembles in the aqueous phase.
  • ATN1-PEG-P (CL-DTC), ATN2-PEG-P (CL -DTC) or cRGD-PEG-P (CL-DTC) as the initial polymer solution, then mixed with the PTX solution, all the other steps are the same as in Example 1, to obtain micelles ATN1-MPTX, ATN2-MPTX of different polypeptide surface densities and cRGD-MPTX, targeting small micellar nanomedicines.
  • targeting micelles were prepared, and the same method as in Example 1 was used to keep the PTX drug loading at 4.8 wt. %.
  • Three series of micellar cRGD-MPTX, ATN1-MPTX and ATN2-MPTX with different targeting molecule densities were prepared respectively.
  • the results of DLS test showed that when the surface peptides did not exceed 5%, the micellar particle size remained basically unchanged with the increase of peptides, and the PDI remained around 0.2 between 31 and 35 nm (Table 4).
  • ATN2-MPTX with a drug loading of 4.8 wt. % of PTX and a peptide density of 5% as an example
  • the effects of PEG350 and DTC on the stability of micelles, the stability of FBS, and the effects of freezing/lyophilized reconstitution on granules were studied.
  • the effect of diameter was found to be the same as that of non-targeted MPTX (Fig. 1 & 2C).
  • the drug release behavior of PTX micelles in the reduction response environment was studied in simulated cells.
  • the PB solution (containing 0.1% Tween 80) containing or not containing 10 mM DTT was used as the release medium.
  • ATN2-MPTX was used as an example to study the Cumulative amount of PTX released into the medium at different time points. It was found that the micelles with a concentration of 0.5 mg/mL could release PTX rapidly in 10 mM DTT, and the cumulative amount of released PTX reached 79.4% in 24 h; while in the environment without DTT, only 17.0% of PTX leaked in 24 h (Fig.
  • the results show that the micelle structure remains relatively stable due to DTC cross-linking, and the drug is always released slowly; under reducing conditions, the sulfhydryl groups generated by the disulfide bond breakage have a certain degree of hydrophilicity, and the micelle is difficult to maintain at a dilute concentration Micellar structure, rapid drug release, showing obvious reduction-responsive controlled drug release behavior.
  • Table 4 Particle size and particle size distribution of micelles with different peptide densities (PTX drug loading is 4.8 wt. %).
  • amphiphilic block polymer PEG 2k -P (CL 1k -DTC 1k ) and the polymer Ta-PEG 2k -P (CL 1k -DTC 1k ) coupled with targeting molecules (weight percent content of 5%) self-assembled in the aqueous phase to form PTX-loaded targeting micelles;
  • Ta is the polypeptide cRGD, ATN1 or ATN2 corresponding to particle sizes of 27 nm, 29 nm and 28 nm, respectively.
  • the invention prepares micelles with high drug loading of PTX, and the prepared micelles with small particle size (20-40 nm) can effectively penetrate deep into the tumor, especially during the drug loading process of the invention, there is no drug precipitation, static There is no drug leakage even after three weeks, and the small molecular weight PGE used does not need to be removed.
  • Example 3 MTT assay to evaluate the cytotoxicity of PTX micelles the cells were selected from mouse highly metastatic triple-negative breast cancer cell line 4T1.
  • the 4T1 cells were cultured by conventional methods and spread on 96-well plates (1.5 ⁇ 10 3 cells/well, cultured After 24 h, the cell confluence rate reached about 60%, and 20 ⁇ L of MPTX, micelles cRGD-MPTX, ATN1-MPTX and ATN2-MPTX containing different peptide densities were added, and the concentration of PTX ranged from 0.002 to 5 ⁇ g/ mL. After incubation for 4 h, the culture medium was discarded, and 100 ⁇ L of fresh medium was added and cultured for 44 h.
  • Triple-negative breast cancer refers to that the expression of estrogen receptor ER, progesterone receptor PR and human epidermal growth factor receptor HER-2 in the tumor of the patient is negative, and the targeted therapy of endocrine therapy, chemotherapy drugs and trastuzumab None of the treatments are effective or sensitive, so the invasiveness and metastases are high, the prognosis of patients is poor, and the five-year survival rate is low. It is urgent to find effective treatments for these patients (accounting for 10%-15%).
  • the present invention takes murine triple-negative breast cancer 4T1 cells as the research object, and uses the MTT method to study the cytotoxicity of three kinds of micelles with different targeting molecule densities.
  • the micelles coupled with targeting molecules showed higher toxicity than MPTX (Fig. 3).
  • the toxicity of the three micelles to 4T1 is not the same.
  • ATN2-MPTX is slightly more toxic than cRGD-MPTX, much higher than ATN1-MPTX; 5% cRGD-MPTX, 20% ATN1-MPTX, 5% ATN2- MPTX had the lowest IC 50 values relative to other peptide density micelles in each group, which were 0.27, 0.46, and 0.21 ⁇ g/mL (Table 5), among which the IC 50 value of 5% ATN2-MPTX was the lowest, even lower than that of free PTX IC 50 value (0.55 ⁇ g/mL) of co-incubation with 4T1 cells for 48 h.
  • cytotoxicity of 5% ATN2-MPTX co-incubated with 4T1 cells for 24 h and 48 h was further investigated (IC 50 values were 0.54 and 0.020 ⁇ g/mL, respectively), and it was found that the IC 50 values were higher than those of micelles without target (1.19 ⁇ g/mL, respectively). and 0.064 ⁇ g/mL) were 2-3 times lower.
  • co-incubation of empty micellar Ms and ATN2-Ms at a concentration of 1 mg/mL for 48 h did not show toxicity to 4T1 cells, which indicated that the nanocarriers prepared by this method had good biocompatibility.
  • Example 4 Study on the endocytic behavior of PTX micelles: Flow cytometry FACS and confocal laser microscopy (CLSM) were used to investigate the uptake of PTX micelles with different targeting densities by 4T1 cells. The micelles themselves are not fluorescent, and the endocytosis of micelles cannot be detected by CLSM and FACS. Cy5-labeled PEG-P (CL-DTC) was obtained by the amidation reaction of NHS-PEG-P (CL-DTC) and Cy5-NH2, and mixed into the original polymer at 1% to obtain Cy5-labeled gel bundle.
  • CL-DTC Cy5-labeled PEG-P
  • mice To establish a subcutaneous tumor model of mouse 4T1-luc triple-negative breast cancer, 5 ⁇ 10 5 cells (50 ⁇ L, containing 30% matrigel) were subcutaneously injected into the upper part of the right hind leg of 12 healthy female Balb/c mice. When the tumor volume reached about 200-250 mm, they were divided into 4 groups, with 3 animals in each group, so that the animals in each group had similar average body weight and tumor size.
  • Cy5/MPTX, Cy5/cRGD-MPTX or Cy5/ATN2-MPTX were injected into the tail vein, and at predetermined time points Near-infrared intravital imaging was performed 15 minutes after intraperitoneal injection of fluorescein potassium salt.
  • mice 12 tumor-bearing mice were injected with PTX, Cy5/MPTX, Cy5/cRGD-MPTX or Cy5/ATN2-MPTX (7.5 mg PTX/kg, 0.4 ⁇ g Cy5/mouse), 4 hours later, intraperitoneal injection of fluorescein potassium salt and then cervical dislocation to sacrifice the animals, within 10 minutes, the tumors and major organs (including heart, liver, spleen, lung, and kidney) of the mice were removed and washed with PBS , dried for ex vivo imaging and weighed. Weigh about 0.1 g of each tissue, add 500 ⁇ L of 1% Triton-100 and incubate overnight.
  • the drawing of the standard curve of PTX in each organ is to add a series of DMF solutions (20 ⁇ L) of known concentrations of PTX to 0.1 g of organs, and then process them according to the above sample processing method, measure HPLC, and draw the peak area
  • the standard curve of PTX in each organ is the ordinate and the concentration is the abscissa.
  • PTX micelles prepared by the invention is small (30-40 nm), and its pharmacokinetics and biodistribution have been studied.
  • the results showed that the pharmacokinetic curves of the three PTX micelles, MPTX, ATN2-MPTX and cRGD-MPTX, all showed an obvious two-phase pattern: a distribution phase in which the concentration of PTX in the blood decreased rapidly and an elimination phase in which the concentration of PTX decreased slowly.
  • the half-lives (t 1/,2 ⁇ ) were 3.85, 3.93 and 4.70 h, respectively (Fig.
  • the accumulation level of drugs in tumor tissues determines the final anti-tumor effect of drug-loaded micelles, and the accumulation in normal tissues will also cause drug side effects.
  • Cy5/MPTX, Cy5/ATN2-MPTX, Cy5/cRGD-MPTX were first injected into 4T1 tumor-bearing mice through the tail vein (7.5 mg PTX/kg) , the distribution of micelles in mice and tumors over time was observed by in vivo fluorescence imaging. It was found that the three PTX micelles were first gradually enriched in the tumor, and the tumor enrichment amount was the largest at 4-6 h, and then decreased slowly (Fig. 6 A&B).
  • Cy5/ATN2-MPTX had the highest tumor enrichment at all time points, and the fluorescence intensity of the tumors at 4 h was that of Cy5/MPTX and Cy5/cRGD-MPTX, respectively. 1.8 times (*p) and 1.2 times, the fluorescence is still strong at 24 h, indicating that ATN2 has a better targeting effect than cRGD. Secondly, the organs were imaged in vitro at 4 h (Fig. 6 C). It can be seen that the fluorescence intensity of Cy5/ATN2-MPTX micelles was the strongest in tumors and the weakest in major organs.
  • the amount of PTX in the organ tissue was tested by HPLC at 4 h.
  • the results showed that ATN2-MPTX had the highest PTX enrichment in the tumor (6.14% ID/g), respectively, and cRGD-MPTX (4.55% ID/g) , MPTX (3.51% ID/g) and PTX (2.84% ID/g) 1.4 times, 1.7 times and 2.2 times (Figure 6D), confirming the significantly higher tumor targeting effect of ATN2-MPTX than the no-target group, And the effect is better than cRGD-MPTX.
  • the ratio of PTX tumor enrichment to normal tissue enrichment can indicate the specific targeting of the drug, and the higher the T/N, the stronger the specificity.
  • Example 6 Study on PTX micelles-induced immunogenic cell death (ICD): The degree of ICD induction was verified by measuring the concentrations of CRT, HMGB1 and ATP produced by 4T1 cells incubated with PTX micelles. 4T1 cells (5 ⁇ 10 4 /well) were seeded in a 24-well plate with glass slides and cultured overnight, and 100 ⁇ L of PTX, MPTX and ATN2-MPTX (PTX concentration of 5 mg/mL) were added and cultured for 12 h.
  • ICD PTX micelles-induced immunogenic cell death
  • micellar nanomedicine of the present invention shows strong cytotoxicity and targeting to tumor cells.
  • MPTX, ATN2-MPTX and free PTX and empty micellar ATN2-Ms were tested in combination with 4T1 cells Exposure of surface CRT and secretion of HMGB-1 and ATP after incubation. First, the cells of each group were stained with CRT primary antibody and goat anti-rabbit Alexa Fluor 633 secondary antibody. CLSM observation (Fig.
  • Example 7 Effects of PTX micelles on primary immune cells: According to routine operations, primary bone marrow cells were extracted from the bone marrow of healthy Balb/c mice, stimulated and cultured in vitro to become BMDM and BMDC. In order to explore the effect of PTX micelles on BMDC, the proliferation of suspension cell BMDC was studied by CCK-8 method. Spread BMDCs on a 96-well plate (1.5 ⁇ 104 /well, 80 mL of 1640 medium) and culture overnight, then add 20 ⁇ L of PTX, MPTX and ATN2-MPTX (PTX concentration: 0.002 to 20 ⁇ g/mL).
  • PTX micelles The effect of PTX micelles on BMDM was studied by MTT method.
  • Spread BMDM cells (5 ⁇ 10 cells/well) in a 96-well plate, culture in 1640 medium for 24 h, add 20 ⁇ L of PTX, MPTX and ATN2-MPTX (PTX concentration: 0.002 to 20 ⁇ g/mL) and incubate After 4 h, culture medium was replaced for 44 h.
  • the subsequent processing methods are the same.
  • PTX is a mitosis inhibitor that stabilizes microtubule polymerization and prevents its disintegration, arresting cell mitosis, but the inhibitory effect is non-specific, and high concentrations of PTX may also damage other cells, such as immune cells.
  • DC is the most effective antigen-presenting cell (APC) recognized by T cells and plays a key role in the host immune system.
  • Tumor-associated macrophages are an important part of tumor-infiltrating immune cells, which are related to the growth, angiogenesis and metastasis of various tumors, and have strong phagocytic function.
  • the effects of PTX nanomedicine on the proliferation and cytokine secretion of DCs and macrophages were studied by MTT and flow cytometry.
  • the mouse bone marrow primary cells were extracted, stimulated into BMDC, and the effects of different concentrations of ATN2-MPTX, MPTX and free PTX on the survival rate and proliferation of BMDC after incubation for 24 and 48 hours were studied.
  • the three PTX drugs had no toxicity to BMDC after 24 and 48 h of incubation, and promoted their proliferation to a certain extent; BMDC after 48 h of incubation The proliferation was higher than 24 h.
  • ATN2-MPTX can induce ICD and release tumor-associated antigen (TAA).
  • TAA tumor-associated antigen
  • the present invention studies the effect of ATN2-MPTX on BMDC maturation, and uses FACS to test the mature BMDC (CD11c + CD86 + CD80 + ) ratio. As shown in Figure 9A&B, compared with the PBS group, free PTX, MPTX and ATN2-MPTX all increased the number of CD11c + BMDCs, consistent with the MTT results. In addition, the mature BMDCs activated by the three were significantly higher than those in the PBS group (***p), which was related to the promotion of BMDC proliferation by the three.
  • ATN2-MPTX is beneficial not only in the number of BMDCs but also in the activation of their maturation.
  • the content of pro-inflammatory cytokines TNF- ⁇ and IL-6 secreted by the three stimulated BMDC maturation was tested by ELISA (Figure 9C&D), and it was also found that the three could induce more TNF- ⁇ than the PBS group ( **p) and IL-6 (***p).
  • Example 8 PTX micelles induced BMDM polarization and cytokine secretion: BMDM (1 ⁇ 10 6 cells/well) were spread in a 24-well plate and cultured in 1640 medium for 24 h to form M0 type macrophages (M0M ), and IL-4 (20 ng/mL) was added to the 1640 medium to stimulate, and M2 macrophages (M2M) were obtained after 24 h.
  • M0M M0 type macrophages
  • M2M M2 macrophages
  • PTX When adding PTX, MPTX or ATN2-MPTX to IL-4-stimulated M2M, it was found that the proportion of M1M could be increased to 23.9%, 40.7% (*p) and 42.9% (**p) respectively (Fig. 10 B) , which demonstrates that ATN2-MPTX is more easily endocytosed by macrophages, polarizing it towards M1M.
  • the contents of pro-inflammatory cytokines TNF- ⁇ and IL-6 induced by M1M in different groups of media were detected by ELISA, and it was found that compared with PBS group and IL-4-stimulated M2M group, PTX preparations could significantly promote TNF- ⁇ .
  • ⁇ and IL-6 (***p), among which ATN2-MPTX and MPTX induced the most, was comparable to that of LPS-stimulated M1M (Fig. 10 C&D).
  • the MTT method detects that different PTX preparations have concentration-dependent cytotoxicity to BMDM, and BMDM is more intolerant to PTX agents, and its cell survival rate is lower than that of BMDC; the IC 50 value of MPTX and ATN2-MPTX is similar, which is twice that of PTX (Fig. 10E).
  • BMDCs (1 ⁇ 10 6 cells/well) were cultured in a 12-well plate for 24 h, then added PBS, PTX, MPTX, and ATN2-MPTX for 24 h, and the final PTX concentration was 5 ⁇ g /mL. After incubation, centrifuge (1500 rpm, 5 min) to separate the upper medium, collect the cells and stain the cells with fluorescently labeled CD11c, CD80 and CD86 antibodies according to the instructions, and finally use FACS to test the amount of total CD11c + BMDC in each group and CD11c + CD80 + CD86 + content of mature BMDC. The concentration of IFN- ⁇ , TNF- ⁇ and IL-6 in the separated culture medium was determined by ELISA kits.
  • ATN2-MPTX can induce ICD, produce a series of tumor antigens, and enable the proliferation and maturation of BMDC.
  • the TAA induced by ATN2-MPTX can be more effectively presented to T cells by these activated APCs, thereby recruiting more T cells.
  • FACS test results showed that, compared with PBS group, ATN2-MPTX could promote the content of total CD11c + BMDC ( Figure 11 A&B); IFN- ⁇ , TNF- and IL-6 concentrations.
  • the increase of cytokines indicates that BMDCs are mature, their antigen presentation ability is enhanced, and they can strongly activate T cells.
  • IFN- ⁇ is a marker cytokine produced after the combination of CDN and STING, which plays a key role in the activation of T cells induced by tumors, as shown in the figure 11 C, D.
  • Example 10 PTX micelles are used in the anti-tumor treatment of mouse TNBC: when the tumor volume of the tumor-bearing mice inoculated with 4T1 (3 ⁇ 10 5 /mouse) is 50-100 mm 3 (day 6 after inoculation) The day of administration was defined as day 0.
  • the mice were randomly divided into 4 groups, 7 in each group, administered every two days, a total of 4 times; the dosage was 7.5 mg PTX/kg and/or 1 mg CDN/kg. They are: PBS, Free PTX (iv), MPTX (iv), ATN2-MPTX (iv).
  • the mouse body weight, tumor volume, and mouse status were monitored every 2 days during the experiment.
  • mice died or the tumor volume was greater than 2000 mm 3 were judged to be dead, and the survival curve was drawn.
  • the anti-tumor effects of ATN2-MPTX and MPTX were systematically compared. See Figure 12. The results showed that the malignant degree of 4T1 tumors was extremely high, and the tumors in the PBS group grew rapidly (Fig. 12 B).
  • the tumor volume was greater than 2000 mm 3 on the 20th day, and the median survival time was 18 days.
  • the tumors grew slowly during the period of administration, and began to grow rapidly after stopping the administration.
  • both ATN2-MPTX and MPTX can significantly inhibit the growth of the tumor, and the tumor basically does not grow within 10 days, but the tumor begins to grow slowly after 10-12 days.
  • MPTX may have a significant EPR effect due to its small particle size, and it can inhibit tumor growth more effectively than PTX (*p).
  • ATN2-MPTX can further restrict tumor growth (***p) than PTX and MPTX (Fig. 12 B), and prolong the survival time of mice, reflecting the targeted anti-tumor effect.
  • the body weight of mice changed little during the treatment period, and tail vein administration of 7.5 mg PTX/kg did not cause toxic side effects on mice (Fig. 12D).
  • Example 11 Evaluation of immune status, side effects and lung metastasis of mice after treatment: As described in Example 10, tumor-bearing mice were randomly divided into 4 groups, 7 in each group. They were: PBS (iv), free PTX (iv), MPTX (iv), ATN-MPTX (iv), administered 4 times at 7.5 mg PTX/kg, once every 2 days. 24 hours after the last administration, 4 mice in each group were sacrificed, and the whole blood of the mice was collected to separate the serum for the determination of IFN- ⁇ and IFN- , IL-6 and TNF- content. The spleen and tumor were collected, ground into a single cell suspension, and counted after cracking.
  • spleen 6 x 106 cells per sample were stained for T cells.
  • each sample was divided into 4 parts, each with 6 ⁇ 10 6 cells, stained for DC, macrophages, and T cells, and analyzed by FACS test.
  • the corresponding fluorescently labeled antibodies of immune cells are: DC: anti-CD11c-FITC, anti-CD80-APC, anti-CD86-PE; macrophage: anti-CD11b-FITC, anti-F4/80-PE, anti- CD206- Alexa Fluor 647; T cells: anti-CD3-APC, anti-CD4-PE, anti-CD8-FITC; Tregs: anti-CD3-FITC, anti-CD4-PE, anti-Foxp3-Alexa Fluor 647.
  • the tumor immune microenvironment is an environment composed of malignant cells, immune cells, blood vessels, extracellular matrix and signaling molecules, which can act alone or jointly affect the sensitivity of immunotherapy. It has been proved above that ATN2-MPTX can target tumor cells to induce ICD, release tumor-associated antigens, and create a favorable immune microenvironment.
  • ATN2-MPTX can target tumor cells to induce ICD, release tumor-associated antigens, and create a favorable immune microenvironment.
  • TME and T cells in the spleen of mice after ATN2-MPTX administration for 24 h, as shown in Figure 13. Firstly, the content of CD80 + CD86 + mature DC and CD206 - M1M in TME was analyzed, and the proportion of mature DC in PTX micelles was higher than that in PBS.
  • PTX can convert macrophages from M2M to M1M.
  • ATN2-MPTX can promote the polarization of M2M to increase CD206 - M1M.
  • the proportion of T cells in the tumor TME of each group was studied, and the proportion of T regs in CD4 + T and CD8 + T/T regs were analyzed.
  • ELISA results showed that the pro-inflammatory cytokine IFN- , IFN- ⁇ , TNF- ⁇ and IL-6 levels were all increased, these results confirm that it can induce a strong anti-tumor immune response.
  • mice On the 22nd day of administration, the mice were intraperitoneally injected with fluorescein potassium salt, and the remaining 3 mice in each group were sacrificed within 10 minutes. Whole blood was collected for routine blood analysis, and serum was separated for blood biochemical analysis. In addition, mice were dissected, and organs such as heart, liver, spleen, lung, and kidney were taken, fixed with tissue fixative, sectioned, and sealed with paraffin. The sections were stained with hematoxylin and eosin (H&E), and the sections were observed under a microscope for histological analysis and judgment of lung metastasis. The ratio of CD4 + and CD8 + T cells to CD45 + total immune cells in the spleen of mice after ATN2-MPTX administration was analyzed. Figure 14 shows that the change trend of CD4 + T and CD8 + T cells in spleen of mice in each group on day 8 after drug administration is consistent with that in tumor.
  • H&E hematoxylin and eosin
  • mice With the development of 4T1 breast cancer, the blood system and organs of mice will be affected to a certain extent; and long-term drug use will also bring cumulative systemic toxicity to mice.
  • 3 mice in each group were sacrificed, and whole blood was taken for blood routine testing and serum was separated for Blood biochemical tests were performed, and major organs were dissected for observation and sectioned, and H&E staining was used for histological analysis.
  • PLT platelets
  • PCT platelet volume
  • MPV mean platelet concentration
  • the quality of the spleen of the mice in the treatment group was positively correlated with the size of the tumor, that is, the smaller the tumor, the smaller the spleen. From the H&E staining of the spleen (Figure 16), it can be seen that the white pulp was significantly reduced and the red pulp was increased in the PBS group, which is one of the signs of the development of the disease in 4T1 mice. The treatment group had abundant white pulp, indicating that the progression of the breast cancer course was prevented. The above results show that the tail vein to 7.5 mg PTX/kg, administered every 2 days for a total of 4 times had no obvious toxicity to mice.
  • TNBC is highly invasive and highly metastatic
  • the inhibition of PTX micelles on lung metastasis of breast cancer bearing 4T1-luc was studied. Twenty-two days after the administration as above, the lungs were dissected, weighed, imaged in vitro, and sliced by H&E staining for histological analysis. Lung imaging and fluorescence quantitative results showed (Figure 18 A&B), mice in PBS and free PTX groups developed severe lung metastasis, and the fluorescence of the lungs in the MPTX group decreased significantly (***p), and ATN2-MPTX also had significant lung metastasis inhibition compared with MPTX (*p).
  • the H&E staining pictures of the lungs showed that there were a large number of tumor cells and inflammatory cell infiltration in the lungs of the PBS group and free PTX mice, and there was almost no alveolar structure; the MPTX group improved, and there was no obvious tumor cell population compared with ATN2-MPTX (Fig. 18D), the results were in good agreement with the bioluminescence map.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dispersion Chemistry (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

