CN110201165B - Photodynamic-chemotherapy combined drug delivery system and preparation method and application thereof - Google Patents
Photodynamic-chemotherapy combined drug delivery system and preparation method and application thereof Download PDFInfo
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- CN110201165B CN110201165B CN201910562571.7A CN201910562571A CN110201165B CN 110201165 B CN110201165 B CN 110201165B CN 201910562571 A CN201910562571 A CN 201910562571A CN 110201165 B CN110201165 B CN 110201165B
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Abstract
The invention provides a linear-tree drug delivery system, wherein pyropheophorbide a or adriamycin is loaded in the linear-tree drug delivery system, and the structure of the linear-tree drug delivery system is shown as a formula I or a formula II; the invention also provides a photodynamic-chemotherapy combined drug delivery system which is prepared by self-assembling the linear-tree-shaped drug delivery system shown in the formula I and the linear-tree-shaped drug delivery system shown in the formula II serving as raw materials. Experimental results show that the linear-tree drug delivery systems shown in the formula I and the formula II can be assembled into a photodynamic-chemotherapy combined drug delivery system with a more stable structure and better monodispersity after being mixed according to a proper proportion, and the photodynamic-chemotherapy combined drug delivery system has a synergistic antitumor effect under the optimal proportion (Ppa: DOX ═ 3:1), and shows an antitumor effect which is remarkably superior to that of the single drug delivery system in-vitro and in-vivo antitumor experiments. The photodynamic-chemotherapy combined drug delivery system has the tumor inhibition rate as high as 99.34 percent and has good application prospect in preparing antitumor drugs.
Description
Technical Field
The invention belongs to the field of high polymer materials, and particularly relates to a photodynamic-chemotherapeutic combined drug delivery system, and a preparation method and application thereof.
Background
Photodynamic Therapy (PDT) is a method in which tumor cells selectively absorb photosensitizers, and then non-thermal laser with a specific wavelength is used to irradiate a diseased region, so that the photosensitizers in tumor tissues undergo a severe photochemical reaction to generate Reactive Oxygen Species (ROS) with cytotoxicity, thereby selectively killing the tumor cells. The mechanisms of the antitumor effect of PDT are mainly divided into three categories: 1) generating singlet oxygen to directly kill tumor cells and induce apoptosis or necrosis; 2) destroy tumor nourishing blood vessels, resulting in tumor ischemia and hypoxia; 3) increase the release of inflammation and immune mediators, even initiate specific immune mechanisms to recognize and destroy distant metastatic lesions. At present, a plurality of photosensitizers are on the market at home and abroad, and the photodynamic therapy achieves better curative effect in the aspect of tumor treatment by the advantages of micro-wound, small side effect and the like, but the conventional photosensitizer which is clinically used at present has the problems of poor water solubility, low cell uptake, slow metabolism, lack of targeting property, delayed phototoxicity and the like. For example, the photosensitizer pyropheophorbide a (Ppa) has excellent photodynamic properties. However, the water solubility of pyropheophorbide a is poor, which limits its wide application.
In order to improve the targeting and enrichment concentration of the photosensitizer to tumor tissues, a drug delivery system carrier is generally adopted to deliver the photosensitizer. However, there are differences in the interaction between the carrier material and the photosensitizer which differ in chemical structure, and this difference can further affect the biological effects of the delivery system, and thus the in vivo circulation, clearance profile and antitumor activity of the delivery system. Therefore, it is very important to find a suitable carrier material.
Chinese patent CN105749280A discloses a tumor targeting nano drug delivery system for synergistic chemotherapy and photodynamic therapy. The drug delivery system is composed of carboxymethyl chitosan, folic acid, photosensitizer chlorin e6 and adriamycin, firstly, the chlorin e6 and folic acid are coupled to a carboxymethyl chitosan chain segment through amido bonds, and then polymer nanoparticles loaded with the adriamycin are obtained through an ion crosslinking method, so that a targeted anti-tumor nano drug delivery system is formed. However, the preparation process of the drug delivery system is complex, and the coupling of the targeting molecular folic acid increases the preparation process; and its tumor-suppressing effect is far from satisfying the demand.
In recent years, researches show that the combination of chemotherapy and photodynamic therapy is expected to achieve more remarkable tumor treatment effect, but due to the complexity of pharmacological action and mechanism of the medicine, the combination of the two medicines can cause antagonism and weaken the treatment effect. Therefore, the photodynamic therapy and chemotherapy combined drug delivery system which has a simple structure, is convenient to prepare and has a remarkable treatment effect on tumors has a wide application prospect.
Disclosure of Invention
The invention aims to provide a combined drug delivery system for photodynamic therapy and chemotherapy, which has the advantages of simple structure, convenient preparation and obvious treatment effect on tumors.
The invention provides a linear-tree drug delivery system, wherein pyropheophorbide a is loaded in the linear-tree drug delivery system, and the structure of the linear-tree drug delivery system is shown as a formula I:
wherein R is1～R16Each independently selected from H or
And is not H at the same time;
n is the degree of polymerization, and n is 444.
Further, in the linear-tree drug delivery system, the drug loading of pyropheophorbide a is 6.00 to 7.00%, preferably 6.19%.
The invention also provides another linear-tree drug delivery system, doxorubicin is loaded in the linear-tree drug delivery system, and the structure of the linear-tree drug delivery system is shown in a formula II:
wherein R is1’～R16' each is independently selected from H or
And is not H at the same time;
n is polymerization degree, and n is 444.
Further, in the linear-tree drug delivery system, the drug loading of the adriamycin is 7.00-8.00%, and is preferably 7.90%.
The drug loading represents (mass of drug/total mass of linear-dendritic drug delivery system) × 100% in the system tested. In the linear-dendritic drug delivery system of the present invention, the drug loading amount of pyropheophorbide a is (mass of pyropheophorbide a/total mass of linear-dendritic drug delivery system) × 100%; doxorubicin load (mass of doxorubicin/total mass of linear-dendrimer) x 100%.
The invention also provides a photodynamic-chemotherapy combined drug delivery system which is prepared by taking the two linear-tree drug delivery systems as raw materials.
