CN113786492A

CN113786492A - Polymer carrier for photodynamic therapy and preparation method and application thereof

Info

Publication number: CN113786492A
Application number: CN202110932558.3A
Authority: CN
Inventors: 罗奎; 罗强; 段振宇; 龚启勇
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: 2021-12-14
Anticipated expiration: 2041-08-13
Also published as: CN113786492B

Abstract

The invention provides a polymer carrier for photodynamic therapy and a preparation method and application thereof, belonging to the technical field of drug delivery systems. Specifically provides a cell membrane-glycosyl polymer, which is formed by wrapping a layer of cell membrane outside the glycosyl polymer with the structure shown in formula I. The polymer has good photodynamic effect, good targeting effect when being used as a photosensitizer, can be gathered at a tumor part, and has good photodynamic treatment effect and strong killing effect on tumor cells. In addition, the polymer has excellent entrapment effect on the drug as a polymer carrier, and the drug loading is high; the tumor drug encapsulated by the coating has excellent tumor treatment effect and excellent application prospect.

Description

Polymer carrier for photodynamic therapy and preparation method and application thereof

Technical Field

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

Background

Cancer (cancer) refers to a malignant tumor that originates in epithelial tissue. The data show that in 2020, there are 1929 new cancer cases worldwide, of which 1006 ten thousand cases in men and 923 ten thousand cases in women; 996 ten thousand cases of cancer death worldwide in 2020, of which 553 thousand in men and 443 ten thousand in women. The incidence rate and the mortality rate of cancer are high, and the cancer becomes one of global diseases seriously threatening human health, and is one of the hot spots and difficulties of the current basic and clinical research. According to the latest data in 2020, the first ten cancers with global incidence are respectively breast cancer, lung cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, cervical cancer, esophageal cancer, thyroid cancer and bladder cancer. The current treatment modes of cancer mainly comprise surgery, radiotherapy, chemotherapy, immunotherapy and the like. Although the early treatment effect of the treatment modes is better, the problems of tumor metastasis, easy relapse, body injury, immune damage and the like still exist.

In recent years, Photodynamic therapy (PDT) has been used as an emerging treatment modality. Photodynamic therapy induces apoptosis of tumor cells and causes DNA damage through Reactive Oxygen Species (ROS) generated by photosensitizers after specific laser irradiation. PDT has the advantages of high selectivity, low systemic toxic and side effects, repeated treatment, and difficulty in generating tolerance, and is receiving more and more attention from researchers. However, most photosensitizers used in PDT have problems of poor water solubility, lack of targeting property, low ROS yield, etc., insufficient accumulation at tumor sites, weak tumor killing effect, and limited application in cancer therapy.

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

Disclosure of Invention

In order to solve the above problems, the present invention provides a polymer carrier useful for photodynamic therapy, a method for preparing the same, and use thereof.

The invention provides a cell membrane-glycosyl polymer, which is formed by wrapping a layer of cell membrane outside a glycosyl polymer with a structure shown in a formula I:

formula I

Wherein the content of the first and second substances,

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

r is selected from

Or

Further, the cell membrane is a cell membrane of a tumor cell;

and/or the glycosyl polymer is prepared from the following raw materials in parts by weight: 100-500 parts of B-gal-SH and 1-100 parts of Maleimide-Ppa 1;

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-gal-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

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 glycosyl polymer is prepared from the following raw materials in parts by weight: B-gal-SH 500 parts, Maleimide-Ppa 75 parts;

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

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

Further, the preparation method of the cell membrane comprises the following steps:

squeezing the cells with a miniature extruder until the cells are destroyed, centrifuging, and collecting the supernatant containing cell membranes;

preferably, the preparation method of the cell membrane comprises the following steps:

preparing PBS cell suspension containing protease inhibitor, adopting Avanit micro extruder without polycarbonate porous membrane to press cells until the cells are destroyed, centrifuging, and taking supernatant containing cell membrane to obtain the product.

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

reacting B-gala-PySS with dithiothreitol to obtain the product.

Further, 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 solvent, reacting under the action of initiator, and lyophilizing to obtain the final product;

preferably, the solvent is a mixed solution of water and methanol;

and/or, oxygen is removed prior to the reaction;

and/or the reaction is a reaction protected from light;

more preferably, the water to methanol volume ratio is 1: 4;

and/or the initiator is VA 044;

and/or the reaction is carried out in an oil bath at the temperature of 40-50 ℃ for 10-12 hours.

Further, the method for preparing the glycosyl polymer comprises the following steps:

(1) dissolving B-gala-SH in water, adding DMSO, and preparing a B-gala-SH/DMSO mixed solution;

(2) dissolving Maleimide-Ppa in DMSO, and adding into B-gal-SH/DMSO mixed solution for reaction;

(3) purifying the reaction liquid obtained in the step (2) to obtain the compound;

preferably, the first and second electrodes are formed of a metal,

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 carried out at room temperature in a dark place.

The present invention also provides a method for preparing the aforementioned cell membrane-glycosyl polymer, which comprises the steps of:

mixing the cell membrane with a glycosyl polymer aqueous solution, and centrifuging to obtain the cell membrane-glycosyl polymer aqueous solution;

preferably, the mixture is stirred overnight at the temperature of 0-4 ℃;

and/or, the centrifugation is 20000g for 30 min.

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

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

The invention also provides the application of the cell membrane-glycosyl polymer in preparing a drug carrier;

preferably, the drug used for entrapment in the drug carrier is an anti-tumor drug;

more preferably, the anti-tumor drug is olaparib.

The invention also provides a medicament which is a preparation prepared by taking the cell membrane-glycosyl polymer as an active ingredient, or taking the cell membrane-glycosyl polymer as a carrier to entrap the medicament 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 cell membrane-glycosyl polymer, which overcomes the defects of insufficient accumulation at a tumor part and weak killing effect on tumors of the existing PDT photosensitizer, can be accumulated at the tumor part, and has good targeting effect; it has photodynamic effect, high ROS generating efficiency when used as photosensitizer for photodynamic therapy, and strong killing effect on tumor cells. Meanwhile, the cell membrane-glycosyl polymer can also be used as a drug delivery system to entrap drugs, in particular tumor drugs, such as olaparib, for tumor treatment, and has excellent effect. The cell membrane-glycosyl polymer has excellent application prospect in preparing medicaments for treating tumors.

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

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

Drawings

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

FIG. 2 shows the structural formula of cathepsin B sensitive functional cross-linking agent MA-GFLGKGLFG-MA and the preparation route thereof.

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

FIG. 4 shows the hydrogen spectrum of the polymer precursor Branche-GFLG-poly D-galactochitosan-PySS (solvent D6-DMSO).

