CN113797350B - Glycosyl polymer and preparation method and application thereof - Google Patents

Abstract

The invention provides a sugar-based polymer and its preparation method and application, belonging to the technical field of drug delivery systems. The structure of the polymer is shown in formula I, wherein the molar ratio of (x+y+z):o:p:n:m is 2.9:0.5:0.5:260:1. The sugar-based polymer overcomes the shortcomings of the existing PDT photosensitizers, such as insufficient accumulation at the tumor site, and a weak tumor killing effect, and can be gathered at the tumor site, and has a good targeting effect; it has a photodynamic effect, and is used as a photosensitizer for Photodynamic therapy has high ROS generation efficiency and strong killing effect on tumor cells. At the same time, the sugar-based polymer can also be used as a drug delivery system to carry drugs, especially tumor drugs, such as olaparib, for tumor treatment, with excellent effects. The glycosyl polymer of the present invention has excellent application prospects in the preparation of drugs for treating tumors.

Description

A kind of sugar base polymer and its preparation method and application

technical field

The invention belongs to the technical field of drug delivery systems, and in particular relates to a sugar-based polymer and its preparation method and application.

Background technique

Cancer refers to malignant tumors originating from epithelial tissue. Data show that in 2020, there will be 1929 new cancer cases worldwide, including 10.06 million males and 9.23 million females; there will be 9.96 million cancer deaths worldwide in 2020, including 5.53 million males and 4.43 million females. With high morbidity and mortality, cancer has become one of the diseases that seriously threaten human health in the world, and it is also one of the hot spots and difficulties in current basic and clinical research. According to the latest data in 2020, the top ten cancers in the world are breast cancer, lung cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, cervical cancer, esophageal cancer, thyroid cancer, and bladder cancer. Current cancer treatments mainly include surgery, radiotherapy, chemotherapy, and immunotherapy. Although the initial treatment effect of these treatment methods is good, there are still problems such as tumor metastasis, easy recurrence, body damage, and immune impairment.

In recent years, photodynamic therapy (PDT) has become an emerging treatment method. Photodynamic therapy induces tumor cell apoptosis and DNA damage through the large amount of reactive oxygen species (Reactive oxygen species, ROS) produced by photosensitizers after specific laser irradiation. PDT has the advantages of high selectivity, low systemic side effects, repeatable treatment, and less tolerance, and has attracted more and more attention from researchers. However, most of the photosensitizers used in PDT have problems such as poor water solubility, lack of targeting and low ROS yield, insufficient accumulation at the tumor site, and weak tumor killing effect, which are limited in the application of cancer therapy.

How to increase the aggregation concentration of photosensitizers in tumor sites, enhance the killing effect of PDT on tumors, and enhance the effect of PDT on cancer treatment are the problems that need to be solved urgently in the current cancer treatment.

Contents of the invention

In order to solve the above problems, the present invention provides a sugar-based polymer and its preparation method and use.

The invention provides a sugar-based polymer, the structure of which is shown in formula I:

in,

The molar ratio of (x+y+z):o:p:n:m is 2.9:0.5:0.5:260:1;

R selected from

Further, the aforementioned sugar-based polymer is prepared from raw materials in the following weight ratio: 100-500 parts of B-gala-SH, 1-100 parts of Maleimide-Ppa;

The structural formula of the Maleimide-Ppa is:

The B-gala-SH is prepared from raw materials in the following weight ratio: 1000-1500 parts of B-gala-PySS, 1000-1500 parts of dithiothreitol;

The B-gala-PySS is prepared from the following raw materials in the weight ratio: 1000-1500 parts of MA-D-Galactosamine, 100-200 parts of MA-PySS, 10-50 parts of MA-GFLGKGLFG-MA, 10-50 parts of MA-GFLGK- CTA 50~100 copies;

The structure of the MA-D-Galactosamine is

The structure of the MA-PySS is

The structure of the MA-GFLGKGLFG-MA is

The structure of the MA-GFLGK-CTA is

Further, the sugar-based polymer is prepared from raw materials in the following weight ratio: 500 parts of B-gala-SH, 75 parts of Maleimide-Ppa;

And/or, the B-gala-SH is prepared from raw materials with the following weight ratio: 1300 parts of B-gala-PySS, 1000 parts of dithiothreitol;

And/or, the B-gala-PySS is prepared from raw materials in the following weight ratio: MA-D-Galactosamine 1446 parts, MA-PySS 100 parts, MA-GFLGKGLFG-MA 23.5 parts, MA-GFLGK-CTA 53 parts .

Further, the preparation method of the B-gala-SH comprises the following steps:

It is obtained by reacting B-gala-PySS with dithiothreitol.

Further, the preparation method of the B-gala-PySS comprises the following steps:

MA-D-Galactosamine, MA-PySS, MA-GFLGKGLFG-MA and MA-GFLGK-CTA are dissolved in a solvent, reacted under the action of an initiator, and freeze-dried to obtain the product.

further,

The solvent is a mixed solution of water and methanol;

And/or, oxygen removal before the reaction;

And/or, the reaction is a light avoidance reaction;

Preferably, the water and methanol volume ratio is 1:4;

And/or, the initiator is VA044;

And/or, the reaction is an oil bath reaction at 40-50° C. for 10-12 hours.

The present invention also provides a method for preparing the aforementioned sugar-based polymer, which comprises the following steps:

(1) Dissolve B-gala-SH in water, add DMSO to prepare B-gala-SH/DMSO mixture;

(2) Dissolve Maleimide-Ppa in DMSO and add it to the B-gala-SH/DMSO mixture to react;

(3) purifying the reaction solution of step (2), to obtain final product;

Preferably,

In step (1), the volume ratio of the water and DMSO is 1: (1-5);

And/or, in step (2), the reaction is at room temperature and protected from light.

The present invention also provides the use of the aforementioned glycosyl polymer in the preparation of photosensitizer medicine;

Preferably, the photosensitizer is a drug for photodynamic therapy of tumors.

The present invention also provides the use of the aforementioned glycosyl polymer in the preparation of a drug carrier;

Preferably, the drug that the drug carrier is used to entrap is an antitumor drug;

More preferably, the antineoplastic drug is olaparib.

