Structural design synthesis and application of biomass carbon dot modified poly L-cysteine-S-S-carboxylic acid amphiphilic polypeptide

Abstract

The invention relates to a structural design, synthesis and application of a biomass carbon dot modified poly L-cysteine-S-S-carboxylic acid amphiphilic polypeptide. The invention provides a structural design, synthesis and application of poly (L-cysteine-S-S-carboxylic acid) amphiphilic polypeptide (P-L-cys) modified by biomass carbon dots (CDs). Comprising synthesis of P-L-cys which contains hydrophobic groups, provides hydrophilic and functionalized carboxyl and provides reduction response by disulfide bonds, synthesis of CDs and CDs modified P-L-cys (CDs (at) P-L-cys), and the macroscopic performance change of the polypeptide is explored from regulation factors of a polypeptide secondary structure and a self-assembly nanostructure assembly mechanism. The P-L-cys and the CDs (at) P-L-cys prepared by the invention have functionalization, fluorescence imaging and dual-response drug release and have particle sizes suitable for drug delivery and enrichment, and the CDs also provide a photo-thermal and photodynamic synergistic treatment effect and have good application prospects in the fields of drug delivery, cancer resistance and the like.

Description

Technical Field

[0001] This application relates to the fields of biomedical material preparation and nanotechnology. Specifically, it relates to a preparation method and application of poly-L-cysteine amphiphilic polypeptide derivatives. Background Art

[0002] The background description provided herein is for the purpose of providing and disclosing the background of this application. The work of the inventors described in the background art and other descriptions, as well as various aspects that cannot be fully regarded as prior art before and after submission, are not explicitly or implicitly recognized as prior art for this application.

[0003] Currently, most drugs have poor water solubility, which often makes it difficult to prepare them into liquid preparations that are easy to absorb, thus greatly limiting the effectiveness of the drugs. To control diseases, it is often necessary to increase the drug dosage intake, and the intake of excessive drugs will cause drug resistance, making the diseases more difficult to cure. Chemotherapy drugs used for disease treatment, especially cancer treatment, often have high toxic and side effects and weak targeted treatment characteristics, bringing great pain to patients during the treatment process. The introduction of nanocarriers can reduce toxicity and improve treatment accuracy, becoming a research hotspot in the biomedical field. Amphiphilic polypeptides are widely used as nanodrug carriers due to their natural protein sequences and excellent biocompatibility. Amphiphilic polypeptides can effectively form micelles with uniform particle size and appropriate size under the combined action of hydrophilic and hydrophobic properties. The abundant functional groups can also effectively load drugs, improve the water solubility of drugs, and achieve precise drug delivery by utilizing the physiological characteristics around the disease.

[0004] At the same time, pure amphiphilic polypeptides usually lack traceability, making it difficult to directly monitor their delivery process in the body. For this reason, experimentalists often add fluorescent proteins or dyes for real-time tracking. However, such fluorescent materials are expensive, prone to photodegradation and bleaching, have poor biocompatibility, and may bring certain toxic and side effects.

[0005] Therefore, it is quite beneficial to prepare an amphiphilic polypeptide drug carrier with low toxicity or non-toxicity that can be applied to biomedicine, achieve real-time tracking, have multiple responses to environmental changes, and exhibit one or more advantages (such as tumor suppression, dental repair, bone repair, antibacterial and anti-inflammatory, ability to cross the blood-brain barrier, etc.). Summary of the Invention

[0006] One aspect of the present application discloses a structural design of covalent connection between biomass carbon dots (CDs) and poly-L-cysteine-S-S-carboxylic acid amphiphilic derivatives (P-L-cys). The method includes the structural design, synthesis and application of a poly-L-cysteine-S-S-carboxylic acid amphiphilic polypeptide (P-L-cys) modified with biomass carbon dots (CDs). It includes the synthesis of P-L-cys containing a hydrophobic group, with carboxyl groups providing hydrophilicity and being functionalizable, and disulfide bonds providing reduction responsiveness, as well as the synthesis of CDs and CDs-modified P-L-cys (CDs@P-L-cys). And micelles or hydrogels of P-L-cys and CDs@P-L-cys are constructed in an aqueous solution.

[0007] Another aspect of the present application discloses P-L-cys and CDs@P-L-cys for real-time tracking and bioimaging. The method includes the selection of chromophores during the construction of P-L-cys molecules, the loading and modification of CDs, so as to form a P-L-cys and CDs@P-L-cys material that generates fluorescence under the irradiation of excitation light.

[0008] Another aspect of the present application discloses a drug carrier with a synergistic photothermal and photodynamic therapy effect. The method includes the selection of anticancer drugs, the binding methods of drugs (including but not limited to covalent binding, electrostatic interaction, hydrogen bond and coordination coupling, hydrophilic and hydrophobic coating), the influence of the covalent connection of CDs on the primary and secondary structures of P-L-cys, using carbon dots to provide photothermal and photodynamic assisted therapy, and quantitative analysis of drug release by fluorescence quenching recovery.

[0009] Another aspect of the present application discloses P-L-cys and CDs@P-L-cys that can be used for gene therapy with gene targeting. The method includes the selection of antibodies loaded on P-L-cys and CDs@P-L-cys (such as anti-Her2 antibody, anti-LAG3 antibody, anti-PD-1 antibody, anti-PD-L1 antibody), and the selection of anticancer drugs. Loading P-L-cys and CDs@P-L-cys with anti-Her 2 monoclonal antibody to achieve targeted therapy for Her 2-positive patients, thereby improving the drug treatment efficiency and reducing the drug toxicity and side effects.

[0010] Another aspect of the present application discloses a drug carrier that can be used for the repair, antibacterial, and anti-inflammatory of tissues such as skin, teeth, and bones. The method includes functionalizing hydroxyapatite and tea polyphenols with P-L-cys and CDs@P-L-cys to construct a drug carrier containing hydroxyapatite and tea polyphenols. The ALN and EGCG carried by P-L-cys and CDs@P-L-cys can jointly care for tissues such as periodontium or bones, that is, promote tissue repair, osteogenesis, anti-inflammatory, and antibacterial. At the same time, the fluorescence effect of P-L-cys and CDs@P-L-cys can be used to further observe the tissue repair situation and realize the feedback on the efficacy of the drug carrier.

[0011] For the combination methods and methods described in the present application, optional features (including but not limited to components, compositions, composition ranges, degrees of polymerization, and conditional steps) are considered and selected from various aspects, examples, and instances provided or mentioned herein.