FIG. 5 shows the hydrogen spectrum of the polymer precursor Branched-GFLG-poly D-galactosamine-SH (solvent D6-DMSO).

FIG. 6 shows the hydrogen spectrum of the Branched-GFLG-poly D-galactosamine-S-Ppa polymer (solvent D6-DMSO).

FIG. 7 is a TEM image of the cell membrane and the CM-BSPO formed after the BSPO is modified by the cell membrane: a is a TEM picture of the cell membrane; b is TEM picture of CM-BSPO formed after BSPO is modified by cell membrane.

FIG. 8 shows the structural formula of Maleimide-hexyl-Ppa compound and its synthetic route.

FIG. 9 shows the hydrogen spectrum of the Branched-GFLG-poly D-galactochitosan-S-hexyl-Ppa polymer (solvent D6-DMSO).

FIG. 10 is a synthetic route for a linear glycopolymer photodynamic therapy system.

FIG. 11 shows the hydrogen spectrum of the polymer precursor Linear-poly D-galactochitosan-PySS (solvent D)₂O)。

FIG. 12 shows the hydrogen spectrum of the polymer precursor Linear-poly D-galactochitosan-SH (solvent D6-DMSO).

FIG. 13 shows the hydrogen spectrum of polymer Linear-poly D-galactochitosan-S-Ppa (solvent D6-DMSO).

Fig. 14 is a synthetic route to a HPMA-based degradable branched/crosslinked polymer photodynamic therapy system.

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

FIG. 16 shows a hydrogen spectrum of Branche-GFLG-poly HPMA-SH polymer precursor (solvent D)₂O)。

FIG. 17 shows the hydrogen spectrum of the Branched-GFLG-poly HPMA-S-Ppa polymer (solvent D)₂O)。

FIG. 18 shows the chemical structures, self-assembly diagrams and TEM results of BSP, BShP and LSP (scale: 200 nm).

Fig. 19 shows DLS detection results of BSP, BShP, and LSP: a is a particle size detection result; b is zeta potential detection result.

FIG. 20 shows the change of fluorescence intensity with increasing laser irradiation time when a singlet oxygen fluorescent probe (SOSG) is added to BSP, BShP, and LSP polymer solutions.

FIG. 21 shows the results of in vitro cytotoxicity experiments with BSP, BShP and LSP.

FIG. 22 shows the statistical results of fluorescence signals of the images and tumor sites of the three groups of BSP, LSP and BShP polymers in the tumor mouse model: a is a distribution image; b is the fluorescent signal statistical result of the tumor part.

FIG. 23 shows the contents of Olaparib in polymer LSP, BSP, BShP and BHSP by HPLC measurement, which are calculated from a standard curve; HRMS results show that the encapsulated olaparib is a raw drug and does not influence the activity of the olaparib.

FIG. 24 is a graph showing the results of the fluorescence intensity of the tumor site after tail vein injection of different polymers at different time points.

FIG. 25 is a graph showing the results of the fluorescence intensity of the tumor site after tail vein injection of different polymers.

FIG. 26 is a tumor treatment flowchart of the mouse tumor model.

FIG. 27 shows the tumor volume changes in groups of mice for different treatment regimens.

Figure 28 summarizes the change in tumor volume in mice after different treatments (. about.. p < 0.01).

FIG. 29 shows tumor inhibition rates of mice of different treatment groups.

Fig. 30 shows the tumor volume magnetic resonance image (red circles indicate tumors), lung CT image (white arrows indicate lung metastases) and lung tissue specimen (black circles indicate lung metastases) for each group of illumination groups.

Fig. 31 is a magnetic resonance image (red circles indicate tumors), a lung CT image (white arrows indicate lung metastases), and a lung tissue specimen (black circles indicate lung metastases) of tumor volumes for each group of non-illuminated groups.

FIG. 32 shows HE staining of lungs of mice in different treatment groups, and red arrows indicate metastasis (scale: 200 μm).

FIG. 33 shows the immunohistochemical staining of tumor CD31 (scale: 200 μm) after treatment of different groups.

FIG. 34 shows Ki-67 immunohistochemical staining of tumors after treatment in different groups (scale: 200 μm).

FIG. 35 is a statistical analysis of the immunohistochemical staining of the tumors Ki-67 in each group (. about.. about.p)<0.01，^nsNo significant difference).

Detailed Description

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

Murine 4T1 breast cancer cell line was purchased from cell bank of Chinese academy of sciences (Shanghai) and cultured in RPMI 1640 medium containing 1% diabody and 10% fetal calf serum under 5% CO₂95% air, 37 ℃ and constant humidity environment. All animal experiments were performed strictly according to the animal study guidelines approved by the ethical committee of the hospital. Experimental female Balb/c mice purchased from great achievementsLaboratory animals Co., Ltd. 4T1 cells were used to establish a subcutaneous tumor model and used for in vivo distribution and imaging studies.

Example 1 preparation of sugar-based polymers according to the invention

The compounds Maleimide-Ppa, MA-PySS and MA-D-galactosamine were synthesized according to the methods reported in

documents

1 and 2.

Literature 1(Pan D, Zheng X, Zhang Q, et al. degraded-Polymer Distuting Cells' Stress Protection by Targeting strategies metals to Metal Vulnerability [ J ]. Advanced Materials,2020,32:1907490.) reports the synthesis of Maleimide-Ppa and MA-PysS, with the following specific synthetic routes:

Maleimide-Ppa：

MA-PySS：

the Synthesis of MA-D-galactosamine is reported in literature 2(Wartchow C A, Wang P, Bednarski M D, et al. carbohydrate Protease Conjugates: Stabilized proteins for Peptide Synthesis [ J ]. The Journal of Organic Chemistry,1995,60: 2216-.

