CN109762099B

CN109762099B - Polymer-antitumor drug conjugate and preparation method and application thereof

Info

Publication number: CN109762099B
Application number: CN201811482988.4A
Authority: CN
Inventors: 罗奎; 蔡豪; 戴幸杭; 龚启勇
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2017-12-06
Filing date: 2018-12-05
Publication date: 2022-03-04
Anticipated expiration: 2038-12-05
Also published as: CN109762099A

Abstract

The invention provides a polymer shown as a formula (I) and a preparation method thereof. The polymer prepared by the invention has a narrow polydispersity index. The invention also provides a polymer-antitumor drug conjugate and a preparation method thereof. The polymer-antitumor drug conjugate prepared by the invention has longer blood circulation time and can obtain larger drug accumulation at tumor sites, so that the drug has better antitumor effect and wide market application prospect.

Description

Polymer-antitumor drug conjugate and preparation method and application thereof

Technical Field

The invention relates to a polymer-antitumor drug conjugate and a preparation method and application thereof.

Background

Polymer-drug conjugates as nanoscale drug delivery systems have now become a major focus in the field of targeted cancer chemotherapy research. Conjugation of low molecular weight anticancer drugs with water soluble polymers with good biocompatibility has become a general strategy to improve the anticancer efficacy of small molecule drugs. This is because the drug delivery system based on polymer-drug conjugates has many unique structures and properties, thus being able to have the potential to improve the solubility and stability of the drug, increase the accumulation of the drug in the tumor through Enhanced Permeability and Retention (EPR) effect, and reduce its side effects. For example, the topoisomerase II inhibitor Doxorubicin (DOX) can induce irreversible single-and double-stranded DNA break transcription and replication, yet has proven difficult to accumulate efficiently at tumor sites due to its short plasma half-life and non-targeting, and it can rapidly spread into the heart, leading to potential cardiotoxicity. To solve these problems, polymer-DOX conjugates prepared by conjugating DOX to a polymer via a linker sensitive to the tumor microenvironment are effective in improving the therapeutic effects thereof. However, despite the great advances in the research of polymers as drug carriers, only a few polymers are currently used for in vivo and clinical studies, mainly due to the inherent toxicity and/or immunogenicity of the polymers.

Therefore, the selection of safe and effective polymeric carriers plays an important role in the construction of polymeric delivery systems.

Disclosure of Invention

In order to solve the above problems, the present invention provides a polymer-antitumor drug conjugate.

The invention firstly provides a polymer shown as a formula (I):

wherein R is

x is 43-265; y is 1 to 8.

Further, the weight average molecular weight of the polymer is 28-35 KDa.

The invention also provides a method for preparing the polymer, which comprises the following steps:

taking a monomer (a), MA-Ala-NHNHBoc, a chain transfer agent and an initiator to react at 45 +/-3 ℃ in the presence of an organic solvent to obtain a compound shown in a formula (I);

further, the monomer (a) is N- (2-hydroxypropyl) methacrylamide or poly (ethylene glycol) methyl ether methacrylate.

Further, when monomer (a) is N- (2-hydroxypropyl) methacrylamide, the molar ratio of monomer (a), MA-Ala-NHNHBoc, chain transfer agent and initiator is 1225: 65: 3-4: 1.

further, when the monomer (a) is poly (ethylene glycol) methyl ether methacrylate, the molar ratio of the monomer (a), MA-Ala-NHNHBoc, the chain transfer agent and the initiator is 315-316: 69-70: 2-3: 1.

further, the organic solvent is methanol aqueous solution; the chain transfer agent is 4-cyanovaleric acid dithiobenzoic acid; the initiator is 2,2' - [ azobis (1-methylethylidene) ] bis [4, 5-dihydro-1H-imidazole ] dihydrochloride.

Further, the methanol in the aqueous methanol solution was 80% v/v.

The invention also provides a polymer-antitumor drug conjugate, which is prepared by the steps of deprotecting the polymer and reacting the deprotected polymer with doxorubicin hydrochloride, and has the following structure:

further, the drug loading rate of the polymer-antitumor drug conjugate in unit weight is 3-12%; preferably 4.5 to 6 percent.

The invention also provides a method for preparing the polymer-antineoplastic drug conjugate, which comprises the following steps: taking the polymer of

claim

1 or 2 to carry out deprotection, and then reacting with doxorubicin hydrochloride to obtain the polymer-antitumor drug conjugate.

The invention also provides the application of the polymer-antitumor drug conjugate in preparing antitumor drugs.

Further, the tumor includes malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, ewing's sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous carcinoma, testicular cancer, stomach cancer, liver cancer.

The invention also provides a pharmaceutical composition, which is prepared into a commonly used pharmaceutical preparation by taking the polymer-antitumor drug conjugate as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

The polymer prepared by the invention has narrow polydispersity index. The polymer-antitumor drug conjugate prepared from the polymer has longer blood circulation time and can obtain larger drug accumulation at tumor sites, so that the drug has better antitumor effect and wide market application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the synthetic steps and chemical structures of pOEGMA-DOX prepared in example 1 and pHPMA-DOX prepared in example 2.

FIG. 2 is a drawing of OEGMA copolymer and pOEGMA-DOX conjugate (dissolved in D) without drug₂In O) of¹H NMR spectrum (a); preparation of drug-free HPMA copolymer and pHPMA-DOX conjugate (dissolved in D2O) prepared in example 2¹H NMR spectrum (B).