Further, in the two linear-dendritic drug delivery systems, the molar ratio of pyropheophorbide a to adriamycin is
Preferably 3:1,1:1 or 1:3, more preferably 3: 1.
the invention also provides a method for preparing the linear-tree drug delivery system, which comprises the following steps:
(1) 3- (tritylthio) propionic acid and Hyperbranched G3-PEG20k-OH are used as raw materials to react to obtain Hyperbranched G3-PEG20 k-STRT;
(2) the Hyperbranched G3-PEG20k-STRT is reacted with Maleimide-Ppa or Maleimide-DOX;
(3) dialyzing the system reacted in the step (2) in ultrapure water, purifying and drying to obtain the compound;
wherein the structure of the Hyperbranched G3-PEG20k-STRt is
The structure of Maleimide-Ppa is
The structure of Maleimide-DOX is
Further, in the step (1), the molar ratio of the 3- (tritylthio) propionic acid to the Hyperbranched G3-PEG20k-OH is (15-25): 1; the reaction is carried out under the action of DCC and DMAP; the reaction solvent is an organic solvent; the reaction condition is that the reaction is carried out at room temperature in a dark place under the nitrogen atmosphere; the reaction time is 24-72 hours;
in the step (2), the molar ratio of the Hyperbranched G3-PEG20k-STRt to the Maleimide-Ppa is 1: (5-15), or the molar ratio of the Hyperbranched G3-PEG20k-STRt to the Maleimide-DOX is 1: (5-15); the reaction solvent is an organic solvent; the reaction condition is that the reaction is carried out at room temperature in a dark place under the nitrogen atmosphere; the reaction time is 24-72 hours;
in the step (3), the dialysis time is 2 days, and the cut-off molecular weight of a dialysis bag used for dialysis is 8000; the purification mode is purification through a size exclusion chromatographic column;
preferably, in the step (1), the molar ratio of the 3- (tritylthio) propionic acid to the Hyperbranched G3-PEG20k-OH is 19.2: 1; the molar ratio of the Hyperbranched G3-PEG20k-OH to the DCC and DMAP is 1: (30-35): (1-2); the reaction solvent is N, N-dimethyl amide; the reaction time is 48 hours;
in the step (2), the molar ratio of the Hyperbranched G3-PEG20k-STRt to the Maleimide-Ppa is 1: 8, or the molar ratio of the Hyperbranched G3-PEG20k-STRt to the Maleimide-DOX is 1: 8; the reaction solvent is N, N-dimethyl amide; the reaction time was 48 hours.
The invention also provides a method for preparing the photodynamic-chemotherapy combined drug delivery system, which comprises the following steps: adding the two linear-tree drug delivery systems into water, and carrying out self-assembly to obtain the spherical nano-particles.
The "spherical shape" of the present invention includes regular and irregular spherical shapes; "nanoparticle" refers to solid or hollow particles on the nanometer scale.
The invention also provides application of the two linear-tree drug delivery systems or the photodynamic-chemotherapy combined drug delivery system in preparation of antitumor drugs, preferably, the tumors are selected from breast cancer, bladder cancer, lung cancer, esophageal cancer, head and neck cancer and skin cancer.
The experimental result shows that the invention designs and constructs two amphiphilic block copolymers: after being mixed according to a proper proportion, the amphiphilic DLD type linear-tree structure photodynamic drug delivery system and the amphiphilic DLD type linear-tree structure chemotherapy drug delivery system which are acid-sensitive in a tumor microenvironment can be assembled into a photodynamic-chemotherapy combined drug delivery system which is more stable in structure and better in monodispersity, and the photodynamic-chemotherapy combined drug delivery system has a synergistic antitumor effect under the optimal proportion (Ppa: DOX is 3:1), and shows an antitumor effect which is obviously superior to that of the single drug delivery system in vitro and in vivo antitumor experiments. The photodynamic-chemotherapy combined drug delivery system has the tumor inhibition rate as high as 99.34 percent and has good application prospect in preparing antitumor drugs.
In the present invention, the DLD type, i.e., dendritic-linear-dendritic, represents a linear-tree structure.
The ratio of G320SP/G320SD is x/y, which means Ppa in G320SP and G320 SD: and the DOX molar ratio is x: y. For example, the ratio of G320SP/G320SD is 3/1, which means Ppa in G320SP and G320 SD: the DOX molar ratio was 3: 1.
The molecular self-assembly is a phenomenon that molecules spontaneously form a molecular aggregate with a specific sequence, stable structure and certain characteristics by utilizing intermolecular or intramolecular non-covalent interaction. Non-covalent interaction force is a key factor of self-assembly, and common non-covalent interaction force is hydrogen bond, electrostatic interaction, pi-pi stacking, van der waals force, hydrophobic effect and the like.
The amphiphilic block copolymer refers to that two or more chain segments with different structures and different hydrophilicity and hydrophobicity exist in a single linear molecule, and the special structure of the hydrophilicity and the hydrophobicity enables the amphiphilic block copolymer to form aggregates with specific shapes and functions, such as micelles, vesicles, nano microspheres and the like, in a selective solvent through self-assembly, and the aggregates have various special shapes, such as spheres, cylinders, rods and the like. The special aggregation form of the amphiphilic block copolymer in the selective solvent endows the self-assembly materials with specific physicochemical properties and biological effects, and the amphiphilic block copolymer is widely concerned by researchers at home and abroad as a novel material and is expected to be widely applied in the fields of biomedicine, nanotechnology and the like.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the synthetic routes of G320SP and G320 SD.
Fig. 2 is a schematic diagram of the self-assembly of the linear-tree photodynamic drug delivery system G320SP (a), the tumor microenvironment acid sensitive linear-tree drug delivery system G320SD (b), and the mixture thereof (c).
FIG. 3 is a nuclear magnetic hydrogen spectrum characterization of Hyperbranched G3-PEG20 k-STRt.
FIG. 4 is a nuclear magnetic hydrogen spectrum characterization of G320 SP.
FIG. 5 is a nuclear magnetic hydrogen spectrum characterization of G320 SD.
FIG. 6 is a standard UV absorption curve of pyropheophorbide a in DMSO.
FIG. 7 is a standard UV absorption curve of doxorubicin in DMSO.
FIG. 8 shows two drug delivery systems in DMSO-d6And D2Nuclear magnetic hydrogen spectrum characterization in O: g320SP (a), G320SD (B).
Fig. 9 is a representation of particle size and morphology by TEM: drug delivery systems G320SP/G320SD drug ratio 1/0(a), G320SP/G320SD drug ratio 3/1(B), G320SP/G320SD drug ratio 1/1(C), G320SP/G320SD drug ratio 1/3(D) and G320SP/G320SD drug ratio 0/1 (E).
Fig. 10 is a characterization of particle size by DLS: the drug delivery system has the drug ratio of G320SP/G320SD being 1/0(A), the drug ratio of G320SP/G320SD being 3/1(B), the drug ratio of G320SP/G320SD being 1/1(C), the drug ratio of G320SP/G320SD being 1/3(D) and the drug ratio of G320SP/G320SD being 0/1(E) in the particle size of pure water.