The present invention also provides a medicine, which is made of the aforementioned sugar-based polymer as the active ingredient, or the aforementioned sugar-based polymer is used as the carrier to carry the medicine as the active ingredient, plus pharmaceutically acceptable adjuvant or auxiliary ingredients prepared preparations;

Preferably, the entrapped drug is an antitumor drug;

More preferably, the entrapped drug is olaparib.

The invention provides a glycosyl polymer BSP, which overcomes the shortcomings of the existing PDT photosensitizers, such as insufficient accumulation at the tumor site, and weak tumor killing effect, can be gathered at the tumor site, and has a good targeting effect ; It has a photodynamic effect, and is used as a photosensitizer for photodynamic therapy with high ROS generation efficiency and strong killing effect on tumor cells. At the same time, the sugar-based polymer can also be used as a drug delivery system to carry drugs, especially tumor drugs, such as olaparib, for tumor treatment, with excellent effects. The glycosyl polymer of the present invention has excellent application prospects in the preparation of drugs for treating tumors.

Apparently, according to the above content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above-mentioned content of the present invention will be further described in detail below through specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 shows the structural formula and preparation route of cathepsin B-sensitive functionalized chain transfer agent MA-GFLGK-CTA.

Fig. 2 is the structural formula and preparation route of cathepsin B-sensitive functionalized crosslinker MA-GFLGKGLFG-MA.

Fig. 3 is a schematic diagram of the synthesis route and structure of Branched-GFLG-poly-D-galactosamine-S-Ppa (BSP).

Figure 4 is the hydrogen spectrum of the polymer precursor Branched-GFLG-poly D-galactosamine-PySS (the solvent is d6-DMSO).

Figure 5 is the hydrogen spectrum of the polymer precursor Branched-GFLG-poly D-galactosamine-SH (solvent is d6-DMSO).

Fig. 6 is the hydrogen spectrum of polymer Branched-GFLG-poly D-galactosamine-S-Ppa (solvent is d6-DMSO).

Figure 7 is the structural formula and synthetic route of the compound Maleimide-hexyl-Ppa.

Fig. 8 is the hydrogen spectrum of polymer Branched-GFLG-poly D-galactosamine-S-hexyl-Ppa (solvent is d6-DMSO).

Figure 9 is the synthetic route of the linear glycopolymer photodynamic therapy system.

Fig. 10 is the hydrogen spectrum of the polymer precursor Linear-poly D-galactosamine-PySS (the solvent is D ₂ O).

Figure 11 is the hydrogen spectrum of the polymer precursor Linear-poly D-galactosamine-SH (the solvent is d6-DMSO).

Fig. 12 is the hydrogen spectrum of polymer Linear-poly D-galactosamine-S-Ppa (solvent is d6-DMSO).

Fig. 13 is a synthetic route of a degradable branched/crosslinked polymer photodynamic therapy system based on HPMA.

Fig. 14 is the hydrogen spectrum of the polymer precursor Branched-GFLG-poly HPMA-PySS (the solvent is D ₂ O).

Fig. 15 is the hydrogen spectrum of the polymer precursor Branched-GFLG-poly HPMA-SH (the solvent is D ₂ O).

Figure 16 is the hydrogen spectrum of the polymer Branched-GFLG-poly HPMA-S-Ppa (the solvent is D ₂ O).

Fig. 17 is the chemical structural formula, self-assembly diagram and TEM results of BSP, BShP and LSP (scale bar: 200nm).

Figure 18 shows the DLS detection results of BSP, BShP and LSP: a is the particle size detection result; b is the zeta potential detection result.

Figure 19 shows the change of fluorescence intensity with the increase of laser irradiation time when singlet oxygen fluorescent probe (SOSG) is added to BSP, BShP and LSP polymer solutions.

Figure 20 is the results of in vitro cytotoxicity experiments of BSP, BShP and LSP.

Figure 21 shows the distribution images of BSP, LSP and BShP three groups of polymers in the tumor mouse model and the statistical results of the fluorescent signals of the tumor sites: a is the distribution image; b is the statistical results of the fluorescent signals of the tumor sites.

Figure 22 is the measurement of the content of olaparib entrapped in polymers LSP, BSP, BShP and BHSP by HPLC, and the content is calculated by the standard curve; HRMS results show that olaparib entrapped is the original drug and does not affect its activity .

Figure 23 shows the detection results of BSPO: a is the TEM image; b is the DLS particle size detection result; c is the zeta potential detection result.

Figure 24 is the fluorescence spectra of BSP, BSPO, Ppa and olaparib.

Figure 25 is the UV spectrum of BSP, BSPO, Ppa and olaparib.

Fig. 26 shows the change of fluorescence intensity of singlet oxygen fluorescent probe (SOSG) added to BSP and BSPO solution with the increase of laser irradiation time.

Detailed ways

The raw materials and equipment used in the specific embodiment of the present invention are all known products, obtained by purchasing commercially available products.

The murine 4T1 breast cancer cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai). It was cultured in RPMI 1640 medium containing 1% double antibody and 10% fetal bovine serum. The incubator conditions were 5% CO ₂ , 95% air, 37 ℃, constant humidity environment. All animal experiments were performed in strict accordance with the animal research guidelines approved by the Hospital Ethics Committee. Female Balb/c mice used in experiments were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. Subcutaneous tumor models were established with 4T1 cells and used for in vivo distribution and imaging studies.

Embodiment 1, the preparation of glycosyl polymer of the present invention

Compounds Maleimide-Ppa, MA-PySS and MA-D-galactosamine were synthesized according to the methods reported in Document 1 and Document 2.

Document 1 (Pan D, Zheng X, Zhang Q, et al.Dendronized-Polymer Disturbing Cells'Stress Protection by Targeting Metabolism Leads to Tumor Vulnerability[J].Advanced Materials,2020,32:1907490.) reported Maleimide-Ppa and MA -PySS synthesis method, the specific synthesis route is as follows:

Maleimide-Ppa:

MA-PySS:

Literature 2 (WARTCHOW C A, Wangnarski M D, Et Al.Carbohydrate ProteaseConjugates: Stabilized Proteases for PepTide Synthesis Mistry, 1995, 60: 2216-2226.) Reported MA-D-Galactosamine synthetic method.

其中Ppa为光敏剂焦脱镁叶绿酸-a(Pyropheophorbide a)结构部分。The structure of Maleimide-Ppa is:

Wherein Ppa is the structural part of the photosensitizer pyropheophorbide-a (Pyropheophorbide a).