FIG. 3 is a particle size distribution, zeta potential and TEM image of pOEGMA-DOX (A, 9.8 nm; B, -2.29 mV; C, about 10-20nm) and a size distribution, zeta potential and TEM image of pHPMA-DOX (D, 10.3 nm; E, -0.32 mV; F, about 10 nm).

FIG. 4 is a plot of particle size and PDI for pOEGMA-DOX and pHPMA-DOX in PBS and cell culture medium as a function of time, with values representing mean. + -. SD, as shown in E and F for pOEGMA-DOX and pHPMA-DOX in 10% FBS-containing PBS, and as shown in FIG. G for the DLS test results for material-free 10% FBS-containing PBS.

FIG. 5 is a release profile of DOX after incubation of pOEGMA-DOX and pHPMA-DOX in phosphate buffer (pH7.4 or 5.4) at 37 ℃ for 60 hours.

FIG. 6 shows the results of cellular uptake of pOEGMA-DOX and pHPMA-DOX, by incubating 4T1 cells with the two materials for 2 hours, 4 hours and 6 hours (A1-C2), respectively (scale bar 10 μm).

FIG. 7 is a flow cytometric analysis of 4T1 cells after incubation with DOX-loaded polymers at various time points (A), cellular uptake of free DOX after 0.5 hour incubation in 4T1 cells (B); nuclei were stained with DAPI (blue), and fluorescence of DOX was set to red (scale bar 10 μm).

FIG. 8 is the cytotoxicity of pOEGMA polymer without drug and pHPMA polymer without drug on 4T1 cells (A), free DOX, pOEGMA-DOX and pHPMA-DOX on 4T1 cells (B).

FIG. 9 is apoptosis detected by flow analysis after labelling of 4T1 cells with annexin V-FITC.

Figure 10 is a mean plasma concentration-time curve of DOX after intravenous administration of DOX, peggma-DOX and pHPMA-DOX at a dose of 4mg/kg in Balb/c mice (n ═ 5).

Figure 11 is an ex vivo fluorescence imaging study (n ═ 5): a1-4 is fluorescence imaging of mice at 6 hours, 12 hours, 24 hours and 36 hours after injection of saline (top row), DOX & HCl (second row), pOEGMA-DOX (third row) and pHPMA-DOX (bottom row), respectively; b, C and D are respectively half-quantitative average fluorescence signals obtained by pOEGMA-DOX, pHPMA-DOX and DOX through a Maestroan in vivo imaging system; e is the mean signal at the tumor site for pOEGMA-DOX, pHPMA-DOX and DOX.

Figure 12 is the in vivo anticancer efficacy of various DOX formulations: (A) tumor growth curves after intravenous injection of saline, DOX · HCl (4 or 8mg/kg), pHPMA-DOX (4 or 8mg/kg) and phegma-DOX (4 or 8mg/kg) in a 4T1 tumor model (n ═ 7); (B) the weight of the mice changes in the experimental process; (C) weighing tumors of each group of mice; (D) tumor inhibition (TGI) for each experimental group.

Detailed Description

Material

Poly (ethylene glycol) methyl ether methacrylate (OEGMA, Mw ═ 500Da), 2,2' - [ azobis (1-methylethylidene) ] bis [4, 5-dihydro-1H-imidazole ] dihydrochloride (VA044), 4-cyanovaleric acid dithiobenzoic acid (CTA), doxorubicin hydrochloride (DOX · HCl) and all other reagents and solvents were purchased from Sigma-Aldrich.

The HPMA reference Ulbrich K,

V,Strohalm J,et al.Polymeric drugs based on conjugates of synthetic and natural macromolecules:I.Synthesis and physico-chemical characterisation[J]the synthesis method was described in Journal of controlled release,2000,64(1): 63-79.

MA-Ala-NHNHBoc reference Etych T, Mrkvan T, Chytil P, et al.N- (2-hydroxypyropy) methylated amide-based polymer conjugates with pH-controlled activation of doxorubicin.I.New synthesis, physiochemical characterization and prediction biological evaluation [ J ] Journal of applied polymer science,2008,109, (5): 3050-3061.

4T1 Breast cancer cells were purchased from the cell bank of the type culture Collection of Chinese academy of sciences (China, Shanghai), cultured in RPMI1640 medium (Life technologies, USA) containing 10% (v/v) fetal bovine serum (FBS, Hyclone) and 1% (v/v) penicillin/streptomycin in a constant temperature and humidity cell culture chamber (5% CO)₂(ii) a Cultured at 37 ℃). Female BALB/c mice (weight 20. + -. 1.2g and 6-8 weeks old) were purchased from Duoduosho Biotech, Inc. All animal experiments were performed according to the rules of the ethical committee of the relevant countries and university of sichuan china.

Use of

Size Exclusion Chromatography (SEC) of FPLC system (GE Healthcare) to measure the average molecular weight and polydispersity index (PDI) of the polymer. Sodium acetate buffer (H) was chosen₂ACN 70:30, v, pH 6.5) as mobile phase. A GE Healthcare Superose 6HR10/30 column was used with a flow rate of 0.4 mL/min. By passing¹H NMR characterizes the polymer structure. The size and zeta potential of the polymer were characterized by Dynamic Light Scattering (DLS). UV-vis spectra were measured using a Varian Cary 400Bio UV-Vis spectrophotometer. Statistical analysis was performed using the Student's t test. Results are expressed as mean ± Standard Deviation (SD). p value<0.05 considered statistically significant difference, p-value<0.01 is considered to have a highly significant difference.