FIG. 11 is a graph of the Critical Micelle Concentration (CMC) of the drug delivery systems G320SP and G320SD in water determined by the addition of pyrene. Calculation of the linear fit curve and CMC values was done by SigmaPlot 12.5 software.
Fig. 12 characterizes the drug delivery system by DLS G320SP/G320SD drug ratios 1/0(a),
3/1(B) for the drug proportion of G320SP/G320SD, 1/1(C) for the drug proportion of G320SP/G320SD, 1/3(D) for the drug proportion of G320SP/G320SD, and 0/1(E) for the drug proportion of G320SP/G320 SD.
Fig. 13 shows uv-vis absorption spectra of drug delivery systems G320SD (a), G310SP (B), DOX (c), ppa (d), Ppa mixed with DOX (E), and G320SD/G310SP in a ratio 3/1(F) after different treatments.
Fig. 14 shows fluorescence emission spectra of drug delivery systems G320SD (a), G310SP (B), DOX (c), ppa (d), Ppa mixed with DOX (E) and G320SD/G310SP in a ratio 3/1(F) after different treatments.
Fig. 15 shows drug release (n-3) at two pH conditions for drug delivery systems G320SD and G320SP + G320SD (mixed drug delivery systems consisting of G320SP and G320SD at a ratio of 3:1 of photosensitizer Ppa to DOX) at 37 ℃.
Figure 16 is an in vitro cytotoxicity study (n-3). The toxicity of drug delivery systems G320SP/G320 SD-1/0, G320SP/G320 SD-3/1, G320SP/G320 SD-1/1, G320SP/G320 SD-1/3 and G320SP/G320 SD-0/1 on 4T1 cells was calculated as the concentration of Ppa (a) and the concentration of DOX (B), respectively.
Fig. 17 is a graph comparing the in vivo anti-tumor effect of the combination of the drug delivery systems G320SP, G320SD, free DOX, free photosensitizer Ppa and photodynamic-chemotherapy with the 4T1 in vivo tumor model, with saline as the control (n-5) (a); after the treatment is over, the tumor is weighed (B); calculating the tumor inhibition rate TGI (C) according to the tumor weight; all tumors were photographed (D).
Detailed Description
The invention has the advantages that the raw materials and equipment are known products and are obtained by purchasing commercial products.
Wherein pyropheophorbide a (Ppa) is purchased from Shanghai Xiaihui pharmaceutical science and technology Limited; hyperbranched G3-PEG30k-OH was purchased from Sigma-Aldrich China.
The SPF mice used in the invention are purchased from laboratory animals of Duoduo biotechnology limited company and are raised in the Central laboratory animal house of the pharmaceutical laboratory animals in Waxi clinical medicine of Sichuan university.
Example 1 preparation of a DLD-type delivery System of the invention
According to the synthetic route shown in fig. 1, the DLD type drug delivery systems G320SP and G320SD according to the present invention were prepared.
(1) Synthesis of Hyperbranched G3-PEG20k-STRT
Hyperbranched G3-PEG20k-OH (800.0mg, 37.0. mu. mol), 3- (Tritylthio) propionic acid (3- (Tritylthio) propionic acid, 247.2mg, 710.4. mu. mol), DCC (dicyclohexylcarbodiimide, 63.0mg, 1276.5. mu. mol) and DMAP (4-dimethylaminopyridine,. 6mg, 71.0. mu. mol) were reacted under nitrogenl) was dissolved in 20mL of ultra dry N, N-dimethylformamide and reacted at room temperature for 48h in the dark. After the reaction was completed, the reaction solution was dialyzed in ultrapure water for 2 days using a dialysis bag having a molecular weight cut-off of 8000, and passed through a size exclusion column (Superose 12HR/16/30column on an)
FPLC system (GEHealthcare) column). Finally, the white solid Hyperbranched G3-PEG20k-STRT with a mass of 621.7mg and a yield of 64.3% was obtained by dialysis again in ultrapure water and freeze-drying. As shown in FIG. 3, compared to the hydrogen spectrum of Hyperbranched G3-PEG20k-OH, we have found that the hydrogen spectrum of Hyperbranched G3-PEG20k-STRT1Characteristic peaks were found in H NMR (400MHz in DMSO)1H NMR (400MHz, DMSO). delta.7.36-7.19 (m,15H),2.29(dd, J ═ 9.5,4.8Hz,4H), shows that the molecular structure is correct, and we successfully synthesized the target molecule Hyperbranched G3-PEG20 k-STRt.
(2) Synthesis of G320SP
The Hyperbranched G3-PEG20k-STRt (300.0mg, 13.0. mu. mol) and Maleimide-Ppa (68.3mg, 104.0. mu. mol) were dissolved in 15mL of ultra-dry N, N-dimethylformamide under nitrogen protection and reacted at room temperature for 48h in the dark. After the reaction was completed, the reaction solution was dialyzed in ultrapure water for 2 days using a dialysis bag having a molecular weight cut-off of 8000, and passed through a size exclusion column (Superose 12HR/16/30column on an)
FPLC system (GEHealthcare) column). Finally, dialysis was performed again in ultrapure water and freeze-dried to obtain G320SP as a black solid with a mass of 277.4mg and a yield of 75.3%. As shown in FIG. 4, compared with the hydrogen spectrum of Hyperbranched G3-PEG20k-STRt, we have at G320SP1The characteristic peaks of Ppa between 10.0 and 4.0, δ 9.75(s,1H),9.47(s,1H),8.91(s,1H),8.24(dd, J ═ 17.8,11.6Hz,1H),6.93(s,1H),6.53(s,1H),6.40(d, J ═ 17.8Hz,1H),6.22(dd, J ═ 11.6,1.3Hz,1H),5.18(m,2H),4.56(t, J ═ 5.5Hz,6H), and the characteristic peaks of perbrand G3-20 k-STrt were found to completely disappear in H NMR (400MHz, DMSO), indicating that we have completely removed the thiol groups and protected the PEG, which indicates that we have completely removed the thiol groupsAnd protecting groups and coupling Ppa through covalent bonds, thereby successfully synthesizing the G320SP target molecule.
