PEG-Peptide linear-tree-shaped drug delivery system and preparation method and application thereof
阅读说明：本技术 一种PEG-Peptide线性-树状给药系统及其制备方法和用途 (PEG-Peptide linear-tree-shaped drug delivery system and preparation method and application thereof ) 是由 罗奎 顾忠伟 郑秀丽 潘达艺 龚启勇 于 2019-06-28 设计创作，主要内容包括：本发明提供了一种线性-树状共聚物,所述线性-树状共聚物的结构如式I所示。本发明还提供了一种给药系统,所述给药系统是上述线性-树状共聚物与光敏剂发生偶联反应后得到的。研究表明,本发明的给药系统PPDP可在选择性溶剂中自组装形成尺寸均一、单分散性良好、稳定性高且带弱负电荷的纳米颗粒,其不仅具有优良的光物理特性,还能有效地产生ROS实现对肿瘤细胞的杀灭；其显著降低了光敏剂的皮肤光毒性；其不仅表现出较好的生物安全性、优异的荧光成像功能、血液长循环功能和长时间和高浓度的肿瘤靶向和富集效应,还具有显著的抗肿瘤活性,在制备抗肿瘤药物中显示出良好的应用前景。<Image he="418" wi="700" file="DDA0002112440740000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention provides a linear-dendritic copolymer, the structure of which is shown in formula I. The invention also provides a drug delivery system, which is obtained by the coupling reaction of the linear-dendritic copolymer and the photosensitizer. Research shows that the drug delivery system PPDP can be self-assembled in a selective solvent to form nano-particles with uniform size, good monodispersity, high stability and weak negative charge, has excellent photophysical characteristics, and can effectively generate ROS to kill tumor cells; it significantly reduces the lightSkin phototoxicity of the sensitizers; the fluorescent probe not only has good biological safety, excellent fluorescence imaging function, long blood circulation function and long-time and high-concentration tumor targeting and enriching effect, but also has obvious antitumor activity and good application prospect in preparing antitumor drugs.)
1. A linear-dendrimer copolymer characterized by: the structure of the linear-dendritic copolymer is shown as the formula I:
n is the polymerization degree, and n is selected from 22-88.
2. The linear-arborescent copolymer of claim 1, wherein: n is 44.
3. A drug delivery system, characterized by: the structure of the drug delivery system is shown as formula II:
wherein R is1、R2、R3、R4Each independently selected from H orAnd is not H at the same time;
n is the polymerization degree, and n is selected from 22-88.
4. The delivery system of claim 3, wherein: the structure of the drug delivery system is shown as formula III:
n is 44.
5. A process for the preparation of a linear-dendrimer according to claim 1 or 2, wherein: the preparation method comprises the following steps:
(1) Fmoc-Lys (Fmoc) -OH and H-Glu (OtBu) -OtBu HCl are subjected to condensation reaction to obtain a compound 1;
(2) removing a-BuOt group of the compound 1 under the action of a deprotection reagent to obtain an intermediate product 1, and then continuing to perform condensation reaction on the intermediate product 1 and H-Glu (OtBu) -OtBu & HCl to obtain a compound 2;
(3) removing-Fmoc group of the compound 2 under the action of a deprotection reagent to obtain an intermediate product 2, and then continuing to perform condensation reaction on the intermediate product 2 and Fmoc-Lys (Fmoc) -OH to obtain a compound 3;
(4) removing a-BuOt group of the compound 3 under the action of a deprotection reagent to obtain an intermediate product 3, and continuing to perform a condensation reaction on the intermediate product 3 and propargylamine to obtain a compound 4;
(5) removing-Fmoc group of the compound 4 under the action of a deprotection reagent to obtain an intermediate product 4, and continuing reacting the intermediate product 4 with mPEGx-N3Reacting to obtain the linear-dendritic copolymer;
wherein the structure of Fmoc-Lys (Fmoc) -OH isThe structure of H-Glu (OtBu) -OtBu isCompound 1 has the structure
Compound 2 has the structure
Compound 3 has the structure
Compound 4 has the structure
mPEGx-N3Represents methoxy polyethylene glycol azide with molecular weight x, wherein x is selected from 1000-4000; preferably, x is 2000.