小胶束纳米药物的制备方法为：将小分子药物、两亲性聚合物/靶向两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将混合溶液加入缓冲溶液中，得到小胶束纳米药物，低聚乙二醇的分子量为200～600，小分子药物可以是紫杉醇。获得的紫杉醇胶束纳米药物，载药量较高，可达23.1wt.％，粒径小于40nm，具有良好的稳定性、还原响应的药物释放性能。

Description

一种小胶束纳米药物及其制备方法与应用 技术领域
本发明属于纳米药物技术，具体涉及一种小胶束纳米药物及其制备方法与应用。
 背景技术
目前恶性肿瘤仍呈高发态势。尽管新的治疗方法和治疗药物频出，以小分子化药如紫衫烷类药物为基础的化学疗法还是最常用的治疗方法。其中，紫杉醇（PTX）是作用于微管蛋白的一种广谱抗肿瘤紫衫烷类药物，用于多种肿瘤的一线或二线治疗。但PTX的水溶性极低，生物利用度低，无法直接使用；其以CremophorEL和无水乙醇为增溶剂的Taxol被批准用于乳腺癌、卵巢癌、非小细胞肺癌等多种肿瘤的治疗，而CremophorEL会引起严重的毒副作用，且对正常细胞和癌细胞无选择性。近年来研究者发展了纳米尺度的药物载体，如脂质体、白蛋白、聚合物胶束和纳米粒等，其中聚合物胶束具有更好的载药性能、循环稳定性以及释放效果，但是针对PTX，现有纳米胶束药物的粒径偏大。
 技术问题
本发明公开了一种小胶束纳米药物及其制备方法与应用，制备了偶联PHSCNK（ATN1）、PhScNK（ATN2）或cRGD多肽的纳米药物ATN1-MPTX、ATN2-MPTX和cRGD-MPTX。实验结果表明，ATN2-MPTX能高效靶向富集到小鼠4T1乳腺癌肿瘤，增加了PTX在肿瘤细胞的浓度，有效延缓了小鼠肿瘤的生长、抑制了乳腺癌的肺转移，延长了小鼠的生存期。
 技术解决方案
本发明采用如下技术方案：一种小胶束纳米药物，其制备方法为，将小分子药物、两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到小胶束纳米药物。
一种靶向小胶束纳米药物，其制备方法为，将小分子药物、两亲性聚合物、靶向两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到靶向小胶束纳米药物。
本发明中，所述低聚乙二醇的分子量（ M n ）为200～600，优选300～500；两亲性聚合物的分子量为2000～15000，靶向两亲性聚合物的分子量为2000～15000。本发明聚合物的分子量为核磁测定的数均分子量（ M n ），单位为Da。
上述技术方案中，小分子药物包括紫杉醇PTX等。
一种小胶束纳米药物冷冻液，其制备方法为，将上述小胶束纳米药物或者靶向小胶束纳米药物在液氮中冷冻后存储于冰箱中，得到小胶束纳米药物冷冻液；冰箱的温度为-80℃～-10℃。
一种小胶束纳米药物冻干粉，其制备方法为，将上述小胶束纳米药物或者靶向小胶束纳米药物与冻干保护剂混合后在液氮中冷冻，然后利用冻干机冻干，得到小胶束纳米药物冻干粉。冻干保护剂优选为蔗糖与甘露醇。
上述技术方案中，两亲性聚合物为PEG-P(CL-DTC)、PEG-P(TMC-DTC)、PEG-P(LA-DTC)等；靶向两亲性聚合物为所述两亲性聚合物偶联靶向分子，优选的，靶向分子为多肽，比如PHSCNK多肽、PhScNK 多肽或cRGD多肽。两亲性聚合物中，PEG的分子量（ M n ）为1000～5000 Da。
上述技术方案中，静置下，将所述混合溶液打入缓冲溶液中，然后搅拌、静置、涡旋、吹打或者倒置；打入为现有技术，比如用注射器或者注射泵；搅拌的转速为100～1000rpm。混合溶液中，小分子药物的浓度为1至10 mg/mL，聚合物的浓度为1～100 mg/mL；聚合物为两亲性聚合物，或者聚合物为两亲性聚合物和靶向两亲性聚合物。
上述技术方案中，混合溶液中含有靶向两亲性聚合物时，靶向两亲性聚合物的用量为两亲性聚合物重量的1～30%，优选2.5～10%。本发明中，两亲性聚合物不偶联靶向分子，靶向两亲性聚合物偶联靶向分子。
上述技术方案中，低聚乙二醇、缓冲溶液的体积比为1∶（5～40），优选1∶（10～30）。
本发明制备了PTX载药量高的胶束，且制备的小粒径胶束（20-40 nm）能够穿透至肿瘤深处。本发明公开了上述小胶束纳米药物或者靶向小胶束纳米药物在制备抗肿瘤药物中的应用，优选肿瘤为三阴性乳腺癌（TNBC）。
本发明设计制备了高效载PTX、双硫交联的胶束MPTX以及三种TNBC主动靶向的胶束纳米药物，载药量高达23.1 wt.%，粒径在30 - 38 nm之间，具有良好的稳定性、还原响应的药物释放性能。细胞层面和动物层面的实验证实，ATN2-MPTX对4T1细胞的靶向内吞作用最强，IC 50值最低，在肿瘤的生物分布最高。此外，ATN2-MPTX可诱导ICD、促进BMDC增值以及成熟、促进BMDM向M1型巨噬细胞极化，产生有利的免疫微环境。能够更好地抑制小鼠TNBC的生长和肺转移的发生，延长小鼠生存期，体现了治疗的优势，为治疗高转移性肿瘤带来了希望。
有益效果
本发明优势主要表现为：1）纳米载体表面积/体积比高，有利于提高PTX等疏水药物的溶解性和装载效率；2）纳米载体能阻止药物快速清除、延长血液循环时间，从而改变药物生物分布，提高生物利用率；3）纳米药物在20～40 纳米，可在肿瘤组织通过增强的渗透性和保留（EPR）效应富集；4）纳米载体可设计性强，通过设计肿瘤微环境刺激响应性载体以及肿瘤主动靶向的纳米载体有助于药物的可控释放，提高特异性。
 附图说明
图1为MPTX和ATN2-MPTX胶束的制备和理化性质（载药量4.8 wt.%）。（A）胶束的制备过程示意图；（B）MPTX和ATN2-MPTX的粒径分布以及MPTX的TEM示意图；（C）载药量为23.1 wt.%的MPTX（I）及相同PTX浓度的PB溶液（II）照片；（D）胶束在室温存放3周、制备后再透析后室温放置1 h的粒径变化；（E）Ms、ATN2-Ms和ncMs以及相同浓度的聚合物溶液（5 mg/mL）的紫外吸收谱图；（F）胶束中芘的荧光吸收谱中I 372/I 383（I 1/I 3）比值随浓度的变化；（G）MPTX和ATN2-MPTX浓度为20 mg/mL的粒径，以及ncMPTX浓度为1 mg/mL、50 mg/mL的粒径及其在室温下放置2天的粒径。
图2为MPTX和ATN2-MPTX在37 度10%FBS中孵育24 h后的粒径分布（A），在10 mM DTT中孵育2 h后的粒径分布（B）；冷冻液-解冻和冻干粉-复溶的粒径分布（C）。（D）ATN2-MPTX在模拟生理环境（PB，pH 7.4）和细胞内还原环境（含10 mM DTT 的PB）下PTX的累积释放（n = 3）。
图3为MTT法（n = 5）测定含不同多肽密度的（A）cRGD-MPTX、（B）ATN1-MPTX和（C）ATN2-MPTX对4T1细胞的毒性；（D）自由PTX先孵育4 h、换新鲜培养基再孵育44 h（4+44 h）及共孵育48 h的细胞毒性；（E）空胶束Ms和ATN2-Ms共孵育48 h的细胞毒性。
图4为流式细胞仪（A）和CLSM（B&C）测试了标记Cy5的MPTX、AT1-MPTX、ATN2-MPTX和cRGD-MPTX被4T1细胞内吞（孵育4 h）的情况。（B）为Cy5半定量荧光强度。受体抑制实验用PhScNK多肽（ATN2）预先和4T1细胞孵育2 h再加Cy5/ATN2-MPTX。Cy5聚合物浓度为2 mg/mL。标尺为25 mm。
图5为cRGD-MPTX、ATN2-MPTX、MPTX和PTX在健康Balb/c小鼠体内的药代动力学曲线（7.5 mg PTX/kg）。
图6为Cy5/MPTX、Cy5/ATN2-MPTX和Cy5/cRGD-MPTX在荷瘤小鼠静脉给药后随时间的体内荧光成像（A）、肿瘤部位荧光定量分析（B）及给药4 h后主要器官和肿瘤的离体荧光成像（C）。MPTX、ATN2-MPTX、cRGD-MPTX和PTX静脉给药4 h后PTX在主要器官及肿瘤的富集（D）及PTX在肿瘤和脏器富集量的比例（T/N）（E）。n = 3。
图7为MPTX、ATN2-MPTX和自由PTX诱导4T1细胞的ICD。（A）CLSM观察细胞表面CRT，标尺为25 mm；（B）为A的荧光强度半定量分析。细胞培养基中ATP（C）和HMGB1（D）的含量。孵育时间12 h，n = 3。
图8为BMDC在ATN2-MPTX、MPTX和自由PTX在孵育24 h（A）和48 h（B）后的增殖情况；（C）和（D）分别是对（A）和（B）曲线中PTX低（0.04 mg/mL）、中（1.25 mg/mL）和高（20 mg/mL）浓度下BMDC增殖情况的对比。n = 5。
图9为BMDC与ATN2-MPTX、MPTX和自由PTX孵育24 h后激活和细胞因子的分泌（n = 3）。（A）流式细胞测定CD11c +总BMDC的比例和（B）CD11c +CD80 +CD86 +成熟BMDC的比例。培养基中分泌的（C）TNF-α和（D）IL-6的浓度。
图10为PTX制剂对M2M调节作用的代表性流式细胞分析（在CD11b +F4/80 +总巨噬细胞圈门内）（A）和CD11b +F4/80 +CD206 -的M1M占总巨噬细胞比例统计（n = 3）（B）。BMDM先与IL-4（20 ng/mL）孵育48 h，换液后再分别与PBS、IL-4（20 ng/mL）、LPS（100 ng/mL）、自由 PTX、MPTX和ATN2-MPTX（PTX浓度为5 mg/mL）孵育24 h。PTX制剂刺激后培养基中TNF-α（C）和IL-6（D）的浓度。（E）自由 PTX、MPTX和ATN2-MPTX对BMDM的毒性。先与BMDM细胞共孵育4 h、换新鲜培养基再孵育44 h（n = 5）。
图11为BMDC的成熟（CD11c +CD86 +CD80 +）和培养基中细胞因子的浓度（n = 3）。ATN2-MPTX和BMDC孵育24 h。（A）BMDC的代表性样品流式细胞仪伪色图（在CD11c +总BMDC圈门内）及其统计学分析（B）。培养基中（C）IL-6、（D）TNF-α的浓度。
图12为ATN2-MPTX胶束治疗荷4T1乳腺癌小鼠（7.5 mg PTX/kg，1 mg CDN/kg）的（A）抗肿瘤治疗方案（本发明仅为蓝色针筒）；（B）肿瘤体积（#号代表肿瘤体积大于2000 mm 3死亡或自然死亡）；（C）小鼠的生存曲线；（D）小鼠体重。n = 7。
图13为ATN2-MPTX给药结束24 h后小鼠肿瘤的流式细胞仪分析（n = 4）。（A）代表性流式细胞术分析和（B）肿瘤中CD3 +CD4 +T细胞（在CD45 +环形门中）的比例。(C）典型的流式细胞术分析和（D）肿瘤中CD3 +CD8 +T细胞（在CD45 +环形门中）的比例。(E）CD4 +T细胞中Tregs（CD3 +CD4 +FoxP3 +，CD45 +）的含量及（F）CD8 +T/Tregs比值。流式细胞术分析（G）成熟DC（CD45 +CD80 +CD86 +）与（H）M1M（CD11b +F4/80 +CD206 -）比值。小鼠血清中细胞因子（I）干扰素-β小鼠和(J）IL-6的含量。
图14为ATN2-MPTX给药结束24 h后小鼠脾脏的流式细胞仪分析。（A）CD3 +CD4 +、CD3 +CD8 + T细胞代表性流式细胞伪色图（在CD45 +总免疫细胞圈门内）以及（B）CD3 +CD4 + 和（C）CD3 +CD8 + T细胞比例统计，n = 4。
图15为自由PTX、MPTX、ATN2-MPTX治疗后第22天小鼠的血常规（A）和血生化（B）分析（n = 3）。
图16为PBS、自由PTX、MPTX、ATN2-MPTX治疗荷瘤小鼠22天后小鼠心、肝、肾、脾切片的H&E染色图（40×，标尺为50 mm）。WP：白髓，RP：红髓。
图17为自由PTX、MPTX、ATN2-MPTX给药第22天小鼠的脾脏图片（A）和质量（B）。n = 3。
图18为荷瘤小鼠在给药22天后肺部离体成像（A）、荧光值半定量（B）和质量（C）。（D）肺H&E染色图。左图为10×，标尺为500 mm；右图为40×，标尺为50 mm。