(3) Synthesis of G320SD
Hyperbranched G3-PEG20k-STRt (200.0mg, 8.7. mu. mol) and Maleimide-DOX (49.3mg, 69.6. mu. mol) were dissolved in 15mL of ultra-dry N, N-dimethylformamide under nitrogen protection and reacted at room temperature for 48h with exclusion of light. After the reaction was completed, the reaction solution was dialyzed in ultrapure water for 2 days using a dialysis bag having a molecular weight cut-off of 8000, and passed through a size exclusion column (Superose 12HR/16/30column on an)
FPLC system (GEHealthcare) column). Finally, dialysis was performed again in ultrapure water and freeze-dried to obtain G320SD as a black solid with a mass of 166.2mg and a yield of 66.4%. As shown in FIG. 5, compared with the hydrogen spectrum of Hyperbranched G3-PEG20k-STRt, we have at G320SD1The characteristic peaks delta 7.93(s,2H),7.67(s,1H) of DOX between 7.5 and 8.0 were found in H NMR (400MHz, DMSO), while the characteristic peaks of Hyperbranched G3-PEG20k-STRt were completely disappeared, which indicates that we have completely removed the thiol protecting group and coupled DOX by covalent bond, and successfully synthesized the G320SD target molecule.
The compounds G320SP and G320SD prepared above were each mixed with pure water to prepare a solution of 200. mu.g/mL, and self-assembled to give two self-assembled systems (schematic diagrams are shown in FIGS. 2(a) and (b)).
Example 2 preparation of a combined photodynamic-chemotherapeutic drug delivery system according to the invention
The compounds G320SP and G320SD obtained in example 1 were mixed at different ratios (Ppa: DOX ═ 1:0,3:1,1:1,1:3,0:1), and added to purified water to prepare a solution of 200 μ G/mL, followed by self-assembly to obtain different self-assembled systems. Wherein, when Ppa: fig. 2(c) shows a schematic diagram of self-assembly when DOX is 3: 1.
The beneficial effects of the present invention are demonstrated by the following experimental examples.
The experimental method adopted by the invention is as follows:
1. magnetic resonance spectroscopy
Weighing about 5mg of sample, fully dissolving the sample by using 0.5mL of heavy water or deuterated dimethyl sulfoxide as a solvent, and testing on a high-resolution superconducting nuclear magnetic resonance spectrometer.
2. Drug load testing
As shown in Table 1, a total of 8 concentrations of the dimethylsulfoxide solution of free photosensitizer Ppa was prepared at a predetermined concentration gradient (10,5,2.5,1,0.5,0.25,0.1, 0. mu.g/mL), and the absorbance thereof was measured at 667nm by UV-visible spectroscopy, and a standard curve was plotted. Then, the absorbance of the drug delivery system G320SP (100. mu.g/mL) was measured at a certain concentration, and the concentration of Ppa in the material was calculated by substituting it into a standard curve, and finally the drug loading was calculated (FIG. 6).
TABLE 1 Absorbance of free photosensitizer Ppa at various concentrations
As shown in Table 2, solutions of DOX in dimethylsulfoxide were prepared in a predetermined concentration gradient (100,50,25,12.5,6.25,3.125, 0. mu.g/mL) to give a total of 7 concentrations, and the absorbance was measured at 667nm using an ultraviolet-visible spectrometer to draw a standard curve. Then, the absorbance of the drug delivery system G320SD (100. mu.g/mL) was measured at a certain concentration, and the concentration of DOX in the material was calculated by substituting it into a standard curve, and finally the drug loading amount was calculated (FIG. 7).
TABLE 2 Absorbance of free DOX at different concentrations
3. Particle size distribution, surface charge and morphology
About 3mg of the drug delivery systems G320SP and G320SD were weighed out, dissolved in ultrapure water, and prepared into solutions of (Ppa: DOX ═ 1:0,3:1,1:1,1:3,0:1) G320SP and G320SD at different drug ratios, with a concentration of 200. mu.g/mL. At normal temperature, a Zetasizer Nano ZS Nano-particle size-potential detector is used for detecting the particle size distribution and surface charge of Nano-particles in aqueous solutions of G320SP and G320SD of various proportional drug delivery systems; the aqueous solutions of the respective dosing systems G320SP and G320SD were dropped onto a carbon film of a copper mesh and, after air-drying, the morphology and size of the respective samples were observed with a transmission electron microscope.
4. Ultraviolet-visible and fluorescent spectra
Free photosensitizer Ppa and free DOX dimethyl sulfoxide solutions with the concentration of 1mg/mL and dimethyl sulfoxide solutions with the drug delivery systems G320SP, G320SD and G320SP/G320SD with the free photosensitizer Ppa concentration of 1mg/mL with the drug ratio of 3/1 are prepared respectively. Then, the five solutions were diluted 20 times with different solutions (DMSO, PBS, DMSO + 0.1% SDS, PBS + 0.1% SDS), and the UV-visible spectrum (wavelength range: 300-800 nm) and the fluorescence emission spectrum (EX: 405nm or 485nm, Em: 500-800 nm) were measured at room temperature.
To evaluate the self-quenching and de-quenching effects of drug delivery systems G320SP, G320SD, and G320SP/G320SD at 3/1, we calculated the fluorescence quenching efficiency of the samples based on the fluorescence intensity measured above in aqueous solution or organic solvent with or without surfactant. The peak intensity was recorded for each set of samples and the percent quenching was determined by the following formula:
wherein F0 is the fluorescence intensity when the sample is quenched in an aqueous solution, and F1 is the fluorescence intensity when the sample is not quenched in a dispersed state in an organic solvent.
5. Critical micelle concentration
Critical Micelle Concentration (CMC) of the drug delivery systems G320SP and G320SD in ultrapure water was measured using pyrene fluorescence.
(1) Determination of critical micelle concentration of delivery system G320SP
First, stock solutions of delivery system G320SP were prepared at a concentration of 1mg/mL and diluted to different concentrations. Secondly, to1mg of pyrene is weighed, dissolved by 5mL of acetone and transferred to a 50mL volumetric flask for constant volume to obtain the product with the concentration of 1.0 multiplied by 10-4And (3) a pyrene acetone solution of mol/L. Then, 5. mu.L of pyrene/acetone solution was pipetted into a series of 5mL volumetric flasks and N was added2Drying acetone, and respectively transferring the solutions of the drug delivery system G320SP with different concentrations into volumetric flasks to obtain a series of pyrene probe-containing micelle solutions with concentrations of 100,60,30,10,6,3,1,0.6,0.3,0.1,0.06 and 0.03 mu G/mL. And finally, placing the micelle solution in a constant temperature oscillator at 37 ℃ for oscillation and dispersion for 2h, and measuring the fluorescence excitation spectrum (Em:668nm, Ex: 310.0-350.0 nm) of pyrene.