The structure of Maleimide-Ppa is:

wherein Ppa is a photosensitizer pyropheophorbide-a (pyropheophorbide a) moiety. The structure of MA-Pyss is:

the structure of the MA-D-galactomannan is as follows:

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 reference 3 (reference 3: Sun L, Li X, Wei X, et al. Stimuli-reactive Biodegradable Polymer-gallium Conjugates as effective and Biocompatible nanoscopic Magnetic Imaging control Agents [ J ]. ACS Applied Materials & Interfaces,2016,8: 10499-. The specific synthesis method comprises the following steps:

MA-GFLG-OH (4.6g, 10mmol), HOBt (1.49g, 11mmol) and HBTU (4.26g, 11mmol) as a condensing agent were placed in a round-bottomed flask, and dissolved by adding ultra-dry DMF (50mL) under nitrogen protection in an ice bath. DIPEA (6.7mL, 40mmol) was added dropwise to the system and reacted for 0.5 h. Weighing H-Lys (O)^tBu) -Fmoc & HCl (4.62g, 10mmol) was added to the system, and the mixture was reacted for 0.5 hour under ice bath and then returned to room temperature for 10 hours. Dissolving in 450mL Ethyl Acetate (EA), washing with saturated sodium bicarbonate solution (100mL × 3), 1M dilute hydrochloric acid (100mL × 3) and saturated sodium chloride solution (100mL × 3), collecting organic phase, drying with anhydrous magnesium sulfate, concentrating, standing at 4 deg.C to obtain crystalline white solid MA-GFLGK (O-GFLGK)^tBu) -NHFmoc (6.51g, 7.51mmol, 75.1% yield).¹H NMR(400MHz，δin ppm)，¹³C NMR(100MHz，d₆-DMSO，δin ppm)，LC-MS(ES+)：m/z＝867.2[M+H]⁺。MALDI-HRMS:m/z＝889.4474[M+Na]⁺。

In ice bath, MA-GFLGK (O)^tBu) -NHFmoc (4.33g, 5mmol) was dissolved with 40mL of a mixed solution of DCM/TFA (v: v ═ 1:9) and reacted back to room temperature. TLC monitoring, after the reaction is completed, the solvent is removed by rotary evaporation, white solid powder is separated out after ether is added, and the obtained white solid powder is washed twice by ether and dried to obtain the product MA-GFLGK (OH) -NHFmoc (3.61g, 4.45mmol, 89.0% yield).¹H NMR(400MHz，δin ppm)，¹³C NMR(100MHz，d₆-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, adopting solid phase polypeptide synthesisThe method synthesizes the functional monomer MA-GFLGK-CTA through the following steps. MA-GFLGK (OH) -NHFmoc (1.62g, 2mmol) and DIPEA (0.67mL, 4mmol) were dissolved in 10mL DMF and reacted with chlorotrityl chloride resin (5.0g, 1.15mmol/g) 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) (100mL) and 3 times with DCM and DMF, respectively. The Fmoc protecting group was treated three times with 50mL of 20% piperidine in DMF and cyanovaleric acid dithiobenzoic acid (CTA-COOH, 2.79g, 10mmol), DIC (1.26g, 10mmol) and HOBt (1.35g, 10mmol) were added and 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 and precipitated and purified by addition to ether, and further purified by high performance liquid chromatography to give MA-GFLGK-CTA (892mg, 1.05mmol, 52.5% yield) as a pink solid product.¹H NMR(400MHz，δin ppm)，¹³C NMR(100MHz，d₆-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 cross-linking agent MA-GFLGKGLFG-MA

The synthesis route of the cathepsin B sensitive functional cross-linking agent MA-GFLGKGLFG-MA is shown in figure 2. MA-GFLGKGLFG-MA was synthesized in a similar manner as reported in reference 3 (reference 3: Sun L, Li X, Wei X, et al. Stimuli-reactive Biodegradable Polymer-gallium Conjugates as effective and Biocompatible nanoscopic Magnetic Imaging compositions [ J ]. ACS Applied Materials & Interfaces 2016,8: 10499-10512.). The specific synthesis method comprises the following steps:

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. DIPEA (3.35mL, 20mmol) was added under ice-bath for 0.5 h. Adding H-Lys (OCH) into the system₃) after-OH.2HCl (583mg, 2.5mmol), the reaction was returned to room temperature for 20 h. The reaction mixture was added to 250mL of ethyl acetate, and washed with a saturated sodium bicarbonate solution (20 mL. times.3), 1M dilute hydrochloric acid (20 mL. times.3), and a saturated sodium chloride solution (20 mL. times.3) in this orderThe organic phase was washed, collected, dried over anhydrous magnesium sulfate, concentrated and crystallized at 4 ℃ to give the product BocNH-GFLGKGLFG-NHBoc (1.62g, 1.46mmol, 58.4% yield) as a white solid.¹H NMR(400MHz，δin ppm)，¹³C NMR(100MHz，δin ppm)，LC-MS(ES+)：m/z＝1109.4[M+H]⁺，MALDI-HRMS：m/z＝1131.6045[M+Na]⁺。

BocNH-GFLGKGLFG-NHBoc (1.33g, 1.2mmol) was placed in a nitrogen protected round bottom flask, 25mL of mixed DCM/TFA (v: v ═ 1:1) solution was added under ice bath, after 12 hours reaction time, solvent was removed by rotary evaporation, and after two additions of anhydrous ether, it was dried by suction to give a white solid intermediate which was dissolved in 100mL of H₂To a mixed solution of O/ACN (v: v ═ 4:1), 1M NaOH solution was added dropwise to adjust pH to 7 to 8 under ice-bath, then methacryloyl chloride (0.3mL, 3mmol) was dissolved in Acetonitrile (ACN), and then added dropwise to the system while slowly adding 1M NaOH solution dropwise to maintain pH at 10, the system was reacted for 1 hour under ice-bath and then left at room temperature for 4 hours, the reaction solution was added to 200mL ethyl acetate, pH was adjusted to 2 to 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 (20mL × 3), finally dried over anhydrous magnesium sulfate and concentrated, and the resulting concentrate was crystallized at 4 ℃ to obtain a white solid product MA-GFLGKGLFG-MA (700mg, 0.68mmol, yield 56.7%).¹H NMR(400MHz，δin ppm)，¹³C 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/cross-linked glycosyl polymer photodynamic therapy system

The synthetic route for the degradable branched/cross-linked glycopolymer photodynamic therapy system is shown in figure 3. The specific synthesis method comprises the following steps:

monomers MA-D-Galactosamine (1446mg, 4.63mmol), MA-PySS (100mg, 0.39mmol), crosslinker MA-GFLGKGLFG-MA (23.5mg, 22.8. mu. mol), chain transfer agent MA-GFLGK-CTA (53.0mg, 62.4. mu. mol) and initiator VA044(6.7mg, 20.8. mu. mol) were dissolved in H₂O/CH₃OH(7.2mL，1:4，vAnd v), placing a polymerization bottle in an ice bath in a dark place, introducing argon gas to carry out bubbling for deoxygenation for 45 minutes, sealing the polymerization bottle, placing the polymerization bottle in the oil bath at 45 ℃ in the dark place for reaction for 12 hours, quenching the reaction by using liquid nitrogen, carrying out low-temperature dialysis and freeze-drying on the reaction liquid, and obtaining a light red solid Branche-GFLG-poly D-galactochitosan-PySS (B-gal-PySS, 1480mg, and the yield is 91.2%). The hydrogen spectrum of the polymer precursor Branche-GFLG-poly D-galactosamine-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 3.5KDa) for 2 days, and the sample was treated with an aqueous phase filter and lyophilized to obtain Branche-GFLG-poly D-galactosamine-SH (B-gal-SH, 1150mg, 88.5% yield) as a white solid powder. The hydrogen spectrum of the polymer precursor Branched-GFLG-poly D-galactosamine-SH (B-gal-SH) is shown in FIG. 5.

