CN113797350B

CN113797350B - Glycosyl polymer and preparation method and application thereof

Info

Publication number: CN113797350B
Application number: CN202110930561.1A
Authority: CN
Inventors: 龚启勇; 段振宇; 罗强; 罗奎
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2021-08-13
Filing date: 2021-08-13
Publication date: 2023-05-05
Anticipated expiration: 2041-08-13
Also published as: CN113797350A

Abstract

The invention provides a glycosyl polymer, a preparation method and application thereof, and belongs to the technical field of drug delivery systems. The structure of the polymer is shown as a formula I, wherein the molar ratio of (x+y+z) o to p to n to m is 2.9:0.5:0.5:260:1. The glycosyl polymer overcomes the defects of insufficient aggregation of the tumor part and weak killing effect on the tumor of the existing PDT photosensitizer, can be aggregated at the tumor part, and has good targeting effect; the compound has photodynamic effect, and can be used as photosensitizer for photodynamic therapy, and has high ROS production efficiency and strong killing effect on tumor cells. Meanwhile, the glycosyl polymer can also be used as a drug delivery system for entrapping drugs, in particular tumor drugs such as Olaparib, and has excellent effect on tumor treatment. The glycosyl polymer has good application prospect in preparing medicines for treating tumors.

Description

Glycosyl polymer and preparation method and application thereof

Technical Field

The invention belongs to the technical field of drug delivery systems, and particularly relates to a glycosyl polymer and a preparation method and application thereof.

Background

Cancer (cancer) refers to a malignant tumor that originates in epithelial tissue. The data shows 1929 global new cancer cases in 2020, with 1006 ten thousand men and 923 ten thousand women; 996 ten thousand cancer deaths occurred worldwide in 2020, with 553 ten thousand men and 443 ten thousand women. The incidence rate and the death rate of cancer are high, and the cancer becomes one of diseases which seriously threaten human health worldwide, and is one of hot spots and difficulties in the current basic and clinical research. According to the latest data in 2020, the first ten cancers of global incidence are breast cancer, lung cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, cervical cancer, esophageal cancer, thyroid cancer and bladder cancer, respectively. The current treatment modes of cancer mainly comprise operation, radiotherapy, chemotherapy, immunotherapy and the like. Although these therapeutic methods have good early-stage therapeutic effects, there are still problems such as tumor metastasis, recurrence, body injury, and immune injury.

In recent years, photodynamic therapy (Photodynamic therapy, PDT) has emerged as an emerging therapeutic modality. Photodynamic therapy induces apoptosis of tumor cells and causes DNA damage by a large amount of reactive oxygen species (Reactive oxygen species, ROS) generated by photosensitizers following specific laser irradiation. PDT has the advantages of high selectivity, low systemic toxic and side effects, repeatable treatment, difficult tolerance generation and the like, and is increasingly focused by researchers. However, most photosensitizers used in PDT have problems of poor water solubility, lack of targeting, low ROS productivity, insufficient aggregation at tumor sites, weak tumor killing effect, and limited application in cancer treatment.

How to increase the aggregation concentration of photosensitizer at the tumor site, enhance the killing effect of PDT on the tumor and enhance the effect of PDT on cancer treatment is a problem which needs to be solved in the current cancer treatment.

Disclosure of Invention

In order to solve the problems, the invention provides a glycosyl polymer and a preparation method and application thereof.

The invention provides a glycosyl polymer, which has a structure shown in a formula I:

wherein, the liquid crystal display device comprises a liquid crystal display device,

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

r is selected from

Further, the glycosyl polymer 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

Further, 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.

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

the B-gal-PySS is reacted with dithiothreitol.

Further, the preparation method of the B-gal-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.

Further, the method comprises the steps of,

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;

preferably, the volume ratio of water to 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.

The invention also provides a method for preparing the glycosyl polymer, which 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) Purifying the reaction liquid obtained in the step (2) to obtain the catalyst;

preferably, the method comprises the steps of,

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.

The invention also provides application of the glycosyl polymer in preparing a medicament of a photosensitizer;

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

The invention also provides application of the glycosyl polymer in preparation of a drug carrier;

preferably, the drug carrier is an antitumor drug;

more preferably, the antitumor drug is olaparib.

The invention also provides a medicine, which is a preparation prepared by taking the glycosyl polymer as an active ingredient or taking the glycosyl polymer as a carrier for encapsulating the medicine as the active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients;

preferably, the entrapped drug is an anti-tumor drug;

more preferably, the entrapped drug is olaparib.

The invention provides a glycosyl polymer BSP, which overcomes the defects of insufficient aggregation of tumor sites and weak killing effect on tumors of the existing PDT photosensitizer, can aggregate at the tumor sites and has good targeting effect; the compound has photodynamic effect, and can be used as photosensitizer for photodynamic therapy, and has high ROS production efficiency and strong killing effect on tumor cells. Meanwhile, the glycosyl polymer can also be used as a drug delivery system for entrapping drugs, in particular tumor drugs such as Olaparib, and has excellent effect on tumor treatment. The glycosyl polymer has good application prospect in preparing medicines for treating tumors.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a structural formula of a cathepsin B-sensitive functionalized chain transfer agent MA-GFLGK-CTA and a preparation route thereof.