The structure of MA-Pyss is:

The structure of MA-D-galactosamine is:

1. Preparation of enzyme-sensitive functionalized chain transfer agent MA-GFLGK-CTA

The synthetic route of the enzyme-sensitive functionalized chain transfer agent MA-GFLGK-CTA is shown in Figure 1. MA-GFLGK-CTA was synthesized according to the similar method reported in literature 3 (document 3: Sun L, Li X, Wei X, et al. Stimuli-Responsive Biodegradable Hyperbranched Polymer–Gadolinium Conjugates as Efficient and Biocompatible Nanoscale Magnetic Resonance Imaging Contrast Agents[J]. ACS Applied Materials & Interfaces, 2016, 8:10499-10512.). The specific synthesis method is as follows:

MA-GFLG-OH (4.6g, 10mmol), HOBt (1.49g, 11mmol) and condensing agent HBTU (4.26g, 11mmol) were placed in a round-bottomed flask under nitrogen protection, and ultra-dry DMF (50mL) was added under ice bath Let it dissolve. DIPEA (6.7mL, 40mmol) was added dropwise into the system to react for 0.5 hours. H-Lys(O ^t Bu)-Fmoc·HCl (4.62g, 10mmol) was weighed and added to the system, and the mixed solution was reacted in an ice bath for 0.5 hours and then returned to room temperature for 10 hours. Add 450mL ethyl acetate (EA) to dissolve, then wash with saturated sodium bicarbonate solution (100mL×3), 1M dilute hydrochloric acid (100mL×3) and saturated sodium chloride solution (100mL×3) successively, collect the organic phase, no After drying over magnesium sulfate, it was concentrated, and after standing at 4°C, a crystalline white solid MA-GFLGK(O ^t Bu)-NHFmoc (6.51 g, 7.51 mmol, yield 75.1%) was precipitated. ¹ H NMR (400 MHz, δ in ppm), ¹³ C NMR (100 MHz, d ₆ -DMSO, δ in ppm), LC-MS (ES+): m/z=867.2 [M+H] ⁺ . MALDI-HRMS: m/z = 889.4474 [M+Na] ⁺ .

Under ice bath, MA-GFLGK(O ^t Bu)-NHFmoc (4.33g, 5mmol) was dissolved in 40mL DCM/TFA (v:v=1:9) mixed solution and returned to room temperature for reaction. TLC monitoring, after the reaction is complete, the solvent is removed by rotary evaporation, and a white solid powder is precipitated after adding ether, and the obtained white solid powder is washed twice with ether and dried to obtain the product MA-GFLGK(OH)-NHFmoc (3.61g, 4.45mmol, rate 89.0%). ¹ H NMR (400MHz, δin ppm), ¹³ C NMR (100MHz, d ₆ -DMSO, δin ppm), LC-MS (ES+): m/z=811.3[M+H] ⁺ , 834.3[M+Na] ⁺ , MALDI-HRMS: m/z = 833.3842 [M+Na] ⁺ .

Using MA-GFLGK(OH)-NHFmoc as raw material, the functional monomer MA-GFLGK-CTA was synthesized by the solid-phase peptide synthesis method through the following steps. MA-GFLGK(OH)-NHFmoc (1.62g, 2mmol) and DIPEA (0.67mL, 4mmol) were dissolved in 10mL of DMF, and chlorotrityl chloride resin (5.0g, 1.15mmol/g) was added to react for 2 hours. The resin was transferred to a polypropylene tube, washed three times with a mixed solution of DCM:MeOH:DIPEA (v:v:v=17:2:1) (100 mL), and then washed three times with DCM and DMF respectively. The Fmoc protecting group was treated three times with 50 mL of DMF containing 20% piperidine, and then cyanovaleric acid dithiobenzoic acid (CTA-COOH, 2.79 g, 10 mmol), DIC (1.26 g, 10 mmol) and HOBt (1.35 g, 10mmol), reacted for 12 hours. The product was treated with TFE/DCM (v:v=3:7) at room temperature for 2 hours and the resin was removed by filtration. The mother liquor was concentrated under reduced pressure, dissolved in a little methanol, added to diethyl ether for precipitation purification, and further purified by high performance liquid chromatography to obtain the pink solid product MA-GFLGK-CTA (892 mg, 1.05 mmol, yield 52.5%). ¹ H NMR (400 MHz, δ in ppm), ¹³ C NMR (100 MHz, d ₆ -DMSO, δ in ppm), LC-MS (ES+): m/z=851.3 [M+H] ⁺ . MALDI-HRMS: m/z = 848.3408 [MH] ^- , 870.3181 [M+Na-2H] ^- .

2. Preparation of enzyme-sensitive functionalized crosslinker MA-GFLGKGLFG-MA

The synthetic route of cathepsin B-sensitive functionalized cross-linker MA-GFLGKGLFG-MA is shown in Figure 2. MA-GFLGKGLFG-MA was synthesized according to the similar method reported in literature 3 (document 3: Sun L, Li X, Wei X, et al. Stimuli-Responsive Biodegradable Hyperbranched Polymer–Gadolinium Conjugates as Efficient and Biocompatible Nanoscale Magnetic Resonance Imaging ContrastAgents[J]. ACS Applied Materials & Interfaces, 2016, 8:10499-10512.). The specific synthesis method is as follows:

BocNH-GFLG-OH (2.46g, 5mmol), HOBt (743mg, 5.5mmol) and HBTU (2.13g, 5.5mmol) were placed in a round bottom flask, and 20mL of ultra-dry DMF was added under nitrogen protection. DIPEA (3.35 mL, 20 mmol) was added under ice-cooling to react for 0.5 hours. H-Lys(OCH ₃ )-OH·2HCl (583 mg, 2.5 mmol) was added to the system, and then returned to room temperature to react for 20 hours. The reaction solution was added to 250 mL of ethyl acetate, washed successively with saturated sodium bicarbonate solution (20 mL×3), 1M dilute hydrochloric acid (20 mL×3) and saturated sodium chloride solution (20 mL×3), and the organic phase was collected. After drying over magnesium sulfate, it was concentrated and crystallized at 4°C to obtain the white solid product BocNH-GFLGKGLFG-NHBoc (1.62g, 1.46mmol, yield 58.4%). ¹ H NMR (400MHz, δin ppm), ¹³ C NMR (100MHz, δin ppm), LC-MS (ES+): m/z=1109.4[M+H] ⁺ , MALDI-HRMS: m/z=1131.6045[M +Na] ⁺ .