[0012] Referring to the following detailed description and drawings, the applications of the examples, the advantages and characteristics of the present application are obvious and worthy of research compared with ordinary researchers. The structural construction of P-L-cys and CDs@P-L-cys is easily affected by different research topics, application environments, and specific examples, which fully demonstrates the advantages of adaptability and modifiability of P-L-cys and CDs@P-L-cys. It should be noted that in the following specific examples, it is only illustrative content and not deliberately limited to the specifically described examples. Brief Description of the Drawings

[0013] Figure 1 Preparation process of polycysteine derivative containing disulfide bond (P-L-cys) Figure 2 Schematic diagram of covalent connection between CDs and naphthyl-L-cysteine-S-benzenesulfinic acid derivative Figure 3 of P-L-cys 1 H NMR spectrum Figure 4 NCDs, RCDs, P 12 , P 30 , NCDs@P 30 and RCDs@P 30 FTIR spectra Figure 5 Using dynamic light scattering method (DLS) to analyze and determine P 12 , P 30 , NCDs@P 30 and RCDs@P 30 critical micelle concentration (CMC) Figure 6 P12 , P 30 , NCDs@P 30 and RCDs@P 30 Particle size-ζ potential diagrams Figure 7 TEM images: a.) P 12 , b.) P 30 , c-d.) NCDs@P 30 , e-f.) RCDs@P 30 . Scale bars are: 200 nm and 100 nm Figure 8 P 12 / CDDP, P 30 / CDDP, NCDs@P 30 / CDDP and RCDs@P 30 In vitro simulated drug release diagrams Figure 9 Cytotoxicity diagrams (half maximal inhibitory concentration) of P12 / CDDP, P30 / CDDP, NCDs@P30 / CDDP and RCDs@P30 / CDDP against free CDDP in HCT116 cells 10 P 12 / CDDP, P 30 / CDDP, NCDs@P 30 / CDDP and RCDs@P 30 Cytotoxicity diagrams (PTT / PDT) of / CDDP, P Figure 11 NCM460 with free CDDP, P 12 / CDDP, P 30 / CDDP, NCDs@P 30 / CDDP and RCDs@P 30 Cytotoxicity diagrams after co-incubation with NCM460 for 24 h, 48 h, 72 h Figure 12 NCDs, RCDs and P 12 , P 30 , NCDs@P 30 and RCDs@P 30 Intracellular fluorescence imaging diagrams Figure 13 P 12 / CDDP, P 30 / CDDP, NCDs@P 30 / CDDP and RCDs@P 30 Intracellular fluorescence imaging diagrams after co-incubation with HCT116 for 0 h and 6 h Figure 14 (a) Tumor tissue volume change diagram of mice during treatment, (b) Body weight change diagram of mice during treatment, (c) Tumor weight diagram of each group after dissection, (d) In vivo fluorescence imaging diagram of mice at different times after injection of drug-loaded micelles Specific implementation manners

[0014] As used herein and unless otherwise specified, "P-L-cys" represents a poly-L-cysteine-S-S-carboxylic acid derivative with L-cysteine as the main chain, containing a hydrophobic group, a disulfide bond and a carboxyl group. CDs@P-L-cys is a unique structural design constructed by covalently connecting biomass carbon dots to the above-mentioned P-L-cys main chain.

[0015] One aspect of the present application discloses a structural design of covalent connection between biomass carbon dots (CDs) and poly-L-cysteine-S-S-carboxylic acid amphiphilic derivatives (P-L-cys). The method includes the structural design synthesis and application of a poly-L-cysteine-S-S-carboxylic acid amphiphilic polypeptide (P-L-cys) modified with biomass carbon dots (CDs). It includes the synthesis of P-L-cys containing a hydrophobic group, a carboxyl group providing hydrophilicity and functionality, and a disulfide bond providing reduction responsiveness, and the synthesis of CDs and CDs-modified P-L-cys (CDs@P-L-cys). And micelles or hydrogels of P-L-cys and CDs@P-L-cys are constructed in an aqueous solution.

[0016] In the present application, the hydrophobic end of P-L-cys mainly uses 1-naphthylamine, and the hydrophilic group uses mercaptopropionic acid. In some embodiments, the hydrophobic end can use aromatic amino compounds such as benzylamine, and alkyl amino compounds such as isobutylamine, octylamine, dodecylamine, hexadecylamine, etc., and the hydrophilic group uses compounds containing a carboxyl group and a mercapto group such as mercaptoacetic acid, mercaptobutyric acid, mercaptoisobutyric acid, etc., to form P-L-cys with pH / reduction dual responsiveness.

[0017] There are environmental conditions in tumor tissues that are significantly different from normal tissues. The tumor tissue has an acidic environment caused by lactic acid accumulation, which leads to protonation of the carboxyl group and changes in the secondary structure of P-L-cys to produce a pH response; and a strong reducing environment of high glutathione (GSH) metabolites, which can effectively promote the cleavage of the disulfide bond in P-L-cys. Under the pH / reduction dual-responsive conditions, the release of drugs in normal tissues is avoided, thereby reducing the toxic and side effects on normal tissues and increasing the enrichment of drugs at the lesion site.

[0018] In each embodiment, the solvent influence removal methods of P-L-cys solid powder include, but are not limited to, the methods mentioned below, such as organic phase extraction, rotary evaporation, long-term dialysis, freeze drying, reducing the solvent usage amount, washing the P-L-cys solid powder with water and centrifuging.

[0019] In each embodiment, the particle size of the P-L-cys micelles is 50 - 300 nm, and a micelle solution with an average diameter of 100 - 200 nm is optionally selected; in each embodiment, the concentration of the P-L-cys micelles for injection should not exceed 10 mg / L; in some embodiments, the P-L-cys hydrogel should be in a liquid gel state, and the P-L-cys concentration should be lower than 10 g / L.

[0020] In each embodiment, the purification method of the P-L-cys micelles and hydrogels should use ultrapure water, sterile water or PBS solution as the dialysis medium; to make the product more pure, a dialysis bag with a similar cut-off molecule should be selected according to the degree of polymerization for dialysis, and at the same time, the dialysis duration and the frequency of changing the dialysis solution can be increased to further purify.

[0021] In each embodiment, the base is a metal hydroxide such as sodium hydroxide, potassium hydroxide, cesium hydroxide, etc. and their combinations.

[0022] Another aspect of the present application discloses P-L-cys and CDs@P-L-cys for real-time tracking and bioimaging. The method includes the selection of a chromophore group during the construction of the P-L-cys molecule and the loading modification of CDs, thereby forming a P-L-cys and CDs@P-L-cys material that produces fluorescence under the irradiation of excitation light.

[0023] In some embodiments, the fluorescent agent is selected as 1-naphthylamine as the hydrophobic group; in some embodiments, the fluorescent agent is selected as carbon dots.

[0024] Another aspect of the present application discloses a drug carrier with a photothermal and photodynamic synergistic therapeutic effect. The method includes the selection of an anticancer drug, the binding method of the drug (including but not limited to covalent binding, electrostatic interaction, hydrogen bond and coordination coupling, hydrophilic and hydrophobic coating), the influence of the covalent connection of CDs on the primary and secondary structures of P-L-cys, using carbon dots to provide photothermal and photodynamic adjuvant therapy, and quantitatively analyzing the drug release by fluorescence quenching recovery.