Branche-GFLG-poly D-galactosamine-S-Ppa (BSP) was prepared as follows: 500mg of B-gala-SH dissolved in 5mL of RO H₂Adding 10mL of DMSO after O to prepare a B-gala-SH/DMSO mixed solution, dissolving 75mg of Maleimide-Ppa in 2mL of DMSO, dropwise adding the dissolved solution into the B-gala-SH/DMSO mixed solution under stirring, reacting overnight in a dark environment at room temperature, dialyzing (MWCO 3.5KDa) for 2 days to remove DMSO, centrifuging (7000rpm multiplied by 5min), taking supernatant, filtering through an aqueous phase filter head (0.45 mu m), and freeze-drying the filtrate to obtain a black-green solid crude product (504mg, yield 87.7%). Dissolving the crude product with 2mL of water, then dropwise adding the solution into 200mL of acetonitrile to generate a large amount of precipitates, centrifuging (10000rpm multiplied by 5min), collecting solid residues, repeatedly treating and purifying for three times, dissolving the obtained solid in 20mL of deionized water again, and freeze-drying to obtain a product (BSP, 420 mg). The hydrogen spectrum of the polymer Branched-GFLG-poly D-galactosamine-S-Ppa (BSP) is shown in FIG. 6.

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

TABLE 1 amino acid analysis results of sugar-based branched/crosslinked Polymer BSP

In general, the polymeric carrier is in the organismThe renal threshold for efficient metabolism in vivo is about 50kDa, and it is therefore necessary to ensure that the molecular weight of the main polymer chain after degradation is below this level. The sugar-based polymer material of the invention is found to be degraded rapidly under the action of enzyme, and the degradation product is obtained after 4 hours (Mn is 32.4X 10)³,Mw＝46.7×10³PDI 1.44) has a better homogeneity with a molecular weight below the renal threshold.

Example 2 preparation of BSP-entrapped drug Olaparib

Prepared by dialysis, 300mg BSP was weighed out and dissolved in 5mL deionized water and 10mL DMSO was added. 30mg of Olaparib (Olaparib) was dissolved in 2mL of DMSO and added dropwise to BSP-containing H₂O/DMSO mixed solution. The reaction was stirred overnight in the dark, dialyzed (3.5kDa MWCO) for 2 days to remove the DMSO solvent, then treated with an aqueous phase filter and lyophilized to give an Olaparib-coated polymer designated BSPO (O for olaparib).

Example 3 preparation of the cell Membrane-glycosyl Polymer of the invention

Normally cultured 4T1 cells (with an adherent density of 80-90%) were scraped and collected in Phosphate Buffered Saline (PBS), and cell pellets were collected by centrifugation (700 g.times.7 min). It was then resuspended in PBS containing a protease inhibitor cocktail. Cells were completely destroyed by pressing 20 times with an Avanit micro-extruder without a polycarbonate porous membrane. The resulting solution was centrifuged at low temperature (1000 g.times.15 min, 3000 g.times.15 min, 10000 g.times.30 min; 4 ℃ C.) in this order, and the cell membrane-containing supernatant was collected (TEM of cell membranes as shown in FIG. 7 a). Mixing the supernatant with an aqueous solution containing BSPO, stirring overnight (4 ℃), centrifuging and collecting (20000g multiplied by 30min), wherein the obtained precipitate is a target product named as CM-BSPO, and observing the morphology of the product by using a TEM after resuspension, as shown in FIG. 7b, the cell membrane structure at the periphery of the glycosyl polymer can be obviously observed, which indicates the successful wrapping of the cell membrane.

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 for the functionalized photosensitizer is shown in FIG. 8.

WeighingMaleimidopropionic acid (Maleimide-COOH, 1.17g, 6.9mmol) and the condensing agent HATU (3.74g, 9.85mmol) were dissolved in 10mL of DMF, DIEA (3.2mL, 18.3mmol) was added under ice-bath to react for 5 minutes, and N-Boc-1, 6-hexanediamine hydrochloride (1.80g, 8.3mmol) was added to the system to react for 2 hours. 50mL of saturated sodium bicarbonate solution and 30mL of DCM were added to the reaction solution, the organic phases were 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 the crude product as a yellow solid. Purifying by column Chromatography (CH)₃DCM ═ 1:20, v/v) and further purified by hplc to give Maleimide-hexyl-NHBoc as a colorless liquid product (1.66g, 65.4% yield).¹H NMR(400MHz，δin ppm)，¹³C 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 (220mg, 0.60mmol) was dissolved in 10mL DCM at 0 deg.C, 10mL TFA was added, the reaction was allowed to proceed overnight, the solvent was removed, and the residue was treated with dry ether to give a solid powder. The solid powder obtained in the above step, pyropheophorbide a (270mg, 0.50mmol) and condensing agent HATU (285mg, 0.75mmol) were dissolved in DCM, DIEA (0.23mL, 1.33mmol) was added, the reaction solution was reacted at room temperature for 1.5 hours, the solvent was removed by rotation, and column purification was performed (DCM: CH)₃OH ═ 30:1-20:1, v/v) gave Maleimide-hexyl-Ppa (237mg, 50.5% yield) as a black solid.¹H 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-galactosamine-S-hexyl-Ppa (BShP)

Branch-GFLG-poly D-galactosamine-S-hexyl-Ppa (BShP) is prepared as follows: 500mg of B-gala-SH dissolved in 5mL of RO H₂Adding 10mL of DMSO to prepare a B-gala-SH/DMSO mixed solution after O, dissolving 80mg of Maleimide-hexyl-Ppa in 2mL of DMSO, dropwise adding the mixture into the B-gala-SH/DMSO mixed solution under stirring to obtain a dark green solution, carrying out a dark reaction at room temperature overnight, and dialyzing (I)Protected from light, MWCO 3.5KDa) to remove DMSO for 2 days, centrifuging (7000 rpm. times.5 min), collecting supernatant, filtering with water phase filter (0.45 μm), and lyophilizing the filtrate to obtain crude black green solid (520mg, 89.7% yield). The crude product was dissolved in 2mL of water and then added dropwise to 200mL of acetonitrile to precipitate a large amount, after centrifugation (9000 rpm. times.5 min), the solid residue was collected and purified repeatedly three times, and the obtained solid was dissolved in 20mL of deionized water again and lyophilized to obtain the product (BShP, 450 mg). The hydrogen spectrum of the polymer Branched-GFLG-poly D-galactosamine-S-hexyl-Ppa (BShP) is shown in FIG. 9.