Put BocNH-GFLGKGLFG-NHBoc (1.33g, 1.2mmol) in a nitrogen-protected round-bottomed flask, add 25mL DCM/TFA (v:v=1:1) mixed solution under ice-cooling, return to room temperature and react for 12 hours The solvent was spun off, and anhydrous ether was added twice, and then dried to obtain a white solid intermediate, which was dissolved in a mixed solution of 100mL H ₂ O/ACN (v:v=4:1), and 1M NaOH solution to adjust the pH to 7-8, then methacryloyl chloride (0.3mL, 3mmol) was dissolved in acetonitrile (ACN) and added dropwise to the system, while slowly adding 1M NaOH solution dropwise to maintain the system at pH =10, reacted under ice bath for 1 hour, then placed it at room temperature for 4 hours, added the reaction solution into 200mL ethyl acetate, adjusted the pH to 2-3 with 1M dilute hydrochloric acid, extracted the aqueous phase with EA three times and combined the organic phase with chlorine Washed with sodium chloride solution (20mL×3), dried over anhydrous magnesium sulfate and concentrated, the obtained concentrate was crystallized at 4°C to obtain the white solid product MA-GFLGKGLFG-MA (700mg, 0.68mmol, yield 56.7%). ¹ H NMR (400MHz, δin ppm), ¹³ C NMR (100MHz, δin ppm), LC-MS (ES+): m/z=516.3[M+2H] ²⁺ , 1031.4[M+H] ⁺ . MALDI-HRMS: m/z = 1031.5506 [M+H] ⁺ , 1053.5377 [M+Na] ⁺ , 1029.5364 [MH] ^- .

3. Construction and preparation of degradable branched/cross-linked glycopolymer photodynamic therapy system

The synthetic route of the degradable branched/cross-linked glycopolymer photodynamic therapy system is shown in Figure 3. The specific synthesis method is as follows:

Monomer MA-D-Galactosamine (1446mg, 4.63mmol), MA-PySS (100mg, 0.39mmol), crosslinker MA-GFLGKGLFG-MA (23.5mg, 22.8μmol), chain transfer agent MA-GFLGK-CTA (53.0 mg, 62.4μmol) and initiator VA044 (6.7mg, 20.8μmol) were dissolved in the mixed solution of H ₂ O/CH ₃ OH (7.2mL, 1:4, v/v), and the polymerization bottle was placed on ice in the dark bubbling argon gas in the bath to deoxygenate for 45 minutes, then seal it tightly, and place it in an oil bath at 45°C in the dark for 12 hours. GFLG-poly D-galactosamine-PySS (B-gala-PySS, 1480 mg, 91.2% yield). The hydrogen spectrum of the polymer precursor Branched-GFLG-poly D-galactosamine-PySS (B-gala-PySS) is shown in Figure 4.

Dissolve 1300mg of B-gala-PySS in 10mL of aqueous solution, add 1000mg of dithiothreitol (DTT) to treat overnight, then dialyze (4°C, MWCO 3.5KDa) for 2 days, the sample is treated with a water phase filter and freeze-dried to obtain a white solid powder Branched-GFLG-poly D-galactosamine-SH (B-gala-SH, 1150 mg, yield 88.5%). The hydrogen spectrum of the polymer precursor Branched-GFLG-poly D-galactosamine-SH (B-gala-SH) is shown in Figure 5.

Branched-GFLG-poly D-galactosamine-S-Ppa (BSP) was prepared as follows: 500mg B-gala-SH was dissolved in 5mL RO H ₂ O and 10mL DMSO was added to prepare B-gala-SH/DMSO mixture, 75mgMaleimide -Ppa was dissolved in 2mL of DMSO and then added dropwise to the B-gala-SH/DMSO mixture under stirring to obtain a dark green solution, reacted overnight at room temperature in the dark and then dialyzed (dark, MWCO 3.5KDa) for 2 days to remove DMSO , after centrifugation (7000rpm×5min), the supernatant was filtered through an aqueous filter head (0.45 μm), and the filtrate was freeze-dried to obtain a black-green solid crude product (504 mg, yield 87.7%). The crude product was dissolved in 2 mL of water and added dropwise to 200 mL of acetonitrile to produce a large amount of precipitation. After centrifugation (10000 rpm × 5 min), the solid residue was collected and purified three times. The obtained solid was dissolved in 20 mL of deionized water again and freeze-dried to obtain the product (BSP, 420mg). The hydrogen spectrum of the polymer Branched-GFLG-poly D-galactosamine-S-Ppa (BSP) is shown in Fig. 6 .

The amino acid analysis results of BSP are shown in Table 1.

Table 1. Amino acid analysis results of glycosyl branched/crosslinked polymer BSP

Generally speaking, the renal threshold for effective metabolism of polymer carriers in vivo is about 50kDa, so it is necessary to ensure that the molecular weight of the polymer main chain after degradation is lower than this level. The study found that the sugar-based polymer material of the present invention can be rapidly degraded under the action of enzymes, and the degradation products (Mn=32.4×10 ³ , Mw=46.7×10 ³ , PDI=1.44) after 4 hours have good uniformity. Its molecular weight is below the renal threshold.

Comparative Example 1, Preparation of Branched-GFLG-poly D-galactosamine-S-hexyl-Ppa (BShP)

1. Preparation of functional photosensitizer Maleimide-hexyl-Ppa

The synthetic route of the functionalized photosensitizer is shown in Figure 7.