6. The method of claim 5, wherein:
in the step (1):
the molar ratio of Fmoc-Lys (Fmoc) -OH to H-Glu (OtBu) -OtBu is 1.0: (1.0 to 2.0), preferably 1.0: 1.5;
the condensation reaction is carried out under the action of a condensing agent and a base, wherein the condensing agent is preferably selected from one or more of HOBt, HBTU, HOAt and HATU, and the base is selected from DIPEA; more preferably, the condensing agent is selected from HOBt and HBTU, and the molar ratio of H-glu (OtBu) -OtBu-HCl to HOBt, HBTU, DIPEA is: 1: 1: 1: (2-3);
the reaction conditions are that the reaction is carried out for 1 hour in an ice bath and then for 24 to 48 hours at room temperature;
the reaction solvent is N, N-dimethyl amide;
and/or, in step (2):
the deprotection reagent is trifluoroacetic acid, and the reaction condition for removing the-BuOt group is that the reaction is carried out for 1h in an ice bath and then for 5h at room temperature; the reaction solvent for removing the-BuOt group is dichloromethane, and the mass-volume ratio of the compound 1 to the reaction solvent and the deprotection reagent is 2.16 g: 10mL of: 10 mL;
the molar ratio of the compound 1 to H-Glu (OtBu) -OtBu-HCl is 1: (2-4), preferably 1: 3;
the condensation reaction is carried out under the action of a condensing agent and a base, wherein the condensing agent is preferably selected from one or more of HOBt, HBTU, HOAt and HATU, and the base is selected from DIPEA; more preferably, the condensing agent is selected from HOBt and HBTU, and the molar ratio of H-glu (OtBu) -OtBu-HCl to HOBt, HBTU, DIPEA is: 1: 1: 1: (2-3);
the condensation reaction condition is that the reaction is carried out for 1 hour under ice bath, and the reaction is continued for 24 to 48 hours at room temperature;
the condensation reaction solvent is N, N-dimethyl amide;
and/or, in step (3):
the deprotection reagent is diethylamine, and the reaction condition for removing the-Fmoc group is reaction for 6 hours at room temperature; the reaction solvent for removing the Fmoc group is dichloromethane, and the mass-volume ratio of the compound 1 to the reaction solvent to the deprotection reagent is 2.08 g: 20mL of: 20 mL;
the molar ratio of the compound 2 to Fmoc-Lys (Fmoc) -OH is 1: (2-4), preferably 1: 3;
the condensation reaction is carried out under the action of a condensing agent and a base, wherein the condensing agent is preferably selected from one or more of HOBt, HBTU, HOAt and HATU, and the base is selected from DIPEA; more preferably, the condensing agent is selected from HOBt and HBTU, and the molar ratio of Fmoc-lys (Fmoc) -OH to HOBt, HBTU, DIPEA is: 1: 1: 1: (2-3);
the condensation reaction condition is that the reaction is carried out for 1 hour in an ice bath, and then the reaction is carried out for 24 to 48 hours at room temperature;
the condensation reaction solvent is N, N-dimethyl amide;
and/or, in step (4):
the deprotection reagent is trifluoroacetic acid, and the reaction condition for removing the-BuOt group is reaction for 12 hours at room temperature; the reaction solvent for removing the-BuOt group is dichloromethane, and the mass-volume ratio of the compound 3 to the reaction solvent and the deprotection reagent is 1.58 g: 10mL of: 10 mL;
the molar ratio of the compound 3 to propargylamine is 1: (5-7), preferably 1: 6;
the condensation reaction is carried out under the action of a condensing agent and a base, wherein the condensing agent is preferably selected from one or more of HOBt, HBTU, HOAt and HATU, and the base is selected from DIPEA; more preferably, the condensing agent is selected from HOBt and HBTU, and the molar ratio of propargylamine to HOBt, HBTU, DIPEA is: 1: 1: 1: (2-3);
the condensation reaction condition is that the reaction is carried out for 1 hour in an ice bath, and then the reaction is carried out for 24 to 48 hours at room temperature;
the condensation reaction solvent is N, N-dimethyl amide;
and/or, in step (5):
the deprotection reagent is diethylamine, and the reaction condition for removing the-Fmoc group is reaction for 12 hours at room temperature; the reaction solvent for removing the Fmoc group is N, N-dimethyl amide, and the mass-volume ratio of the compound 4 to the reaction solvent to the deprotection reagent is 0.2 g: 5mL of: 5 mL;
the compound 4 and mPEG2k-N3Is 1.0: (7.0 to 8.0), preferably 1.0: 7.2;
the compound 4 and mPEG2k-N3Is carried out in CuSO4·5H2Performed under the action of sodium O and L-ascorbate, mPEG2k-N3With CuSO4·5H2O, L sodium ascorbateIn a molar ratio of 1: 0.67: 1.34; the reaction condition is that the reaction is carried out for 48 hours at room temperature in a dark place; the reaction solvent is DMSO: h2And the volume ratio of O is 4: 1.