(2) Determination of critical micelle concentration of delivery system G320SD
First, stock solutions of delivery system G320SD were prepared at a concentration of 1mg/mL and diluted to different concentrations. Secondly, accurately weighing 1mg of pyrene, dissolving the pyrene with 5mL of acetone, transferring the solution to a 50mL volumetric flask for constant volume to obtain the pyrene with the concentration of 1.0 multiplied by 10-4And (3) a pyrene acetone solution of mol/L. Then, 5. mu.L of pyrene/acetone solution was pipetted into a series of 5mL volumetric flasks and N was added2Drying acetone, and respectively transferring the solutions of the drug delivery system G320SD with different concentrations into volumetric flasks to obtain a series of pyrene probe-containing micelle solutions with concentrations of 100,60,30,10,6,3,1,0.6,0.3,0.1,0.06 and 0.03 mu G/mL. And finally, placing the micelle solution in a constant temperature oscillator at 37 ℃ for oscillation and dispersion for 2h, and measuring the fluorescence emission spectrum (Ex:330nm, Em: 350.0-500.0 nm) of pyrene.
6. Colloidal stability
Colloidal stability was assessed by monitoring the dimensional changes of the drug delivery systems G320SP and G320SD at various drug ratios (Ppa: DOX ═ 1:0,3:1,1:1,1:3,0:1) under simulated physiological conditions. Drug delivery systems G320SP and G320SD at different drug ratios (Ppa: DOX ═ 1:0,3:1,1:1,1:3,0:1) were added to ultrapure water containing 10% FBS and PBS containing 10% FBS, respectively, at a concentration of 200 μ G/mL. Changes in particle size distribution, surface charge, and degree of dispersion of the drug delivery systems G320SP and G320SD in ultrapure water containing 10% FBS and PBS containing 10% FBS were examined at different time points for different drug ratios (Ppa: DOX ═ 1:0,3:1,1:1,1:3,0:1) using a Zetasizer Nano ZS Nano particle size-potential detector at room temperature.
7. In vitro Release assay
Drug delivery system G320SD and mixed drug delivery system G320SP + G320SD (mixed drug delivery system consisting of G320SP and G320SD at a ratio of 3:1 of photosensitizer Ppa to DOX) were dissolved in 2mL of phosphate buffer solutions of different pH values (pH 5.2, pH 5.2/10mM DTT, pH 7.4/10mM DTT), and the solutions were placed in dialysis bags with a molecular weight cut-off of 2000 Da. The dialysis bag was clamped with a dialysis clamp, immersed in 30mL of the above phosphate buffer solution, placed in a horizontal constant temperature oscillator at 37 ℃ for dynamic dialysis (frequency 170r/min), and the time was started. 500 mul of dialysate is taken at intervals, and after every 500 mul of dialysate is taken, corresponding equal volume of buffer solution is needed to be added. Preparing doxorubicin hydrochloride (DOX) standard solution, measuring the light absorption value by using a microplate reader, wherein the excitation light wavelength is 480nm, the emission light wavelength is 550nm, averaging (n is 3), and drawing a standard curve. And measuring the content of the dialysate DOX by using an enzyme-labeling instrument, calculating the accumulated release amount, and drawing an in-vitro release curve.
8. Cell lines and cytotoxicity assays
The cell line adopted in the research work of this chapter is 4T1 mouse breast cancer cell (purchased from cell bank of China academy of sciences type culture Collection), and the maintenance culture conditions are all improved 1640 medium (Hyclone) and are at 37 ℃ and 5% CO2Culturing in the environment. Before culturing the cells, 10% fetal bovine serum (FBS, Excell, north america) and 1% diabody (penicillin and streptomycin) were added to the medium. To examine the cytotoxicity of the combined photodynamic-chemotherapy drug delivery system, a mixture of the drug delivery systems G320SD and G310SP with the same concentration gradient and different drug ratios was added to 4T1 cells in the logarithmic phase of growth. 3 duplicate wells were set for each concentration, and a control group without drug treatment and culture wells containing 100. mu.L of medium were set as blank control groups. For the experimental group needing illumination, the experimental group needs to be incubated for 24 hours in a dark place in advance, and then the experimental group needs to be irradiated by 660nm laser according to the same unit area power. Subsequently, the wells were further incubated for 24h in the absence of light, washed 2 times with 10mmol/L, pH in 7.4 PBS, and incubated for 2h with 100. mu.L of medium containing 10% CCK-8 reagent, followed by microplate reader (II)BioTek, USA) determined the OD value at 450 nm. And finally, obtaining the optical density OD values of different experimental holes according to the same method. Cell viability (cell viability) was calculated using the following formula:
Cell viability(％)＝(ODt-OD0)/(ODc-OD0)×100％
in the formula, ODt: experimental group optical density; OD0: blank set optical density; ODc: control group optical density.
Calculating the half Inhibitory Concentration (IC) of each group based on the cell viability obtained from each experimental group50Value according to IC50The value of the combination index CI (combination index) is calculated according to the following formula:
(D)1: the drug concentration at which 50% inhibition is calculated as drug 1 in the combination;
(D)2: the drug concentration at which 50% inhibition is calculated as drug 2 in the combination;
(D50)1: drug 1 alone inhibited the required drug concentration by 50%;
(D50)2: drug 2 alone inhibited the required drug concentration by 50%.
CI <1 indicates synergy, CI ═ 1 indicates only additive effect, and CI >1 indicates antagonism.
9. Evaluation of antitumor Effect in vivo
In the experiment, a 4T1 tumor model is taken as a research object, and a female BALB/c mouse with the age of 6-8 weeks is taken as an inoculation object to establish a mouse breast cancer subcutaneous transplantation tumor model. The cultured 4T1 cells were resuspended in PBS buffer at 5X 105The inoculum/was injected subcutaneously at the intersection of the hind limb and back of the mice. When the tumor volume reaches 50mm3At the time, the model mice were randomly divided into 7 groups of 5 mice each, namely, saline control, free photosensitizer Ppa treatment group, free DOX treatment group, free photosensitizer Ppa and free photosensitizer PpaA DOX combination treatment group, a drug delivery system G320SD treatment group, a drug delivery system G320SP treatment group and a photodynamic-chemotherapy combination drug delivery system G320SP/G320SD treatment group. The treatment scheme is as follows: the same dose of photosensitizer Ppa and chemotherapeutic agent DOX was injected into the tail vein of each group of mice every four days from day 1 of treatment initiation. 150J cm on day 2 after administration2The tumor site of the mouse was irradiated with 660nm laser at a dose of 0.26W/cm28min), once every 4 days for a total of 3 treatments. On day 17 after the first dose, mice were euthanized and dissected, and their major organs (tumor, heart, liver, spleen, lung, kidney) were collected, fixed thoroughly with 10% neutral formaldehyde buffer, and immunohistochemical analysis was performed. Meanwhile, taking a picture of the stripped Tumor, recording and weighing the Tumor, and calculating the Tumor inhibition rate (TGI) according to the following formula:
in the formula (I), the compound is shown in the specification,
the average tumor weight of the control group was,
mean tumor weights of experimental groups.