Weigh maleimide-COOH (Maleimide-COOH, 1.17g, 6.9mmol) and condensing agent HATU (3.74g, 9.85mmol) dissolved in 10mL DMF, add DIEA (3.2mL, 18.3mmol) under ice cooling to react 5 Minutes later, N-Boc-1,6-hexanediamine hydrochloride (1.80 g, 8.3 mmol) was added to the system and reacted for 2 hours. 50mL of saturated sodium bicarbonate solution and 30mL of DCM were added to the reaction solution, the organic phase was collected and the aqueous phase was extracted three times with DCM, the combined organic phase was washed twice with saturated sodium bicarbonate solution, dilute hydrochloric acid and saturated sodium chloride solution, After drying over anhydrous magnesium sulfate, the solvent was removed to obtain the crude product as a yellow solid. After column purification (CH ₃ OH:DCM=1:20, v/v) and further separation and purification by high performance liquid chromatography, the product Maleimide-hexyl-NHBoc (1.66 g, yield 65.4%) was obtained as a colorless liquid. ¹ H NMR (400MHz, δin ppm), ¹³ C NMR (100MHz, δin ppm), LC-MS (ES+): m/z=368.3m/z[M+H] ⁺ , 390.2m/z[M+Na ] ⁺ . MALDI-HRMS: m/z = 390.2000 [M+Na] ⁺ .

At 0°C, Maleimide-hexyl-NHBoc (220 mg, 0.60 mmol) was dissolved in 10 mL of DCM and 10 mL of TFA was added. After overnight reaction, the solvent was removed, and the residue was treated with anhydrous ether to obtain a solid powder. The solid powder obtained in the previous step, pyropheophorbide a (270mg, 0.50mmol) and condensing agent HATU (285mg, 0.75mmol) were dissolved in DCM and DIEA (0.23mL, 1.33mmol) was added, and the reaction solution was heated at room temperature After reacting for 1.5 hours, the solvent was spun off, and purified by column (DCM:CH ₃ OH=30:1-20:1, v/v) to obtain a black solid powder Maleimide-hexyl-Ppa (237 mg, yield 50.5%). ¹ HNMR (400 MHz, δin ppm), LC-MS (ES+): m/z = 784.4 m/z [M+H] ⁺ . MALDI-HRMS: m/z = 806.3995 [M+H] ⁺ , m/z = 822.3678 [M+K] ⁺ .

2. Preparation of Branched-GFLG-poly D-galactosamine-S-hexyl-Ppa (BShP)

Branched-GFLG-poly D-galactosamine-S-hexyl-Ppa (BShP) was prepared as follows: 500mg B-gala-SH was dissolved in 5mL RO H ₂ O and 10mL DMSO was added to prepare B-gala-SH/DMSO mixture , 80mg of Maleimide-hexyl-Ppa was dissolved in 2mL of DMSO and added dropwise to the B-gala-SH/DMSO mixture under stirring to obtain a dark green solution, reacted overnight at room temperature in the dark and then dialyzed (dark, MWCO 3.5KDa) for 2 days After removing DMSO, after centrifugation (7000rpm×5min), the supernatant was filtered through an aqueous filter head (0.45μm), and the filtrate was freeze-dried to obtain a black-green solid crude product (520mg, yield 89.7%). The crude product was dissolved in 2 mL of water and added dropwise to 200 mL of acetonitrile, resulting in a large amount of precipitation. After centrifugation (9000 rpm×5 min), the solid residue was collected and purified three times. The obtained solid was dissolved in 20 mL of deionized water again and freeze-dried to obtain the product (BShP, 450mg). The hydrogen spectrum of the polymer Branched-GFLG-poly D-galactosamine-S-hexyl-Ppa (BShP) is shown in Fig. 8 .

Comparative Example 2, Preparation of Linear Glycopolymer Photodynamic Therapy System

The synthetic route of the linear glycopolymer photodynamic therapy system is shown in Figure 9:

As a control, a linear glycosyl polymer photodynamic therapy system was designed and synthesized. Monomer MA-D-Galactosamine (722mg, 2.31mmol), MA-PySS (50mg, 0.20mmol), chain transfer agent 4-cyano-4-(thiobenzoyl)valeric acid (CTA, 5.3mg, 19.0 μmol) and initiator VA044 (2.1mg, 6.5μmol) were dissolved in a mixed solution of H ₂ O/CH ₃ OH (3.5mL, 1:4, v/v), and the polymerization bottle was placed in an ice bath in the dark and After bubbling with argon to remove oxygen for 45 minutes, seal it up, and place it in an oil bath at 45°C for 12 hours in the dark. galactosamine-PySS (L-gala-PySS: 725 mg, yield 93.3%). The hydrogen spectrum of the polymer precursor Linear-poly D-galactosamine-PySS is shown in Figure 10.

Dissolve 620mg of L-gala-PySS in 10mL of aqueous solution, add 600mg of DTT overnight, and then dialyze (4°C, MWCO 3.5KDa) for 2 days. The sample is treated with a water phase filter and freeze-dried to obtain a white solid powder Linear-poly D-galactosamine- SH (L-gala-SH, 560 mg, 93.3% yield). The hydrogen spectrum of the polymer precursor Linear-poly D-galactosamine-SH is shown in Figure 11.

The preparation of Linear-poly D-galactosamine-S-Ppa (LSP) is similar to that of the degradable branched/cross-linked glycopolymer photodynamic therapy system: 500mg L-gala-SH is dissolved in 5mL RO H ₂ O Then add 10mL DMSO to make L-gala-SH/DMSO mixed solution, dissolve 80mg Maleimide-Ppa in 2mL DMSO and add dropwise to L-gala-SH/DMSO mixed solution under stirring to obtain a dark green solution. After the light reaction overnight, dialyze (protect from light, MWCO3.5KDa) for 2 days to remove DMSO, centrifuge (7000rpm×5min), take the supernatant and filter it through an aqueous filter head (0.45μm), and freeze-dry the filtrate to obtain a black-green solid crude product (512mg , yield 88.3%). The crude product was dissolved in 2 mL of water and added dropwise to 200 mL of acetonitrile to produce a large amount of precipitation. After centrifugation (10000 rpm × 5 min), the solid residue was collected and purified three times. The obtained solid was dissolved in 20 mL of deionized water again and freeze-dried to obtain the product (LSP, 496mg). The hydrogen spectrum of the polymer Linear-poly D-galactosamine-S-Ppa (LSP) is shown in Fig. 12 .