7. A method of preparing a delivery system according to any of claims 3 to 4, wherein: the method comprises the following steps: the linear-dendrimer of claim 1 or 2, which is obtained by coupling reaction with a photosensitizer.
8. The method of claim 7, wherein: in the reaction, the photosensitizer is pyropheophorbide a;
and/or, the molar ratio of the linear-dendrimer copolymer to the photosensitizer is 1: (3-15), preferably 1: 8;
and/or, the coupling reaction is completed under the action of a condensing agent and a base; preferably, the condensing agent is selected from one or more of HOBt, HBTU, HOAt and HATU, and the base is selected from DIPEA; more preferably, the condensing agent is selected from the group consisting of HOAt and HATU, and the molar ratio of photosensitizer to HOAt, HATU and DIPEA is 1: 1.1: 1.1: (0.5 to 0.6);
and/or the reaction conditions are that the reaction is carried out for 1 hour in an ice bath, and then the reaction is carried out for 48 hours at room temperature in a dark place;
and/or the reaction solvent is N, N-dimethyl amide.
9. A self-assembling system, comprising: the self-assembly system is spherical nanoparticles obtained by self-assembly of the drug delivery system of any one of claims 3 to 4 in water.
10. Use of the delivery system according to any of claims 3 to 4 or the self-assembling system according to claim 9 for the preparation of an anti-tumor drug, preferably the tumor is selected from the group consisting of breast cancer, bladder cancer, lung cancer, esophageal cancer, head and neck cancer, skin cancer.
Technical Field
The invention belongs to the field of high molecular materials, and particularly relates to a PEG-Peptide linear-tree drug delivery system, and a preparation method and application thereof.
Background
Currently, chemotherapy is one of the major clinical approaches to the treatment of malignant tumors. However, the traditional chemotherapy drugs have large toxic and side effects, not only lack tumor selectivity, but also easily cause multidrug resistance of tumors after long-term administration, and the inevitable defects are important reasons for chemotherapy failure. Therefore, how to seek a low-toxicity, high-efficiency and economic treatment means for malignant tumors by improving the defects of poor curative effect, high toxic and side effects and the like of the traditional chemotherapeutic drugs is a hot research direction in the current oncology field.
As an emerging tumor treatment means, Photodynamic therapy (PDT) brings a new opportunity for realizing high-efficiency and low-toxicity treatment of malignant tumors. The method is different from traditional methods such as surgery, radiotherapy and chemotherapy, and mainly utilizes tumor cells to selectively absorb photosensitizer, and then utilizes non-thermal laser with specific wavelength to irradiate the tumor part, so that the photosensitizer in the tumor tissue generates violent photochemical reaction to generate ROS (reactive oxygen species) with cytotoxicity, thereby selectively killing the tumor cells. In addition, the existing research shows that the medicine resistance problem is not easy to cause by utilizing the photodynamic therapy to treat malignant tumor, and a plurality of researches also report that the photodynamic therapy can effectively inhibit tumor cells which generate chemotherapy resistance, and the treatment effect of the photodynamic therapy is obviously better than that of chemotherapeutic drugs. Therefore, the photodynamic therapy of the tumor has potential research value and wide application prospect in the field of novel tumor therapy research.