[0025] In each embodiment, the drug concentration of the drug-loaded group for tumor treatment should be exactly the same as that of the free drug group, while ensuring that the solvent volume of each group is the same, and the blank group should use the same volume of pure solvent.

[0026] In each embodiment, the solvent of the drug-loaded micelles is selected as sterile PBS solution or sterile sodium chloride injection.

[0027] In some embodiments, the mass ratio of carbon dots to P-L-cys is 1:10 to 1:2. In each embodiment, the particle size of the carbon dots is 20 nm or less, and carbon dots with a particle size less than 10 nm and not easily agglomerated are preferred.

[0028] In some embodiments, the hydrophilicity / hydrophobicity and fluorescence properties of the carbon dots are selected according to specific requirements.

[0029] Another aspect of the present application discloses P-L-cys and CDs@P-L-cys that can be used in gene therapy with gene targeting. The method includes the selection of P-L-cys and the antibody loaded on CDs@P-L-cys (such as anti-Her2 antibody, anti-LAG3 antibody, anti-PD-1 antibody, anti-PD-L1 antibody), and the selection of anti-cancer drugs. Loading P-L-cys and CDs@P-L-cys with anti-Her2 monoclonal antibody can achieve targeted therapy for Her2-positive patients, thereby improving the drug treatment efficiency and reducing the drug toxicity and side effects.

[0030] Another aspect of the present application discloses a drug carrier that can be used for the repair of tissues such as skin, teeth, and bones, as well as antibacterial and anti-inflammatory effects. The method includes functionalizing P-L-cys and CDs@P-L-cys with hydroxyapatite and tea polyphenols to construct a drug carrier containing hydroxyapatite and tea polyphenols. At the same time, the fluorescence effect of P-L-cys and CDs@P-L-cys can be used to further observe the tissue repair situation and achieve feedback on the efficacy of the drug carrier. In various embodiments, drugs for tumor suppression can be selected from cisplatin, anti-Her2 antibody, anti-LAG3 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CLDN18.1 antibody, 4-hydroxytamoxifen, paclitaxel, doxorubicin, folic acid, gold nanoparticles, and stored in a suitable temperature and liquid environment.

[0031] In various embodiments, the mineralized drugs for periodontal treatment and bone repair can be freely combined from the following groups: hydroxyapatite (ALN), fluorine-substituted hydroxyapatite, silicon-doped hydroxyapatite, carbonate hydroxyapatite, biphasic calcium phosphate, nano-hydroxyapatite, collagen, hyaluronic acid composite materials. In various embodiments, the drugs for anti-inflammatory and bactericidal effects can be freely combined from the following groups: kaempferol, luteolin, artemisinin, apigenin, hesperidin, rutin, naringenin, myricetin, tetracycline, penicillin. Example

[0032]

[0033] This example provides a preparation process of P-L-cys containing a disulfide bond with an alkyl / naphthyl group as the hydrophobic group and a propionic acid group as the hydrophilic group.

[0034] (1) Under ice bath and N2 atmosphere, dissolve 20 mmol of L-cysteine hydrochloride in 20 mL of 2 M hydrochloric acid solution, and stir for 30 min to remove dissolved oxygen. Subsequently, slowly add dropwise 10 mL of 0.02 M aqueous NaNO2 solution until the solution turns blood red, and continue the reaction for 1 h. Then, slowly drip 20 mL of 40 mmol aqueous sodium benzenesulfinate solution. Stop stirring after the system changes from pink to milky white. Seal the system and place it in a 5 °C refrigerator overnight. The obtained milky white suspension is filtered, washed three times with 100 mL of ultrapure water in sequence, and then washed three times with 100 mL of absolute ethanol. The washed white solid is transferred to 2 L of ultrapure water, heated to boiling for dissolution, filtered while hot and cooled. Finally, a pale yellow crystalline L-cysteine-S-benzenesulfinic acid derivative is obtained, which is filtered and dried for later use.

[0035] (2) Take 12 mmol of the product from step (1) and dissolve it with 8 mmol of triphosgene in 60 mL of THF. Place the reaction system at 50 °C under N2 protection and react fully for 3 h. The obtained solution is deoxygenated by dissolving and precipitating THF and n-hexane in multiple cycles to remove unreacted triphosgene and by-products, and a solid powder of L-cysteine-S-benzenesulfinic acid cyclic anhydride derivative is obtained.

[0036] (3) Completely dissolve 37.5 mmol of the product from step (2) with 2.84 mmol and 1.82 mmol of 1-naphthylamine or n-dodecylamine in 120 mL of anhydrous DMF. React fully for 72 h under ice bath and N2 protection, and then dialyze for 72 h using a 1000 Da dialysis bag to obtain alkyl / naphthyl-L-cysteine-S-benzenesulfinic acid derivatives with two degrees of polymerization. Use 1 1H NMR to calculate its average degree of polymerization. Redissolve it in DMF and redialyze and lyophilize it using a dialysis bag with the corresponding molecular weight cut-off to obtain solid powders with degrees of polymerization of 12 and 30.

[0037] (4) Completely dissolve 1 mmol of the product from step (3) in DMF, and add 12 mmol and 30 mmol of 3-mercaptopropionic acid according to its degree of polymerization. React fully for 4 h at room temperature, then dialyze for 72 h using a 1000 Da dialysis bag, and lyophilize to obtain solid powders of P 12 ,P 30 with degrees of polymerization of 12 and 30.

[0038] In the examples, the degrees of polymerization of 12 and 30 are the results obtained through detailed experiments. The amino substances can be added or reduced according to requirements to decrease or increase the degree of polymerization of P-L-cys. Generally speaking, the larger the degree of polymerization, the more carboxyl groups, and the stronger the hydrophilicity; the smaller the degree of polymerization, the smaller the carboxyl groups, and the stronger the hydrophobicity. Therefore, P-L-cys can also be designed into amphiphilic polypeptides with different hydrophilic and hydrophobic properties according to different requirements, reflecting the controllability of the structure.

[0039] Through 1 Characterizations such as 1H NMR, FTIR, and XPS can be used to indicate the successful preparation of P-L-cys.

[0040]

[0041] This example provides a preparation process of biomass carbon dots and the preparation process of covalently connecting the obtained carbon dots with the P-L-cys formed in Example 1.