 本发明的实施方式
甲氧基聚乙二醇（MeO-PEG-OH， M n = 2.0 kg/mol）、马来酰亚胺和琥珀酰亚胺酯官能化的聚乙二醇（Mal/NHS-PEG-OH， M n = 3.5 kg/mol）均购自北京键凯科技有限公司，经甲苯共沸蒸馏后使用。ε-己内酯（ε- CL, 99%，Alfa Aesar）经氢化钙干燥、减压蒸馏后使用。1,2-二硫戊环三亚甲基碳酸酯（DTC）由本实验室合成并提纯。磷酸二苯酯（DPP，> 99%，TCI）使用前真空干燥30分钟。c(RGDfC)（cyclic peptide (Arg-Gly-Asp-D-Phe-Cys)）、Ac-PHSCNK-NH 2（ATN-1）和Ac-PhScNK-NH 2（ATN-2）的纯度都大于 98%，均购自上海强耀生物。高效液相色谱（HPLC）用试剂均从Sigma Aldrich (USA) 购买得到。紫杉醇（PTX，> 99%，上海金和生物制药有限公司）、甲氧基低聚乙二醇（PEG 350， M n = 350 g/mol，Sigma）、谷胱甘肽（GSH，> 99%，Roche）和Cy5-NH 2（Lumiprobe）等试剂均是购买后直接使用。Micro BCA试剂盒（Pierce，Thermo Scientific）、增强型ATP检测试剂盒（S0027，碧云天）、小鼠高迁移率族蛋白B1（HMGB-1）（E-EL-M0676c， Elabscience）、小鼠白细胞介素-6（IL-6）ELISA检测试剂盒（JN-029562-S，上海研域）、小鼠干扰素-
Figure 948243dest_path_image002
、干扰素-
Figure 737207dest_path_image004
（IFN-
Figure 370314dest_path_image005
、IFN-
Figure 877519dest_path_image004
）和细胞坏死因子-（TNF-
Figure 355905dest_path_image006
）ELISA检测试剂盒（Invivogen）、钙网蛋白（CRT）抗体（Ab2907，Abcam）、以及小鼠荧光标记的各种抗体（Biolegend）CD45-PerCP/Cyanine5.5、CD80-APC、CD86-PE、CD11c-FITC、CD11b-FITC、CD206- Alexa Fluor 647、F4/80-PE、CD4-PE、CD8-FITC、FoxP3- Alexa Fluor 647和山羊抗兔-Alexa Fluor 633等试剂盒和抗体均购买后按说明书使用。小鼠乳腺癌细胞株4T1是从中科院上海细胞库购买。
本发明的原料都是现有产品，具体操作方法以及测试方法为本领域常规方法。本发明所有数据均是以平均值呈现，组间差异性由ANOVA单因素方差分析评估，*p < 0.05视为具有显著性差异，将**p < 0.01和***p < 0.001视为高显著性差异。
本发明实施例中，比较了自由PTX、MPTX、ATN2-MPTX治疗的效果，其他技术方案不影响本发明技术效果的实现。
制备例：PEG-P(CL-DTC) 嵌段共聚物的合成是以MeO-PEG-OH ( M n = 2.0 kg/mol) 为大分子引发剂、磷酸二苯酯（DPP）为催化剂引发DTC和CL的开环聚合而得。具体地，在惰性气体手套箱里，向25 mL密闭反应器中依次加入MeO-PEG-OH (800 mg, 0.4 mmol)、DPP (1.0 g，4 mmol)、DTC (400 mg，2.1 mmol) 、CL (400 mg，3.5 mmol) 和无水DCM（3.2 mL），搅拌溶解后密封，移出手套箱，置于40 ºC 油浴中磁力搅拌反应48 h。反应结束后，加入冰醋酸终止反应，在30倍体积的冰乙醚中沉淀2次，抽滤、真空干燥24 h后得到PEG 2k-P(CL 1k-DTC 1k)，制备路线如下所示：
Figure 264955dest_path_image007
。
其氢核磁谱显示了聚合物的各特征峰：PEG：δ 3.38 (C H 3 O-)和3.64 (-C H 2 C H 2 O-)；DTC：δ 3.00 (-C(C H 2 SSC H 2 )C-)和4.14 (-OCOC H 2 (CH 2SSCH 2)C H 2 O-)；CL：δ 1.04 (-O(CH 2) 2C H 2 (CH 2) 2CO-)、1.66(-OCH 2C H 2 CH 2C H 2 CH 2CO-)、2.31(-O(CH 2) 4C H 2 CO-)和4.05(-OC H 2 (CH 2) 4CO-)。以核磁图中PEG亚甲基氢（δ 3.64）的积分面积为参照，按PEG和DTC（δ 3.00）、CL（δ 1.04和2.31）特征峰的积分比值算得PEG-P(CL-DTC)的分子量为2k-0.9k-1.1k，GPC测得分子量为7.1 kg/mol，分子量分布为1.2（表1）。
用分子量为5000 Da的MeO-PEG-OH为引发剂、DPP为催化剂，根据上述方法可控开环聚合CL和DTC得到PEG-P(CL-DTC)，核磁分子量为5k-4k-3k或者5k-4k-2k。
用上述相同方法：不加DTC合成了PEG-PCL；以Mal-PEG-OH或NHS-PEG-OH（ M n = 3.5 kg/mol）替换MeO-PEG-OH ( M n = 2.0 kg/mol)作为引发剂合成了Mal-PEG-P(CL-DTC)或NHS-PEG-P(CL-DTC)。
ATN-PEG-P(CL-DTC) 通过PHSCNK或PhScNK多肽与NHS-PEG-P(CL-DTC)通过酰胺化反应得到。简言之，在氮气保护下，将NHS-PEG-P(CL-DTC)（132 mg，0.024 mmol）溶解在1 mL无水DMSO中，PHSCNK多肽或PhScNK多肽（20 mg，0.028 mmol）溶解在0.5 mL无水DMSO中。将聚合物溶液滴加至30℃的多肽溶液中，30 min滴加结束后再加入7 mL的三乙胺调节体系pH在8.0，密闭反应器后再30度反应48 h。将反应液装入透析袋（MWCO 3500 Da）在DMSO中透析4 h、DCM中透析2 h，然后在30倍体积的冰乙醚/无水乙醇（9/1，v/v）中沉淀2次，离心、真空干燥24 h后得到ATN-PEG-P(CL-DTC)。 1H NMR（600 MHz，DMSO- d 6 ）测试得到谱图可进行聚合物结构和分子量分析，用Micro BCA蛋白试剂盒测试可计算多肽的官能化度。反应如下：
Figure 803383dest_path_image008
。
其核磁图出现NHS特征峰（δ 2.59）并且其积分值和PEG主峰的比值接近理论值。此外，两种不同构型的多肽PHSCNK（ATN1）和PhScNK（ATN2）分别通过其C端赖氨酸的伯胺和聚合物的NHS端基发生酰胺化反应分别得到ATN1-PEG-P(CL-DTC)和ATN2-PEG-P(CL-DTC)。PhScN是PHSCN中组氨酸和半胱氨酸的D型（D-His和D-Cys）同分异构体，研究发现二者对乳腺癌细胞和前列腺癌细胞的结合位点相同，但在抑制细胞的基底膜浸润能力方面存在差别。在C端引入了一个赖氨酸K，可通过其伯胺将多肽高效键合到聚合物末端。本发明研究两种多肽对胶束内吞的影响。以ATN2-PEG-P(CL-DTC)的氢核磁图为例，发现NHS特征峰显著变小，PhScNK特征峰（δ 1.7-2.1和δ 7.0-7.1）出现。通过Micro BCA微量蛋白试剂盒测定聚合物多肽的含量，计算出PhScNK和PHSCNK的官能化度分别为71.2%和79.6%。
cRGD-PEG-P(CL-DTC) 由c(RGDfC) 多肽的巯基和Mal-PEG-P(CL-DTC) 通过迈克尔加成反应制得，其步骤、聚合物的提纯和表征同上。反应如下：
Figure 532305dest_path_image009
。
其氢核磁图上Mal的特征峰（δ 7.0）的出现及其积分和PEG主峰（δ 3.51）比值接近理论值，说明成功合成了Mal-PEG-P(CL-DTC)、Mal官能团未受影响。后者再与cRGD多肽的巯基发生迈克尔加成反应得到cRGD-PEG-P(CL-DTC)，核磁图显示，Mal的特征峰（δ 7.0）的消失，cRGD特征峰（δ 7.2）出现。通过cRGD特征峰（δ 7.2）与PEG主峰的积分比值，计算得到cRGD的官能化度约为89.2%；用Micro BCA试剂测试该聚合物所含多肽的量，得到cRGD的官能化度约为86.6%。
表1 各聚合物的表征。
Figure 876699dest_path_image010
。
将PTX和PEG 2k-P(CL 1k-DTC 1k) 按照质量比（1/20）溶于PEG 350中，其中聚合物浓度保持1 mg/mL。常规超声5 min后，在37℃取100 mL的该混合溶液打入900 mL的PB溶液底部（pH 7.4，10 mM），过程中静置不搅拌。打入之后用移液枪贴液面吹打5次，即得到PTX载药胶束，为非靶向小胶束纳米药物，DLS测定其粒径为27.8nm，粒径分布（PDI）为0.18。在上述方法基础上：将聚合物更换为PEG 5k-P(CL 4k-DTC 3k)，同样方法得到的PTX载药胶束，DLS测定其粒径为55.1nm，粒径分布（PDI）为0.27。如果混液过程中常规搅拌（600 rpm），其余不变，得到的PTX载药胶束粒径达到168 nm，且存放1小时即出现药物析出。将PEG 350更换为DMF或者DMSO，同样方法得到的PTX载药胶束，粒径较小，但是稳定性差，常规环境下存放4小时后，出现大量沉淀，说明药物明显析出，较本发明存放20天以上无药物析出差得多。
实施例一 PTX胶束的制备及表征：将PTX和PEG 2k-P(CL 1k-DTC 1k) 按照不同质量比（5/100、10/100、20/100、30/100）溶于PEG 350中，其中聚合物浓度在50 mg/mL。常规超声5 min后，在37℃取100 mL的该混合溶液打入900 mL的PB溶液底部（pH 7.4，10 mM），过程中静置不搅拌。打入之后用移液枪贴液面吹打5次，即得到理论PTX载药量为4.8 wt.%～23.1 wt.%的胶束MPTX，为非靶向小胶束纳米药物。DLS测定其粒径及粒径分布（PDI），TEM测定胶束形貌。用含20 mM DTT的乙腈溶解MPTX（聚合物浓度为0.1 mg/mL），再用HPLC测试其中PTX浓度，计算载药量和载药效率。用DLS在不同时间点监测PTX胶束在25和37℃存放、稀释至低浓度（20 mg/mL）及在含10%FBS溶液中粒径和粒径分布的变化，见表2。
作为对照，用相同方法也制备了基于PEG-PCL的载PTX胶束（ncMPTX）。
现有技术多数文献中报道的PTX纳米载体载药量均较低。本发明利用少量PEG350来溶解聚合物PEG-P(CL-DTC)和PTX，将混合物加入到水相中（PEG350的最终体积含量10%），通过疏水作用聚合物形成核壳结构的胶束把PTX包裹在疏水核中，而核中DTC可快速自交联，能更稳定地把PTX固定在胶束核中，得到PTX胶束纳米药物MPTX（图1 A）。这里使用的PEG350无毒，获FDA批准使用。本发明通过该方法制备了载药量从4.8 wt.%到23.1 wt.%的胶束MPTX，粒径范围在30-38 nm，粒径分布0.13-0.17（表2）。例如，DLS测试结果表明，载4.8 wt.%的MPTX粒径为32.7 nm，PDI为0.16，TEM图显示出其具有较规则的实心小球的形状（图1B）。此外，即使是理论载药量达23.1 wt.%时，MPTX胶束依然保持澄清透明，粒径为38.1 nm，PDI为0.13（表2）。由于PTX的水溶性很低（水中溶解度为5.56 mg/L），将相同量PTX用PEG350 溶解后直接加入到水中，PTX迅速析出，而载23.1 wt.% PTX的MPTX能保持澄清，说明PTX全装载在胶束里面（图1 C）。使用该方法制备胶束纳米药物过程简单、重现性好、胶束的稳定性好，在室温存放三周后胶束的粒径和粒径分布也基本不变（图1 D），没有沉淀；这比基于PEG-PDLLA聚合物制备的Genexol-PM稳定得多，Genexol-PM在2～4 h内粒径会增大并且产生很多晶状沉淀。本发明制备的MPTX比文献中报道的脂质体和多数纳米载体及Genexol-PM（PTX载药量10 wt.%）具有更高的载药量。