The key to photodynamic therapy of malignant tumor is the reasonable matching of oxygen, irradiation light source and photosensitizer. Among them, photosensitizers are the core of photodynamic therapy and are important factors in determining the therapeutic effect and toxic side effects thereof. The ideal photosensitizer has the characteristics of good water solubility, high singlet quantum yield, stable physicochemical property, tumor cell targeting property, easy in-vivo clearance, low dark toxicity and the like, but the photosensitizer which is clinically applied and is researched at present still generally has the problems of poor water solubility, slow clearance, poor targeting property, easy generation of delayed phototoxicity and the like. Compared with clinical application of gold standard-first generation photosensitizerThe second-generation photosensitizer pyropheophorbide a (Ppa) has strong absorption at longer wavelength (about 670nm), deeper tissue penetration during phototherapy, higher singlet oxygen quantum yield and obvious clinical application potential. However, the composition also has the defects of difficult water solubility, high toxicity, slow clearance, poor targeting property and the like, and the clinical development and application of the composition are severely limited. In this regard, Cooper et al designed and developed Photochlor (HPPH), a chemical modification of pyropheophorbide a, and entered clinical phase I/II trials. However, HPPH still cannot completely solve the problems of poor targeting and high toxicity of photosensitizer, especially strong skin phototoxicity and the like, so that the clinical test of the HPPH is very slow in progress.
In addition, in order to improve the targeting and concentration of the photosensitizer to tumor tissues, a drug delivery system carrier is generally required 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, distribution and antitumor activity of the delivery system.
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, wherein the chlorin e6 and folic acid are coupled to a carboxymethyl chitosan chain segment through amido bonds, and polymer nanoparticles loaded with the adriamycin are obtained through an ion crosslinking method to form a targeted anti-tumor nano drug delivery system. However, the preparation process of the drug-loaded system is complex, and the coupling of the targeting molecular folic acid increases the preparation process; the combination of doxorubicin and a photosensitizer further complicates the drug system.
Therefore, the photodynamic drug delivery system which has simple structure, convenient preparation and obvious treatment effect on the tumor has wide application prospect by reasonably selecting carrier materials, optimally designing molecular structures and skillfully constructing a drug delivery system.
Disclosure of Invention
The invention aims to provide a photodynamic drug delivery system which has a simple structure, is convenient to prepare and has a remarkable treatment effect on tumors.
The invention provides a linear-dendritic copolymer, the structure of which is shown as formula I:
n is the polymerization degree, and n is selected from 22-88.
Preferably, n is 44.
The invention also provides a drug delivery system, which has a structure shown in the formula II:
wherein R is1、R2、R3、R4Each independently selected from H orAnd is not H at the same time;
n is the polymerization degree, and n is selected from 22-88.
Further, the structure of the drug delivery system is shown as formula III:
n is 44.
The drug loading represents (mass of drug/total mass of linear-dendritic drug delivery system) × 100% in the system tested. For example, the drug loading of pyropheophorbide a is (mass of pyropheophorbide a/total mass of the administration system) × 100%.
In the administration system of the present invention, the drug loading of pyropheophorbide a was 16.01%.