[0042] (1) Preparation of carbon dots: The peeled industrial hemp stalks are ground into powdery solids by an industrial grinder. Subsequently, they are fully mixed with 0.1 M NaOH solution at a mass ratio of 1:50 and heated to boiling for 5 min to promote the decomposition of crude fiber into small molecule cellulose, lignin, etc. in an alkaline environment. The mixture is left to stand at room temperature for 3 days to ensure the full degradation of the crude fiber. Subsequently, it is filtered through medium-speed quantitative filter paper, and the industrial hemp leachate is collected and stored in the refrigerator for later use. Take 50 mL of the industrial hemp leachate, add 0.5 g of urea as the nitrogen doping source, fully dissolve and mix evenly, then transfer it to a hydrothermal autoclave and react at 200 °C for 6 h. The filling rate of the hydrothermal autoclave is about 50%. The resulting brown solution is filtered, centrifuged, dialyzed, and freeze-dried to finally obtain the NCDs solid powder. Take 10 mg of the NCDs solid powder and 20 mg of o-phenylenediamine and dissolve them in 100 mL of methanol solution. Slowly add 60 mL of 0.1 M NaOH solution dropwise, fully mix evenly, and then slowly add 5 mL of 30% H2O2. The reaction continues at room temperature for 24 h. During this period, the solution gradually changes from light yellow to dark red, indicating the successful complexation of o-phenylenediamine with NCDs. Subsequently, the resulting dark red solution is further reacted in a microwave synthesizer (P100, 6 min). Finally, the obtained RCDs solution is filtered, centrifuged, dialyzed, and freeze-dried to obtain the RCDs solid powder.

[0043] (2) Covalently connect the P-L-cys with a degree of polymerization of 30 in Example 1 (3) to the carbon dots (NCDs, RCDs) after oxalyl chloride acylation to form amide bonds in a DMF environment.

[0044] (3) Obtain CDs@P-L-cys containing disulfide bonds in the same manner as in Example 1 (4).

[0045] In the examples, the synthesis of carbon dots is just the most common "bottom-up method" among many carbon dot synthesis routes. Here, the synthesis route of carbon dots does not limit their synthesis methods and processes.

[0046] Through 1 Characterizations such as 1H NMR, FTIR, and XPS can be used to indicate the successful preparation of CDs@P-L-cys.

[0047] This example provides a preparation process for P-L-cys to form micelles and hydrogels in an aqueous solution environment.

[0048] The P-L-cys and CDs@P-L-cys solid powders obtained in Examples 1 and 2 are used to further construct micelles and hydrogels.

[0049] (1) Preparation of micelles: Mix the P-L-cys and CDs@P-L-cys solid powders at a concentration of 1 g / L with PBS (0.01 M, pH 7.4), and adjust the pH to about 8.0 with 0.5 M NaOH to completely dissolve them. Let the solution stand for 2 h and it can self-assemble into a micelle solution.

[0050] (2) Preparation of hydrogels: Mix the P-L-cys and CDs@P-L-cys solid powders at a concentration of 100 g / L with PBS (0.01 M, pH 7.4), and adjust the pH to about 8.0 with 0.5 M NaOH to completely dissolve them. Let the solution stand for 2 h, then adjust its pH to about 5.5 with 0.5 M HCl, mix well, and let it stand for another 2 h to self-assemble into a hydrogel.

[0051] Through the critical micelle concentration (CMC), TEM, Size-Zeta, and physical pictures, it can strongly prove the successful preparation of P 12 , P 30 , NCDs@P 30 , RCDs@P 30 micelles and hydrogels (as Figures 5-7 ). 4. Comparative example: Attempt to construct NCDs@P 12 , RCDs@P 12 micelles and hydrogels Covalently connect NCDs / RCDs with P 12 in the same manner as in Examples 1 and 2, and the same solid powder product as P 12 can be obtained. It is speculated that due to the shorter chain length of P 12 , the covalent connection of NCDs / RCDs increases the steric hindrance, making NCDs@P 12 , RCDs@P 12It is unable to effectively form micelles and hydrogels.

[0052] P-L-cys is an amphiphilic polypeptide derivative constructed from L-cysteine. The carbonyl group and secondary amine in the peptide bond can form more complex secondary structures through intramolecular and intermolecular hydrogen bonding in aqueous solution. The formation of the secondary structure of amphiphilic polypeptides is determined by the combined influence of their amino acid sequence, hydrophilic-hydrophobic interactions, intramolecular and intermolecular interactions, and environmental conditions. Due to the abundance of amide bonds (-CONH-) on the polypeptide backbone, where the secondary amine (-NH-) serves as the hydrogen bond donor and the carbonyl group (-C=O) serves as the hydrogen bond acceptor, and intermolecular and intramolecular hydrogen bonds are formed in aqueous solution, further promoting the polypeptide chain to generate different secondary structures.

[0053] (1) P-L-cys and CDs@P-L-cys solutions were prepared at a solution concentration of 0.1 g / L at different pH values (5.5, 6.2, 7.4, 8.5) and characterized by combining FTIR and circular dichroism spectroscopy.

[0054] This example provides a preparation process for P-L-cys drug-loaded micelles using CDDP as an anticancer model drug.

[0055] (1) The P-L-cys and CDs@P-L-cys micelles obtained according to Example 3 were coordinated and loaded with an equal mass of CDDP in an aqueous solution environment. And its release amount and release situation were speculated by in vitro simulation.

[0056] (2) The P-L-cys and CDs@P-L-cys drug-loaded micelles were irradiated with near-infrared light (NIR, 650 nm) to observe the photothermal and photodynamic effects (PTT / PDT) to determine whether the loading of NCDs / RCDs can effectively improve the PTT / PDT synergistic effect.

[0057] (3) The anticancer effectiveness of the P-L-cys and CDs@P-L-cys drug-loaded micelles and the PTT / PDT synergistic treatment effect were systematically and comprehensively analyzed through cytotoxicity, cell morphology, cell migration, flow cytometry, and in vivo experiments on tumor-bearing mice.

[0058] (4) Cytotoxicity: Well - conditioned HCT116 cells (human colorectal cancer cells) were seeded into 96 - well plates at a density of 4000 cells / well. After the cells adhered and grew to a confluence of 50 - 60%, the cells were treated with different concentrations and different experimental groups for 24 h, 48 h, and 72 h. The grouping was as follows: blank control group (PBS), blank micelle group (P - L - cys and CDs@P - L - cys), free CDDP group, and drug - loaded carrier group of CDDP (P - L - cys / CDDP and CDs@P - L - cys / CDDP). Finally, according to the treatment concentrations of free CDDP (①0.6 μg / mL, ②1.5 μg / mL, ③3 μg / mL, ④4.5 μg / mL, ⑤6 μg / mL), the concentrations of different drug - loaded carrier groups and blank micelle groups were configured. On this basis, a near - infrared light irradiation group was set up to explore the effect of near - infrared light - assisted treatment. The treatment method was that the normal drug - administration group was treated in the dark for 24 h, while the light - irradiation group was treated with near - infrared light (680 nm, 0.6 W / cm - 2, 5 min) at the end. Finally, their effects were compared. The above - mentioned micelles and drug - loaded micelles were configured into 100 mL of PBS solution and mixed with 1900 mL of complete DMEM medium (DMEM:FBS:antibiotics, 100:10:1), and then 200 μL per well was used to treat HCT116 cells.