Example 1 Synthesis of pHPMA-DOX Polymer drug conjugates

The synthesis procedure is shown in FIG. 1.

1. preparation of pHPMA-NHNHBoc

The chain transfer agents CTA (7.0mg,0.025mmol), monomeric HPMA (1400.0mg, 9.8mmol), MA-Ala-NHNHBoc (140.0mg, 0.52mmol) were added proportionally to a 15mL small-necked round-bottomed flask and argon was purged (repeated 3 times). In ice bath, solvent (H) containing initiator VA044(2.7mg,0.008mmol) dissolved therein was added₂O/CH₃OH 1:4, 7.0mL) was added to the flask with a syringe and bubbling with argon was continued for 30 minutes. Then transferring the round bottom bottle to 45 ℃ for photophobic reaction for 17 hours, and quenching the reactionThen, the reaction solution was dropped into a mixed solution of acetone and ether (300mL, acetone/ether ═ 1: 1), the precipitate was collected, filtered and dried under vacuum, the crude product was purified by FPLC column chromatography, the mobile phase containing the product fraction was collected, the collected mobile phase was put into a dialysis bag (MWCO 2000) for dialysis for 1.5 days using deionized water as a medium, and the pHPMA-nhboc (Mw ═ 34kDa, PDI ═ 1.08) was obtained by freeze-drying.

2、pHPMA-NHNH₂Preparation of

pHPMA-NHNHBoc (750.0mg) was weighed into a 50mL round-bottomed flask, 10mL dichloromethane was added with ice-bath stirring, and 10mL TFA was added slowly. Reacting at room temperature for 10h, performing reduced pressure suspension evaporation to remove solvent, dissolving the residue with deionized water, dialyzing in dialysis bag (MWCO 3500) for 1.5 days, and freeze drying to obtain pHPMA-NHNH without Boc group₂。

3. preparation of pHPMA-DOX

The product pHPMA-NHNH is obtained₂(545.0mg) was dissolved in ammonium acetate buffer (20mL,0.1M) at pH 5.7, and doxorubicin hydrochloride (DOX. HCl, 400.0mg) was added under ice-cooling to conduct a reaction for two days with exclusion of light. And (3) taking deionized water as a medium, putting the reaction solution into a dialysis bag (MWCO 2000) for dialysis until the deionized water turns colorless, collecting a product, and freeze-drying to obtain the pHPMA-DOX. And detecting the content of the adriamycin by using an ultraviolet spectrophotometer to obtain the drug-loading rate of the polymer conjugate of 4.7%.

Example 2 Synthesis of pOEGMA-DOX Polymer drug conjugates

The synthesis procedure is shown in FIG. 1.

1. preparation of pPEG-NHNHBoc

The chain transfer agents CTA (9.2mg,0.033mmol), OEGMA (2.05g, 4.1mmol) and MA-Ala-NHBoc (243.9mg, 0.9mmol) were added in proportion to a small-mouth round-bottom flask and argon was purged. In ice bath, solvent (H) in which initiator VA044(3.5mg, 0.013mmol) was dissolved₂O/CH₃OH 1:4, 10.4mL) was added to the flask with a syringe and argon bubbling was continued for half an hour. The flask was then transferred to 45 ℃ and reacted for 17 hours in the dark. After quenching reaction, the reaction solution was dialyzed in dialysis bag (MWCO 3500) for 1 day to remove excess solvent and small molecules that did not react completely,purifying the crude product by FPLC column chromatography, collecting mobile phase containing product part, dialyzing in dialysis bag (MWCO 3500) with deionized water as medium for 1.5 days, and lyophilizing to obtain pPEG-NHBoc (Mw is 32kDa, and PDI is 1.10).

2、pOEGMA-NHNH₂Preparation of

pOEGMA-NHNHBoc (800mg) was added to a 50mL round bottom flask, 10mL dichloromethane was added with ice bath stirring, and 10mL TFA was added slowly. Reacting at room temperature for 10h, performing reduced pressure suspension evaporation to remove solvent, dissolving the residue with deionized water, dialyzing in dialysis bag (MWCO 3500) for 1.5 days, and freeze drying to obtain pOEGMA-NHNH without Boc group₂。

3. preparation of pOEGMA-DOX

The product pOEGMA-NHNH is added₂(600mg) was dissolved in ammonium acetate buffer (20mL,0.1M) at pH 5.7, and doxorubicin hydrochloride (DOX. HCl, 400mg) was added under ice-cooling to conduct a reaction for two days with exclusion of light. And (3) taking deionized water as a medium, putting the reaction solution into a dialysis bag (MWCO 2000) for dialysis until the deionized water becomes colorless, collecting a product, and freeze-drying to obtain pOEGMA-DOX. And detecting the content of the adriamycin by using an ultraviolet spectrophotometer to obtain the drug-loading rate of the polymer drug conjugate of 5.7%.

Test example 1, confirmation of chemical Structure and molecular weight

1) Experimental Material

pHPMA-NHNH prepared in example 1₂And pHPMA-DOX; pOEGMA-NHNH prepared in example 2₂And pOEGMA-DOX.