Comparative example 3. Preparation of degradable branched/crosslinked polymer photodynamic therapy system based on HPMA

As a control of sugar-based materials, a degradable branched/cross-linked polymer photodynamic therapy system based on HPMA was designed and synthesized. The synthetic route of the degradable branched/crosslinked polymer photodynamic therapy system based on HPMA is shown in Figure 13. The specific synthesis method is as follows:

Monomer HPMA (722mg, 5.04mmol), MA-PySS (50mg, 0.20mmol), crosslinker MA-GFLGKGLFG-MA (11.8mg, 11.4μmol), chain transfer agent MA-GFLGK-CTA (26.5mg, 31.2μmol ) and initiator VA044 (3.4 mg, 10.4 μmol) were dissolved in a mixed solution of H ₂ O/CH ₃ OH (3.6 mL, 1:4), and the polymerization bottle was placed in an ice bath in the dark and bubbled with argon After deoxygenation for 45 minutes, it was airtight, protected from light, and placed in an oil bath at 45°C for 12 hours. After the reaction was quenched with liquid nitrogen, the reaction solution was dialyzed and freeze-dried at low temperature to obtain a light red solid Branched-GFLG-poly HPMA-PySS (B - HPMA-PySS, 740 mg, 91.2% yield). The hydrogen spectrum of the polymer precursor Branched-GFLG-poly HPMA-PySS is shown in Figure 14.

Dissolve 640mg of B-HPMA-PySS in 10mL of aqueous solution, add 1000mg of DTT to treat overnight, and then dialyze (4°C, MWCO 3.5KDa) for 2 days. The sample is treated with a water phase filter and freeze-dried to obtain a white solid powder Branched-GFLG-polyHPMA-SH (B-HPMA-SH, 600 mg, 93.8% yield). The hydrogen spectrum of the polymer precursor Branched-GFLG-poly HPMA-SH is shown in Fig. 15.

Branched-GFLG-poly-HPMA-S-Ppa (BHSP) was prepared as follows: 500mg B-HPMA-SH was dissolved in 5mLRO H ₂ O and 10mL DMSO was added to make B-HPMA-SH/DMSO mixture, 80mg Maleimide-hexyl -Ppa was dissolved in 2mL of DMSO and then added dropwise to the B-HPMA-SH/DMSO mixture under stirring to obtain a dark green solution. After overnight reaction in the dark at room temperature, it was dialyzed (protected from light, MWCO 3.5KDa) for 2 days to remove DMSO. After centrifugation (7000rpm×5min), the supernatant was filtered through an aqueous filter head (0.45μm), and the filtrate was freeze-dried to obtain a black-green solid crude product (500mg, yield 86.2%). The crude product was dissolved in 2 mL of water and added dropwise to 200 mL of acetone, resulting in a large amount of precipitation. After centrifugation (9000 rpm×5 min), the solid residue was collected and purified three times. The obtained solid was dissolved in 20 mL of deionized water again and freeze-dried to obtain the product (BHSP, 460mg). The hydrogen spectrum of the polymer Branched-GFLG-poly HPMA-S-Ppa (BHSP) is shown in FIG. 16 .

Prove the beneficial effect of the present invention below by concrete test example:

Test example 1, the characterization of polymer

1. Determination of particle size and potential of polymer

BSP, BShP, and LSP were weighed, dissolved in pure water and diluted to a final concentration of 1.0 mg/mL, and the particle size and Zata potential of the sample were characterized using a particle size meter (each measurement was repeated three times), and the final data was analyzed using GraphPad Prism software. At the same time, the sample with a concentration of 200 μg/mL was dropped onto the copper grid and air-dried naturally, and the particle size and shape were observed with a transmission electron microscope.

The morphology of each group of materials observed by transmission electron microscopy is shown in Figure 17. The assembly of BShP was very uniform round nanoparticles; in comparison, the nanoparticles formed by BSP were not too regular, but a similar round shape could also be observed; those without branched/crosslinked structures LSP is distributed in a loose filamentary form without aggregation to form a special morphology.

As shown in Figure 18: the particle sizes of BSP, BShP and LSP measured by DLS are about 238nm, 164nm and 374nm, respectively, that is, the particles formed by BShP are the most dense, while BSP is relatively loose but still maintains the shape of nanoparticles. The Zeta potentials of the three groups of materials are -16.81mV, -19.23mV and -21.97mV respectively, all of which are electronegativity.

2. In vitro ROS generation assay of polymers

BSP, BShP and LSP were dissolved and diluted to a photosensitizer pyropheophorbide a (Pyropheophorbide a, Ppa) concentration of 5.0 μg/mL, and SOSG detection reagent was added to a final concentration of 2.5 μM. Pipette 100 μL of the solutions of each group into a 96-well plate, and set up 3 duplicate wells, and then irradiate with a 660nm laser (power density: 10mW/cm ² , duration: 15min). At the time points of 0.5, 1, 1.5, 2, 3, 5, 10, and 15 min, the fluorescence intensity was measured with a multi-functional microplate reader (the excitation wavelength was 490 nm, and the emission wavelength was 525 nm), and the fluorescence intensity of each sample at a wavelength of 525 nm was recorded. PBS was used as blank control.

Ppa can absorb photons of specific wavelengths to transition from the ground state to the excited state, and then transfer the absorbed energy to nearby oxygen molecules to generate singlet oxygen. The ROS generated by Ppa after light excitation can be detected by the kit. Among them, the singlet oxygen sensor green (SOSG) is a highly selective detection reagent for singlet oxygen. The indicator initially has a weak blue fluorescence, and it will emit light after interacting with singlet oxygen. Fluorescein is similar to green fluorescence (the maximum excitation/emission wavelength is about 504/525nm), and the intensity of green fluorescence is positively correlated with the amount of ROS produced. As shown in Figure 19, after absorbing 660nm excitation energy, the fluorescence intensity produced by BSP is the strongest, followed by LSP, and the weakest by BShP, and the fluorescence intensity of BSP is significantly higher than that of LPS and BShP. It shows that the amount and efficiency of ROS produced by BSP in vitro are higher, while the production efficiency of ROS by BShP and LSP is relatively low. It further shows that BSP has a better killing effect on tumors.

3. Determination of polymer IC50 value in vitro

Inoculate 4T1 cells in a 96-well plate at a concentration of 5× ¹⁰ cells/well, discard the medium after the cells adhere to the wall (incubate for about 12 hours), and add BSP, LSP and BShP prepared in the medium Gradient concentration (Ppa concentration: 50, 20, 10, 5, 2, 1, 0.5, 0.1, ^0.01 μg/mL) solution and then continue to incubate for 6 hours. Hours later, use the CCK8 reagent according to the kit instructions, and detect the absorbance at about 450 nm with a microplate reader. GraphPad Prism (vison 8.0) software was used for graphical analysis of the measurement results.