[0059] (5) MCF - 7 (human breast cancer cells) tumor - bearing model mice were established by subcutaneous injection of 1 × 10 6Female BALB / C-nu mice at 5 weeks of age were constructed by injecting V MCF-7 cells (80 μL of normal saline with 25% Matrigel). Since MCF-7 cells are estrogen-positive cells, not only female mice were used as research models, but 17β-estradiol benzoate (0.8 mg / kg / mouse) should be intraperitoneally injected into the mice 1 - 2 days before MCF-7 cell injection to induce the proliferation and growth of MCF-7 cells. The monitoring of the tumor volume and the tumor mass minus the body weight of the mice started from 3 days after cell injection. According to the pre-grouping situation, when the average volume of the tumor-bearing mice ≥ 80 mm3, the mice were randomly divided into 6 groups (n = 4), and named as the control group, free CDDP group, drug-loaded micelle groups (P-L-cys / CDDP and CDs@P-L-cys / CDDP), and NIR irradiation group (NIR-CDs@P-L-cys / CDDP). The administration method was intraperitoneal injection (150 μL), and the injection was performed on days 1, 3, 5, 7, and 9. Since CDDP has relatively large side effects, the drug was injected in a way of increasing concentration (the solvent was sterile normal saline), with the injection method of 1 mg / kg / mouse for the first time, 2 mg / kg / mouse for the second and third times, and 3 mg / kg / mouse for the fourth and fifth times, aiming to make the mice adapt to the side effects brought by CDDP. Among them, 2 h after the drug injection in Group 6, NIR irradiation (660 nm, 0.5W / cm-2, 2 min) was performed, and the infrared imager was used to photograph and record the local temperature change of the tumor. The distance between the light source and the tumor was 30 cm, aiming to reduce the pain of the mice being burned or scalded under continuous red light irradiation. When the 5-time drug administration was completed, the drug administration was stopped, and the mice were continuously fed for 3 days before reaching the experimental end point. The tumors and important organs (heart, liver, spleen, lung, kidney) of each group of mice were taken out for the next experimental detection. During the whole experiment, the body temperature, body weight of the mice, and the size of the tumors were measured at the same time period every day. Among them: the volume of the tumor ( = short diameter a 2 × long diameter b / 2 , unit: mm), and the body weight of the mice minus the tumor weight ( m = m2 (body weight of the tumor-bearing mice)- m 1 (tumor volume × 10 -3 ), unit: g).

[0060] After the above various analytical characterizations, the anti-cancer effectiveness of P-L-cys and CDs@P-L-cy as drug-loaded micelles can be verified, and whether they can effectively reduce the toxic and side effects of CDDP on normal tissues.

[0061] This example provides the preparation processes of P-L-cys and CDs@P-L-cys for real-time tracking and bioimaging.

[0062] (1) Incubate the P-L-cys micelles and CDs@P-L-cys micelles constructed in Examples 3 and 6 with live cells. Then, use a live cell workstation and a laser confocal microscope to capture the fluorescence intensity inside the cells, and perform quantitative analysis of the fluorescence intensity through ImageJ to infer the optimal imaging micelle group.

[0063] (3) Meanwhile, CDDP is an excellent fluorescence quencher. The absorption of CDDP in the ultraviolet-visible light range produces a light shielding effect, reducing the energy transfer of the excitation light. In addition, as a heavy metal complex, CDDP may cause the micelle spacing to shrink to 1-10 nm, inducing the fluorescence resonance energy transfer (FRET) effect and enhancing non-radiative transitions. At the same time, the high light absorption of CDDP may trigger the inner filter effect, interfering with fluorescence detection. The change in the micelle structure and the aggregation effect of the fluorophore further exacerbate fluorescence quenching.

[0064] (4) Therefore, incubate the CDDP-loaded drug micelles with tumor cells or apply them to tumor treatment in mice. By timing (such as 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, etc., including but not limited to these time periods), take in vivo fluorescence images of the tumor cells or the tumors on the surface of the mice. The release of CDDP can be qualitatively and quantitatively analyzed based on the intensity of the fluorescence.

[0065] This example provides the preparation processes of P-L-cys and CDs@P-L-cys for gene therapy with gene targeting properties.

[0066] (1) Take P-L-cys and CDs@P-L-cys prepared in Examples 1 to 3 as examples, including but not limited to using naphthyl as the hydrophobic group, using 3-mercaptopropionic acid to construct disulfide bonds and introducing carboxyl groups.

[0067] (2) Covalently link the obtained solid powders of P-L-cys and CDs@P-L-cys with anti-Her 2 monoclonal antibody in an alkaline solution through the EDC / NHS coupling chemical method, introducing monoclonal antibody substances with active targeting for breast cancer without destroying the activity of the monoclonal antibody.

[0068] (3) Perform an esterification reaction between the solid powders of P-L-cys and CDs@P-L-cys loaded with Her 2 monoclonal antibody and 4-hydroxytamoxifen to effectively load anticancer drugs.

[0069] In this embodiment, the selection of targets is not limited to Her 2 monoclonal antibodies, and anti-LAG3 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CD20 antibodies, anti-CLDN18.1 antibodies, anti-CD44 antibodies, anti-CD34 antibodies, etc. can also be selected; and the selected anti-tumor drugs include but are not limited to 4-hydroxytamoxifen, and platinum anticancer drugs, paclitaxel, doxorubicin, nanogold, graphene, carbon dots, quantum dots, etc. can also be selected.

[0070] This embodiment provides a process for preparing a drug carrier for repairing tissues such as teeth or bones and for antibacterial and anti-inflammatory effects.

[0071] The limitation of periodontal and bone repair is that the lesions cannot effectively capture free Ca 2+ , which makes it difficult to repair bones or teeth. Inflammation and infection at the lesion site often cause the surrounding immune cells to be in a highly activated state, which can easily inhibit the repair of the lesion site. At the same time, due to the deep location of bone damage and lesions, it is often impossible to directly and clearly see the intuitive repair effect of the drug carrier on them, so it is often necessary to add certain fluorescent tracer substances for tracking and observation. Such fluorescent substances often have to be combined with bone characteristics or bone-targeted substances to avoid the fluorescent substances combining with normal cells, resulting in the inability to effectively judge the repair of bones or teeth.

[0072] (1) Taking the PL-cys and CDs@PL-cys micelles and hydrogels prepared in Examples 1 to 3 as examples, including but not limited to using naphthyl as a hydrophobic group, using 3-mercaptopropionic acid to construct a disulfide bond and introduce a carboxyl group, and using carbon dots as a fluorescent imaging tracer substance.