10. Statistical analysis
All experimental data were statistically processed using SigmaPlot 12.5 software, GraphPad Prism 5 software, and Microsoft Excel 2010 software. All results are expressed as means + -SEM and Student's t test was used to analyze the differences among the groups of data, with P < 0.05 being statistically significant.
Experimental example 1 drug Loading test of DLD-type drug delivery System of the present invention
The detection result of an ultraviolet-visible spectrophotometer shows that the drug loading rate of the drug delivery system G320SP to Ppa is higher than 6.19%, and the drug loading rate of the drug delivery system G320SD to DOX is 7.90%, so that the requirements of photodynamic therapy and chemotherapy can be met.
Experimental example 2 characterization of self-Assembly of DLD-type drug delivery systems of the invention
1. Structural characterization of self-assembled systems
To verify the self-assembly behavior of the delivery systems G320SP and G320SD in aqueous solution, respectively, we used D2O or DMSO-d6The solvent fully dissolves both drug delivery systems and tests are performed on a high resolution superconducting nmr spectrometer. As shown in FIG. 8A, delivery system G320SP is shown at D2In the hydrogen spectrum with O as a solvent, the characteristic peak of the photosensitizer Ppa completely disappeared. As shown in FIG. 8B, delivery system G320SD is shown at D2In the hydrogen spectrum of O as a solvent, the characteristic peaks of DOX completely disappear.
This indicates that the drug delivery systems G320SP and G320SD can form stable nanostructures by self-assembly in aqueous solution, encapsulating the photosensitizer Ppa and DOX in the inner hydrophobic region, and thus the characteristic peaks of both drugs are completely suppressed.
2. Topography characterization of self-assembled systems
We used Transmission Electron Microscopy (TEM) electron microscopy to characterize particle size and morphology of the unmixed drug delivery system as well as the drug delivery system mixed in different proportions. As shown in fig. 9, the drug loading of the drug delivery systems G320SP and G320SD is close, but the hydrophobicity of DOX is lower than that of photosensitizer Ppa, so the structure formed by the assembly of the drug delivery system G320SD is not as regular as that of G320SP, and a regular spherical structure is not easy to form. However, the administration system G320SP also has a problem that the particle size distribution is not sufficiently concentrated. The morphology of the drug delivery systems G320SP and G320SD after mixing is obviously better than that of the single drug delivery system, and the larger the particle size of the mixed assembly is, the more spherical the morphology is, with the increase of the components of the drug delivery system G320 SP.
3. Particle size characterization of self-assembled systems
Further characterization of the particle size of the aqueous phase by a dynamic light scattering particle sizer (DLS) showed similar results as described above (as shown in figure 10). The single drug delivery system G320SP has a larger particle size and a wider distribution, while the single drug delivery system G320SD has a smaller particle size. After mixing, the mixed particle size gradually increases with the increase of the components of the drug delivery system G320SP, and the particle size distribution of the drug delivery systems G320SP and G320SD is most concentrated and the dispersity is best according to the ratio of the photosensitizer Ppa to the DOX of 3:1, which is consistent with the result of TEM characterization.
4. Critical Micelle Concentration (CMC) characterization of self-assembled systems
We also examined further the assembly behavior by determining the critical micelle concentration. As shown in FIG. 11, the CMC value of the drug delivery system G320SP was 7.23. mu.g/mL, and the CMC value of the drug delivery system G320SD was 46.1. mu.g/mL, both of which were much lower than the concentrations required in practical applications, as determined by fluorometry of pyrene probes and calculation of a linear fit curve equation, indicating that both drug delivery systems are susceptible to aggregation by self-assembly in an aqueous environment.
The above results indicate that Ppa can be assembled into a delivery system with a more stable structure and a more focused particle size distribution, possibly due to interaction between the two upon mixing, and that the delivery system has the best particle size distribution and monodispersity when the ratio of photosensitizer Ppa to DOX is 3: 1.
5. Critical Micelle Concentration (CMC) characterization of self-assembled systems
To further study the stability of unmixed single drug delivery systems and mixed drug delivery systems in different proportions in a body fluid environment, we simulated the body fluid environment in vivo by adding 10% Fetal Bovine Serum (FBS) in pure water and dissolved the different drug delivery systems in the above solution to perform the assay of the particle size of the aqueous phase. The results are shown in figure 12, where both single delivery systems G320SP and G320SD are susceptible to FBS and disrupt the original particle size distribution, but after mixing, the mixed delivery system is relatively less susceptible to FBS, and the best stability of the mixed delivery system is achieved when the delivery systems G320SP and G320SD are at a ratio of 3:1 of photosensitizer Ppa to DOX.
Combining the above experimental results, the drug delivery systems G320SP and G320SD can be combined to form a mixed drug delivery system with more stable structure and more concentrated particle size distribution, and when the ratio of the photosensitizer Ppa to DOX is 3:1, nanoparticles with the most uniform size, the most monodispersity and the highest stability can be formed in an aqueous environment.
Experimental example 3 optical Properties of photodynamic-chemotherapeutic combination drug delivery System of the present invention
In the above experiment, we have confirmed that the dosage systems G320SP and G320SD have the optimal size and morphology according to the composition of the mixture when the ratio of photosensitizer Ppa to DOX is 3:1, and therefore we focused on the uv-vis absorption spectrum and fluorescence emission spectrum of the mixed dosage system obtained by mixing the above ratios in different solvent environments, and investigated whether the optical properties of photosensitizer Ppa and DOX are affected by the mixture of G320SP and G320 SD.
As a result, as shown in fig. 13, the free photosensitizer Ppa, the free DOX, the drug delivery systems G320SP and G320SD, and the mixture thereof in the organic solvent were in a completely dispersed state, and their characteristic absorption wavelengths were kept consistent. Whereas in the aqueous environment of PBS, the absorption intensity of the drug delivery system, free photosensitizer Ppa, and free DOX were all significantly lower than in the organic phase. However, after the addition of the surfactant Sodium Dodecyl Sulfate (SDS), the aggregation structures of the drug delivery systems G320SP and G320SD and the mixture of the two were destroyed, and the absorption intensity of the characteristic absorption peak was enhanced. Free photosensitizer Ppa and free DOX were solubilized by SDS resulting in an increase in the absorption intensity.