FIG. 2 is a structural formula of a cathepsin B sensitive functionalized cross-linking agent MA-GFLGKGLFG-MA and a preparation route thereof.

FIG. 3 is a schematic diagram showing the synthetic route and structure of Branched-GFLG-poly-D-galactose-S-Ppa (BSP).

FIG. 4 is a hydrogen spectrum of a polymer precursor, branched-GFLG-poly D-galactose-PySS (solvent D6-DMSO).

FIG. 5 is a hydrogen spectrum of a polymer precursor, branched-GFLG-poly D-galactose-SH (D6-DMSO as solvent).

FIG. 6 is a hydrogen spectrum of the polymer Branched-GFLG-poly D-galactose-S-Ppa (D6-DMSO as solvent).

FIG. 7 is a structural formula of a compound Maleimide-hexyl-Ppa and a synthetic route thereof.

FIG. 8 is a hydrogen spectrum of the polymer Branched-GFLG-poly D-galactose-S-hexyl-Ppa (D6-DMSO as solvent).

FIG. 9 is a synthetic route to a linear sugar based polymer photodynamic therapy system.

FIG. 10 shows the hydrogen spectrum of a polymer precursor Linear-poly D-galactose-PySS (solvent D ₂ O)。

FIG. 11 is a hydrogen spectrum of a polymer precursor Linear-poly D-galactose-SH (solvent D6-DMSO).

FIG. 12 is a hydrogen spectrum of polymer Linear-poly D-galactose-S-Ppa (D6-DMSO as solvent).

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

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

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

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

FIG. 17 shows the chemical structural formulae, self-assembly schematic, and TEM results (scale: 200 nm) of BSP, BShP, and LSP.

Fig. 18 shows DLS detection results of BSP, bshps, and LSPs: a is the particle size detection result; b is the zeta potential detection result.

FIG. 19 shows the change in fluorescence intensity with increasing laser irradiation time when a singlet oxygen fluorescent probe (SOSG) was added to the BSP, BShP, and LSP polymer solutions.

FIG. 20 shows the results of in vitro cytotoxicity experiments on BSP, BShP and LSP.

FIG. 21 shows the fluorescence signal statistics of the distribution images of three groups of polymers BSP, LSP and BShP in tumor mouse model and tumor sites: a is a distribution image; b is the fluorescent signal statistics of the tumor site.

FIG. 22 shows the measurement of the content of Olaparib entrapped in polymer LSP, BSP, BShP and BHSP by HPLC, calculated by standard curve; HRMS results show that the entrapped Olaparib is a raw medicine and does not affect the activity of the Olaparib.

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

FIG. 24 is a graph of fluorescence spectra of BSP, BSPO, ppa and Olaparib.

Fig. 25 is an ultraviolet spectrum of BSP, BSPO, ppa and olaparib.

FIG. 26 shows the change in fluorescence intensity of BSP and BSPO solutions with the addition of singlet oxygen fluorescent probes (SOSG) as the laser irradiation time increases.

Detailed Description

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

Murine 4T1 breast cancer cell lines were purchased from China academy of sciences cell Bank (Shanghai) and cultured in RPMI 1640 medium containing 1% diabody and 10% fetal bovine serum under 5% CO in an incubator ₂ 95% air, 37 ℃ and constant humidity environment. All animal experiments were performed strictly in accordance with the guidelines for animal studies approved by the ethics committee of hospitals. Female Balb/c mice were purchased from Chengdu laboratory animal Co. Subcutaneous tumor models were established with 4T1 cells and used for in vivo distribution and imaging studies.

EXAMPLE 1 preparation of the sugar-based Polymer of the invention

The compounds Maleimide-Ppa, MA-PySS and MA-D-galactose were synthesized as reported in literature 1 and literature 2.

The synthetic methods of Maleimide-Ppa and MA-PySS are reported in 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.), the specific synthetic routes being as follows:

Maleimide-Ppa：

MA-PySS：

a method for synthesizing MA-D-galactose is reported in document 2 (Wartchow C A, wang P, bednarski M D, et al, carbohydrate Protease Conjugates: stabilized Proteases for Peptide Synthesis [ J ]. The Journal of Organic Chemistry,1995, 60:2216-2226.).