[0073] (2) The obtained PL-cys and CDs@PL-cys solid powders are combined with the calcifier hydroxyapatite (ALN) and the antibacterial and anti-inflammatory agent epigallocatechin-3-glucose (EGCG), which simultaneously achieve the dual effects of bone or tooth repair and bactericidal and anti-inflammatory effects.

[0074] (3) At the same time, by utilizing the fluorescence characteristics, fluorescence can be effectively generated by irradiating with specific excitation light, and fluorescence images can be taken to further speculate the effectiveness of the PL-cys drug delivery system for tooth or bone repair.

[0075] This embodiment provides a preparation process of PL-cys that can break through the blood-brain barrier.

[0076] The blood-brain barrier (BBB) is a specific physiological barrier in the brain and the central nervous system (CNS), which can selectively regulate the exchange of substances between the blood and the brain tissue. Under normal circumstances, the existence of the BBB protects the brain from harmful substances and maintains the stability of the brain internal environment.

[0077] However, the existence of the BBB causes drugs to be unable to enter the brain microenvironment. The tight arrangement of brain microvascular endothelial cells results in almost closed intercellular gaps, preventing passive transport and free diffusion of macromolecules. At the same time, the low pinocytosis activity of brain microvascular endothelial cells leads to reduced active transport, and the presence of P-glycoprotein (P-gp) and multidrug resistance protein (MRP) also makes it more difficult for drugs to cross the BBB. To make drugs transparent in the BBB, specific transport proteins (such as transferrin) or cell-penetrating peptides must be introduced into the drug carrier. (1) Taking the P-L-cys micelles and hydrogels prepared in Examples 1 to 3 as examples, including but not limited to using naphthyl as the hydrophobic group, using 3-mercaptopropionic acid to construct disulfide bonds and introduce carboxyl groups, and using carbon dots as fluorescent imaging tracer substances.

[0078] (2) Effectively covalently bind or couple the prepared P-L-cys solid powder with blood-brain barrier breakthrough carriers (including but not limited to transferrin receptor, low-density lipoprotein receptor-related protein, carbon dots, fluorescein, cell-penetrating peptides, magnetic targeting nanoparticles, integrin, CD47 antibody).

[0079] (3) Combine the obtained P-L-cys solid powder loaded with blood-brain barrier breakthrough carriers with drugs, such as anticancer drugs: doxorubicin, paclitaxel, cisplatin, carboplatin, etc., and apply them to human subjects or zebrafish to observe whether they can effectively break through the blood-brain barrier.

[0080] (a) Based on the experimental process routes of Examples 1 to 3, poly-L-cysteine-S-S-propionate derivatives with naphthyl as the hydrophobic end were constructed. At the same time, industrial hemp biomass carbon dots reacted with the secondary amines on the P-L-cys main chain and covalently connected through the formation of amide bonds. Finally, P 12 ,P 30 ,NCDs@P 30 ,RCDs@P 30 ,four different properties of P-L-cys were constructed.

[0081] The prepared P-L-cys solid powder was analyzed by X-ray diffraction (XRD), nuclear magnetic resonance hydrogen spectrum ( 1It was characterized by nuclear magnetic resonance (¹H NMR), Fourier transform infrared spectrometer (FTIR), X-ray photoelectron spectrometer (XPS) or various characterization methods equivalent to the above. Through Figure 3 1 The characterization by ¹H NMR clearly showed that the peak positions at each chemical shift corresponded to the structure of P-L-cys as expected. At the same time, Figure 4 The characterization by FTIR further supplemented the characterization results, fully demonstrating the successful preparation of P-L-cys (b) Preparation of P-L-cys micelles: Dissolve P 12 , P 30 , NCDs@P 30 , RCDs@P 30 in ultrapure water or PBS solution at a concentration of 1 g / L, slowly adjust to pH 8.0 with 0.5 M NaOH, dissolve the solid particles by mixing, ultrasonic treatment, etc., and let stand for 2 h to obtain the P-L-cys micelle solution.

[0082] (c) Preparation of P-L-cys hydrogel: Dissolve P 12 , P 30 , NCDs@P 30 , RCDs@P 30 in ultrapure water or PBS solution at a concentration of 100 g / L, slowly adjust to pH 8.0 with 0.5 M NaOH, dissolve the solid particles by mixing, ultrasonic treatment, etc., and let stand for 2 h. Then continue to add 0.5 M HCl to adjust the pH to about 5.5 and let stand for 2 h to obtain the P-L-cys hydrogel.

[0083] The prepared P-L-cys micelle solution was characterized and analyzed by critical micelle concentration (CMC), particle size-ζ potential, and transmission electron microscope (TEM). Figures 5 to 7 The characterization results showed that the particle size of P-L-cys micelles was 100 - 200 nm, which met the optimal particle size mentioned in this application, and P-L-cys could form micelles at a relatively low concentration in aqueous solution.

[0084] From Comparative Example 4, attempts were made to construct NCDs@P 12 , RCDs@P 12 micelles and hydrogels According to the same method as in Examples 1 and 2, NCDs / RCDs were covalently linked to P 12 to obtain the same solid powder product as P 12 , but in aqueous solution, due to P 12The shorter chain length enhances the hydrophobicity. The covalent connection between NCDs / RCDs increases the steric hindrance, resulting in NCDs@P 12 , RCDs@P 12 being unable to effectively form micelles and hydrogels. Therefore, Comparative Example 4 verifies that P-L-cys with a low degree of polymerization (less than or equal to 12) cannot effectively form micelles and hydrogels under the condition of covalent connection between NCDs / RCDs.

[0085] The P-L-cys used in Example 2 has a uniform particle size constructed in Example 1 and meets the optimal state mentioned in the examples. Based on Examples 6 and 7, Example 2 constructs P-L-cys drug-loaded micelles with fluorescence quenching recovery response and pH / reduction dual response. And cytotoxicity, cell imaging, cell morphology, flow cytometry, apoptosis and in vivo experiments on mice and other experiments are carried out to fully verify its effectiveness as a drug carrier, and it can highlight that the P-L-cys drug carrier loaded with carbon dots can almost eliminate the tumor tissue on the surface of mice through the synergistic treatment effect of photothermal and photodynamic therapy.

[0086] Specific experimental results can be obtained from Figures 8-14 to draw the following conclusions: For clearer identification, in the following figures or text descriptions, the P-L-cys drug carrier will be divided into (P 12 / CDDP, P 30 / CDDP, NCDs@P 30 / CDDP, RCDs@P 30 / CDDP).