Comparative example 2 preparation of Linear glycosyl Polymer photodynamic therapy System

The synthetic route for the linear glycosyl polymer photodynamic therapy system is shown in figure 10:

as a control, a linear glycopolymer photodynamic therapy system was designed and synthesized. Monomers MA-D-Galactosamine (722mg, 2.31mmol), MA-PySS (50mg, 0.20mmol), chain transfer agent 4-cyano-4- (thiobenzoyl) pentanoic acid (CTA, 5.3mg, 19.0. mu. mol) and initiator VA044(2.1mg, 6.5. mu. mol) were dissolved in H₂O/CH₃In a mixed solution of OH (3.5mL, 1:4, v/v), placing a polymerization bottle in an ice bath in a dark place, introducing argon gas, bubbling, deoxidizing for 45 minutes, sealing, placing in an oil bath at 45 ℃ in a dark place, reacting for 12 hours, quenching the reaction with liquid nitrogen, dialyzing the reaction liquid at low temperature, and freeze-drying to obtain light red solid Linear-poly D-galactosamine-PySS (L-gal-PySS: 725mg, yield 93.3%). The hydrogen spectrum of the polymer precursor Linear-poly D-galactochitosan-PySS is shown in FIG. 11.

620mg of L-gal-PySS was dissolved in 10mL of an aqueous solution, treated with 600mg of DTT overnight and dialyzed (MWCO 3.5KDa at 4 ℃ C.) for 2 days, and the sample was treated with an aqueous phase frit and lyophilized to obtain Linear-poly D-galactosamine-SH (L-gal-SH, 560mg, 93.3% yield) as a white solid powder. The hydrogen spectrum of the polymer precursor Linear-poly D-galactochitosan-SH is shown in FIG. 12.

The preparation of Linear-poly D-galactosamine-S-Ppa (LSP) is similar to the preparation method of degradable branched/crosslinked glycosyl polymer photodynamic therapy system: 500mgL-gala-SH in 5mL RO H₂O, then 10mL DMSO is added to prepare L-gala-SH/DMSO mixed solution, 80mg Maleimide-Ppa is dissolved in 2mL DMSO, and then stirring is carried outThe resulting solution was added dropwise to the mixture of L-gal-SH/DMSO to give a dark green solution, which was reacted overnight at room temperature in the dark, then dialyzed (MWCO 3.5KDa) for 2 days to remove DMSO, centrifuged (7000 rpm. times.5 min), and the supernatant was filtered through a water phase filter (0.45 μm), and the filtrate was lyophilized to give a crude dark green solid (512mg, 88.3% yield). Dissolving the crude product with 2mL of water, then dropwise adding the solution into 200mL of acetonitrile to generate a large amount of precipitates, centrifuging (10000rpm multiplied by 5min), collecting solid residues, repeatedly treating and purifying for three times, dissolving the obtained solid in 20mL of deionized water again, and freeze-drying to obtain a product (LSP, 496 mg). The hydrogen spectrum of the polymer Linear-poly D-galactochitosan-S-Ppa (LSP) is shown in FIG. 13.

COMPARATIVE EXAMPLE 3 preparation of HPMA-based degradable branched/crosslinked Polymer photodynamic therapy System

As a control for sugar-based materials, HPMA-based degradable branched/cross-linked polymer photodynamic therapy systems were designed and synthesized. The synthetic route for the HPMA-based degradable branched/crosslinked polymer photodynamic therapy system is shown in fig. 14.

The specific synthesis method comprises the following steps:

monomer HPMA (722mg, 5.04mmol), MA-PySS (50mg, 0.20mmol), crosslinker MA-GFLGKGLFG-MA (11.8mg, 11.4. mu. mol), chain transfer agent MA-GFLGK-CTA (26.5mg, 31.2. mu. mol) and initiator VA044(3.4mg, 10.4. mu. mol) were dissolved in H₂O/CH₃In a mixed solution of OH (3.6mL, 1:4), a polymerization bottle is placed in an ice bath in a dark place, argon is introduced for bubbling to remove oxygen for 45 minutes, then the polymerization bottle is sealed, placed in an oil bath at 45 ℃ in a dark place for reaction for 12 hours, and the reaction solution is subjected to low-temperature dialysis and freeze-drying after being quenched by liquid nitrogen to obtain a light red solid Branche-GFLG-poly HPMA-PySS (B-HPMA-PySS, 740mg, and the yield is 91.2%). The hydrogen spectrum of the polymer precursor Branched-GFLG-poly HPMA-PySS is shown in FIG. 15.

640mg of B-HPMA-PySS was dissolved in 10mL of an aqueous solution, treated with 1000mg of DTT overnight and dialyzed (MWCO 3.5KDa at 4 ℃ C.) for 2 days, and the sample was treated with an aqueous phase filter 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. 16.

Of Branched-GFLG-poly-HPMA-S-Ppa (BHSP)The preparation method comprises the following steps: 500mg of B-HPMA-SH dissolved in 5mL of RO H₂Adding 10mL of DMSO after O to prepare a mixed solution of B-HPMA-SH/DMSO, dissolving 80mg of Maleimide-hexyl-Ppa in 2mL of DMSO, dropwise adding the dissolved solution into the mixed solution of B-HPMA-SH/DMSO while stirring to obtain a dark green solution, reacting overnight at room temperature in a dark environment, dialyzing (MWCO 3.5KDa) for 2 days to remove DMSO, centrifuging (7000rpm multiplied by 5min), taking supernatant, filtering through an aqueous phase filter head (0.45 mu m), and freeze-drying the filtrate to obtain a crude black green solid (500mg, yield 86.2%). The crude product was dissolved in 2mL of water and then added dropwise to 200mL of acetone to cause a large amount of precipitation, after centrifugation (9000 rpm. times.5 min), the solid residue was collected and purified by repeating the treatment three times, and the obtained solid was dissolved in 20mL of deionized water again and then lyophilized to obtain a product (BHSP, 460 mg). The hydrogen spectrum of the polymer Branched-GFLG-poly HPMA-S-Ppa (BHSP) is shown in FIG. 17.

The following specific test examples prove the beneficial effects of the invention:

test example 1 characterization of sugar-based Polymer

1. Particle size and potential measurement of sugar-based Polymer

BSP, BShP and LSP are weighed, dissolved and diluted by pure water to a final concentration of 1.0mg/mL, a particle size analyzer is used for representing the particle size and the Zata potential of a sample (the measurement is repeated three times each time), and the final data are analyzed by GraphPad Prism software. Meanwhile, a sample with the concentration of 200 mug/mL is dripped on a copper net for natural air drying, and the size and the shape of the particle size are observed by using a transmission electron microscope.