The structure of Maleimide-Ppa is:

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

The MA-Pyss has the structure:

the MA-D-galactose has the structure:

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

The synthetic route for the enzyme-sensitive functionalized chain transfer agent MA-GFLGK-CTA is shown in FIG. 1. MA-GFLGK-CTA was synthesized in a similar manner as reported in document 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 (4.6 g,10 mmol), HOBt (1.49 g,11 mmol) and condensing agent HBTU (4.26 g,11 mmol) were placed in a round bottom flask and dissolved by adding extra dry DMF (50 mL) under nitrogen protection in an ice bath. DIPEA (6.7 mL,40 mmol) was added dropwise to the system and reacted for 0.5 h. Weighing H-Lys (O) ^t Bu) -Fmoc. HCl (4.62 g,10 mmol) was added to the system and the mixed solution was reacted for 0.5 hours in an ice bath and then returned to room temperature for 10 hours. Adding 450mL Ethyl Acetate (EA), dissolving, sequentially washing with saturated sodium bicarbonate solution (100 mL×3), 1M diluted hydrochloric acid (100 mL×3) and saturated sodium chloride solution (100 mL×3), collecting organic phase, drying with anhydrous magnesium sulfate, concentrating, standing at 4deg.C, and precipitating crystalline white solid MA-GFLGK (O) ^t Bu) -NHFmoc (6.51 g,7.51mmol, 75.1% yield). ¹ H NMR(400MHz，δin ppm)， ¹³ C NMR(100MHz，d ₆ -DMSO，δin ppm)，LC-MS(ES+)：m/z＝867.2[M+H] ⁺ 。MALDI-HRMS:m/z＝889.4474[M+Na] ⁺ 。

MA-GFLGK (O) ^t Bu)-NHFmoc(4.33g，5mmol)Dissolved in 40mL of a mixture of DCM/TFA (v: v=1:9) and reacted back to room temperature. TLC monitoring, after the reaction was completed, the solvent was removed by rotary evaporation, diethyl ether was added to precipitate a white solid powder, and the obtained white solid powder was washed twice with diethyl ether and dried to obtain the product MA-GFLGK (OH) -NHFmoc (3.61 g,4.45mmol, yield 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] ⁺ 。

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

2. Preparation of enzyme-sensitive functional crosslinking agent MA-GFLGKGLFG-MA

The synthetic route of the cathepsin B sensitive functional cross-linking agent MA-GFLGKGLFG-MA is shown in FIG. 2. MA-GFLGKGLFG-MA was synthesized in a similar manner as reported in document 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:

BocNH-GFLG-OH (2.46 g,5 mmol), HOBt (743 mg,5.5 mmol) and HBTU (2.13 g,5.5 mmol) were placed in a round bottom flask and 20mL of ultra dry DMF was added under nitrogen. DIPEA (3.35 mL,20 mmol) was added and reacted for 0.5 hours on ice. Addition of H-Lys (OCH) to the System ₃ ) after-OH 2HCl (583 mg,2.5 mmol) was allowed to react for 20 hours at room temperature. The reaction solution was added to 250mL of ethyl acetate, washed with saturated sodium hydrogencarbonate solution (20 mL. Times.3), 1M diluted hydrochloric acid (20 mL. Times.3) and saturated sodium chloride solution (20 mL. Times.3) in this order, and the organic phase was collected, dried over anhydrous magnesium sulfate and concentrated, and crystallized at 4℃to give the product BocNH-GFLGKGLFG-NHBoc (1.62 g,1.46mmol, yield 58.4%) as a white solid. 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] ⁺ 。

BocNH-GFLGKGLFG-NHBoc (1.33 g,1.2 mmol) was placed in a nitrogen-protected round bottom flask, 25mL of a DCM/TFA (v: v=1:1) mixture was added under ice and allowed to react for 12 hours at room temperature before spinning off the solvent, and after twice the addition of anhydrous diethyl ether was pumped to give a white solid intermediate which was dissolved in 100mL H ₂ In a mixed solution of O/ACN (v: v=4:1), 1M NaOH solution was added dropwise under ice bath to adjust pH to 7-8, then methacryloyl chloride (0.3 mL,3 mmol) was dissolved in Acetonitrile (ACN) and then added dropwise to the system while slowly adding 1M NaOH solution to maintain the system at ph=10, the reaction was allowed to react at room temperature for 1 hour under ice bath, the reaction solution was added to 200mL ethyl acetate, pH was adjusted to 2-3 with 1M diluted hydrochloric acid, the aqueous phase was extracted three times with EA, the combined organic phases were washed with sodium chloride solution (20 ml×3), finally dried with anhydrous magnesium sulfate and concentrated, and the resulting concentrate was crystallized at 4 ℃ to give MA-GFLGKGLFG-MA (700 mg,0.68mmol, yield 56.7%) as a white solid. 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[M-H] ^- 。