The invention also provides a preparation method of the linear-dendritic copolymer, which comprises the following steps:
(1) Fmoc-Lys (Fmoc) -OH and H-Glu (OtBu) -OtBu HCl are subjected to condensation reaction to obtain a compound 1;
(2) removing a-BuOt group of the compound 1 under the action of a deprotection reagent to obtain an intermediate product 1, and then continuing to perform condensation reaction on the intermediate product 1 and H-Glu (OtBu) -OtBu & HCl to obtain a compound 2;
(3) removing-Fmoc group of the compound 2 under the action of a deprotection reagent to obtain an intermediate product 2, and then continuing to perform condensation reaction on the intermediate product 2 and Fmoc-Lys (Fmoc) -OH to obtain a compound 3;
(4) removing a-BuOt group of the compound 3 under the action of a deprotection reagent to obtain an intermediate product 3, and continuing to perform a condensation reaction on the intermediate product 3 and propargylamine to obtain a compound 4;
(5) removing-Fmoc group of the compound 4 under the action of a deprotection reagent to obtain an intermediate product 4, and continuing reacting the intermediate product 4 with mPEGx-N3Reacting to obtain the linear-dendritic copolymer;
wherein the structure of Fmoc-Lys (Fmoc) -OH isThe structure of H-Glu (OtBu) -OtBu isKnot of Compound 1Is constructed asCompound 2 has the structureCompound 3 has the structureCompound 4 has the structure
mPEGx-N3Represents methoxy polyethylene glycol azide with molecular weight x, wherein x is selected from 1000-4000; preferably, x is 2000.
Further, the air conditioner is provided with a fan,
in the step (1):
the molar ratio of Fmoc-Lys (Fmoc) -OH to H-Glu (OtBu) -OtBu is 1.0: (1.0 to 2.0), preferably 1.0: 1.5;
the condensation reaction is carried out under the action of a condensing agent and a base, wherein the condensing agent is preferably selected from one or more of HOBt, HBTU, HOAt and HATU, and the base is selected from DIPEA; more preferably, the condensing agent is selected from HOBt and HBTU, and the molar ratio of H-glu (OtBu) -OtBu-HCl to HOBt, HBTU, DIPEA is: 1: 1: 1: (2-3);
the reaction conditions are that the reaction is carried out for 1 hour in an ice bath and then for 24 to 48 hours at room temperature;
the reaction solvent is N, N-dimethyl amide;
and/or, in step (2):
the deprotection reagent is trifluoroacetic acid, and the reaction condition for removing the-BuOt group is that the reaction is carried out for 1h in an ice bath and then for 5h at room temperature; the reaction solvent for removing the-BuOt group is dichloromethane, and the mass-volume ratio of the compound 1 to the reaction solvent and the deprotection reagent is 2.16 g: 10mL of: 10 mL;
the molar ratio of the compound 1 to H-Glu (OtBu) -OtBu-HCl is 1: (2-4), preferably 1: 3;
the condensation reaction is carried out under the action of a condensing agent and a base, wherein the condensing agent is preferably selected from one or more of HOBt, HBTU, HOAt and HATU, and the base is selected from DIPEA; more preferably, the condensing agent is selected from HOBt and HBTU, and the molar ratio of H-glu (OtBu) -OtBu-HCl to HOBt, HBTU, DIPEA is: 1: 1: 1: (2-3);
the condensation reaction condition is that the reaction is carried out for 1 hour under ice bath, and the reaction is continued for 24 to 48 hours at room temperature;
the condensation reaction solvent is N, N-dimethyl amide;
and/or, in step (3):
the deprotection reagent is diethylamine, and the reaction condition for removing the-Fmoc group is reaction for 6 hours at room temperature; the reaction solvent for removing the Fmoc group is dichloromethane, and the mass-volume ratio of the compound 1 to the reaction solvent to the deprotection reagent is 2.08 g: 20mL of: 20 mL;
the molar ratio of the compound 2 to Fmoc-Lys (Fmoc) -OH is 1: (2-4), preferably 1: 3;
the condensation reaction is carried out under the action of a condensing agent and a base, wherein the condensing agent is preferably selected from one or more of HOBt, HBTU, HOAt and HATU, and the base is selected from DIPEA; more preferably, the condensing agent is selected from HOBt and HBTU, and the molar ratio of Fmoc-lys (Fmoc) -OH to HOBt, HBTU, DIPEA is: 1: 1: 1: (2-3);
the condensation reaction condition is that the reaction is carried out for 1 hour in an ice bath, and then the reaction is carried out for 24 to 48 hours at room temperature;