Fluorescence emission spectroscopy results (shown in FIG. 14) show that both 405nm and 480nm can excite DOX, the emission intensity of 480nm excitation is higher than that of 405nm excitation, but free photosensitizer Ppa can be efficiently excited to generate emission only at 405 nm. Thus, DOX and free photosensitizer Ppa can be excited simultaneously at 405nm, which facilitates the study of the mixture of G320SP and G320 SD. Meanwhile, the drug delivery systems G320SP and G320SD and the mixture of the two can still generate fluorescence quenching effects with different degrees in different solution environments. Since both are completely dispersed in organic solvent, there is no effect on the fluorescence emission spectra of covalently coupled photosensitizer Ppa and DOX, but in the aqueous environment of PBS, fluorescence quenching occurs, except that the quenching rate of drug delivery system G320SP is highest, the quenching rate of G320SD is lowest, the quenching rate of mixing is between the two, and the quenching rate of mixing upon excitation at 405nm is also significantly higher than 480nm, probably because the stability of drug delivery system G320SP is significantly higher than that of G320SD (table 3). After addition of SDS, all fluorescence was gradually recovered, and both Ppa and DOX fluorescence were significantly recovered to a greater extent in the drug delivery system than the free drug.
The above studies show that the drug delivery systems G320SP and G320SD and the mixture of the two can induce fluorescence quenching by self-assembly aggregation in an aqueous environment, and when the aggregation structure is destroyed, the fluorescence can be recovered, while the optical properties of the photosensitizer Ppa and DOX are not affected by the mixture of G320SP and G320 SD.
TABLE 3 fluorescence quenching rates of drug delivery systems G320SP, G320SD and mixed drug delivery systems in different solvent environments
Experimental example 4 drug Release from the photodynamic-chemotherapeutic combination drug delivery System of the present invention
In order to examine whether the nano-mixed drugs G320SP and G320SD combined with photodynamic-chemotherapy can realize the responsive controlled drug release function under a specific pH condition, we compared the drug release behaviors of G320SD and G320SP + G320SD (a mixed drug delivery system consisting of G320SP and G320SD in a ratio of 3:1 of photosensitizer Ppa to DOX) under two pH conditions (pH 5.2 corresponds to the pH value in endocytosis or lysosome, and pH 7.4 corresponds to the pH value in blood).
The results show (fig. 15) that G320SD and G320SP + G320SD both release slowly under physiological conditions at pH 7.4, with G320SD released at 8h significantly higher than G320SP + G320SD, and the cumulative release at 48h was 44.65 ± 1.09, 35.88 ± 1.47, respectively. Under the weak acidic condition (pH is 5.2), the release rates of the two drug delivery systems are not obviously different, the release amount is obviously higher than that under the physiological pH condition, and the cumulative release amount at 48 hours is not obviously different to 89.12 +/-1.99 and 87.65 +/-2.29 respectively.
The results of the above studies indicate that the mixed drug delivery system G320SP + G320SD has good pH-responsive release characteristics, maintains better stability at physiological pH, and releases free DOX at lower pH, compared to G320 SD. Therefore, the addition of G320SP enhances the stability of G320SD in blood without affecting the drug release characteristics in the weakly acidic environment of tumor tissues or cells.
Experimental example 5 in vitro cell evaluation of photodynamic-chemotherapy combination drug delivery System of the present invention
To examine whether the combination of the drug delivery systems G320SP and G320SD has synergistic effect of photodynamic therapy and chemotherapy on tumor cell inhibition, we studied the cytotoxicity of the single drug delivery systems G320SP and G320SD and the mixed drug delivery system composed of the two in different proportions on the breast cancer cells of 4T1 mice by the CCK-8 method.
As a result, as shown in fig. 16 and table 4, the drug delivery systems G320SP and G320SD had synergistic effects on inhibiting 4T1 cells only with the mixed drug delivery system consisting of photosensitizer Ppa and DOX in a ratio of 3:1 (CI ═ 0.93< 1). However, no synergistic effect can be generated after the drug delivery systems G320SP and G320SD are mixed according to the ratio of 1:1 and 1:3 of the photosensitizer Ppa to DOX, and the combination indexes CI of the drug delivery systems are respectively 1.55 and 1.61 which are both larger than 1, which shows that the mixture prepared according to the ratios generates larger antagonistic action and the purpose of synergistic effect of photodynamic-chemotherapy is difficult to achieve.
Based on the above research results, the combined photodynamic-chemotherapy drug delivery system composed of the drug delivery systems G320SP and G320SD according to the ratio of the photosensitizer Ppa to DOX of 3:1 can generate obvious synergistic effect on tumor cell inhibition, and is significantly better than a single treatment mode, due to the advantages of physicochemical properties such as most uniform size, best monodispersity, highest stability and the like.
TABLE 4 Combination Index (CI) and IC50 values
Experimental example 6 evaluation of in vivo antitumor Activity of photodynamic-chemotherapy combination drug delivery System of the present invention
As proved by the drug delivery system combined by photodynamic and chemotherapy, which is formed when the drug delivery systems G320SP and G320SD are mixed according to a proper proportion, the drug delivery system has the optimal physicochemical property, can also generate a remarkable synergistic effect on tumor cell inhibition, and in order to further verify the in-vivo anti-tumor effect, a mouse breast cancer transplantation tumor model is established subcutaneously in female BALB/c mice by taking 4T1 mouse breast cancer cells as research objects. The in vivo antitumor effects of the free photosensitizer Ppa-treated group, the free DOX-treated group, the free photosensitizer Ppa and free DOX combination-treated group, the drug delivery system G320 SP-treated group, the drug delivery system G320 SD-treated group, and the hybrid treatment group of photodynamic-chemotherapy combination (G320SP/G320SD drug ratio: 3/1) were compared with the Saline (saine) -injected mouse as a control group.
The results are shown in fig. 17, where the combined photodynamic-chemotherapy drug delivery system has the best antitumor effect, significantly better than the single treatment modality, and the treatment effect is also significantly better than the combined treatment effect of free photosensitizer Ppa and free DOX. In the hybrid treatment group combining photodynamic therapy and chemotherapy, the tumors of 2 mice completely disappear after treatment, and the tumors of the other mice are also obviously reduced. Meanwhile, the photodynamic-chemotherapy combination has a significantly higher tumor inhibition rate (TGI) of 99.34%. The tumor inhibition rates of the drug delivery systems G320SP and G320SD were also relatively high, 94.45% and 88.54%, respectively. The tumor inhibition rate of the free photosensitizer Ppa treatment group is only 47.21%, that of the free DOX treatment group is 42.28%, and that of the free photosensitizer Ppa and free DOX combination treatment group is 66.80%.