2) Experimental methods and results

By passing¹H NMR Spectroscopy analysis of drug-free copolymer (pHPMA-NHNH)₂And pOEGMA-NHNH₂) And the chemical structure of the polymer drug conjugates (pOEGMA-DOX and pHPMA-DOX). As shown in FIG. 2, with a copolymer containing no drug¹H NMR spectra were compared and DOX signals were observed at positions 7.4, 7.2, 6.8 and 5.5ppm for the polymer drug conjugate, indicating successful coupling of DOX to the polymer support.

Use of

Size Exclusion Chromatography (SEC) of FPLC system (GE Healthcare) to measure the average molecular weight and polydispersity index (PDI) of the polymer. Sodium acetate buffer (H) was chosen₂ACN 70:30, v, pH 6.5) as mobile phase. A GE Healthcare Superose 6HR10/30 column was used with a flow rate of 0.4 mL/min. The results showed that the two polymer drug conjugates (pOEGMA-DOX and pHPMA-DOX) were prepared with similar molecular weights and narrow molecular weight distributions (Table 1).

Table 1: characterization of the synthesized pOEGMA-DOX conjugates and pHPMA-DOX conjugates.

Test example 2 evaluation of particle size distribution, zeta potential, morphology and stability

1) Experimental Material

The polymer drug conjugates pOEGMA-DOX and pHPMA-DOX prepared in examples 1 and 2.

2) Experimental methods and results

The size and zeta potential of the polymer were characterized by Dynamic Light Scattering (DLS). Respectively dissolving pOEGMA-DOX and pHPMA-DOX in deionized water (the concentration is 0.5 mg. multidot.mL)^-1) The size distribution and zeta potential of the copolymers were determined by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) instrument and the results were processed using DTS software version 3.32. As can be seen in figures 3A and D, the phegma-DOX conjugates have an average size of about 9.8nm, whereas the pHPMA-DOX conjugates have a similar size (about 10.3 nm); as can be seen from FIGS. 3B and E, both pHPMA-DOX and pOEGMA-DOX conjugates have a slightly negatively charged surface (-0.32mV and-2.29 mV), which minimizes their interaction with serum proteins in the blood circulation, thereby extending blood half-life and increasing accumulation at the tumor site.

Measurement of changes in particle size and PDI at 37 ℃ in PBS (pH7.4), PBS containing 10% Fetal Bovine Serum (FBS) and cell culture medium over time by the same method as described above for pOEGMA-DOX and pHPMA-DOX, it was difficult to obtain useful information in PBS containing 10% FBS (FIGS. 4E, F, G) because the size of FBS was very close to the size of the two polymer nanoparticles. However, both polymers showed good stability within 48 hours in the PBS and cell culture media groups (fig. 4A, B, C, D). These results indicate that both conjugates have appropriate nanoscale and good stability under physiological conditions and that efficient accumulation at the tumor site is possible through the EPR effect.

The morphology of the two polymer drug conjugates was further observed using Transmission Electron Microscopy (TEM). As seen in FIGS. 3C and F, pOEGMA-DOX and pHPMA-DOX have similar morphology and relatively uniform distribution in the dry state, consistent with DLS results.

Test example 3 evaluation of Release ability of DOX

1) Experimental Material

2) Experimental methods and results

3mg of the polymer conjugate (pOEGMA-DOX and pHPMA-DOX) were dissolved in triplicate in 1mL of PBS (0.1M, pH7.4 or 5.4), respectively, and the solution was placed in a dialysis bag (MWCO 3500Da), immersed in 30mL of PBS (0.1M, pH7.4 or 5.4), and incubated at 37 ℃ for 60 hours with constant temperature shaking (200 rpm). At a specific time point, 1.2mL of solution was removed and analyzed. At the same time, an equal amount of fresh medium with the same pH was added. Samples taken at different time points were analyzed by uv-vis spectrophotometry for the concentration of DOX.

As can be seen from FIG. 5, the drug release profile of the polymer drug conjugate pHPMA-DOX has a clear pH-dependent profile. Under physiological conditions (pH7.4), after 60 hours, the release amount of adriamycin in the polymer drug conjugates pHPMA-DOX and pOEGMA-DOX is less than 17%, which means that the stability of the two polymer drug conjugates in the blood circulation system in vivo is relatively good. In contrast, when the pH value is reduced to 5.4, the release rate of the adriamycin is rapidly increased like the acid environment in lysosomes of tumor cells, particularly after the polymer drug conjugate pHPMA-DOX is incubated for 24 hours, the release rate of the adriamycin exceeds 80 percent, and the release rate of the adriamycin of pOEGMA-DOX is about 40 percent.