研究了制剂中的PEG350对MPTX载药量和稳定性的影响，将1 mL新制的载药量为4.8 wt.%的MPTX（1 mg/mL）的一半室温存储，另一半装入透析袋（MWCO 3500 Da）中，放入50 mL PB溶液（pH 7.4，10 mM）中透析3 h（每30分钟换一次缓冲液）。测定透析后粒径及PDI变化和PTX载药量的变化。结果发现，除去PEG350后胶束的PTX载药量稍微降低，但包封率保持在87%以上，说明该方法能把PTX紧密包在胶束里；但是透析后胶束粒径减小6-8 nm，PDI变大（表3），室温下放置1 h就逐渐出现浑浊和PTX析出，在200-1000 nm出现聚集体（图1 D），这与没有除去PEG350样品的稳定形成鲜明对比。
研究了聚合物中DTC对形成的PTX胶束载药量和稳定性的影响，相同条件制备的基于PEG-PCL载PTX不交联胶束ncMPTX和载4.8 wt.% PTX的PEG-P(CL-DTC)（MPTX），一半室温存放、一半透析，测定两组各种透析前后粒径及PDI变化和PTX载药量的变化，研究比较了ncMs和Ms两种胶束的紫外吸收、临界胶束浓度（CMC）以及两种载药胶束的稳定性。纳米粒中DTC的二硫戊环的330 nm的紫外吸收值降低很多，说明发生硫硫交联反应得到交联纳米粒。而本胶束在制备中由于没有透析，PEG350仍然存在，是否在制备后也能在胶束核内形成交联是不可预知的。本发明首先测定了两种胶束和相同浓度的对应聚合物溶液（5 mg/mL）的紫外吸收，发现PEG-PCL聚合物溶液及其胶束ncMs无明显的吸收峰。而Ms中DTC在326 nm出现二硫戊环的紫外吸收峰，但是其强度低于对应的聚合物溶液中DTC的强度（图1 E），下降了40.7%。这结果说明Ms胶束中部分二硫戊环开环，形成核内自交联。其次，用芘为荧光染料来确定两种胶束的临界胶束浓度（CMC），发现ncMs的CMC为15.6 mg/mL，但Ms在聚合物浓度为1.2 mg/mL至 2.5 mg/mL范围内I 1/I 3没有发现明显的转变（图1 F），即没有CMC，胶束在稀释到CMC以下时也不会解离成单分子。在实验中也发现将MPTX胶束浓度稀释到20 mg/mL测DLS时，胶束仍然完整，粒径呈正态分布，而ncMPTX胶束稀释至50 mg/mL时测试就出现聚集现象，PDI也迅速变大（图1 G）。此外，ncMPTX在室温下放置2天后变得不稳定，出现PTX析出以及大分子聚集（图1 G）。这些实验结果都证实了本发明制备的MPTX胶束是双硫交联的，这有助于进一步稳定PTX。综上，本发明基于PEG-P(CL-DTC) 的胶束MPTX比基于T g高得多的PEG-PDLLA的Genexol-PM还稳定得多。
表2不同载药量的MPTX的粒径以及冷冻液/冻干粉复溶后的粒径。
Figure 575009dest_path_image011
。
表3 MPTX透析前后粒径及载药量变化。
Figure 612235dest_path_image012
。
测试了胶束的还原响应行为，将MPTX（50 mg/mL，1 mL）在氮气保护下加入还原谷胱甘肽（GSH）溶液（10 mM），在37ºC、200 rpm的摇床中放置，DLS监测胶束粒径和PDI的变化。为研究PTX体外释放（n = 3），将ATN2-MPTX（50 mg/mL，1 mL），分别装入透析袋（MWCO 14 kDa）中，浸于25 mL含和不含10 mM GSH的PB溶液（pH 7.4，10 mM，含0.1% 吐温80）中，置于37ºC、200 rpm摇床中孵育。在指定时间点取透析液（5 mL），并补等体积相应新鲜介质。样品冷冻干燥后加500 mL乙腈/水（4/1, v/v）溶解，用HPLC测定PTX的浓度。MPTX在含10%FBS的PB缓冲液中（模拟体内血液环境）保持稳定，24 h粒径保持不变（图2 A）；然而在含10 mM DTT的PB溶液（模拟细胞质内还原环境）中其粒径在2 h时很快出现500-1000 nm的大颗粒，4 h时出现十纳米的小颗粒（图2 B），这是由于其疏水核中的二硫键在还原条件下逐渐解交联、胶束溶胀、在低聚合物浓度下会解离成单分子的缘故。这表明该胶束具有快速还原响应性能。
为便于运输和长期储存，制备了胶束冷冻液和冻干粉。冷冻液是将装PTX胶束（1 mg/mL，1 mL）玻璃瓶浸入液氮中冷冻10分钟，转移至-20度冰箱中储存，临用前在室温解冻。冻干粉是在PTX胶束（4 mg/mL，200 mL）中加入110 mL的由蔗糖与甘露醇（1/1，80 mg/mL）混合而成的冻干保护剂，混匀后浸入液氮中冷冻10分钟，之后用冻干机冻干24 h得到，使用时加入800 mL二次水复溶即可得到1 mg/mL的PTX胶束（保护剂占1.1% w/v）。分别测定PTX胶束冷冻/冻干前及解冻/复溶后的粒径和PDI的变化，HPLC测定载药量的变化。胶束通过解冻/复溶、测试DLS发现，MPTX依然保持澄清透明，除高载药量（23.1 wt.%）胶束冻干复溶后粒径和PDI变大之外，其余胶束的冷冻液和冻干粉的粒径及PDI基本保持不变（表2）。
实施例二 多种PTX胶束的制备及性质研究：装载PTX的靶向胶束是由两亲性嵌段聚合物PEG 2k-P(CL 1k-DTC 1k)和偶联靶向分子的聚合物Ta-PEG-P(CL-DTC)（Ta为多肽cRGD、ATN1或ATN2）在水相中自组装而成。在PEG-P(CL-DTC) 中分别混合重量百分含量为2.5%、5%、7.5%、10%或20%的ATN1-PEG-P(CL-DTC)、ATN2-PEG-P(CL-DTC)或cRGD-PEG-P(CL-DTC) 作为起始的聚合物溶液，然后和PTX溶液混合，其余步骤同实施例一，得到不同多肽表面密度的胶束ATN1-MPTX、ATN2-MPTX和cRGD-MPTX，为靶向小胶束纳米药物。
PEG-(CL-DTC)和不同比例的偶联cRGD、PHSCNK或PhScNK的靶向聚合物混合后，制备靶向胶束，采用实施例一相同方法、保持PTX载药量为4.8 wt.%，分别制备了三个系列具有不同靶向分子密度的胶束cRGD-MPTX、ATN1-MPTX和ATN2-MPTX。DLS测试结果表明，在表面多肽不超过5%时，随着多肽的增加，胶束粒径基本不变，在31到35 nm之间，PDI保持在0.2左右（表4）。此外，以PTX载药量为4.8 wt.%、多肽密度为5%的 ATN2-MPTX为例，研究了其中PEG350及DTC对胶束稳定性、FBS稳定性，以及冷冻/冻干复溶对粒径的影响，发现结果与非靶向MPTX相同（图1&2C）。研究了PTX胶束在模拟细胞中还原响应环境的药物释放行为，分别用含或不含10 mM DTT的PB溶液（含0.1%吐温80）作为释放介质，以ATN2-MPTX为例研究了在不同时间点释放到介质中的PTX的累积量。发现浓度为0.5 mg/mL的胶束在10 mM DTT中能够快速释放PTX，24 h的释放PTX累积量达79.4%；而在不含DTT环境中PTX 24 h仅泄漏17.0%（图2 D），结果表明胶束结构因为DTC交联而保持相对稳定、药物始终释放缓慢；而在还原条件下，双硫键断裂生成的巯基有一定的亲水性，胶束在较稀浓度下很难维持胶束结构，药物快速释放，呈现出明显的还原响应性药物控释行为。
表4 不同多肽密度胶束的粒径和粒径分布（PTX载药量为 4.8 wt.%）。
Figure 766136dest_path_image013
。
采用同样的步骤，由两亲性嵌段聚合物PEG 2k-P(CL 1k-DTC 1k)和偶联靶向分子的聚合物Ta-PEG 2k-P(CL 1k-DTC 1k)（重量百分含量为5%）在水相中自组装而成装载PTX的靶向胶束；Ta为多肽cRGD、ATN1或ATN2对应的粒径分别为27 nm、29 nm 和28nm。
本发明制备了PTX载药量高的胶束，且制备的小粒径胶束（20～40 nm），能够有效穿透至肿瘤深处，尤其是本发明载药过程中无药物析出，静置三周也无药物泄露，采用的小分子量PGE无需去除。
实施例三 MTT实验评估PTX胶束的细胞毒性：细胞选用小鼠高转移性三阴乳腺癌细胞株4T1。4T1细胞用常规方法培养，铺于96孔板（1.5×10 3个/孔，培养基80 mL），24 h后细胞汇合率达到60%左右，加入20 μL的MPTX、含不同多肽密度的胶束cRGD-MPTX、ATN1-MPTX和ATN2-MPTX，PTX浓度范围为0.002至5 μg/mL。孵育4 h后，弃掉培养基，加100 μL新鲜培养基后继续培养44 h。加入10 µL MTT溶液（5 mg/mL）孵育4 h，弃掉上清液，加150 µL DMSO来溶解活细胞和MTT产生的紫色甲瓒晶体。10分钟后用酶标仪测定570 nm的紫外吸收。加PBS的细胞作为对照组（100%），细胞的存活率为各孔吸光度与空白组吸光度的比值。每个实验平行5次（n=5），最终呈现的结果为平均值±SD。对4T1细胞的半数抑制浓度（IC 50）通过非线性回归计算得到。在受体抑制实验中，将4T1细胞预先用自由PhScNK多肽处理2小时后，再加入5%ATN2-MPTX孵育，后面的实验步骤和测试相同。
三阴乳腺癌是指患者的肿瘤雌激素受体ER、孕激素受体PR以及人表皮生长因子受体HER-2的表达均为阴性，内分泌治疗、化疗药物以及曲妥珠单抗的靶向治疗均无效或不敏感，因此侵袭性、转移性高，患者愈后较差，五年生存率低，亟需找到这类患者（占10%-15%）的有效治疗方法。本发明以鼠源三阴乳腺癌4Tl细胞为研究对象，用MTT法研究了不同靶向分子密度的三种胶束的细胞毒性。结果表明，MPTX对4T1的毒性具有浓度依赖性，IC 50为0.6 μg/mL，明显低于PTX（IC 50为1.12 μg/mL），主要是由于PTX胶束小、比表面积大，更容易被细胞摄取。而偶联靶向分子的胶束均表现了比MPTX更高的毒性（图3）。三种胶束对4T1的毒性不尽相同，整体来看，ATN2-MPTX比cRGD-MPTX毒性略高，远高于ATN1-MPTX；5%cRGD-MPTX、20%ATN1-MPTX、5%ATN2-MPTX在各组中相对其他多肽密度胶束具有最低的IC 50值，分别为0.27、0.46、0.21 μg/mL（表5），其中5%ATN2-MPTX的IC 50值最低，甚至低于自由PTX与4T1细胞共孵育48 h的IC 50值（0.55 μg/mL）。
表5 不同靶向分子密度胶束的IC 50值(μg/mL)总结。
Figure 914220dest_path_image014
。
进一步考察了5%ATN2-MPTX与4T1细胞共孵育24 h和48 h的细胞毒性（IC 50值分别为0.54 和0.020 μg/mL），发现该IC 50值均比无靶胶束（分别为1.19和0.064μg/mL）的低2-3倍。此外，空胶束Ms和ATN2-Ms在浓度1 mg/mL时共孵育48 h对4T1细胞均没有显示出毒性，这说明该方法制备的纳米载体具有良好的生物相容性。