In summary, the invention designs and constructs an amphiphilic DLD type linear-tree structure photodynamic drug delivery system and a tumor microenvironment acid-sensitive amphiphilic DLD type linear-tree structure chemotherapy drug delivery system, and after mixing according to a proper proportion (Ppa/DOX), the two systems can be assembled into a combined drug delivery system with a more stable structure and better monodispersity, and the combined drug delivery system has a synergistic anti-tumor effect under the optimal proportion (Ppa: DOX is 3:1), shows an anti-tumor effect which is obviously better than that of the single drug delivery system in vitro and in vivo anti-tumor experiments, and shows a good application prospect in preparing anti-tumor drugs.
Claims (16)
1. A linear-tree drug delivery system, comprising: the linear-tree drug delivery system is loaded with pyropheophorbide a, and the structure of the linear-tree drug delivery system is shown as a formula I:
wherein R is1～R16Each independently selected from H or
And is not H at the same time;
n is the degree of polymerization, and n is 444.
2. The linear-tree delivery system of claim 1, wherein: in the linear-tree drug delivery system, the drug loading rate of pyropheophorbide a is 6.00-7.00%;
the drug loading of pyropheophorbide a was ═ 100% (mass of pyropheophorbide a/total mass of linear-dendrimer drug delivery system).
3. The linear-tree delivery system of claim 2, wherein: in the linear-dendritic drug delivery system, the drug loading of pyropheophorbide a was 6.19%.
4. A linear-tree delivery system loaded with doxorubicin, said linear-tree delivery system characterized by: the structure of the linear-tree drug delivery system is shown as the formula II:
wherein R is1～R16Each independently selected from H or
And is not H at the same time;
n is polymerization degree, and n is 444.
5. The linear-tree delivery system of claim 4, wherein: in the linear-tree drug delivery system, the drug loading rate of the adriamycin is 7.00-8.00%;
doxorubicin load (mass of doxorubicin/total mass of linear-dendrimer) x 100%.
6. The linear-tree delivery system of claim 5, wherein: in the linear-tree drug delivery system, the drug loading of the adriamycin is 7.90%.
7. A combined photodynamic-chemotherapy drug delivery system, comprising: the photodynamic-chemotherapy combined drug delivery system is prepared by taking the linear-tree drug delivery system of any one of claims 1 to 3 and the linear-tree drug delivery system of any one of claims 4 to 6 as raw materials.
8. The combined photodynamic-chemotherapy delivery system according to claim 7, wherein: the linear-dendritic delivery system of any one of claims 1 to 3 and the linear-dendritic delivery system of any one of claims 4 to 6 wherein the molar ratio of pyropheophorbide a to doxorubicin is in the range
9. The combined photodynamic-chemotherapy delivery system according to claim 8, wherein: the molar ratio of pyropheophorbide a to adriamycin is 3:1,1:1 or 1: 3.
10. The combined photodynamic-chemotherapy delivery system according to claim 9, wherein: the molar ratio of pyropheophorbide a to adriamycin is 3: 1.
11. a method of preparing a linear-dendritic drug delivery system according to any of claims 1 to 6, wherein: the method comprises the following steps:
(1) 3- (tritylthio) propionic acid and Hyperbranched G3-PEG20k-OH are used as raw materials to react to obtain Hyperbranched G3-PEG20 k-STRT;
(2) the Hyperbranched G3-PEG20k-STRT is reacted with Maleimide-Ppa or Maleimide-DOX;
(3) dialyzing the system reacted in the step (2) in ultrapure water, purifying and drying to obtain the compound;
wherein the structure of the Hyperbranched G3-PEG20k-STRt is
wherein-STRt is
The structure of Maleimide-Ppa is
The structure of Maleimide-DOX is
12. The method of claim 11, wherein: in the step (1), the molar ratio of the 3- (tritylthio) propionic acid to the Hyperbranched G3-PEG20k-OH is (15-25): 1; the reaction is carried out under the action of DCC and DMAP; the reaction solvent is an organic solvent; the reaction condition is that the reaction is carried out at room temperature in a dark place under the nitrogen atmosphere; the reaction time is 24-72 hours;
in the step (2), the molar ratio of the Hyperbranched G3-PEG20k-STRt to the Maleimide-Ppa is 1: (5-15), or the molar ratio of the Hyperbranched G3-PEG20k-STRt to the Maleimide-DOX is 1: (5-15); the reaction solvent is an organic solvent; the reaction condition is that the reaction is carried out at room temperature in a dark place under the nitrogen atmosphere; the reaction time is 24-72 hours;
in the step (3), the dialysis time is 2 days, and the cut-off molecular weight of a dialysis bag used for dialysis is 8000; the purification mode is purification through a size exclusion chromatographic column.
13. The method of claim 12, wherein: in the step (1), the molar ratio of the 3- (tritylthio) propionic acid to the Hyperbranched G3-PEG20k-OH is 19.2: 1; the molar ratio of the Hyperbranched G3-PEG20k-OH to the DCC and DMAP is 1: (30-35): (1-2); the reaction solvent is N, N-dimethyl amide; the reaction time is 48 hours;
in the step (2), the molar ratio of the Hyperbranched G3-PEG20k-STRt to the Maleimide-Ppa is 1: 8, or the molar ratio of the Hyperbranched G3-PEG20k-STRt to the Maleimide-DOX is 1: 8; the reaction solvent is N, N-dimethyl amide; the reaction time was 48 hours.
14. A method for preparing a combined photodynamic-chemotherapeutic drug delivery system as claimed in any one of claims 7 to 10, wherein: the method comprises the following steps: adding the linear-dendritic drug delivery system of any one of claims 1 to 3 and the linear-dendritic drug delivery system of any one of claims 4 to 6 into water, and self-assembling to obtain spherical nanoparticles.
15. Use of a linear-dendritic drug delivery system according to any one of claims 1 to 6 or a combined photodynamic-chemotherapy drug delivery system according to any one of claims 7 to 10 for the preparation of an anti-tumor drug.
16. Use according to claim 15, characterized in that: the tumor is selected from breast cancer, bladder cancer, lung cancer, esophageal cancer, head and neck cancer, and skin cancer.
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