3. Construction and preparation of degradable branched/crosslinked glycosyl polymer photodynamic therapy system

The synthetic route for the degradable branched/crosslinked glycosyl polymer photodynamic therapy system is shown in figure 3. The specific synthesis method is as follows:

monomer MA-D-Galactosamine (1446 mg,4.63 mmol), MA-PySS (100 mg,0.39 mmol), crosslinker MA-GFLGKGLFG-MA (23.5 mg, 22.8. Mu. Mol), chain transfer agent MA-GFLGK-CTA (53.0 mg, 62.4. Mu. Mol) and initiator VA044 (6.7 mg, 20.8. Mu. Mol) were dissolved in H ₂ O/CH ₃ In a mixed solution of OH (7.2 mL,1:4, v/v), a polymerization bottle was placed in an ice bath in a dark place and bubbling argon gas for deoxidization for 45 minutes, then the bottle was closed, the bottle was placed in an oil bath at 45 ℃ in a dark place for reaction for 12 hours, the reaction solution was quenched with liquid nitrogen, and then the reaction solution was lyophilized by low-temperature dialysis to obtain a pale red solid, branched-GFLG-poly D-galactose-PySS (B-gala-PySS, 1480mg, yield 91.2%). The hydrogen spectrum of the polymer precursor, branched-GFLG-poly D-galactose-PySS (B-gal-PySS) is shown in FIG. 4.

1300mg of B-gal-PySS was dissolved in 10mL of an aqueous solution, treated with 1000mg of Dithiothreitol (DTT) overnight and dialyzed (4 ℃ C., MWCO 3.5 kDa) for 2 days, and the sample was treated with an aqueous filter and lyophilized to give a white solid powder of Branched-GFLG-poly D-gal-SH (B-gal-SH, 1150mg, yield 88.5%). The hydrogen spectrum of the polymer precursor Branched-GFLG-poly D-galactose-SH (B-gal-SH) is shown in FIG. 5.

Branched-GFLG-poly D-galactose-S-Ppa (BSP) was prepared as follows: 500mg of B-gala-SH are dissolved in 5mL of RO H ₂ After O, 10mL of DMSO was added to prepare a B-gal-SH/DMSO mixture, 75mg of Maleimide-Ppa was dissolved in 2mL of DMSO, and the resulting dark green solution was added dropwise to the B-gal-SH/DMSO mixture with stirring, reacted overnight in a dark environment at room temperature, dialyzed (dark, MWCO 3.5 kDa) for 2 days to remove DMSO, centrifuged (7000 rpm. Times.5 min), and the supernatant was collected and filtered through an aqueous filter head (0.45 μm), and the filtrate was lyophilized to give a crude dark green solid (504 mg, yield 87.7%). The crude product was dissolved in 2mL of water and added dropwise to 200mL of acetonitrile to cause a large amount of precipitation, and after centrifugation (10000 rpm. Times.5 min), the solid residue was collected and purified by repeated treatmentsThree times, the resulting solid was redissolved in 20mL deionized water and lyophilized to give the product (BSP, 420 mg). The hydrogen spectrum of the polymer Branched-GFLG-poly D-galactose-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, the renal threshold for effective metabolism of the polymer carrier in an organism is about 50kDa, and thus it is desirable to ensure that the molecular weight of the degraded polymer backbone is below this level. The sugar-based polymer material of the present invention was found to degrade rapidly under the action of an enzyme, and degradation products after 4 hours (mn=32.4×10 ³ ,Mw＝46.7×10 ³ Pdi=1.44) has better uniformity, with a molecular weight below the renal threshold.

Comparative example 1 preparation of Branched-GFLG-poly D-galactose-S-hexyl-Ppa (BShP)

1. Preparation of functionalized photosensitizer Maleimide-hexyl-Ppa

The synthetic route for the functionalized photosensitizer is shown in FIG. 7.

Maleimide propionic acid (Maleimide-COOH, 1.17g,6.9 mmol) and condensing agent HATU (3.74 g,9.85 mmol) were weighed out in 10mL DMF, DIEA (3.2 mL,18.3 mmol) was added under ice bath to react for 5min, and N-Boc-1, 6-hexamethylenediamine hydrochloride (1.80 g,8.3 mmol) was added to the system to react for 2 h. 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 organic phases were combined and washed twice with saturated sodium bicarbonate solution, dilute hydrochloric acid and saturated sodium chloride solution, and the solvent was removed after drying over anhydrous magnesium sulfate to give a yellow solid crude product. Purification by Column (CH) ₃ OH: DCM=1:20, v/v) followed by further isolation and purification by high performance liquid chromatography gave the product, maleimide-hexyl-NHBoc (1.66 g, 65.4% yield) 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] ⁺ 。

Maleimide-hexyl-NHBoc (220 mg,0.60 mmol) was dissolved in 10mL DCM at 0deg.C and 10mL TFA was added and after overnight reaction the solvent was removed and the residue was treated with anhydrous ether to give a solid powder. The solid powder obtained in the above step, pyropheophorbide a (270 mg,0.50 mmol) and condensing agent HATU (284 mg,0.75 mmol) were dissolved in DCM and DIEA (0.23 mL,1.33 mmol) was added, the solvent was removed by spinning after the reaction solution was reacted at room temperature for 1.5 hours, and the mixture was purified by column chromatography (DCM: CH) ₃ OH=30:1-20:1, v/v) to give a black solid powder, maleimide-hexyl-Ppa (237 mg, yield 50.5%). ¹ H NMR(400MHz，δin ppm)，LC-MS(ES+)：m/z＝784.4m/z[M+H] ⁺ 。MALDI-HRMS：m/z＝806.3995[M+H] ⁺ ，m/z＝822.3678[M+K] ⁺ 。