Comparison of _IC50 values was performed by in vitro cell experiments (Figure 20). Under the same Ppa concentration gradient, light dose and culture conditions, the IC50 values of the three groups of polymers were BSP 0.25 μg/mL, LSP 0.91 μg/mL, and BShP 4.8 μg/mL, indicating that BSP has the strongest killing effect on tumor cells.

4. Signal detection of tumor site in the polymer body

1×10 ⁶ 4T1 cells were inoculated into the flank of Balb/c mice (20 g, 6-8 weeks old), and the experiment was started when the tumor grew to a diameter of about 5 mm. The mice were randomly divided into 3 groups (5 in each group), and different groups of materials (BSP, BShP and LSP) with a Ppa concentration of 5 mg/kg were injected through the tail vein respectively. After the injection was completed at different time points (5min, 30min, 1h, 3h, 6h, 12h and 24h), the fluorescence signal of the tumor site was observed through the in vivo fluorescence imaging system, and the data was processed and analyzed through the data analysis system.

The results showed that the fluorescence intensity of the tumor site of the BSP material gradually increased after injection, and reached the highest at about 6 hours, and the fluorescence intensity of the tumor site at each time point was much higher than that of the LSP and BShP groups (Figure 21).

Under the same concentration of Ppa, BSP was higher than LSP and BShP in terms of the amount of ROS produced in vitro, the toxicity to tumor cells and the degree of accumulation in tumor tissue, which ensured the effect of photodynamic therapy. BSP can be used as a photosensitizer for photodynamic therapy of tumors with excellent results.

Test Example 2. Polymer Encapsulated Olaparib and Its Characterization

1. Polymer loaded olaparib

Polymer-encapsulated olaparib was prepared by dialysis, and 300 mg of LSP, BSP, BShP and BHSP were weighed and dissolved in 5 mL of deionized water, and 10 mL of DMSO was added to obtain a H ₂ O/DMSO mixed solution containing materials. 30 mg of Olaparib (Olaparib) was dissolved in 2 mL of DMSO and added dropwise to the H ₂ O/DMSO mixed solution containing the materials. Stir overnight in the dark, dialyze the reaction solution (3.5kDa MWCO) for 2 days to remove the DMSO solvent, then treat it with an aqueous phase filter membrane and freeze-dry to obtain a polymer loaded with olaparib. The polymers are named LSPO, BSPO, BShPO and BHSPO (O stands for olaparib).

2. Determination of the loading capacity of olaparib

The entrapment capacity of LSP, BSP, BShP and BHSP for olaparib was calculated from the results of HPLC. Drug loading (wt%)=(mass of entrapped drug/total mass of polymer and entrapped drug)×100%. The materials of each group were dissolved in the mobile phase and then ultrasonicated, and the filtrate after the treatment of the organic phase filter head was subjected to HPLC detection. The column model is (Column name: Shim-pack GLST, C18, 5μm.Size: 250×4.6mm ID), the test column temperature is 30°C, the flow rate is 1.0mL/min, the injection volume is 20μL, and the sample concentration is about 100 μg/mL, the detector was selected as UV light (276nm), and the mobile phase was a mixed solution of CH ₃ OH/H ₂ O (v:v=1:1).

The results are shown in Table 2. The entrapment capacity of BSP polymer was the highest, reaching 4.03%; the entrapment capacity of BShP was 2.17%, which was worse than that of BSP. In comparison, the control materials LSP and BHSP could not effectively entrap small molecule drugs, and their entrapment amounts were only 0.33% and 0.13%, respectively. It shows that the "proper" assembly and stacking form of BSP can carry more small molecule drugs. The loaded small molecule drugs were collected by HPLC for detection, and HRMS results showed that olaparib was still the active drug (Figure 22).

Table 2. The Ppa content of each group of polymers and the corresponding Olaparib loading

3. Relevant characterization of BSPO

The particle size and zeta potential of the polymer BSPO (1.0 mg/mL) loaded with olaparib were measured by DLS, and its morphology was further investigated by TEM (sample concentration was 200 μg/mL). The ultraviolet spectra of the samples BSP, BSPO, Ppa and Olaparib were detected within the range of 200-800nm by a UV-visible spectrophotometer, and the fluorescence spectra of BSP, BSPO, Ppa and Olaparib were measured by a fluorescence spectrometer. The change of the in vitro singlet oxygen generation efficiency of BSP and BSPO was evaluated by the SOSG detection reagent, and the specific method was the same as the conditions and methods for the determination of in vitro ROS generation in Test Example 1.

As shown in Figure 23, the DLS results show that the particle size of the BSPO water phase is about 178nm, and its surface is also negatively charged; while the TEM results show that it forms nanoparticles with relatively uniform sizes. This indicates that compared with BSP, the structure of BSPO loaded with small molecule olaparib may be denser.

Fluorescent spectral properties of BSPO: As shown in Figure 24, under the same concentration of Ppa, there is no significant difference in the fluorescence intensity of BSP and BSPO at around 680nm, indicating that the entrapped olaparib does not affect the fluorescent properties of Ppa.

At the same time, in the ultraviolet spectrum (Figure 25), the ultraviolet spectra of BSP and BSPO at around 330-800nm basically overlap, indicating that the ultraviolet spectrum does not change in this wavelength range; while at about 300nm, the ultraviolet spectrum is slightly different, That is, the spectrum of BSPO around 300nm has changed. The ultraviolet absorption peak of olaparib is around 276nm. This change is caused by the overlapping of the ultraviolet peak of Ppa and that of olaparib. This result shows to some extent that BSP can effectively contain olaparib. And the inclusion of olaparib will not affect the ability of the polymer to generate ROS.

As shown in Figure 26, compared with BSP, there is no significant difference in the amount and efficiency of ROS production in vitro between BSPO and BSP, indicating that olaparib can still efficiently produce ROS after being entrapped.

The above test results show that: the glycosyl polymer BSP can be used as a photosensitizer for photodynamic therapy, which has a good aggregation effect on the tumor site and has a good killing effect on the tumor; at the same time, the glycosyl polymer BSP can be used as a drug Carrier-coated drugs, such as olaparib, are further used in disease treatment. The drug-loading capacity of the encapsulated drug is high, and the effect as a drug delivery system is excellent.