实施例四 PTX胶束的细胞内吞行为研究：用流式细胞术FACS和激光共聚焦显微镜（CLSM）来考察4T1细胞对不同的靶向密度PTX胶束的摄取情况，由于聚合物和PTX两者本身都不带荧光，用CLSM和FACS无法检测到胶束的内吞行为。通过NHS-PEG-P(CL-DTC)与Cy5-NH2的酰胺化反应得到Cy5标记的PEG-P(CL-DTC)，并将其按照1%混合到原聚合物中可得到Cy5标记的胶束。FACS实验中，将4T1细胞（3×10 5/孔）铺于6孔板并培养24 h，加入1 wt.% Cy5标记的胶束Cy5/MPTX、Cy5/cRGD-MPTX和Cy5/ATN2-MPTX培养4 h。之后加胰酶消化细胞，用培养基终止，1000 rpm离心3分钟，再用PBS清洗两遍，最终分散至500 µL PBS中用于FACS测试，用FCS Express 5软件分析数据。CLSM实验中，将4T1细胞（5×10 4/孔）铺于24孔板中的载玻片上培养24 h后，加入100 μL上述Cy5胶束培养4 h。移去培养基，用4%的多聚甲醛溶液固定细胞15分钟，再用DAPI染细胞核5分钟，每步处理之后都用PBS清洗三遍。最后甘油封片，通过CLSM观察细胞内荧光并拍照。在内吞抑制实验中，将4T1细胞预先用自由PhScNK多肽处理2小时后，再加入5%ATN2-MPTX孵育，后面的实验步骤相同。通过NHS-PEG-(CL-DTC)和Cy5-NH 2的酰胺化反应，制备了荧光标记的聚合物Cy5-PEG-(CL-DTC)（标记效率> 99%），并在组成胶束聚合物成分中混入1 wt.%。用水合法制备即得到Cy5标记的胶束，不同靶向密度均选用表5中的最佳靶向密度。
FACS测试结果显示（图4 A），和Cy5/cRGD-MPTX、Cy5/ATN2-MPTX孵育4 h后的4T1细胞的荧光强度是对照无靶Cy5/MPTX组细胞的2.1倍和2倍，而Cy5/ATN1-MPTX与无靶组相比只有1.2倍。通过CLSM观察到，4T1细胞经含Cy5胶束孵育4 h后，各靶向胶束的荧光强度均高于无靶组，其中Cy5/ATN2-MPTX组最高，与PBS组 和Cy5/cRGD-MPTX都存在显著性差异（图4 B，*p），且Cy5/ATN2-MPTX组的Cy5荧光出现在每个细胞的细胞质中。而Cy5/cRGD-MPTX和Cy5/ATN1-MPTX组中均有部分细胞的细胞质中没有Cy5荧光（图4 C）。
实施例五 PTX胶束的药代动力学和生物分布研究：通过尾静脉将MPTX、ATN2-MPTX和cRGD-MPTX和自由PTX（7.5 mg PTX/kg）注入健康Balb/c小鼠体内（n = 3），其中自由PTX用Cremphor和无水乙醇体积比1:1为助溶剂制备。在预定时间点，用肝素化毛细采血管从小鼠内眦静脉从取血70 µL，立即离心（5000 rpm，20 min），取20 µL血清加入600 µL含20 mM DTT的DMF溶液，37度200 rpm摇床过夜萃取PTX。涡旋震荡后离心取上清液，过滤后用HPLC测定PTX的含量。PTX浓度对时间作图得药代动力学曲线，利用Origin8拟合半衰期（t 1/2 β）和曲线下面积（AUC）。
为建立小鼠4T1-luc三阴乳腺癌皮下瘤模型，给12只健康雌性Balb/c小鼠的右后腿上部皮下注射5×10 5个细胞（50 µL，含30%的基质胶）。当肿瘤体积达到200-250 mm 3左右时分组，共4组，每组3只，使每组动物具有相似的平均体重和肿瘤大小。为了观察PTX胶束在小鼠体内的生物分布，将Cy5/MPTX、Cy5/cRGD-MPTX或Cy5/ATN2-MPTX（7.5 mg PTX/kg，0.4 µg Cy5/只）尾静脉注射，在预定时间点腹腔注射荧光素钾盐15分钟后用近红外活体成像。
为了定量测定PTX胶束在不同器官中的富集，给12只荷瘤小鼠尾静脉注射PTX、Cy5/MPTX、Cy5/cRGD-MPTX或Cy5/ATN2-MPTX（7.5 mg PTX/kg，0.4 µg Cy5/只），4 h后先腹腔注射荧光素钾盐再颈椎脱臼处死动物，10分钟内取出小鼠的肿瘤及主要脏器（包括心、肝、脾、肺、肾），用PBS清洗、拭干进行离体成像并称重。各组织均称取约0.1 g，分别加500 µL的1%的Triton-100过夜孵育。之后剪碎、用匀质机（20000 rpm）研磨组织，加入1 mL含20 mM DTT的DMF溶液萃取PTX。48 h后离心（6000 rpm，15 min）取上清，过滤后用HPLC测量PTX的含量。PTX在各器官中的标准曲线的绘制是将一系列已知浓度PTX的DMF溶液（20 µL）加入至0.1 g的器官中，然后按照上面样品的处理方法处理后，测HPLC，绘制以峰面积为纵坐标、浓度为横坐标的PTX在各器官中的标准曲线。
纳米药物是否能有效进入肿瘤细胞对于其抗肿瘤效果有极大的影响，而这很大程度上取决于其药代动力学性能以及在肿瘤组织的富集情况。本发明制备的PTX胶束粒径较小（30-40 nm），研究了其药代动力学和生物分布。结果显示，MPTX、ATN2-MPTX和cRGD-MPTX三种PTX胶束的药代动力学曲线均呈现明显的两相模式：血液中PTX浓度快速下降的分布相和缓慢下降的消除相，其消除相半衰期（t 1/,2 β）分别为3.85、3.93和4.70 h（图5），均显著长于用Cremophor ® EL和无水乙醇（1:1）为溶剂制备的自由PTX（0.67 h）。这些PTX胶束的t 1/2 β都比Genoxel-PM（0.21 h，10 mg PTX/kg）和Nanoxel-PM（2.1 h，13 mg DTX/kg）要长得多。这表明不仅胶束表面的PEG能将胶束疏水内核及PTX和血液中蛋白成分阻隔开来，而且胶束交联的核使其在血液循环中更稳定，循环时间更长。
药物在肿瘤组织的富集水平，决定了载药胶束最终的抗肿瘤效果，而在正常组织的累积也会造成药物毒副作用。为了探究PTX胶束在小鼠肿瘤及正常组织中的分布，首先将Cy5/MPTX、Cy5/ATN2-MPTX、Cy5/cRGD-MPTX经尾静脉注入荷4T1瘤小鼠体内（7.5 mg PTX/kg），通过活体荧光成像观察胶束在小鼠体内和肿瘤的分布随时间的变化情况。结果发现，三种PTX胶束均先逐渐富集至肿瘤处，在4 -6 h时肿瘤富集量最多，之后缓慢减少（图6 A&B）。Cy5/ATN2-MPTX相较于Cy5/MPTX以及Cy5/cRGD-MPTX在所有时间点肿瘤的富集量均最多，其中4 h时其肿瘤的荧光强度分别为Cy5/MPTX和Cy5/cRGD-MPTX的1.8倍（*p）和1.2倍，在24 h时荧光还是很强，说明ATN2比cRGD具有更优异的靶向作用。其次，4 h时器官进行离体成像（图6 C）可以看出，Cy5/ATN2-MPTX胶束在肿瘤荧光强度最强，在主要脏器最弱。再次，HPLC测试4 h脏器组织中的PTX的量，结果表明， ATN2-MPTX在肿瘤有最高的PTX富集量（6.14% ID/g），分别是cRGD-MPTX（4.55% ID/g）、MPTX（3.51% ID/g）和PTX（2.84% ID/g）的1.4倍、1.7倍和2.2倍（图6 D），证实了ATN2-MPTX比无靶组显著高的肿瘤靶向作用，且效果优于cRGD-MPTX。此外，PTX的肿瘤富集量和正常组织富集量的比例（T/N）可说明药物的特异靶向性，T/N 越高特异性就越强。计算结果发现，ATN2-MPTX组的T/N均显著高于cRGD-MPTX和MPTX（图6 E），说明了ATN2-MPTX靶向4T1肿瘤的特异性最高，对正常组织的毒性最小。综合细胞实验和生物分布实验结果，5%ATN2-MPTX表现最佳，后面的实验如无特殊说明，靶向胶束就指的是这个组成。
实施例六 PTX胶束诱导免疫原性细胞死亡（ICD）研究：通过测定4T1细胞与PTX胶束孵育后产生的CRT、HMGB1及ATP的浓度来验证其诱导ICD的程度。将4T1细胞（5×10 4/孔）种于放置载玻片的24孔板中培养过夜，加100 μL的PTX、MPTX和ATN2-MPTX（PTX浓度为5 mg/mL）培养12 h。吸走培养基，用4%的多聚甲醛溶液固定细胞15分钟，用含有0.1% 吐温20的PBS封闭30 分钟，然后加入CRT抗体孵育60分钟，再加入Alexa Fluor-633二抗孵育30分钟，最后用DAPI染核5分钟。每步处理之后都用PBS洗涤三次，最后用甘油封片后用CLSM观察、拍摄。和PTX胶束孵育12 h后吸取的培养基分别用试剂盒按照使用说明测试ATP（碧云天）和HMGB-1（Elabscience）的浓度。
PTX等化药不仅可以直接杀死肿瘤细胞，还可以通过诱导肿瘤细胞的ICD来诱导内源性死细胞抗原（尤其是肿瘤抗原）的产生，进而引发机体的免疫应答。本发明的胶束纳米药物对肿瘤细胞显示了强细胞毒性和靶向性，为了验证其是否能有效诱导ICD，测试了MPTX、ATN2-MPTX和自由PTX以及空胶束ATN2-Ms在与4T1细胞孵育后表面CRT的暴露以及HMGB-1和ATP的分泌情况。首先，用CRT一抗及山羊抗兔的Alexa Fluor 633二抗来染色各组细胞，CLSM观察（图7 A&B）发现，孵育12 h后不同剂型诱导CRT的暴露有显著区别，空胶束也能诱导一定CRT的暴露，自由PTX和MPTX组的CRT暴露都比PBS组显著增加（***p），ATN2-MPTX组细胞表面的CRT表达最高（***p），且细胞存量最少，细胞皱缩，细胞核裂分，但是几乎每个细胞的表面都有强荧光。其次，Elisa法测试的4T1细胞培养基中释放的ATP和HMGB-1浓度（图7 C&D）结果显示，与CRT暴露的趋势相近：ATN2-MPTX组释放的ATP和HMGB-1最高，其中ATP显著高于MPTX组（**p）和自由PTX组（***p），HMGB-1比MPTX组高，也显著高于自由PTX组（**p）；空胶束和PBS相当，不诱导ATP和HMGB-1。这证明ATN2-MPTX由于能靶向进入细胞、更快杀死肿瘤细胞，因此其诱导ICD的能力不仅没有由于胶束包裹而比自由PTX有所下降，反而有明显上升。
实施例七 PTX胶束对原代免疫细胞的影响：按照常规操作，从健康Balb/c小鼠骨髓中提取原代骨髓细胞，体外刺激培养成为BMDM和BMDC。为探究PTX胶束对BMDC的影响，使用CCK-8法来研究悬浮细胞BMDC的增值情况。将BMDC铺于96孔板（1.5×10 4个/孔，1640培养基80 mL）培养过夜后，加入20 μL的PTX、MPTX和ATN2-MPTX（PTX浓度：0.002至20 μg/mL）。孵育24 h或48 h后，加入5 µL的CCK-8溶液孵育4 h，用酶标仪测定各孔在450 nm处的紫外吸收。加PBS的细胞作为100%，细胞的存活率为各孔吸光度与PBS组吸光度的比值。每个实验平行5次（n = 5），最终呈现的结果为平均值±SD。
用MTT法研究PTX胶束对BMDM的影响。将BMDM细胞（5×10 3个/孔）铺于96孔板中，1640培养基培养24 h后，加入20 μL的PTX、MPTX和ATN2-MPTX（PTX浓度：0.002至20 μg/mL）孵育4 h后，换新培养基后继续培养 44 h。后面的处理方法相同。
肿瘤组织里除了有大量的肿瘤细胞，还存在一定量的免疫细胞。PTX是有丝分裂抑制剂，可稳定微管聚合并防止其分解，使细胞有丝分裂停滞，但是该抑制作用是非特异性的，高浓度的PTX也可能会损害其他细胞，比如免疫细胞。DC是T细胞能识别的最有效的抗原呈递细胞（APC），在宿主免疫系统中起关键作用。肿瘤相关巨噬细胞（TAM）是肿瘤浸润免疫细胞的重要组成部分，与多种肿瘤的生长、血管生成和转移有关，具有强吞噬功能。为此，通过MTT和流式细胞仪研究了PTX纳米药物对DC和巨噬细胞的增殖及细胞因子分泌的影响。首先提取了小鼠骨髓原代细胞、刺激成BMDC，研究了不同浓度的ATN2-MPTX、MPTX和自由PTX在孵育24和48小时后对BMDC存活率和增殖的影响。如图8所示，三种PTX药物除20 mg/mL的自由PTX（48 h）外，孵育24、48 h都对BMDC无毒性，在一定程度上还促进了其增殖；孵育48 h后BMDC的增殖要高于24 h。这和Pandha等人报道的PTX会影响DC的增殖相吻合。此外，ATN2-MPTX在高浓度（20 mg/mL）和低浓度（0.04 mg/mL）区间，均比自由PTX和MPTX使BMDC增值要多（图8 C D）。尤其是自由PTX在浓度20 mg/mL孵育48h毒性增强，BMDC存活率显著下降（到50%左右）。形成鲜明对比的是，两种PTX胶束在该浓度下仍然能促进BMDC的增殖（***p），说明胶束能稳定装载PTX，使PTX的暴露量大大下降，增加了BMDC的耐受能力。这将有助于PTX胶束治疗实体瘤时，杀伤肿瘤细胞的同时使体内免疫微环境中的DC增殖，这对于联合APC刺激或调节剂进行高效肿瘤治疗非常有益。