The morphology of each group of materials observed by transmission electron microscopy is shown in fig. 18. The assembly of BShP is a very uniform round nanoparticle; in contrast, the nanoparticles formed by BSP are not regular, but a rounded-like morphology is observed; while LSP without branched/cross-linked structure is in loose filamentous distribution without aggregation to form special morphology.

As shown in fig. 19: the particle sizes of BSP, BShP and LSP measured by DLS were about 238nm, 164nm and 374nm, respectively, i.e., BShP formed the most dense particles, while BSP was relatively loose but still retained in the nanoparticle form. The Zeta potentials of the three groups of materials are respectively-16.81 mV, -19.23mV and-21.97 mV, which are all electronegativity.

2. In vitro ROS production assay for carbohydrate-based polymers

BSP, BShP and LSP were dissolved and diluted to a photosensitizer 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 the solutions was pipetted at 100. mu.L into a 96-well plate and provided with 3 multiple wells, followed by irradiation with 660nm laser (power density: 10 mW/cm)²The duration is as follows: 15 min). Fluorescence intensity (excitation wavelength 490nm, emission wavelength 525nm) was measured with a multifunctional microplate reader at time points 0.5, 1, 1.5, 2, 3, 5, 10, 15min and the fluorescence intensity of each sample at the wavelength 525nm was recorded. PBS was used as blank control.

Ppa are capable of absorbing photons of a particular 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 generated by light excitation can be detected by the kit. The Singlet oxygen fluorescent probe (SOSG) is a detection reagent with high selectivity on Singlet oxygen, the indicator initially has weak blue fluorescence, and can emit green fluorescence (the maximum excitation/emission wavelength is about 504/525nm) similar to fluorescein after the action of Singlet oxygen, and the intensity of the green fluorescence is in positive correlation with the amount of generated ROS. As shown in FIG. 20, BSP produced the strongest fluorescence after absorption of 660nm excitation energy, the second LSP, the weakest BShP, and the BSP fluorescence intensity was significantly higher than that of LPS and BShP. Indicating that the ROS production amount and efficiency of BSP in vitro are higher, while the ROS production efficiency of BShP and LSP is relatively lower. Further shows that BSP has better killing effect on tumors.

3. In vitro determination of IC50 values for carbohydrate-based polymers

4T1 cells were plated at 5X 10⁴The cell concentration per well was seeded in a 96-well plate, the medium was aspirated off after the cells were attached (about 12 hours of incubation), the incubation was continued for 6 hours after addition of a solution prepared from the medium and containing a gradient of BSP, LSP and BShP (Ppa concentration: 50, 20, 10, 5, 2, 1, 0.5, 0.1, 0.01. mu.g/mL), and 2J/cm was given after completion of the incubation²After the incubation for 12 hours, the reagent CCK8 is used according to the instruction of the kit, and the absorbance of the reagent is detected by a microplate reader at about 450 nm. Soft using GraphPad Prism (vison 8.0)The test results were plotted and analyzed.

IC was performed by in vitro cell experiments₅₀Comparison of values (fig. 21). Under the same Ppa concentration gradient, illumination dose and culture condition, the IC50 values of the three groups of polymers are respectively 0.25 mu g/mL of BSP, 0.91 mu g/mL of LSP and 4.8 mu g/mL of BShP, which indicates that the killing effect of BSP tumor cells is strongest.

4. Glycosyl polymer in vivo tumor site signal detection

1×10⁶ A4T 1 cell was inoculated into the flank of Balb/c mice (20 g, 6-8 weeks old) and the experiment was started until the tumor grew to about 5mm in diameter. Mice were randomly divided into 3 groups (5 per group) and each group was injected via tail vein with Ppa concentrations of 5mg/kg of material (BSP, BShP, and LSP). The tumor site fluorescence signals were observed by a live fluorescence imaging system at different time points (5min, 30min, 1h, 3h, 6h, 12h and 24h) at which the injection was completed, and the data were processed and analyzed by a data analysis system.

The results show that the fluorescence intensity of the BSP material at the tumor site gradually increased after injection, and reached the highest in about 6 hours, and the fluorescence intensity at the tumor site at each time point was much higher than that of the LSP and BShP groups (FIG. 22).

At the same Ppa concentration, BSP, whether ROS production in vitro, toxic effects on tumor cells, and accumulation in tumor tissues, are higher than LSP and BShP, which warrant photodynamic therapy. BSP can be used as photosensitizer, and has excellent effect for photodynamic therapy of tumor.

Test example 2 sugar-based Polymer-Encapsulated Olaparib and characterization thereof

1. Polymer-encapsulated olaparib

The polymer-coated olaparib is prepared by a dialysis method, 300mg of LSP, BSP, BShP and BHSP are weighed and respectively dissolved in 5mL of deionized water, 10mL of DMSO is added, and H containing the material is obtained₂O/DMSO mixed solution. 30mg Olaparib (Olaparib) was dissolved in 2mL DMSO and added dropwise to H containing material₂O/DMSO mixed solution. Stirring overnight in the dark, dialyzing the reaction solution (3.5kDa MWCO) for 2 days to remove DMSO solvent, treating with water phase filter membrane, and lyophilizing to obtain polymerization coated with OlaparibThe polymers were designated LSPO, BSPO, BShPO and BHSPO (O stands for olaparib) in this order.

2. Determination of Olaparib entrapment

The loading of LSP, BSP, BShP and BHSP for olaparib was calculated by HPLC results. Drug loading (wt%) × 100% (mass of drug loaded/total mass of polymer and drug loaded). Dissolving the materials in the mobile phase, performing ultrasonic treatment, and performing HPLC detection on the filtrate obtained after the organic phase filter head is treated. Column model number (Column name: Shim-pack GLST, C18, 5 μm. size: 250X 4.6mm I.D.), test Column temperature 30 ℃, flow rate 1.0mL/min, sample size 20 μ L, sample concentration about 100 μ g/mL, detector selected as UV light (276nm), mobile phase CH₃OH/H₂And (v: v ═ 1:1) mixed solution.

The results are shown in Table 2. The highest BSP polymer entrapment amount reaches 4.03%; the BShP loading was 2.17% worse than BSP. In contrast, the control materials LSP and BHSP were not able to effectively encapsulate small molecule drugs, and the encapsulation amount was only 0.33% and 0.13%, respectively. Indicating that the BSP is in a proper assembled stacked form to be capable of loading more small molecule drugs. The entrapped small molecule drug was collected by HPLC for detection and HRMS results indicated that olaparib was still the active drug (fig. 23).