the condensation reaction solvent is N, N-dimethyl amide;
and/or, in step (4):
the deprotection reagent is trifluoroacetic acid, and the reaction condition for removing the-BuOt group is reaction for 12 hours at room temperature; the reaction solvent for removing the-BuOt group is dichloromethane, and the mass-volume ratio of the compound 3 to the reaction solvent and the deprotection reagent is 1.58 g: 10mL of: 10 mL;
the molar ratio of the compound 3 to propargylamine is 1: (5-7), preferably 1: 6;
the condensation reaction is carried out under the action of a condensing agent and a base, wherein the condensing agent is preferably selected from one or more of HOBt, HBTU, HOAt and HATU, and the base is selected from DIPEA; more preferably, the condensing agent is selected from HOBt and HBTU, and the molar ratio of propargylamine to HOBt, HBTU, DIPEA is: 1: 1: 1: (2-3);
the condensation reaction condition is that the reaction is carried out for 1 hour in an ice bath, and then the reaction is carried out for 24 to 48 hours at room temperature;
the condensation reaction solvent is N, N-dimethyl amide;
and/or, in step (5):
the deprotection reagent is diethylamine, and the reaction condition for removing the-Fmoc group is reaction for 12 hours at room temperature; the reaction solvent for removing the Fmoc group is N, N-dimethyl amide, and the mass-volume ratio of the compound 4 to the reaction solvent to the deprotection reagent is 0.2 g: 5mL of: 5 mL;
the compound 4 and mPEG2k-N3Is 1.0: (7.0 to 8.0), preferably 1.0: 7.2;
the compound 4 and mPEG2k-N3Is carried out in CuSO4·5H2Performed under the action of sodium O and L-ascorbate, mPEG2k-N3With CuSO4·5H2O, L-sodium ascorbate in a molar ratio of 1: 0.67: 1.34; the reaction condition is that the reaction is carried out for 48 hours at room temperature in a dark place; the reaction solvent is DMSO: h2And the volume ratio of O is 4: 1.
The invention also provides a preparation method of the drug delivery system, which comprises the following steps: and (3) carrying out coupling reaction on the linear-dendritic copolymer and a photosensitizer to obtain the photosensitive emulsion.
Further, in the reaction, the photosensitizer is pyropheophorbide a;
and/or, the molar ratio of the linear-dendrimer copolymer to the photosensitizer is 1: (3-15), preferably 1: 8;
and/or, the coupling reaction is completed under the action of a condensing agent and a base; preferably, the condensing agent is selected from one or more of HOBt, HBTU, HOAt and HATU, and the base is selected from DIPEA; more preferably, the condensing agent is selected from the group consisting of HOAt and HATU, and the molar ratio of photosensitizer to HOAt, HATU and DIPEA is 1: 1.1: 1.1: (0.5 to 0.6);
and/or the reaction conditions are that the reaction is carried out for 1 hour in an ice bath, and then the reaction is carried out for 48 hours at room temperature in a dark place;
and/or the reaction solvent is N, N-dimethyl amide.
The invention also provides a self-assembly system, which is spherical nanoparticles obtained by self-assembly of the drug delivery system in water.
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 the application of the drug delivery system or the self-assembly system in preparing an anti-tumor drug, preferably, the tumor is selected from breast cancer, bladder cancer, lung cancer, esophageal cancer, head and neck cancer and skin cancer.
Experimental results show that the invention designs and constructs the amphipathic LD-PEG-Peptide linear-dendrimer-pyropheophorbide a photodynamic drug delivery system PPDP, the drug delivery system can be self-assembled in a selective solvent to form nanoparticles with uniform size, good monodispersity, high stability and weak negative charge, the nanoparticles not only have excellent photophysical characteristics, but also can effectively generate ROS to kill tumor cells; it significantly reduces the skin phototoxicity of the photosensitizer; the fluorescent probe not only has good biological safety, excellent fluorescence imaging function, long blood circulation function and long-time and high-concentration tumor targeting and enriching effect, but also has obvious antitumor activity and good application prospect in preparing antitumor drugs.