另一方面，ATN2-MPTX能诱导产生ICD，释放肿瘤相关抗原（TAA），本发明研究了ATN2-MPTX对BMDC成熟的影响，用FACS测试PTX制剂与BMDC孵育24 h后成熟BMDC（CD11c +CD86 + CD80 +）的比例。如图9A&B所示，与PBS组相比，自由PTX、MPTX和ATN2-MPTX均能使CD11c + 的BMDC数量增加，与MTT结果一致。另外，三者激活的成熟BMDC比PBS组显著高（***p），这和三者促进BMDC增殖有关。所以，ATN2-MPTX不仅在BMDC数量上还在激活其成熟上都有利。此外，用ELISA法测试了三者刺激BMDC成熟，分泌的促炎细胞因子TNF-α、IL-6的含量（图9C&D），也发现三者比PBS组能诱导产生更多的TNF-α（**p）和IL-6（***p）。
实施例八 PTX胶束诱导BMDM极化和分泌细胞因子：将BMDM（1×10 6个/孔）铺于24孔板中，在1640培养基中培养24 h，为M0型巨噬细胞（M0M），而向1640培养基中加入IL-4（20 ng/mL）刺激，24 h后得到M2型巨噬细胞（M2M）。将M2M的培养基换成新鲜培养基后，加PTX、MPTX和ATN2-MPTX（PTX浓度：5 µg/mL）孵育24 h后，收集上清液以检测TNF-α和IL-6含量；并消化细胞，加CD11b、F4/80、CD206荧光标记的抗体，FACS测试M1M (CD11b +F4/80 +CD206 -)和M2M (CD11b +F4/80 +CD206 +)含量。
为了研究了M2M与PBS、PTX、MPTX和ATN2-MPTX孵育24 h对M2M的调节作用（图10），以加入IL-4的细胞为M2M阳性对照，加LPS为M1M阳性对照。先用FITC-CD11b和PE-F4/80抗体标记巨噬细胞（CD11b +F4/80 +），再用Alexa Fluor 647-CD206抗体标记M2M（CD206 +）和M1M（CD206 -），计算其占总巨噬细胞得比例。FACS检测结果发现，与IL-4共孵育48 h后24 h不刺激，M1型比例在19.3%，说明IL-4刺激48 h能维持M2表型；而后又加入IL-4孵育24 h能刺激BMDM更多地极化为M2型，M1M比例降低至14.0%（图10A&B）。加入LPS孵育24 h使刺激成M2M部分极化为M1M，M1M的比例增加至40.1%（*p）。而当向IL-4刺激的M2M中加入PTX、MPTX或ATN2-MPTX，发现能使M1M比例能分别增加至23.9%、40.7%（*p）和42.9%（**p）（图10 B），这证明ATN2-MPTX更容易被巨噬细胞内吞，使之向M1M极化。用ELISA法检测了不同组培养基中M1M诱导的促炎细胞因子TNF-α、IL-6的含量，发现和PBS组和IL-4刺激的M2M组相比，PTX制剂均能显著促进TNF-α和IL-6的分泌（***p），其中ATN2-MPTX和MPTX诱导的最多，与LPS刺激的M1M的分泌量相当（图10 C&D）。而MTT法检测不同PTX制剂对BMDM有浓度依赖性的细胞毒性，BMDM更不耐受PTX药剂，其细胞存活率要低于BMDC；MPTX和ATN2-MPTX的IC 50值相近，是PTX的2倍（图10 E）。
 实施例九PTX胶束激活BMDC的研究：BMDC（1×10 6个/孔）在12孔板中培养24 h后加入PBS、PTX、MPTX、ATN2-MPTX孵育24 h，最终PTX浓度为5 µg/mL。孵育结束后离心（1500 rpm，5 min），分离出上层培养基，收集细胞后用荧光标记的CD11c、CD80和CD86抗体按说明书来染细胞，最后用FACS测试各组中总CD11c + BMDC的量和CD11c +CD80 +CD86 +成熟BMDC的含量。分离的培养基用ELISA试剂盒分别测定IFN-β、TNF-α和IL-6的浓度。
ATN2-MPTX可诱导ICD，产生一系列肿瘤抗原，并能使BMDC增殖与成熟，ATN2-MPTX诱导的TAA能更有效地被这些激活的APC呈递给T细胞，从而招募更多的T细胞。FACS测试结果表明，相比于PBS组，ATN2-MPTX能促进总CD11c +的BMDC含量（图11 A&B）；测定了BMDC培养基中IFN-β、TNF-
Figure 469967dest_path_image015
和IL-6的浓度。细胞因子的增多表明BMDC成熟，抗原呈递能力增强，可极强地激活T细胞，其中IFN-β是CDN与STING结合后产生的标志性细胞因子，对肿瘤引发T细胞启动具有关键作用，见图11 C、D。
实施例十 PTX胶束用于小鼠TNBC的抗肿瘤治疗：在接种4T1（3×10 5/只）的荷瘤小鼠的肿瘤体积在50-100 mm 3（接种后第6天）开始给药，给药当天为第0天。小鼠随机分4组，每组7只，每两天给药，共4次；给药剂量为7.5 mg PTX/kg和/或1 mg CDN/kg。分别为：PBS、自由PTX（i.v.）、MPTX（i.v.）、ATN2-MPTX（i.v.）。实验过程中每2天监测小鼠体重、肿瘤体积和小鼠状态。小鼠死亡或肿瘤体积大于2000 mm 3判定死亡，绘制生存曲线。给荷瘤（肿瘤体积约50 mm 3）小鼠每2天尾静脉注射ATN2-MPTX，并把药量降低到7.5 mg/kg，共给药4次（图12A），研究该方案对小鼠肿瘤尺寸、体重和生存期的影响，当肿瘤体积大于2000 mm 3时，判定小鼠死亡（#号表示）。系统比较ATN2-MPTX、MPTX的抗肿瘤效果。参见图12。结果显示，4T1肿瘤的恶性程度极高，PBS组肿瘤快速增长（图12 B），到第20天肿瘤体积均大于2000 mm 3，中位生存期为18天。自由PTX组在给药期间肿瘤缓慢生长，停药后开始快速生长。而ATN2-MPTX、MPTX均能显著抑制肿瘤的生长，10天内肿瘤基本不长，但10-12天开始肿瘤都开始缓慢生长。比较第22天肿瘤体积可知，MPTX可能由于小粒径而使其EPR效应显著，比PTX能更有效抑制肿瘤生长（*p）。而ATN2-MPTX则能比PTX和MPTX更进一步限制肿瘤生长（***p）（图12 B），延长小鼠的生存时间，体现了靶向抗肿瘤疗效。此外，小鼠在治疗期间体重变化很小，尾静脉给7.5 mg PTX/kg没有对小鼠产生毒副作用（图12 D）。
实施例十一 治疗后评估小鼠免疫状态、毒副作用和肺转移：同实施例十描述，荷瘤小鼠随机分4组，每组7只。分别为：PBS（i.v.）、自由PTX（i.v.）、MPTX（i.v.）、ATN-MPTX（i.v.），按照7.5 mg PTX/kg给药4次，每2天一次。最后一次给药后24 h每组处死4只小鼠，取小鼠全血分离血清测定IFN-β、IFN-
Figure 678094dest_path_image016
、IL-6和TNF-
Figure 319291dest_path_image015
的含量。取脾脏和肿瘤，研磨成单细胞悬液，裂红后计数。对脾脏，每个样品6×10 6个细胞进行T细胞染色。对肿瘤，每个样品分为4份，每份6×10 6个细胞，进行DC、巨噬细胞、T细胞染色，通过FACS测试分析。免疫细胞相应的荧光标记的抗体为：DC：anti-CD11c-FITC、anti-CD80-APC、anti-CD86-PE；巨噬细胞：anti-CD11b-FITC、anti- F4/80-PE、anti-CD206- Alexa Fluor 647；T细胞：anti-CD3-APC、anti-CD4-PE、anti-CD8-FITC；Tregs：anti-CD3-FITC、anti-CD4-PE、anti-Foxp3- Alexa Fluor 647。
肿瘤免疫微环境（TME）是由恶性细胞、免疫细胞、血管、细胞外基质和信号分子组成的环境，可单独发挥作用，也可共同影响免疫疗法的敏感性。上文已证明，ATN2-MPTX可靶向进入肿瘤细胞诱导ICD、释放肿瘤相关抗原，产生有利的免疫微环境。这里将分析ATN2-MPTX给药24 h后小鼠TME以及脾脏中T细胞的影响，见图13。首先分析了TME中CD80 +CD86 + 成熟DC和CD206 -的M1M的含量，PTX胶束成熟DC比例都要比PBS高。前面细胞实验中，PTX可使巨噬细胞从M2M转化为M1M。在TME中，ATN2-MPTX能促进了M2M极化为CD206 -的M1M增多。研究了各组肿瘤TME中T细胞的比例，分析了T regs在CD4 + T中的占比及CD8 + T/T regs。ELISA结果表明，ATN2-MPTX的促炎细胞因子IFN-
Figure dest_path_image017
、IFN-γ、TNF-α和IL-6含量均增加，这些结果证实了其能诱导强烈的抗肿瘤免疫反应。
在给药第22天，给小鼠腹腔注射荧光素钾盐，10分钟内处死每组剩余的3只小鼠，取全血进行血常规分析、分离出血清进行血生化分析。此外，解剖小鼠，取和心、肝、脾、肺、肾等器官，用组织固定液固定、切片、石蜡封切。切片用苏木精和伊红（H&E）染色，显微镜观察切片进行组织学分析和肺转移判断。分析了ATN2-MPTX给药后小鼠脾脏中CD4 +和CD8 + T细胞占CD45 +总免疫细胞的比例。图14显示，各组药物给药后第8天小鼠脾中的CD4 + T和CD8 + T细胞的变化趋势和肿瘤中的一致。
随着4T1乳腺癌病情的发展，小鼠的血液系统以及器官会受到一定影响；而长期用药也会给小鼠带来全身累计毒性。为了研究本发明的PTX胶束对荷瘤小鼠的全身累计影响或毒副作用的改善，在如上给药第22天，每组牺牲3只小鼠，取全血进行血常规检测、分离血清进行血生化检测，并解剖取主要脏器观察并切片，用H&E染色进行组织学分析。血常规结果显示（图15 A），荷瘤小鼠血液（PBS组）中白细胞（WBC）数大量增多，是正常Balb/c小鼠的91倍（**p），与文献报道一致。其中的中性粒细胞数（Neut）（*p）和淋巴细胞数（Lymph）（**p）升高最显著，这可能是由于肿瘤的生长和转移，以及出现了一些炎症导致的。红细胞数（RBC）、血红蛋白浓度（HGB）、红细胞压积（HCT）较正常小鼠有所下降，这主要是由于荷瘤小鼠出现贫血。此外，PBS组小鼠血液中的血小板数（PLT）、血小板压积（PCT）和平均血小板浓度（MPV）均有增加，这将增加血栓发生的概率。有研究表明，血小板有助于肿瘤细胞的存活和转移，因此血小板的增加也促使了4T1乳腺癌发生转移。和PBS组小鼠相比，自由PTX治疗小鼠的血常规指标基本无缓解，ATN2-MPTX治疗的小鼠血常规指标均有所缓解。血生化检测表明，主要肝（碱性磷酸酶ALP、谷氨酰基转移酶GGT、天门冬氨酸氨基转移酶AST、丙氨酸氨基转移酶ALT）、肾（肌酐CRE、尿素URE）功能的各项指标与正常Balb/c小鼠的无显著性差异（图15B）。
荷瘤小鼠主要脏器的H&E染色切片（图16）显示，各组小鼠心、肝、肾没有出现明显的组织损伤。但PBS组小鼠心脏有少许炎症，而PTX制剂各组小鼠心脏正常；除ATN2-MPTX组，其他组小鼠肝脏均有轻微炎症发生。此外，PBS组小鼠脾脏显著增大、质量增加（图17），Yong等人也发现荷4T1乳腺癌Balb/c小鼠有脾肿大现象。治疗组小鼠脾脏的质量和肿瘤大小呈现正相关，即肿瘤越小，脾越小。从脾的H&E染色（图16）可以看到，PBS组白髓明显减少，红髓变多，这是4T1小鼠病程发展的标志之一。治疗组具有丰富的白髓，说明阻止了乳腺癌病程的发展。以上结果说明，尾静脉给7.5 mg PTX/kg，每2天给药、共4次的方式对小鼠没有明显毒性。
由于TNBC具有高浸润性及高转移性，研究了PTX胶束对荷4T1-luc乳腺癌肺转移的抑制。如上给药后22天，解剖取肺部、称重以及离体成像、切片H&E染色做组织学分析。肺部成像及荧光定量结果显示（图18 A&B），PBS和自由PTX组小鼠发生了严重的肺转移，MPTX组肺部荧光有显著下降（***p），其中ATN2-MPTX较MPTX也具有明显的肺转移抑制（*p）。此外，肺部H&E染色图片显示，PBS组和自由PTX小鼠肺部存在大量肿瘤细胞和炎症细胞的浸润，几乎没有肺泡结构；MPTX组有所改善，ATN2-MPTX相比没有明显的肿瘤细胞群（图18 D），结果和生物发光图吻合。