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

Test example 3 Effect study of cell Membrane-glycosyl Polymer

Statistical Analysis of multiple comparisons was performed in this study using Analysis of variance (ANOVA for short), indicating a very significant difference in p <0.01, a significant difference in p <0.05, and no significant difference in ns.

First, fluorescence detection of tumor site aggregation living body

1. Test method

Will be 1 × 10⁶4T1 cells were inoculated into Balb/c mice(20 g, 6-8 weeks old) Back until tumor growth reaches a volume of about 50mm³The experiment was started from the left and right. Tumor model mice were randomly divided into 4 groups of 5 mice each, and Ppa concentrations of 5mg/kg of different groups of material (free Ppa, BSP prepared in example 1, BSPO prepared in example 2, and CM-BSPO prepared in example 3) were injected separately via tail vein. After completion of the injection, tumor site fluorescence signals were observed by a live fluorescence imaging system (IVIS, PerkinElmer, usa) at various time points (1 hour, 4 hours, 8 hours, 1 day, 2 days, 4 days, and 7 days), and the data were processed and analyzed by a data analysis system.

2. Test results

As shown in fig. 24, the highest fluorescence signal intensity was observed about 4 hours after injection of free Ppa into mice, and then the fluorescence signal intensity at the tumor site gradually decreased at time points, and the fluorescence signal at the tumor site was not substantially observed about 2 days later. The free Ppa molecular weight of the small molecule photosensitizer is about 548, and the small molecule photosensitizer can be rapidly discharged out of the body through renal metabolism in vivo; at the same time, due to the absence of nanostructures and size, efficient aggregation at the tumor site by passive targeting is not possible, so a rapid decrease and disappearance of the fluorescence signal is observed. After the BSP and BSPO groups are injected into mice, the fluorescence signals of tumor tissues gradually increase within the first 8 hours and then slowly decrease, but higher fluorescence signals can still be detected on the 7 th day, which indicates that the BSP and BSPO can be accumulated and effectively retained at the tumor sites of the mice. Compared with BSP and BSPO, the fluorescence signal of CM-BSPO with the surface wrapped by tumor cell membranes at the tumor site of the mouse is obviously higher in the first hours, and the duration of the high fluorescence intensity signal is longer. It was found by semi-quantifying the fluorescence signal and plotting the signal change (see fig. 25), that the signal change curve of free Ppa is relatively steep, and after 4 hours, it rapidly decreases until it disappears, while the BSP and BSPO group signals do not start to slowly decrease until about 1 day, and after 7 days, the fluorescence signal can still be detected. The CM-BSPO group showed higher signal intensity at each time point than BSP and BSPO, indicating that it was more significant at the tumor site. Compared with free Ppa, the in vivo metabolism time of the polymer drug delivery system is obviously prolonged, which not only increases the aggregation intensity of the photosensitizer and the olaparib at the tumor site, but also prolongs the window period of photodynamic therapy, and effectively improves the action time of the combined application of the tumor photodynamic therapy and the olaparib.

Second, anti-tumor therapy in vivo in mouse tumor model

1. Test method

A tumor treatment flow chart for the mouse tumor model is shown in fig. 26. Will be 1 × 10⁶4T1 cells were inoculated onto the backs of Balb/c mice (20 g, 6-8 weeks old) until tumors grew to a volume of about 50mm³The experiment was started on the left and right (about 7 days). Tumor model mice were randomly divided into 10 groups of 5 mice each:

the illumination group (+ L) was divided into 5 groups: CM-BSPO + L, BSPO + L, BSP + L, Ppa + L and Saline + L;

the non-illuminated group (-L) has 5 groups, and is divided into: CM-BSPO-L, BSPO-L, BSP-L, Ppa-L and Saline-L.

Separately, Ppa BSP, BSPO, CM-BSPO and physiological saline with the concentration of 5mg/kg were injected via tail vein. 12 hours after the injection, the illumination group is illuminated by a 660nm laser, and the illumination dose is 2J/cm²Photodynamic therapy is carried out every 2 days for 3 times, and the tumor volume is measured and monitored in the treatment process.

Firstly, the tumor treatment effect is detected by image means such as MRI, CT and the like

The size of the tumor is measured by Magnetic Resonance Imaging (MRI) while photodynamic therapy is performed. MR images of the tumor site were acquired by a 7T MRI scanner. The parameters of the MRI sequence are as follows: TR 100ms, TE 2.5ms, FOV: 40mm × 40mm, matrix: 256 × 256, scan time 128 s. After 20 days of concurrent treatment, breast scans of the mice were performed by Micro-CT apparatus to observe the metastasis of the tumor in both lungs of the mice.

HE staining and immunohistochemical staining of tumor tissue section

After the treatment (day 19 of the experiment), lung and tumor tissues were collected after euthanasia of mice, fixed in paraformaldehyde (4% in PBS pH 7.4) for 24 hours, embedded in paraffin and sectioned. The lungs were imaged after HE staining to obtain images. Tumor tissues were treated and stained for CD31 and Ki67 immunohistochemistry and photographed for image acquisition.

2. Test results

(1) Tumor volume

The tumor volume changes in each mouse are shown in fig. 27 for the different treatment groups, and the illumination group (+ L) is divided into: CM-BSPO + L, BSPO + L, BSP + L, Ppa + L and Saline + L; also, the non-illuminated group (-L) was divided into: CM-BSPO-L, BSPO-L, BSP-L, Ppa-L and Saline-L. The tumor volumes of mice in different treatment groups were counted, as shown in fig. 28, the tumor volumes of the saline group without light treatment and light treatment only and Ppa groups of mice were not significantly different (ns), and were all much larger than those of CM-BSPO, BSPO and BSP groups with light treatment, and the tumor sizes increased with time during the observation period. Groups not exposed to light, of which CM-BSPO, BSPO and BSP groups, although abundant Ppa was accumulated at the tumor site, failed to generate ROS to kill tumor cells without light, and thus had no significant antitumor effect. In each group subjected to the light treatment, the normal saline group which does not have Ppa as a photosensitizer can not generate ROS to kill tumor cells, and has no obvious anti-tumor effect. The residence time of free Ppa at the tumor site was only a few hours, and the amount of free Ppa remaining at the tumor site with light exposure 24 hours after dosing was insufficient to generate significant amounts of ROS to kill tumor cells. Compared with free Ppa, the polymer Ppa has relatively longer aggregation time in a tumor body, provides longer treatment window period and obviously improves the treatment effect of the tumor.