[0087] (a) In the HCT116 cytotoxicity graph, it can be seen that although the cytotoxicity of the P-L-cys drug carrier is not as high as that of free CDDP, rapid high toxicity often easily induces drug-resistant cells, and free CDDP has no targeting property. Therefore, from the cytotoxicity graph of NCM460, we can see that the cytotoxicity of free CDDP to cells reaches about 20% at 72 h, while the cytotoxicity of the P-L-cys drug carrier is lower than 8%. This is because the pH / reduction dual response constraint of the P-L-cys drug carrier on CDDP makes CDDP unable to be directly released into normal cells, thus effectively reducing the cytotoxicity of normal cells.

[0088] (b) In the cell imaging and the cell imaging before and after drug release, it can be seen that the P-L-cys blank micelles can produce recognizable fluorescence inside the cells, and the carbon dot-loaded P-L-cys micelles effectively increase the fluorescence intensity of the P-L-cys micelles in the cells. At the same time, it is also proved that the P-L-cys micelles can be successfully taken up by the cells and distributed in the cytoplasm to produce fluorescence. Due to the fluorescence quenching effect of CDDP, the P-L-cys drug carrier can hardly observe fluorescence when incubated with cells at the beginning, but recognizable fluorescence can be seen inside the cells after incubating with cells for a period of time, indicating that CDDP has been successfully released into the cells. (c) The exploration of drugs, drug delivery systems, etc. in the biomedical field needs to be supported and developed by the results of animal experiments. If it only stays at the cell experiment stage, the pharmacokinetic properties of drugs cannot be effectively evaluated, and the potential toxic and side effects of drugs in the human body in the future cannot be evaluated.

[0089] In the in vivo experiment of mice, Group 1 is usually used to represent the blank control group, which is only injected with an equal volume of normal saline; Group 2 represents the free CDDP group; Group 3 represents the P 30 / CDDP drug-loaded micelle group; Group 4 represents the RCDs@P 30 / CDDP drug-loaded micelle group; Group 5 represents the NCDs@P 30 / CDDP drug-loaded micelle group; Group 6 represents the NIR-RCDs@P 30 / CDDP drug-loaded micelle group, where NIR refers to near-infrared light irradiation.

[0090] After 5 times of treatment every other day, it can be seen from the figure that only the tumor tissue of Group 1 injected with normal saline continues to grow, and the remaining treatment groups all show effective inhibitory effects. Especially under the synergistic treatment of NIR irradiation, the tumor of Group 6 hardly proliferates. After stopping the treatment, the tumor reduction rate of Group 4 slows down, indicating that continuous treatment is needed to maintain the anti-cancer effect. On the contrary, after stopping the treatment, the tumors of Group 2, Group 3 and Group 5 not only do not decrease or stop growing, but instead relapse, indicating that these groups cannot effectively control the growth of tumors and only continuous treatment can avoid tumor recurrence. This result further shows that the sustained release effect of Group 4 is excellent, and even 3 days after stopping drug injection, it can still effectively control tumor growth.

[0091] By monitoring the body weights of mice, it was found that only the body weights of mice in the free CDDP group decreased, indicating that the direct injection of free CDDP had relatively large drug toxic and side effects, leading to anorexia and indigestion in mice, and ultimately a decrease in body weight. In contrast, the body weights of mice in other groups gradually increased over time, reflecting their good health status and indicating that the drug-loaded micelles effectively reduced the toxic and side effects of CDDP and alleviated the pain of mice. At the same time, in Group6, since the tumor hardly grew anymore, the pain of the mice was significantly reduced. Therefore, the body weight change of the mice in this group was the most ideal, further verifying the excellent anti-cancer effect of the treatment plan in Group6.

[0092] The mice were sacrificed by euthanasia and the tumor tissues of each group were removed for size and weight measurement. It was found that the tumors of the mice in Group6 almost completely disappeared, and the tumor tissue of one mouse completely disappeared, fully demonstrating the high anti-cancer effect under PTT / PDT combined treatment. Although Group4 did not receive NIR irradiation, its treatment effect was still better than that of other groups, further verifying the potential of Group4 to inhibit tumor growth in in vivo experiments. In contrast, although Group5, which showed the best performance in in vitro experiments, failed to exhibit the same anti-cancer characteristics in in vivo experiments, which may be related to its relatively high drug release rate. The relatively fast drug release may lead to tumor recurrence after drug withdrawal and may induce drug-resistant cells, thus reducing the anti-cancer effect of chemotherapy drugs.

[0093] Since CDDP can cause fluorescence quenching of the drug-loaded micelles, we performed in vivo fluorescence imaging on the mice at the time of drug injection and 3 h and 4 h after drug injection. No fluorescence in the mice could be observed at the initial stage of drug injection because the loading of CDDP caused fluorescence quenching and the fluorescence could not be shown. As the drug entered the tumor tissue through the body fluid circulation and began to release the drug, CDDP detached from the micelles and the fluorescence of the micelles was restored, verifying our hypothesis. 3 h after drug injection, fluorescence could be observed in the tumor tissues of the mice, and the fluorescence was only visible in the tumor tissues, which not only proved the successful release of CDDP but also indicated that the drug was only released in the tumor tissues. According to the previous conclusion, the drug release rate of Group4 should be lower than that of the drug-loaded micelles of Group5 and Group3, but here the drug release amount of Group4 was greater than that of Group5 and Group3. We speculated that this phenomenon was not caused by the difference in drug release amount but because the fluorescence of NCDs@P 30 and P 30 was in the blue-green wavelength band, which was easily absorbed by organisms and overlapped with the fluorescence of organisms themselves, resulting in ineffective observation. While the fluorescence of RCDs@P 30 was less likely to be absorbed by organisms due to its excellent red light characteristics, thus ensuring excellent fluorescence characteristics in organisms.

[0094] The embodiments described above are some, but not all, of the embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application claimed, but merely represents selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts shall fall within the scope of protection of the present application.

Claims

1. Structural design, synthesis and application of a biomass carbon dot (CDs) modified poly L-cysteine-SS-carboxylic acid amphiphilic peptide (PL-cys), including PL-cys containing a hydrophobic group, carboxyl groups providing hydrophilicity and functionalization, disulfide bonds providing reduction response, and CDs and CDs modified PL-cys (CDs@PL-cys).

2. According to claim 1, the synthesis method of PL-cys and CDs@PL-cys comprises: (1) Through the intracyclic anhydride synthesis method and the ring-opening polymerization method, a hydrophobic amino-based polymerization initiator is used to promote the polymerization of L-cysteine ​​intracyclic anhydride, and finally a disulfide-bonded amphiphilic polypeptide PL-cys is constructed by mercapto acid (such as mercaptopropionic acid). At the same time, the degree of polymerization of PL-cys can be controlled by the amount of polymerization initiator. The molar ratio of polymerization initiator to L-cysteine ​​intracyclic anhydride is selected in the range of 1:40 to 1:

5. (2) The biomass carbon source is modified and activated, and precursors with different fluorescent properties and hydrophilic and hydrophobic properties are constructed according to the needs, and CDs rich in functional groups such as carboxyl and amino groups are synthesized by the "bottom-up method". (3) The CDs obtained in step (2) are subjected to an amide reaction to form an amide covalent bond between the carboxyl group of CDs and the secondary amine of PL-cys obtained in step (1), and finally CDs modified with CDs@PL-cys are synthesized.