Claims (10)

  1. 一种小胶束纳米药物，其特征在于，所述小胶束纳米药物的制备方法为，将小分子药物、两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到小胶束纳米药物；所述低聚乙二醇的分子量为200～600。
  2. 根据权利要求1所述小胶束纳米药物，其特征在于，小分子药物为紫杉醇；两亲性聚合物为PEG-P(CL-DTC)、PEG-P(TMC-DTC)、PEG-P(LA-DTC)中的一种或几种。
  3. 权利要求1所述小胶束纳米药物的制备方法，其特征在于，将小分子药物、两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到小胶束纳米药物。
  4. 一种靶向小胶束纳米药物，其特征在于，所述靶向小胶束纳米药物的制备方法为，将小分子药物、两亲性聚合物、靶向两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到靶向小胶束纳米药物；所述低聚乙二醇的分子量为200～600。
  5. 权利要求1所述小胶束纳米药物或者权利要求4所述靶向小胶束纳米药物在制备抗肿瘤药物中的应用。
  6. 一种靶向小胶束纳米药物的制备方法，其特征在于，包括以下步骤，将小分子药物、两亲性聚合物、靶向两亲性聚合物加入低聚乙二醇中，得到混合溶液，再将所述混合溶液加入缓冲溶液中，得到靶向小胶束纳米药物；所述低聚乙二醇的分子量为200～600。
  7. 根据权利要求6所述靶向小胶束纳米药物的制备方法，其特征在于，靶向两亲性聚合物为所述两亲性聚合物偶联靶向分子；两亲性聚合物为PEG-P(CL-DTC)、PEG-P(TMC-DTC)、PEG-P(LA-DTC)中的一种或几种。
  8. 根据权利要求6所述靶向小胶束纳米药物的制备方法，其特征在于，靶向两亲性聚合物的用量为聚合物总重量的1～30%。
  9. 一种小胶束纳米药物冷冻液，其特征在于，所述小胶束纳米药物冷冻液的制备方法为，将权利要求1所述小胶束纳米药物或者权利要求4所述靶向小胶束纳米药物在液氮中冷冻后存储于冰箱中，得到小胶束纳米药物冷冻液；冰箱的温度为-80℃～-10℃。
  10. 一种小胶束纳米药物冻干粉，其特征在于，所述小胶束纳米药物冻干粉的制备方法为，将权利要求1所述小胶束纳米药物或者权利要求4所述靶向小胶束纳米药物与冻干保护剂混合后在液氮中冷冻，然后利用冻干机干燥，得到小胶束纳米药物冻干粉。
PCT/CN2022/097029 2021-06-04 2022-06-03 一种小胶束纳米药物及其制备方法与应用 WO2022253342A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110626601.3A CN113332241A (zh) 2021-06-04 2021-06-04 一种小胶束纳米药物及其制备方法与应用
CN202110626601.3 2021-06-04