In the invention, 1-hydroxybenzotriazole (HOBt), benzotriazole-N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HBTU), 2- (7-oxybenzotriazole) -N, N, N ', N ' -tetramethyluronium Hexafluorophosphate (HATU), 1-hydroxy-7-azobenzotriazol (HOAt), N, N ' -Diisopropylethylamine (DIPEA), 2-propylnulamine (propargylamine), mPEG-N3Namely methoxypolyethylene glycol azide; sodium ascorbate is sodium L-ascorbate.
In the present invention, the LD type, i.e., Linear-Dendritic, represents a Linear tree structure.
Self-assembly, refers to a technique in which basic building blocks (molecules, nanomaterials, substances on the micrometer or larger scale) spontaneously form an ordered structure. During the self-assembly process, the basic building blocks spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance under the interaction based on non-covalent bonds.
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 is a scheme of the PPDP synthesis of the delivery system of the present invention.
FIG. 2 is a schematic view of the self-assembly of a PPDP delivery system of the present invention.
FIG. 3 is a nuclear magnetic hydrogen spectrum characterization of Compound 1.
FIG. 4 is an ESI-TOF MS characterization of Compound 1.
FIG. 5 is a nuclear magnetic hydrogen spectrum characterization of Compound 2.
FIG. 6 is an ESI-TOF MS characterization of Compound 2.
FIG. 7 is a nuclear magnetic hydrogen spectrum characterization of Compound 3.
FIG. 8 is an ESI-TOF MS characterization of Compound 3.
FIG. 9 is a nuclear magnetic hydrogen spectrum characterization of Compound 4.
FIG. 10 is an ESI-TOF MS characterization of Compound 4.
FIG. 11 is MALDI-TOF MS characterization of Compound 5
FIG. 12 is Compound 6 (PPDP).
FIG. 13 is a standard UV absorption curve of pyropheophorbide a in DMSO.
FIG. 14 shows PPDP in DMSO-d6(A) And D2Nuclear magnetic hydrogen spectrum characterization in o (b).
FIG. 15 is a representation of particle size and morphology by transmission electron microscopy: carrier material (PPD) (a) and administration system ppdp (b).
Fig. 16 is a characterization of particle size by DLS: administration system PPDP particle size in pure water (A) and PBS, respectively (B).
Figure 17 is a graph of Critical Micelle Concentration (CMC) of PPDP in water for the drug delivery system determined by the addition of pyrene, wherein the calculation of the linear fit curve and the CMC values was done by SigmaPlot 12.5 software.
FIG. 18 is a graph showing the stability of particle size by DLS, particle size (A) of drug delivery system PPDP in pure water and PBS buffer with 10% FBS added, respectively, and PDI (B) of drug delivery system PPDP in pure water and PBS buffer with 10% FBS added, respectively, with time.
Figure 19 shows the color of solutions of PPDP and free photosensitizer Ppa in purified water, PBS and DMSO, respectively, for the drug delivery system.
FIG. 20 shows the UV-VIS absorption spectra (A and B) and fluorescence emission spectra (B) of the drug delivery system PPDP and free photosensitizer Ppa after different treatments.
FIG. 21 shows the photostability of the delivery system PPDP (B) and the free photosensitizer Ppa (A) in different solvents.
Fig. 22 is a plot of DMA consumption and conversion fit of the drug delivery system PPDP and free photosensitizer Ppa.
Fig. 23 shows cytotoxicity of 4T1 cells (a), LO2 cells under dark conditions (B), and LO2 cells by PPD (a) and free photosensitizer Ppa, as delivery systems, after exposure to 660nm laser light, and toxicity of the carrier material PPD (C), where n is 3 and IC50 values were calculated by GraphPad Prism 5.01 software.
FIG. 24 is a graph of cellular uptake (A) after 1h, 3h and 6h incubation of drug delivery system PPDP and free photosensitizer Ppa in 4T1 cells by confocal laser microscopy (CLSM); cellular uptake of the drug delivery system PPDP and free photosensitizer Ppa after various periods of incubation in 4T1 cells (B) was examined by flow cytometry, with a scale bar representing 25 microns.