The experimental results showed that BSP + L, BSPO + L and CM-BSPO + L group Ppa were highly concentrated at the tumor site, and in addition to light, they generated a large amount of ROS, thereby killing tumor cells. Volume monitoring revealed significantly smaller tumor volumes in mice, and no significant increase in tumor volume during treatment. In the BSPO + L group loaded with olaparib, olaparib can enhance DNA damage of tumor cells, thereby increasing the effect of killing tumor cells, so that the tumor volume is significantly reduced compared with the BSP group, and the tumor volume is statistically different (p <0.01, n ═ 5). The CM-BSPO + L with the surface wrapped by the cell membrane has the advantages that the accumulation degree of the tumor part is increased, the cell uptake is increased, the treatment effect is obviously improved, the tumor volume is not obviously increased in the whole treatment period, and the tumor volume after treatment is obviously statistically different compared with the BSPO + L group.

(2) Tumor inhibition rate

The tumor inhibition rate of tumor treatment can be calculated by comparing the other groups with the group without adding light by normal saline, as shown in fig. 29, the tumor inhibition rate is sequentially as follows: CM-BSPO + L87.66%, BSPO + L81.34%, BSP + L69.55%, Ppa + L36.99%, Saline + L26.18%, and the tumor inhibition rate of the rest groups without illumination is only about 12%. It can be seen that the therapeutic effect of CM-BSPO plus light is most pronounced.

(3) Magnetic resonance imaging

Tumor scans were performed every other week during treatment by magnetic resonance imaging, i.e. on

days

1, 7, 14 and 19 of treatment, and the results are shown in fig. 30 and 31. The magnetic resonance image reflects the same therapeutic effect. The BSP + L, BSPO + L and CM-BSPO + L groups showed significant reduction in tumor volume. On day 19 of treatment, mice in each treatment group were subjected to pulmonary CT scan, and mice were observed for pulmonary metastasis while lung tissue was taken out for tissue imaging and HE staining.

The CT scan results in FIGS. 30 and 31 show that no distinct metastases were observed in the lungs of the CM-BSPO + L and BSPO + L group of mice, and no abnormal tissue was observed by visual observation of the specimen tissue. In other groups, one or more high-density isolated nodules can be observed in the CT lung image, and nodular protrusions can be observed in the general specimen observation; and HE staining (fig. 32) observed deeply enlarged nuclei, with heterogeneity of cell morphology with surrounding normal lung tissue.

(4) Immunohistochemical staining

The tumor tissue sections were subjected to CD31 immunohistochemical staining to observe the vascularity of the tumor sites, as shown in fig. 33, the expression levels of CD31 in CM-BSPO + L, BSPO + L and BSP + L tumors with obvious tumor treatment effects were lower than those in other groups, indicating that the tumor treatment effects were better and the number of new vessels in the tumors was less.

Ki-67 immunohistochemical staining was also performed to observe cell proliferation in the tumors, as shown in FIG. 34. The semi-quantitative statistical analysis result is shown in FIG. 35, the Ki-67 expression levels in tumors between the CM-BSPO + L, BSPO + L group and the BSP + L group are not significantly different and are all lower than those of the Ppa + L group and the Saline + L group, and the statistical differences are achieved. The results show that the intracellular proliferation of the three groups of tumors, CM-BSPO + L, BSPO + L and BSP + L, is inhibited, and the treatment effect is better than that of other control groups.

The above test results show that: the glycosyl polymer prepared by the invention, especially the cell membrane-glycosyl polymer has good photodynamic effect, can be used as a photosensitizer for photodynamic therapy, and has excellent killing effect on tumors. Meanwhile, the glycosyl polymer has good effect as a drug carrier and high drug loading, and the treatment effect is further enhanced after drug loading.

In conclusion, the invention provides a cell membrane-glycosyl polymer, which overcomes the defects of insufficient accumulation at the tumor part and weak killing effect on the tumor of the existing PDT photosensitizer, can be accumulated at the tumor part, and has good targeting effect; it has photodynamic effect, high ROS generating efficiency when used as photosensitizer for photodynamic therapy, and strong killing effect on tumor cells. Meanwhile, the cell membrane-glycosyl polymer can also be used as a drug delivery system to entrap drugs, in particular tumor drugs, such as olaparib, for tumor treatment, and has excellent effect. The cell membrane-glycosyl polymer has excellent application prospect in preparing medicaments for treating tumors.

Claims

1. A cell membrane-sugar based polymer characterized by: the glycosyl polymer with the structure shown in formula I is coated with a layer of cell membrane:

wherein the content of the first and second substances,

(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 cell membrane-glycosyl polymer according to claim 1, wherein: the cell membrane is the cell membrane of a tumor cell;

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

the MA-D-Galactosamine has the structure

The structure of the MA-PySS is

The structure of the MA-GFLGKGLFG-MA is

The structure of the MA-GFLGK-CTA is

3. The cell membrane-glycosyl polymer according to claim 2, wherein: the glycosyl polymer is prepared from the following raw materials in parts by weight: B-gal-SH 500 parts, Maleimide-Ppa 75 parts;

4. The cell membrane-glycosyl polymer according to claim 1, wherein: the preparation method of the cell membrane comprises the following steps:

and/or, the preparation method of the B-gal-SH comprises the following steps:

reacting B-gala-PySS with dithiothreitol to obtain the product.

5. The cell membrane-glycosyl polymer according to claim 2 or 3, wherein: the preparation method of the B-gala-PySS comprises the following steps:

preferably, the solvent is a mixed solution of water and methanol;

and/or, oxygen is removed prior to the reaction;

and/or the reaction is a reaction protected from light;

more preferably, the water to methanol volume ratio is 1: 4;

and/or the initiator is VA 044;

6. The cell membrane-glycosyl polymer according to claim 2 or 3, wherein: the method of the glycosyl polymer comprises the following steps:

preferably, the first and second electrodes are formed of a metal,

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

7. A method for preparing the cell membrane-sugar based polymer according to any one of claims 1 to 6, wherein: it comprises the following steps:

preferably, the mixture is stirred overnight at the temperature of 0-4 ℃;

and/or, the centrifugation is 20000g for 30 min.

8. Use of the cell membrane-sugar based polymer according to any one of claims 1 to 6 for the preparation of a photosensitizer;

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

9. Use of the cell membrane-glycosyl polymer of any one of claims 1 to 6 in the preparation of a pharmaceutical carrier;

more preferably, the anti-tumor drug is olaparib.

10. A medicament, characterized by: the preparation is prepared by taking the cell membrane-glycosyl polymer as an active ingredient according to any one of claims 1 to 6, or taking the cell membrane-glycosyl polymer as a carrier to entrap a drug 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.