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

Branched-GFLG-poly D-galactose-S-hexyl-Ppa (BShP) was prepared as follows: 500mg of B-gala-SH are dissolved in 5mL of RO H ₂ After O, 10mL of DMSO is added to prepare a B-gal-SH/DMSO mixed solution, 80mg of Maleimide-hexyl-Ppa is dissolved in 2mL of DMSO, and then the mixture is added dropwise to the B-gal-SH/DMSO mixed solution under stirring to obtain a dark green solution, after the dark reaction at room temperature overnight, the solution is dialyzed (MWCO 3.5 kDa) for 2 days to remove DMSO, after centrifugation (7000 rpm multiplied by 5 min), the supernatant is taken and filtered by an aqueous phase filter head (0.45 mu m), and the filtrate is freeze-dried to obtain a dark green solid crude product (520 mg, yield 89.7%). The crude product was dissolved in 2mL of water and added dropwise to 200mL of acetonitrile to cause a large amount of precipitation, and after centrifugation (9000 rpm. Times.5 min), the solid residue was collected and purified three times by repeated treatments, and the obtained solid was dissolved again in 20mL of deionized water and lyophilized to give the product (BShP, 450 mg). The hydrogen spectrum of the polymer Branched-GFLG-poly D-galactose-S-hexyl-Ppa (BShP) is shown in FIG. 8.

Comparative example 2 preparation of Linear sugar based Polymer photodynamic therapy System

The synthetic route for the linear sugar based polymer photodynamic therapy system is shown in figure 9:

as a control, a linear glycosyl polymerization was designed and synthesizedA physical photodynamic therapy system. Monomer MA-D-Galactosamine (720 mg,2.31 mmol), MA-PySS (50 mg,0.20 mmol), chain transfer agent 4-cyano-4- (thiobenzoyl) pentanoic acid (CTA, 5.3mg, 19.0. Mu. Mol) and initiator VA044 (2.1 mg, 6.5. Mu. Mol) were dissolved in H ₂ O/CH ₃ In a mixed solution of OH (3.5 mL,1:4, v/v), a polymerization bottle was placed in an ice bath in a dark place and bubbling argon gas for deoxidization for 45 minutes, then the bottle was closed, the bottle was placed in an oil bath at 45 ℃ in a dark place for reaction for 12 hours, and after the reaction was quenched with liquid nitrogen, the reaction solution was lyophilized by low-temperature dialysis to obtain a pale red solid Linear-poly D-glactosamine-PySS (L-gala-PySS: 725mg, yield 93.3%). The hydrogen spectrum of the polymer precursor Linear-poly D-methylacetamine-PySS is shown in FIG. 10.

620mg of L-gal-PySS was dissolved in 10mL of an aqueous solution, and after overnight treatment with 600mg of DTT, dialyzed (4 ℃ C., MWCO 3.5 kDa) for 2 days, the sample was treated with a water phase filter and lyophilized to give Linear-poly D-glactosamine-SH (L-gal-SH, 560mg, yield 93.3%) as a white solid powder. The hydrogen spectrum of the polymer precursor Linear-poly D-galactose-SH is shown in FIG. 11.

Preparation of Linear-poly D-galactose-S-Ppa (LSP) was similar to the preparation of degradable branched/crosslinked glycosyl polymer photodynamic therapy systems: 500mg of L-gala-SH are dissolved in 5mL of RO H ₂ After O, 10mL of DMSO was added to prepare an L-gala-SH/DMSO mixture, 80mg of Maleimide-Ppa was dissolved in 2mL of DMSO, and the mixture was stirred and added dropwise to the resulting dark green solution, which was reacted overnight in a dark environment at room temperature, dialyzed (dark, MWCO 3.5 kDa) for 2 days to remove DMSO, centrifuged (7000 rpm. Times.5 min), and the supernatant was collected and filtered through an aqueous filter head (0.45 μm), and the filtrate was lyophilized to give a crude dark green solid (512 mg, yield 88.3%). The crude product was dissolved in 2mL of water and added dropwise to 200mL of acetonitrile to cause a large amount of precipitation, and after centrifugation (10000 rpm. Times.5 min), the solid residue was collected and purified three times by repeated treatments, and the obtained solid was dissolved again in 20mL of deionized water and lyophilized to obtain the product (LSP, 496 mg). The hydrogen spectrum of the polymer Linear-poly D-methylacetamine-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 for the glycosyl material, a degradable branched/crosslinked polymer photodynamic therapy system based on HPMA was designed and synthesized. The synthetic route for the HPMA-based degradable branched/crosslinked polymer photodynamic therapy system is shown in fig. 13. The specific synthesis method is as follows:

monomer HPMA (72mg, 5.04 mmol), MA-PySS (50 mg,0.20 mmol), crosslinker MA-GFLGKGLFG-MA (11.8 mg, 11.4. Mu. Mol), chain transfer agent MA-GFLGK-CTA (26.5 mg, 31.2. Mu. Mol) and initiator VA044 (3.4 mg, 10.4. Mu. Mol) were dissolved in H ₂ O/CH ₃ In a mixed solution of OH (3.6 mL, 1:4), a polymerization bottle is placed in an ice bath in a dark place, argon is bubbled for deoxidization for 45 minutes, the polymerization bottle is closed, the polymerization bottle is placed in an oil bath at 45 ℃ in a dark place for reaction for 12 hours, the reaction liquid is quenched by liquid nitrogen, and then the reaction liquid is dialyzed and freeze-dried at a low temperature to obtain a pale red solid, namely, the Branched-GFLG-poly HPMA-PySS (B-HPMA-PySS, 740mg, yield is 91.2%). The hydrogen spectrum of the polymer precursor, branched-GFLG-poly HPMA-PySS, is shown in FIG. 14.

640mg of B-HPMA-PySS was dissolved in 10mL of aqueous solution, treated overnight with 1000mg of DTT, dialyzed (4 ℃ C., MWCO 3.5 kDa) for 2 days, and the sample was treated with aqueous filter head and lyophilized to give a white solid powder of Branched-GFLG-poly HPMA-SH (B-HPMA-SH, 600mg, 93.8% yield). The hydrogen spectrum of the polymer precursor Branched-GFLG-poly HPMA-SH is shown in FIG. 15.

The preparation of the Branched-GFLG-poly-HPMA-S-Ppa (BHSP) was as follows: 500mg of B-HPMA-SH are dissolved in 5mL of RO H ₂ Adding 10mL of DMSO after O to prepare a B-HPMA-SH/DMSO mixed solution, dissolving 80mg of Maleimide-hexyl-Ppa in 2mL of DMSO, dripping the mixture into the dark green solution obtained in the B-HPMA-SH/DMSO mixed solution under stirring, performing dialysis (MWCO 3.5 kDa) for 2 days after light shielding reaction overnight at room temperature, removing DMSO, centrifuging (7000 rpm multiplied by 5 min), taking supernatant, filtering by an aqueous phase filter head (0.45 mu m), and freeze-drying the filtrate to obtain a dark green solid crude product (500 mg, yield 86.2%). The crude product was dissolved in 2mL of water and added dropwise to 200mL of acetone to give a large amount of precipitate, and after centrifugation (9000 rpm. Times.5 min), the solid residue was collected and purified three times by repeated treatments, and the obtained solid was dissolved again in 20mL of deionized water and lyophilized to give the product (BHSP, 460 mg). The hydrogen spectrum of the polymer Branched-GFLG-poly HPMA-S-Ppa (BHSP) is shown in FIG. 16.

The following proves the beneficial effects of the invention through specific test examples:

test example 1 characterization of Polymer

1. Particle size and potentiometric measurement of Polymer

BSP, BShP, LSP was weighed, diluted in pure water to a final concentration of 1.0mg/mL, the particle size of the sample and the Zata potential were characterized using a particle size meter (three replicates per measurement), and the final data was analyzed using GraphPad Prism software. Meanwhile, a sample with the concentration of 200 mug/mL is dripped on a copper net for natural air drying, and a transmission electron microscope is used for observing the particle size and the morphology.

The morphology of each group of materials was observed by transmission electron microscopy as shown in fig. 17. The assembly of BShP is quite uniform round nano particles; the BSP-formed nanoparticles are not so regular, but circular-like morphology is observed; whereas LSPs without branched/crosslinked structure are loosely filiform in distribution and do not aggregate to form a special morphology.

As shown in fig. 18: the particle sizes of BSP, BShP and LSP were about 238nm, 164nm and 374nm, respectively, as measured by DLS, i.e., the particles formed by BShP were the most dense, but the BSP was relatively loose but still retained in nanoparticle form. The Zeta potentials of the three groups of materials are respectively-16.81 mV, -19.23mV and-21.97 mV, and are all electronegative.

2. In vitro ROS production assay for polymers

BSP, BShP and LSP were dissolved and diluted to a photosensitizer pyropheophorbide-a (Pyropheophorbide a, ppa) concentration of 5.0. Mu.g/mL, while SOSG detection reagent was added to a final concentration of 2.5. Mu.M. Each set of solutions was pipetted into 100. Mu.L to 96-well plates and 3 multiplex wells were provided, followed by irradiation with 660nm laser (power density: 10 mW/cm) ² Duration of: 15 min). Fluorescence intensities (excitation wavelength 490nm, emission wavelength 525 nm) were measured with a multifunctional microplate reader at time points of 0.5, 1, 1.5, 2, 3, 5, 10, 15min and the fluorescence intensities of the respective samples at 525nm were recorded. PBS was used as a blank.