Fig. 25 is (a): 4T1 cells were assayed for ROS production by DCFH-DA probes and pictures were taken by inverted fluorescence microscopy, the cells were treated differently as follows: a) controls without probe and light; b) control with probe and no light; c) control of probe and light addition; d) control with added probe and live hydrogen donor; e) adding a probe, light and a drug delivery system PPDP; f) adding probe, 1J/cm2660nm illumination and free photosensitizer Ppa; (B) the method comprises the following steps Measuring ROS generation condition of 4T1 cells after different treatments by a flow cytometer, wherein PBS is a control without a probe and light; DCFH-DA, control with probe alone; DCFH-DA + laser, probe addition and light control; DCFH-DA + H2O2 control with added probe and active hydrogen donor; ppa adding probe, light and dissociating Ppa; PPDP, adding probe, light and drug delivery system PPDP; wherein the light dose is 1J/cm2660nm, scale bar represents 200 microns.
FIG. 26 shows the morphology and aggregation of erythrocytes treated with different concentrations of PPDP of the drug delivery system and PPD of the carrier material (A), and erythrocytes treated with PBS as a control (B).
FIG. 27 shows the hemolysis of erythrocytes treated with different concentrations of PPDP (A) as a drug delivery system and PPD as a carrier material, and the calculation of the hemolysis rate (B).
FIG. 28 shows the skin toxicity (A) and the counting statistics (B) of the degree of toxicity in healthy mice after injection of the administration system PPDP and free photosensitizer Ppa in the back area.
FIG. 29 is a graph comparing the distribution of PPDP and free photosensitizer Ppa in tumor model mice by in vivo fluorescence imaging experiments, at a dose of 5mg Ppa/kg per mouse for each group.
Fig. 30 compares tumor-targeted aggregation of the drug-delivered systemic PPDP and free photosensitizer Ppa in 4T1 tumor model mice (n ═ 3) at various time points (1d,3d,5d,7d,10d,14d) by ex vivo fluorescence imaging experiments, at each group at a dose of 5mg Ppa/kg per mouse.
Fig. 31 is a microscopic image of tumor distribution of the administered systemic PPDP and free photosensitizer Ppa in 4T1 tumor model mice at each time point (1d,3d,5d,7d,10d,14d) by fluorescent staining of frozen sections of tumors, wherein the color red: photosensitizer, green: blood vessels, blue: cell nucleus; the scale bar represents 50 microns.
FIG. 32 is a graph showing the quantitative determination of the blood content (A) of PPDP and free photosensitizer Ppa in the drug delivery system in normal mice at each time point, and the dose of each group was 5mg of Ppa/kg per mouse.
Fig. 33 is a graph comparing the in vivo anti-tumor effect of administered systemic PPDP and free photosensitizer Ppa by a 4T1 in vivo tumor model, with saline as a control (n ═ 6, dose per administration: 5mg Ppa/kg per) (a); after the treatment, the tumors were dissected and weighed (B); calculating the tumor inhibition rate (C) according to the tumor weight; and all tumors were photographed (D).
FIG. 34 is a graph of the effect on tumor apoptosis levels and inhibition of angiogenesis by TUNEL and CD-31 staining of tumors comparing the treatment with systemic PPDP and free photosensitizer Ppa, and the scale bar represents 50 microns.
FIG. 35 is a graph of the in vivo antitumor effect of PPDP in the drug delivery system and Ppa free photosensitizer in comparison to a drug resistance model established by the A549-R drug resistant strain, in saline control (n-5, dose per administration: 5mg of Ppa/kg per each) (A); after the treatment, the tumors were dissected and weighed (B); calculating the tumor inhibition rate (C) according to the tumor weight; and 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; H-Glu (OtBu) -OtBu HCl, Fmoc-Lys (Fmoc) -OH, available from Gill Biochemical (Shanghai) Co., Ltd.
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.