These results indicate that the polymer drug conjugate pHPMA-DOX contains a pH responsive linkage, and can rapidly release the drug linked through a hydrazone bond after reaching tumor cells. It should be noted that the faster doxorubicin release rate and more doxorubicin release from the polymer drug conjugate pHPMA-DOX, as compared to the polymer drug conjugate pOEGMA-DOX, may be responsible for the different polymer carrier structures. Although the two polymer drug conjugates have the same backbone and similar molecular weights, their side chains are completely different. pOEGMA-DOX contains an OEG side chain that is longer than that of HPMA, which results in a more complex overlap integration, which may be more compact. Thus, doxorubicin in the polymer drug conjugate pOEGMA-DOX is more sterically hindered than doxorubicin in the polymer drug conjugate pHPMA-DOX due to the different steric structure, particularly the entanglement and encapsulation of the highly flexible oligoethylene glycol. This explains that the polymer drug conjugate pOEGMA-DOX has a relatively poor doxorubicin release rate compared to the polymer drug conjugate pHPMA-DOX. Test example 4 evaluation of cell uptake Performance

1) Experimental Material

2) Experimental methods and results

A4T 1 cell suspension (1X 104cells/mL) was seeded in a 35X 12mm glass plate, and after 24 hours of incubation, pOEGMA-DOX and pHPMA-DOX (prepared at a concentration corresponding to an doxorubicin concentration of 5. mu.g.mL) were added, respectively^-1) RPMI1640 medium of (1). Incubation in the incubator was continued. The cells were incubated in the incubator for 2, 4, and 6 hours, respectively. The medium was removed and the cells were washed three times with PBS (pH7.4) before fixation with 1% paraformaldehyde. Ten minutes before photographing, the film was stained with 4, 6-diamidino-2-pHenylindole 4', 6-diamidino-2-pHenylindole (DIPA, blue) dye. The photographs were observed by Confocal Laser (CLSM) after 3 washes with PBS.

As seen in fig. 6a1-C2, the intracellular fluorescence intensity of both conjugates gradually increased with increasing incubation time, showing time-dependent internalization and cellular uptake. Notably, at the same incubation time, the pOEGMA-DOX conjugate had a weaker fluorescence intensity than pHPMA-DOX, indicating that pOEGMA-DOX had a slower rate of cellular uptake. Flow cytometry analysis further confirmed the results: quantitative studies (FIG. 7A) showed that pHPMA-DOX was taken up by cells to a greater extent than pOEGMA-DOX at any selected time point. Specifically, pPHPMA-DOX uptake at 6 hours was about 2-fold higher than that of pOEGMA-DOX. Furthermore, as shown in fig. 7B, high fluorescence intensity was observed after incubating free DOX with 4T1 cells for 0.5 hours, indicating that the cells can rapidly take up small molecule DOX due to passive diffusion.

Test example 5 evaluation of cytotoxicity in vitro

1) Experimental Material

2) Experimental methods and results

4T1 cells were incubated in a 96-well plate (cell concentration: 5X 103cells/well), and after 24 hours of incubation in RPMI1640 medium, the medium was removed, and media containing various concentrations (concentration equivalent to doxorubicin concentration: 0.1 to 90. mu.g.mL-1) of free drug doxorubicin, pOEGMA-DOX and pHPMA-DOX were added, respectively. After another 48-hour incubation, the medium was removed and washed three times with PBS (pH 7.4). After adding 100. mu.L of newly prepared culture medium containing 10% CCK-8(1v/v) and incubating for 3 hours in the dark, the absorbance of each sample was measured by a microplate reader, and the cell viability was calculated according to the instructions.

As shown in FIG. 8A, the results show that the two drug-free copolymers at different concentrations (50-800. mu.g/mL) have no significant cytotoxicity to 4T1 cells (survival rate above 90%), indicating that the polymer carrier has good biocompatibility. In contrast, as shown in FIG. 8B, when 4T1 cells were cultured with DOX, pOEGMA-DOX and pHPMA-DOX for 48 hours, a significant decrease in cell viability was observed with increasing concentration. IC of pHPMA-DOX₅₀The value was 0.90. mu.g/mL, which is lower than pOEGMA-DOX (2.86. mu.g/mL). Free DOX exhibits minimal IC, since small DOX molecules are able to permeate the cell membrane by passive diffusion₅₀Value (0.18. mu.g/mL). This may be thatBecause the cytotoxicity generated by the conjugate requires a series of processes such as endocytosis and stimuli-responsive drug release.

Test example 6 in vitro apoptosis assay

1) Experimental Material

2) Experimental methods and results

Apoptosis of doxorubicin hydrochloride, pOEGMA-DOX and pHPMA-DOX was determined by FACS Calibur flow cytometer using a flow cytometer. After the 4T1 cells were incubated in a 6-well plate for 24 hours (cell concentration: 1.5X 105cells/well), the medium was purged, and medium containing free drug doxorubicin at a concentration equivalent to the doxorubicin concentration (0.3. mu.g/mL), pOEGMA-DOX and pHPMA-DOX were added, respectively. After 24 hours of incubation, the medium was removed, washed three times with PBS (pH7.4), trypsinized, centrifuged to collect cells, treated with Annexin V-FITC apoptosis test kit as described, and then examined by flow cytometry, the results were processed using WinMDI 2.9 software.

As shown in fig. 9, the quantitative assay showed that DOX, pOEGMA-DOX and pHPMA-DOX induced apoptosis and necrosis of similar 4T1 cells, respectively, with values of 59.9%, 54.2% and 60.6%, respectively, which coincided with the cytotoxicity assay. Together, these results also indicate that the drug can act rapidly in tumor cells after release from the conjugate. Although both DOX conjugates exhibited concentration-dependent cytotoxicity, we noted that the IC of pHPMA-DOX₅₀Is 3.2 times lower than pOEGMA-DOX. The lower cytotoxicity of cells treated with pHPMA-DOX compared to pPEGEG-DOX may be due to slower cellular uptake and slower drug release rates. In particular, as previously observed, the side chains of pOEGMA-DOX are short poly (oligo (ethylene glycol) (pOEG) chains, which are more flexible than HPMA-based polymers, and thus, OEGMA-based polymer conjugates are more likely to form intact nanostructures, the high density of polyoxyethylene side chains can shield the positive charge of DOX, create a high degree of steric hindrance, significantly inhibit the interaction between the macromolecular carrier and the cells, and thus result in a reduced degree of cellular internalizationOne step affects the pH-responsive release of DOX, a result that has been confirmed by previous in vitro drug release studies when the conjugate is internalized by cells, all of which ultimately lead to two drug conjugates. Have different cytotoxicity.