3. According to claims 1 and 2, the structural modification of PL-cys can regulate its microstructure (primary structure, secondary structure) and have a significant impact on its self-assembly structure, thereby changing the critical gelation concentration and the stability of micelles and hydrogels, and affecting its macroscopic structural properties.

4. According to claims 1 to 3, in order to change the hydrophilicity and hydrophobicity of PL-cys and introduce new functions, the hydrophobic group of PL-cys can be changed and the degree of polymerization can be increased to adjust the hydrophilicity and hydrophobicity; new functions can be introduced by introducing different functional groups on the carboxyl group. At the same time, CDs with different hydrophilicity and hydrophobicity can be modified to regulate the self-assembly structure of PL-cys.

5. According to claims 1 to 4, the hydrophobic group of PL-cys can be selected from the following substances as the hydrophobic group: benzylamine, o-chloroaniline, benzylamine, aniline, p-tert-butylaniline, n-dodecylamine, cyclohexylamine, octylamine, tert-butylamine, diisopropylamine, etc.

6. According to claims 1 to 4, the functional groups can be selected from the following substances and combinations thereof: various biomass carbon dots (biomass carbon dots prepared from coffee waste, coconut shells, chestnut shells, and silkworm feces), quantum dots, graphene, active targeting antibodies (anti-Her2 antibodies, anti-LAG3 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies), anticancer drugs (cisplatin, 4-hydroxytamoxifen, paclitaxel, doxorubicin), mineralizers: (hydroxyapatite, fluorine-substituted hydroxyapatite, silicon-doped hydroxyapatite, carbonated hydroxyapatite) antibacterial agents: (gallic acid, quercetin, tea polyphenols, artemisinin, apigenin, naringenin, tetracycline, penicillin).

7. According to claims 1 to 4, the particle size of CDs is about 20 nm or less, and the optimal particle size is about 10 nm or less, and the CDs are evenly dispersed in the solution and are not easy to agglomerate.

8. According to the method described in any of the preceding claims, nitrogen-doped biomass carbon dots are designed to improve the fluorescence intensity and hydrophilicity of the biomass carbon dots; and o-phenylenediamine-composite biomass carbon dots are designed to increase the hydrophobicity of the biomass carbon dots and have red fluorescence.

9. The method according to claim 8, according to different requirements, the nitrogen doping method can be urea, ethylenediamine, polyethyleneimine, melamine, aniline, polyaniline, o-phenylenediamine, m-phenylenediamine, p-phenylenediamine, amino acids and combinations thereof; o-phenylenediamine as a complexing agent can also be replaced by aniline, polyaniline, o-phenylenediamine, m-phenylenediamine, p-phenylenediamine and combinations thereof.

10. The PL-cys or CDs@PL-cys according to any of the above claims is dissolved in ultrapure water, PBS solution, or sterile water at a concentration not exceeding 10 g / L, and the pH value is adjusted to 8.0 by alkaline dissolution to completely dissolve the solid, and the solution is allowed to stand for 2 h to obtain a micellar solution. When the concentration is greater than 10 g / L and less than 200 g / L, an alkaline solution is used to adjust the solid to completely dissolve, the solution is allowed to stand for 2 h, and the pH is adjusted to about 5.0 by an acidic solution, or a solution containing Ca is added. 2+ The hydrogel can be obtained by standing for 2 h with the addition of metal cations.

11. PL-cys and CDs@PL-cys for secondary structure change research, the method comprising the method of claim 2 and any of the above claims, preparing PL-cys with two or more polymerization degrees, and covalently linking them with CDs with different hydrophilic and hydrophobic properties to obtain CDs@PL-cys. And exploring the secondary structure changes and mutual conversion under the same concentration and different pH solvent environments.

12. According to the method of claim 11, the mass ratio of PL-cys to biomass carbon dots can be 100:1 to 1:1, and the optimal ratio range is 20:1 to 5:

1.

13. The method according to claim 12, wherein the concentration of PL-cys in the solution used to explore the changes in the secondary structure is 0.2 g / L or even lower.

14. PL-cys and CDs@PL-cys for real-time tracking and biological imaging, wherein the PL-cys and CDs@PL-cys obtained by the methods of claims 1 to 12 have fluorescence properties. When introduced into cells, experimental animals or human subjects, fluorescence can be observed under certain excitation light, thereby achieving biological imaging and real-time tracking.

15. A drug carrier with photothermal-photodynamic synergistic therapeutic effect, wherein the PL-cys and CDs@PL-cys obtained by the methods of claims 1 to 12 are loaded with anti-tumor drugs and constructed as drug carriers. The drugs are administered to experimental animals or even human subjects by oral administration, intraperitoneal injection, intravenous injection, etc., and the photothermal-photodynamic synergistic therapeutic effect generated under near-infrared light irradiation (650-1100 nm) inhibits tumor proliferation.

16. According to the method of claim 15, PL-cys and CDs@PL-cys will produce a fluorescence quenching effect after being loaded with cisplatin. When cisplatin is released in tumor cells or tumor tissues, the fluorescence will be restored. Through real-time tracking of fluorescence, qualitative and quantitative analysis of drug release can be achieved.

17. According to the method of claim 16, the following fluorescence quenchers can also be selected to modify the drug to achieve qualitative and quantitative analysis of drug release: gold nanoparticles, graphene oxide, QSY type, BHQ type.

18. A gene therapy drug carrier with gene targeting, wherein the PL-cys and CDs@PL-cys obtained by the methods of claims 1 to 12 are loaded with monoclonal antibodies to achieve gene targeting functionalization. For example, if PL-cys and CDs@PL-cys are loaded with anti-Her 2 monoclonal antibodies, targeted treatment can be achieved for Her 2-positive patients, thereby improving the efficiency of drug treatment and reducing drug toxicity and side effects. At the same time, according to claims 6 and 15, anti-tumor drugs are loaded to achieve synergistic treatment.

19. Used for repairing skin, teeth, bones and other tissues and for antibacterial and anti-inflammatory effects. According to the method described in claims 1 to 12, the obtained PL-cys and CDs@PL-cys are functionalized with hydroxyapatite and tea polyphenols as described in claim 6 to construct a hydrogel material containing mineralization and antibacterial components to promote tissue repair.

20. The method according to any of the above claims, wherein the obtained micelles are spherical micelles with a size of 80-300 nm and a zeta potential of -50 mV to -5 mV, preferably micelles with a particle size of 100-200 nm and a zeta potential of less than or equal to -20 mV.

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