Ppa are capable of absorbing photons of a specific wavelength to transition from a ground state to an excited state, and subsequently transferring the absorbed energy to nearby oxygen molecules to produce singlet oxygen. Ppa ROS produced by illumination excitation can be detected by the kit. Among them, the singlet oxygen fluorescent probe (Singlet oxygen sensor green, SOSG) is a detection reagent having high selectivity for singlet oxygen, the indicator initially has weak blue fluorescence, and when acted on by singlet oxygen, it emits green fluorescence (maximum excitation/emission wavelength is about 504/525 nm) similar to fluorescein, and the intensity of green fluorescence is positively correlated with the amount of ROS generated. As shown in FIG. 19, after absorbing 660nm excitation energy, BSP generated fluorescence intensity was strongest, LSP was weakest, and BSP fluorescence intensity was significantly higher than LPS and BShP. Indicating that BSP produces more ROS in vitro and more efficiently, while BShP and LSP produce less ROS more efficiently. Further, the BSP has better killing effect on tumors.

3. In vitro IC50 value determination of Polymer

4T1 cells were plated at 5X 10 ⁴ The cell concentration per well was inoculated into a 96-well plate, the medium was aspirated after the cell had adhered to the wall (about 12 hours of incubation), and incubation was continued for 6 hours after adding a gradient concentration (Ppa concentration: 50, 20, 10, 5, 2, 1, 0.5, 0.1, 0.01. Mu.g/mL) solution containing BSP, LSP and BShP prepared with the medium, and after the completion of incubation, 2J/cm was administered, respectively ² After 12 hours of incubation, the absorbance at about 450nm was measured by an enzyme-labeled instrument using CCK8 reagent according to the kit instructions. The assay results were plotted using GraphPad Prism (vison 8.0) software.

IC was performed by in vitro cell experiments ₅₀ Comparison of the values (fig. 20). Under the same Ppa concentration gradient, illumination dose and culture conditions, the IC50 values of the three groups of polymers are respectively 0.25 mug/mL of BSP, 0.91 mug/mL of LSP and 4.8 mug/mL of BShP, which shows that the BSP has the strongest killing effect on tumor cells.

4. Polymer in vivo tumor site signal detection

1×10 ⁶ The 4T1 cells were inoculated into the flank of Balb/c mice (20 g, 6-8 weeks old) and experiments were started after the tumor had grown to a diameter of about 5 mm. Mice were randomly divided into 3 groups (5 each) and each were injected via the tail vein with Ppa of the different groups of material (BSP, BShP and LSP) at a concentration of 5 mg/kg. At different time points (5 min, 30min, 1h, 3h, 6h, 12h and 24 h) when injection is completed, fluorescence of tumor part is observed through a living body fluorescence imaging systemSignals, and processes and analyzes the data by a data analysis system.

The results show that the fluorescence intensity of the BSP material gradually increased at the tumor site after injection, 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 LSP and BShP groups (FIG. 21).

At the same Ppa concentration, BSP is higher than LSP and BShP in terms of in vitro ROS production, toxicity to tumor cells and tumor tissue aggregation, which ensures the effect of photodynamic therapy. BSP can be used as photosensitizer and has excellent effect for photodynamic therapy of tumor.

Test example 2 Polymer-entrapped Olaparib and characterization thereof

1. Polymer-entrapped Olaparib

The polymer-entrapped Olaparib is prepared by dialysis, 300mg LSP, BSP, BShP and BHSP are weighed and dissolved in 5mL deionized water respectively, and 10mL DMSO is added to obtain H containing material ₂ O/DMSO mixed solution. 30mg of Olaparib (Olaparib) was dissolved in 2mL of DMSO and added dropwise to H containing material ₂ O/DMSO mixed solution. After stirring overnight in the dark, the reaction solution was dialyzed (3.5 kDa MWCO) for 2 days to remove the DMSO solvent, and then lyophilized after treatment with an aqueous filter membrane to obtain an Olaparib-entrapped polymer, which was designated as LSPO, BSPO, BShPO and BHSPO (O stands for olapearbib) in that order.

2. Measurement of Olaparib pack load

LSP, BSP, BShP and BHSP loadings for olaparib were calculated by HPLC results. Drug loading (wt%) = (mass of drug entrapped/total mass of polymer and drug entrapped) ×100%. Each group of materials is dissolved by mobile phase and then is sonicated, and the filtrate after the organic phase filter head treatment is subjected to HPLC detection. The Column type was (Column name: shim-pack GLST, C18,5 μm.Size: 250X 4.6mm I.D.), the Column temperature was 30 ℃, the flow rate was 1.0mL/min, the sample loading was 20. Mu.L, the sample concentration was about 100. Mu.g/mL, the detector was chosen to be UV light (276 nm), and the mobile phase was CH ₃ OH/H ₂ O (v: v=1:1).