The invention synthesizes glycosyl polymer carriers BSP, BShP and LSP with different structures, and a drug carrier BSHP based on pHPMA. The experimental results showed that in the case of the same photosensitizer pyropheophorbide a (Pyropheophorbide a, Ppa) concentration, compared with BShP and LSP, BSP had the highest ROS generation efficiency after light irradiation. Tumor cytotoxicity experiments found that the half maximal inhibitory concentrations (Half maximal inhibitory concentration, IC50) of BSP, BShP and LSP three polymer carriers were 0.25 μg/mL, 4.8 μg/mL and 0.91 μg/mL, BSP had more obvious tumor Cell killing effect.

The present invention also prepares BSPO, a dual-drug delivery system loaded with olaparib. The research on the ability to entrap the drug olaparib found that BSP has the strongest ability to entrap the drug and is the best drug carrier.

It can be seen that the glycosyl polymer carrier BSP prepared by the present invention has a high aggregation concentration at the tumor site, has a good photodynamic therapy effect, has a strong tumor killing effect, and is a good photosensitizer; and as a drug carrier, it has a large loading capacity, It is an excellent drug delivery system.

To sum up, the present invention provides a glycosyl polymer BSP, which overcomes the shortcomings of the existing PDT photosensitizers, such as insufficient accumulation at the tumor site, and weak tumor killing effect, can be gathered at the tumor site, and has a good Targeting effect; it has a photodynamic effect, and is used as a photosensitizer for photodynamic therapy with high ROS generation efficiency and strong killing effect on tumor cells. At the same time, the sugar-based polymer can also be used as a drug delivery system to carry drugs, especially tumor drugs, such as olaparib, for tumor treatment, with excellent effects. The glycosyl polymer of the present invention has excellent application prospects in the preparation of drugs for treating tumors.

Claims (17)

1. A sugar-based polymer characterized by: the structure of the polymer is shown in a formula I:

wherein,,

(x+y+z) o: p: n: m in a molar ratio of 2.9:0.5:0.5:260:1;

r is selected from

2. The sugar-based polymer of claim 1, wherein: the composite material is prepared from the following raw materials in parts by weight: 100-500 parts of B-gala-SH and 1-100 parts of Maleimide-Ppa;

the structural formula of the Maleimide-Ppa is as follows:

the B-gala-SH is prepared from the following raw materials in parts by weight: 1000-1500 parts of B-gala-PySS and 1000-1500 parts of dithiothreitol;

the B-gala-PySS is prepared from the following raw materials in parts by weight: 1000-1500 parts of MA-D-Galactosamine, 100-200 parts of MA-PySS, 10-50 parts of MA-GFLGKGLFG-MA and 50-100 parts of MA-GFLGK-CTA;

the MA-D-Galactosamine has the structure that

The MA-PySS has the structure that

The MA-GFLGKGLFG-MA has the structure that

The MA-GFLGK-CTA has the structure that

3. The sugar-based polymer of claim 2, wherein: the glycosyl polymer is prepared from the following raw materials in parts by weight: 500 parts of B-gala-SH and Ppa parts of Maleimide;

and/or the B-gala-SH is prepared from the following raw materials in parts by weight: 1300 parts of B-gala-PySS and 1000 parts of dithiothreitol;

and/or the B-gala-PySS is prepared from the following raw materials in parts by weight: MA-D-Galactamine 1446 parts, MA-PySS 100 parts, MA-GFLGKGLFG-MA 23.5 parts, MA-GFLGK-CTA 53 parts.

4. A sugar-based polymer according to claim 2 or 3, characterized in that: the preparation method of the B-gala-SH comprises the following steps:

the B-gal-PySS is reacted with dithiothreitol.

5. A sugar-based polymer according to claim 2 or 3, characterized in that: the preparation method of the B-gala-PySS comprises the following steps:

dissolving MA-D-Galactosamine, MA-PySS, MA-GFLGKGLFG-MA and MA-GFLGK-CTA in a solvent, reacting under the action of an initiator, and freeze-drying to obtain the final product.

6. The sugar-based polymer of claim 5, wherein:

the solvent is a mixed solution of water and methanol;

and/or, the pre-reaction oxygen is removed;

and/or, the reaction is a light-shielding reaction.

7. The sugar-based polymer of claim 6, wherein:

the volume ratio of the water to the methanol is 1:4;

and/or, the initiator is VA044;

and/or the reaction is oil bath reaction at 40-50 ℃ for 10-12 hours.

8. A process for preparing a sugar-based polymer according to any one of claims 2 to 7, characterized in that: it comprises the following steps:

(1) Dissolving B-gala-SH in water, and adding DMSO to prepare a B-gala-SH/DMSO mixed solution;

(2) Dissolving Maleimide-Ppa in DMSO, and adding the DMSO into a B-gala-SH/DMSO mixed solution for reaction;

(3) And (3) purifying the reaction liquid obtained in the step (2) to obtain the catalyst.

9. The method according to claim 8, wherein:

in the step (1), the volume ratio of the water to the DMSO is 1: (1-5);

and/or, in the step (2), the reaction is a light-shielding reaction at room temperature.

10. Use of a glycosyl polymer according to any one of claims 1 to 7 in the manufacture of a medicament for a photosensitizer.

11. Use according to claim 10, characterized in that: the photosensitizer is a medicine for photodynamic therapy of tumors.

12. Use of a glycosyl polymer according to any one of claims 1 to 7 in the manufacture of a pharmaceutical carrier.

13. Use according to claim 12, characterized in that: the medicine carrier is used for encapsulating an antitumor medicine.

14. Use according to claim 13, characterized in that: the antitumor drug is Olaparib.

15. A medicament, characterized in that: the preparation is prepared by taking the glycosyl polymer as an active ingredient in any one of claims 1 to 7 or taking the glycosyl polymer as a carrier for encapsulating medicines as an active ingredient in any one of claims 1 to 7 and adding pharmaceutically acceptable auxiliary materials.

16. A medicament as claimed in claim 15, wherein: the entrapped medicine is an anti-tumor medicine.

17. A medicament as claimed in claim 16, wherein: the entrapped drug is olaparib.

Images