Test example 7 pharmacokinetic evaluation

1) Experimental Material

2) Experimental methods and results

Normal female BALB/c mice (20-22g) were randomly divided into three groups (n-40, 5 mice per time point). DOX, pOEGMA-DOX and pHPMA-DOX were injected separately via the tail vein (DOX equivalent dose: 4mg/kg mice). At predetermined time points, 200 μ L blood samples were taken from each mouse and collected in heparinized tubes, which were then stored at-20 ℃. Next, the blood sample was thawed at room temperature, and 1mL of a mixed solution of acetonitrile and water (ACN/H) was added₂O ═ 4:1, v/v), vortexed for 5 minutes and left overnight at 4 ℃. Then, the blood sample was taken, vortexed for 1 minute, and then centrifuged at 14,000rpm for 5 minutes. Thereafter, 200 μ L of the supernatant was collected and added to a black 96-well plate. The fluorescence intensity (Ex/Em: 485/590nm) was measured using VarioskanWalsh (Thermo Scientific, MA, USA). Blood pharmacokinetic parameters such as T1/2, AUC, MRT and CL, etc. were analyzed by non-compartmental models using Skanit software 2.4.3.

As shown in fig. 10, blood levels in the DOX group rapidly dropped to very low levels within 1 hour after injection, consistent with previously published reports of rapid distribution of free DOX into organs. An increase in blood residence time of both polymer conjugates compared to DOX was observed. Notably, pOEGMA-DOX has significantly higher blood residence times than pHPMA-DOX, probably because the short oligo (ethylene glycol) chains enriched in the pOEGMA-DOX structure reduce its adsorption and thus prolong its blood circulation time. Pharmacokinetic parameters were calculated using a non-compartmental assay and the results are summarized in table 2. Average half-lives (t) of pOEGMA-DOX and pHPMA-DOX_1/2) 20.05h and 9.56h respectively, higher than DOX group (3.88 h). pOEGMA-DOX and pHPMA-DOX (The areas under the plasma concentration time curve (AUC0- ∞, 185.35. mu.g/mL · h and 30.95. mu.g/mL · h) were 32 times and 5 times (6.14. mu.g/mL · h) as much as DOX, respectively. The mean Clearance (CL) of pOEGMA-DOX and pHPMA-DOX was significantly reduced compared to DOX (4.45L/h/kg and 26.65L/h/kg, respectively, relative to 134.28L/h/kg, p, of the DOX group<0.05), whereas the mean residence times (MRT, 28.68h and 12.05h) of pOEGMA-DOX and pHPMA-DOX are significantly longer than DOX (4.65 h). These results indicate that both polymer-drug conjugates can prolong the residence time of DOX in the blood circulation compared to free DOX and can have better accumulation at the tumor site by the EPR effect. Furthermore, pOEGMA-DOX may increase half-life (t1/2), AUC0- ∞ and MRT compared to pHPMA-DOX, which may be attributed primarily to their different polymeric carrier structures. It has previously been reported that the intravascular half-life of HPMA-based polymeric drug delivery systems depends on the molecular weight of the polymeric carrier, since higher molecular weights can prolong blood circulation time in vivo. For pHPMA-DOX, aiming at the balance between targeting and metabolism, HPMA copolymer with relatively low molecular weight is selected as a carrier, and the blood circulation time in vivo is reduced to a certain extent. In contrast, although pOEGMA-DOX has a similar molecular weight to pHPMA-DOX, the high density of polyoxyethylene side chains is effective in preventing the nanoparticles from being rapidly cleared from the body. This is probably due to the intrinsic properties of PEG which reduce material recognition by macrophages and the reticuloendothelial system. In addition, the softer the side chain, the more likely the DOX anthracycles will self-assemble via pi-pi interactions into tighter nanostructures, thereby enhancing the stability of pOEGMA-DOX in vivo circulation. Thus, when the polymeric carriers have similar lower molecular weights, the polymer carrier based on ppeegma is more effective in improving the plasma stability of the polymeric drug conjugate than the HPMA-based polymer carrier.

Table 2 pharmacokinetic parameters (n-5) after intravenous administration of pHPMA-DOX and pOEGMA-DOX and free DOX (4mg/kg DOX) in Balb/c female mice obtained by non-compartmental model fitting data.

Test example 8 evaluation of in vitro imaging

1) Experimental Material

2) Experimental methods and results

4T1 cells were suspended in 70. mu.L PBS (1.4X 10)⁵Individual cells) and injected subcutaneously into the right hind leg of female BALB/c mice. Tumor volumes were calculated as follows: v ═ lxw²0.5, wherein L refers to the longest diameter and W refers to the shortest diameter. When the tumor reaches about 100mm³When mice were randomly divided into 4 groups of 5), and each group was injected with free DOX, phegma-DOX or pHPMA-DOX (equivalent to 5mg DOX/kg mice) via tail vein. Groups of mice were euthanized at 6h, 12h, 24h, 36h post-dose. The heart, liver, spleen, lung, kidney and tumor were then isolated separately and subjected to In vitro fluorescence imaging on a Maestro In-Vivo imaging system. Mice treated with physiological saline served as a control group.