The results are shown in Table 2. The highest inclusion amount of the BSP polymer reaches 4.03%; the bshps have a packet size of 2.17% and are worse than BSPs. In contrast, the control materials LSP and BHSP were not effective in encapsulating small molecule drugs, with only 0.33% and 0.13% of the encapsulation capacity, respectively. Indicating that the "proper" assembled stacked configuration of the BSP is capable of entrapping more small molecule drugs. The detection was performed by HPLC collection of the entrapped small molecule drug, and HRMS results indicated that olaharib was still the active drug substance (fig. 22).

TABLE 2 Ppa content of the polymers of each group and the corresponding coating amount of Olaparib

3. Correlation characterization of BSPO

The aqueous phase particle size and zeta potential of the polymer BSPO (1.0 mg/mL) entrapped with olaparib was measured with DLS and further examined for morphology by TEM (sample preparation concentration 200 μg/mL). The ultraviolet spectra of sample BSP, BSPO, ppa and Olaparib were measured with an ultraviolet-visible spectrophotometer in the range of 200-800nm, and the fluorescence spectra of BSP, BSPO, ppa and Olaparib were measured by a fluorescence spectrometer. The change in the in vitro singlet oxygen production efficiency of BSP and BSPO was evaluated by SOSG assay reagents in the same manner as in test example 1.

As shown in fig. 23, DLS results indicate that the BSPO aqueous phase particle size is about 178nm, and the surface is also negatively charged; whereas TEM results indicate that they form nanoparticles of relatively uniform size. This suggests that the BSPO structure may be denser after the small molecule olaparib is entrapped compared to BSP.

Fluorescence spectral properties of BSPO: as shown in fig. 24, at the same Ppa concentration, the fluorescence intensity of BSP and BSPO was not significantly different at about 680nm, indicating that the fluorescence properties of Ppa were not affected after the entrapment of olaparib.

Meanwhile, in the ultraviolet spectrogram (fig. 25), the ultraviolet spectrums of the BSP and the BSPO are basically overlapped at about 330-800nm, which indicates that the ultraviolet spectrums are not changed in the wavelength range; the ultraviolet spectrum is slightly different around 300nm, namely, the spectrum of BSPO changes around 300 nm. The ultraviolet absorption peak of the Olaparib is about 276nm, the change is caused by overlapping of the ultraviolet peak of Ppa and the ultraviolet peak of the Olaparib, and the result shows that the BSP can effectively encapsulate the Olaparib to a certain extent, and the encapsulating of the Olaparib does not influence the ROS generating capability of the polymer.

As shown in FIG. 26, there was no significant difference in the in vitro ROS production and efficiency of BSPO compared to BSP, indicating that ROS can still be efficiently produced after the encapsulation of Olaparib.

The test results above demonstrate that: the glycosyl polymer BSP can be used as a photosensitizer for photodynamic therapy, has good aggregation effect on tumor sites and has good killing effect on tumors; meanwhile, the glycosyl polymer BSP can be used as a drug carrier for wrapping drugs, such as Olaparib, and further used for treating diseases. The medicine has high medicine carrying capacity and excellent effect as medicine feeding system.

The invention synthesizes glycosyl polymer carriers BSP, BShP and LSP with different structures, and a drug carrier BSHP based on pHPMA. Experimental results indicate that the ROS production efficiency after BSP illumination is highest compared with BShP and LSP at the same concentration of the photosensitizer pyropheophorbide-a (Pyropheophorbide a, ppa). Tumor cytotoxicity experiments show that the half inhibition concentrations (Half maximal inhibitory concentration, IC 50) of the three polymer carriers of BSP, BShP and LSP are respectively 0.25 mug/mL, 4.8 mug/mL and 0.91 mug/mL, and the BSP has more obvious tumor cell killing effect.

The invention also prepares a dual drug delivery system BSPO of the entrapped Olaparib. The study on the capacity of the medicine-entrapped Olaparib shows that the BSP has the strongest medicine-entrapped capacity and is optimal as a medicine carrier.

The glycosyl polymer carrier BSP prepared by the invention has high aggregation concentration at tumor sites, good photodynamic treatment effect and strong tumor killing effect, and is a good photosensitizer; and the medicine carrier has large coating capacity, and is an excellent drug delivery system.

In summary, the invention provides a glycosyl polymer BSP, which overcomes the defects of insufficient aggregation of tumor sites and weak tumor killing effect of the existing PDT photosensitizer, can aggregate at the tumor sites, and has good targeting effect; the compound has photodynamic effect, and can be used as photosensitizer for photodynamic therapy, and has high ROS production efficiency and strong killing effect on tumor cells. Meanwhile, the glycosyl polymer can also be used as a drug delivery system for entrapping drugs, in particular tumor drugs such as Olaparib, and has excellent effect on tumor treatment. The glycosyl polymer has good application prospect in preparing medicines for treating tumors.

Claims

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

(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 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;

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:

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:

(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);

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.