Publications (1)

Publication Number Publication Date
WO2022253342A1 true WO2022253342A1 (zh) 2022-12-08

Family

ID=77474415

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/097029 WO2022253342A1 (zh) 2021-06-04 2022-06-03 一种小胶束纳米药物及其制备方法与应用

Country Status (2)

Country Link
CN (1) CN113332241A (zh)
WO (1) WO2022253342A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116509801A (zh) * 2023-05-06 2023-08-01 复旦大学 一种适配胰腺癌微环境的乏氧响应递药胶束及制备方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113332241A (zh) * 2021-06-04 2021-09-03 苏州大学 一种小胶束纳米药物及其制备方法与应用
CN113476402B (zh) * 2021-07-05 2023-08-01 苏州大学 一种多西他赛胶束纳米药物及其制备方法与应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107281141A (zh) * 2017-06-26 2017-10-24 苏州大学 生物可降解交联纳米药物冻干粉的制备方法
CN108245483A (zh) * 2016-12-28 2018-07-06 中国人民解放军第二军医大学 一种包载难溶性抗肿瘤药物的聚合物纳米胶束体系
CN108478531A (zh) * 2018-05-21 2018-09-04 中国医学科学院生物医学工程研究所 叶酸靶向还原敏感载药聚合物纳米胶束及其制备方法和应用
CN113332241A (zh) * 2021-06-04 2021-09-03 苏州大学 一种小胶束纳米药物及其制备方法与应用
CN113350283A (zh) * 2021-06-04 2021-09-07 苏州大学 一种化疗免疫联合药物及其制备方法与应用
CN113476402A (zh) * 2021-07-05 2021-10-08 苏州大学 一种多西他赛胶束纳米药物及其制备方法与应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107096038B (zh) * 2017-04-12 2021-06-18 苏州大学 基于主动反应型一步法的交联纳米药物的制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108245483A (zh) * 2016-12-28 2018-07-06 中国人民解放军第二军医大学 一种包载难溶性抗肿瘤药物的聚合物纳米胶束体系
CN107281141A (zh) * 2017-06-26 2017-10-24 苏州大学 生物可降解交联纳米药物冻干粉的制备方法
CN108478531A (zh) * 2018-05-21 2018-09-04 中国医学科学院生物医学工程研究所 叶酸靶向还原敏感载药聚合物纳米胶束及其制备方法和应用
CN113332241A (zh) * 2021-06-04 2021-09-03 苏州大学 一种小胶束纳米药物及其制备方法与应用
CN113350283A (zh) * 2021-06-04 2021-09-07 苏州大学 一种化疗免疫联合药物及其制备方法与应用
CN113476402A (zh) * 2021-07-05 2021-10-08 苏州大学 一种多西他赛胶束纳米药物及其制备方法与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RIDOLFO ROXANE, EDE BENJAMIN C., DIAMANTI PARASKEVI, WHITE PAUL B., PERRIMAN ADAM W., VAN HEST JAN C. M., BLAIR ALLISON, WILLIAMS : "Biodegradable, Drug-Loaded Nanovectors via Direct Hydration as a New Platform for Cancer Therapeutics", SMALL, WILEY, vol. 14, no. 32, 1 August 2018 (2018-08-01), pages 1703774, XP093009681, ISSN: 1613-6810, DOI: 10.1002/smll.201703774 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116509801A (zh) * 2023-05-06 2023-08-01 复旦大学 一种适配胰腺癌微环境的乏氧响应递药胶束及制备方法

Also Published As

Publication number Publication date
CN113332241A (zh) 2021-09-03

Similar Documents

Publication Publication Date Title
Ma et al. Bioresponsive immune-booster-based prodrug nanogel for cancer immunotherapy
Wang et al. Cancer nanomedicines stabilized by π-π stacking between heterodimeric prodrugs enable exceptionally high drug loading capacity and safer delivery of drug combinations
WO2022253342A1 (zh) 一种小胶束纳米药物及其制备方法与应用
Belhadj et al. Multifunctional targeted liposomal drug delivery for efficient glioblastoma treatment
KR102190093B1 (ko) 난소암을 특이적으로 표적하는 생분해성 양친성 폴리머, 이로부터 제조된 폴리머 배시클 및 용도
WO2022253080A1 (zh) 一种化疗免疫联合药物及其制备方法与应用
Liang et al. Π electron-stabilized polymeric micelles potentiate docetaxel therapy in advanced-stage gastrointestinal cancer
Shang et al. Enhancing cancer chemo-immunotherapy by biomimetic nanogel with tumor targeting capacity and rapid drug-releasing in tumor microenvironment
CN113679670B (zh) 一种载氯喹化合物的囊泡纳米药物及其制备方法与应用
CN112516109A (zh) 一种基于间充质干细胞的融合癌细胞膜仿生纳米粒及其制备方法
Cheng et al. Hydrogen-bonded supramolecular micelle-mediated drug delivery enhances the efficacy and safety of cancer chemotherapy
Shao et al. Targeting fluorescence imaging of RGD-modified indocyanine green micelles on gastric cancer
WO2023280128A1 (zh) 一种多西他赛胶束纳米药物及其制备方法与应用
Wu et al. Promote Intratumoral Drug Release and Penetration to Counteract Docetaxel‐Induced Metastasis by Photosensitizer‐Modified Red Blood Cell Membrane‐Coated Nanoparticle
Zhang et al. Zwitterionic choline phosphate conjugated folate-poly (ethylene glycol): a general decoration of erythrocyte membrane-coated nanoparticles for enhanced tumor-targeting drug delivery
WO2022152298A1 (zh) 载 plk1 抑制剂的聚合物囊泡药物及其制备方法与应用
US11260031B2 (en) Protein particle with a poorly water-soluble drug encapsulated therein and preparation method thereof
Li et al. Multifunctional liposomes remodeling tumor immune microenvironment for tumor chemoimmunotherapy
Gou et al. Non‐Pore Dependent and MMP‐9 Responsive Gelatin/Silk Fibroin Composite Microparticles as Universal Delivery Platform for Inhaled Treatment of Lung Cancer
Jin et al. A comparative study on the effect of docetaxel-albumin nanoparticles and docetaxel-loaded PEG-albumin nanoparticles against non-small cell lung cancer
Sudareva et al. Doxorubicin delivery systems based on polypeptide nanoparticles for subcutaneous administration in cancer therapy
Wang et al. Hemin-lipid assembly as an artemisinin oral delivery system for enhanced cancer chemotherapy and immunotherapy
CN107028882B (zh) 一种物理包裹的肿瘤靶向纳米递药系统及制备方法和应用
Fan et al. Polysialic acid self-assembled nanocomplexes for neutrophil-based immunotherapy to suppress lung metastasis of breast cancer
Xu et al. Bufalin-loaded CaP/DPPE-PEG-EGF nanospheres: preparation, cellular uptake, distribution, and anti-tumor effects

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22815381

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22815381

Country of ref document: EP

Kind code of ref document: A1