As shown in fig. 11, tumors and major organs of mice treated with DOX had very weak fluorescence intensity throughout the experiment, indicating that free DOX can be rapidly excreted from the body. In contrast, fluorescence was observed widely distributed in major organs and tumors 6 hours after injection of pOEGMA-DOX or pHPMA-DOX. Over time, a stronger signal of pOEGMA-DOX or pHPMA-DOX was observed in the tumor tissue, indicating a gradual accumulation in the tumor tissue by the EPR effect. The distribution of the medicine in the body is further determined by the quantitative analysis result. As shown in FIG. 11, pOEGMA-DOX and pHPMA-DOX have much higher mean signals in tumor tissues than DOX and reach a maximum at 24 hours, indicating that these drugs remain at high concentrations at the tumor site for a long time, resulting in better antitumor effect.

It is noted that although the fluorescence intensity of pOEGMA-DOX and pHPMA-DOX gradually increased within 24 hours, the average signal of pOEGMA-DOX was higher than that of pHPMA-DOX. One of the main reasons that may be attributed is that the structure of their polymeric carriers is different. Compared to HPMA-based conjugates, OEGMA-based conjugates have a more complete spatial structure due to the flexibility of the OEG side chain. Furthermore, covalently bound DOX on the conjugate can reduce the uptake by the reticuloendothelial system (RES) due to the protective effect of PEG, resulting in prolonged blood circulation time. These observations are in close agreement with the results of the pharmacokinetic studies. All of the above factors ultimately improve the accumulation of pOEGMA-DOX at the tumor site.

Test example 9 evaluation of in vivo antitumor Effect

1) Experimental Material

2) Experimental methods and results

The 4T1 tumor model was established as described above. When tumors of female BALB/c mice reach about 60-80mm³When mice were divided into 7 groups (n-7), each of which was: (1) a physiological saline group, (2) a free DOX group (4mg DOX/kg mouse), (3) free DOX (8mg DOX/kg mouse), (4) pOEGMA-DOX (4mg DOX/kg mouse), (5) pOEGMA-DOX (8mg DOX/kg mouse), (6) pHPMA-DOX (4mg DOX/kg mouse) and (7) pHPMA-DOX (8mg DOX/kg mouse). Mice were treated every 4 days with the above formulation by tail vein injection for a total of 4 administrations. At the same time, the body weight and tumor volume of each mouse were recorded every 2 days. All mice were euthanized on day 21, and heart, liver, spleen, lung, kidney, and tumor were dissected out, respectively. Tumors from each group were weighed and tumor inhibition rate (TGI) was calculated using the formula: TGI ═ 1-W₁/W₂) X 100% where W₁And W₂Mean tumor weights for the treated and control groups are indicated.

As shown by the tumor growth curves of each group (fig. 12A), all of the pharmaceutical formulations showed varying degrees of efficacy in inhibiting tumor growth compared to the rapid growth of tumors in mice treated with physiological saline. Both pOEGMA-DOX and pHPMA-DOX showed better tumor treatment efficiency with increasing DOX dose. Among them, the mice in the pOEGMA-DOX8mg/kg DOX group showed the highest antitumor efficacy, while the mice in the pHPMA-DOX group at 8mg DOX/kg showed similar tumor growth inhibition to the mice in the pOEGMA-DOX group at 4mg DOX/kg. Combined with the results of fluorescence imaging studies, the better antitumor effect of pOEGMA-DOX may be due to its higher accumulation at the tumor site. It can be noted that DOX (4mg/kg mouse) showed a slight antitumor efficacy, probably because the low dose of DOX was rapidly excreted from the body and thus did not reach a sufficient concentration at the tumor site. However, for free DOX, although the antitumor activity improved significantly when the dose of DOX was increased to 8mg/kg, mice showed sustained weight loss (> 20%) 13 days after injection (fig. 11B), some mice began to die after day 15, indicating that high doses of DOX had severe systemic toxicity. In our previous studies, free DOX had significant cardiotoxicity in mice dosed with 4mg DOX/kg. In contrast, no significant weight reduction was detected in the treated mice with zhoegma-DOX or pHPMA-DOX conjugate, indicating that both polymeric drug delivery systems have good biocompatibility.

On day 21 after the administration, all mice were sacrificed, and the main organs such as heart, liver, spleen, lung, kidney, tumor, etc. of each group of mice were excised. Tumors were weighed to calculate Tumor Growth Inhibition (TGI). As shown in FIG. 12D, in the pOEGMA-DOX (8mgDOX/kg mouse), pOEGMA-DOX (4mgDOX/kg mouse), pHPMA (8mgDOX/kg mouse), pHPMA-DOX (equivalent to 4mgDOX/kg mouse) and DOX (4mgDOX/kg mouse) groups, TGI was 80%, 62%, 60%, 44% and 22%, respectively. These results are consistent with tumor growth curves. Among them, mice treated with pOEGMA-DOX (8mg/kg) showed much higher antitumor activity. When the dose was 4mg DOX/kg mouse, the TGI of the mice treated with pHPMA-DOX and pOEGMA-DOX was 2 times and 2.7 times that of the DOX-treated group, respectively. These data further indicate that polymer conjugates based on HPMA or OEGMA can be effective in increasing antitumor efficiency and reducing side effects compared to free DOX.

It is noteworthy that although pHPMA-DOX showed better drug release and faster cellular uptake than pOEGMA-DOX in the in vitro results, higher anticancer efficacy was observed in the in vivo studies. This can be explained by the fact that pOEGMA-DOX has a better pharmacokinetic behavior than pHPMA-DOX, enabling the accumulation of more drug at the tumor site by the EPR effect.

In conclusion, the present invention synthesizes two polymer-DOX conjugates based on HPMA and OEGMA by RAFT polymerization to investigate the effect of the side chain of the polymer on drug delivery properties in cancer treatment. DOX is bound to the polymer backbone through a pH sensitive hydrazone bond. The flexible hydrophilic polymer backbone provides a stable internal environment for hydrophobic drugs, and the pH sensitive linker can achieve extracellular stability of the drugs in blood circulation and specific release in tumor cells. In vitro anticancer evaluation showed that the copolymer-DOX conjugate had similar cytotoxicity to 4T1 cells compared to free DOX, while in vivo anticancer studies showed that both ppeegma-DOX and pHPMA-DOX potentiated the antitumor efficacy of DOX without significant side effects. It is noteworthy that, although pHPMA-DOX has better drug release properties and more efficient cellular uptake than do oegma-DOX, phegma-DOX has a longer blood circulation time and can achieve greater drug accumulation at the tumor site, which ultimately leads to better antitumor effect (TGI 80% vs 60%). These results may provide reference for the study of HPMA and OEGMA based polymeric drug delivery systems.

Claims

1. A polymer-antineoplastic drug conjugate, characterized by: the conjugate is a polymer-antitumor drug conjugate obtained by reacting a polymer shown in a formula (I) with doxorubicin hydrochloride after deprotection, and has the following structure:

；

the structure of the polymer shown in the formula (I) is as follows:

（Ⅰ）

wherein R is

，

；

x is 43-265; y is 1-8;

the weight average molecular weight of the polymer shown in the formula (I) is 28-35 kDa.

2. The polymer-antitumor drug conjugate according to claim 1, wherein: the drug loading rate of the polymer-antitumor drug conjugate in unit weight is 3-12%.

3. The polymer-antitumor drug conjugate according to claim 2, wherein: the drug loading rate of the polymer-antitumor drug conjugate in unit weight is 4.5-6%.

4. The polymer-antitumor drug conjugate according to claim 1, wherein: the preparation method of the polymer shown in the formula (I) comprises the following steps:

taking the monomer (a), MA-Ala-NHNHBoc, a chain transfer agent and an initiator to react at 45 +/-3 ℃ in the presence of an organic solvent to obtain the compound shown in the formula (I).

5. The polymer-antitumor drug conjugate according to claim 4, wherein: the monomer (a) is N- (2-hydroxypropyl) methacrylamide or poly (ethylene glycol) methyl ether methacrylate.

6. The polymer-antitumor drug conjugate according to claim 5, wherein: when monomer (a) is N- (2-hydroxypropyl) methacrylamide, the molar ratio of monomer (a), MA-Ala-NHNHBoc, chain transfer agent and initiator is 1225: 65: 3-4: 1.

7. the polymer-antitumor drug conjugate according to claim 5, wherein: when the monomer (a) is poly (ethylene glycol) methyl ether methacrylate, the molar ratio of the monomer (a), the MA-Ala-NHNHBoc, the chain transfer agent and the initiator is 315-316: 69-70: 2-3: 1.

8. the polymer-antitumor drug conjugate according to any one of claims 4 to 7, wherein: the organic solvent is methanol water solution; the chain transfer agent is 4-cyanovaleric acid dithiobenzoic acid; the initiator is 2,2' - [ azobis (1-methylethylidene) ] bis [4, 5-dihydro-1H-imidazole ] dihydrochloride.

9. The polymer-antineoplastic drug conjugate of claim 8, wherein: in the aqueous methanol solution, the concentration of methanol was 80% v/v.

10. A method for preparing the polymer-antitumor drug conjugate according to any one of claims 1 to 9, wherein: the method comprises the following steps: and (3) deprotecting the polymer shown in the formula (I), and reacting with doxorubicin hydrochloride to obtain the polymer-antitumor drug conjugate.

11. Use of the polymer-antitumor drug conjugate according to any one of claims 1 to 9 for preparing an antitumor drug.

12. Use according to claim 11, characterized in that: the tumor comprises malignant lymphoma, breast cancer, bronchogenic carcinoma, ovarian cancer, soft tissue sarcoma, osteogenic sarcoma, rhabdomyosarcoma, Ewing's sarcoma, blastoma, neuroblastoma, bladder cancer, thyroid cancer, prostate cancer, head and neck squamous carcinoma, testicular cancer, gastric cancer, and liver cancer.

13. A pharmaceutical composition characterized by: the polymer-antitumor drug conjugate as claimed in any one of claims 1 to 9 is used as an active ingredient, and is added with pharmaceutically acceptable auxiliary materials or auxiliary ingredients to prepare a pharmaceutically common preparation.