CN113198022A - Multifunctional core-shell dendrimer copper complex and preparation and application thereof - Google Patents

Multifunctional core-shell dendrimer copper complex and preparation and application thereof Download PDF

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CN113198022A
CN113198022A CN202110468062.5A CN202110468062A CN113198022A CN 113198022 A CN113198022 A CN 113198022A CN 202110468062 A CN202110468062 A CN 202110468062A CN 113198022 A CN113198022 A CN 113198022A
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nhac
cstd
peg
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沈明武
宋聪
欧阳智俊
高悦
史向阳
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Donghua University
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Abstract

The invention relates to a multifunctional core-shell dendrimer copper complex and preparation and application thereof, wherein the preparation method and application comprise the following steps: (1) is divided intoThe shell component of the dendrimer (Ad-G3. NH) which is terminal at the amino group2) Three functionalized shell component dendrimers are obtained by modifying pyridine, a transmembrane polypeptide (DER) and a targeting polypeptide (RGD): Ad-G3.NH2‑Pyr、Ad‑G3.NH2‑PEG‑DER、Ad‑G3.NH2-PEG-RGD; (2) core component dendrimers (CD-G5. NH)2) Forming a host-guest nucleocapsid dendrimer nano material with three functionalized shell component dendrimers through supermolecule self-assembly and carrying out acetylation treatment; (3) and (3) directly complexing copper ions with the acetylated multifunctional core-shell dendrimer obtained in the step (2) to form a multifunctional core-shell dendrimer copper complex nano platform (M-CSTD. NHAc/Cu (II)). The invention has the advantages of easily obtained raw materials, simple synthesis process and easy operation, can cross the blood brain barrier of mice for brain tumor MR imaging and chemokinetic treatment, and has diagnosis and treatment integration performance and good application prospect.

Description

Multifunctional core-shell dendrimer copper complex and preparation and application thereof
Technical Field
The invention belongs to the technical field of functional hybrid nano platform preparation, and relates to a multifunctional core-shell dendrimer copper complex, and preparation and application thereof.
Background
In the field of nanomedicine, various nanoplatforms have been widely used for imaging and treatment of cancer. Among them, the polyamidoamine dendrimers have attracted much attention and much attention from researchers because of their characteristics of perfect spherical structure, monodispersity, good water solubility, nonimmunity and easy functionalization. However, since the size of the dendrimer of a single generation is small (e.g., the dendrimer of 5 generation is only 5.4nm), the dendrimer still has many disadvantages, such as limited drug loading, limited tumor passive targeting based on EPR effect, and lack of versatility of stimulating response. In order to overcome the limitations of single generation dendrimers, it has become the latest research direction of researchers to construct a nano platform based on a superstructure dendrimer with higher complexity, controllable size and structure by using the dendrimer as a reactive module or member, and to apply the nano platform to the advanced cancer nano-medicine field.
As is well known, Glioma (GBM) is one of the most common malignant intracranial tumors that severely harm humans worldwide, and is highly malignant, growing mostly in a wide invasive manner, aggressive and at high risk of recurrence. Moreover, brain gliomas are difficult to treat, mainly because of the unique substance exchange regulation mechanism, the Blood Brain Barrier (BBB), which protects the central nervous system. The BBB maintains central nervous system homeostasis, preventing penetration of most macromolecular substances or drugs with potential imaging or therapeutic effects, which greatly reduces drug or other substance accumulation at brain gliomas, thereby limiting diagnosis and treatment of brain gliomas. Therefore, in order to implement accurate diagnosis and efficient treatment of brain glioma, a reasonable diagnosis and treatment system is necessary to be designed, so that the blood brain barrier can be broken through in the conveying process, and enrichment in tumor tissues can be realized, thereby achieving the purpose of exerting efficient diagnosis and treatment effects.
The research on the integration of diagnosis and treatment by using multifunctional core-shell dendrimer complex copper ions is not found in the domestic and foreign literature retrieval.
Disclosure of Invention
The invention aims to provide a multifunctional core-shell dendrimer copper complex, and preparation and application thereof, which can cross the blood brain barrier of a mouse and be used for brain tumor MR imaging and chemokinetic treatment, and has diagnosis and treatment integration performance, good application prospect and the like.
The purpose of the invention can be realized by the following technical scheme:
one of the technical schemes of the invention provides a preparation method of a multifunctional core-shell dendrimer copper complex, which comprises the following steps:
(1) under the condition of solution, activating adamantane acetic acid (Ad-COOH), and adding into 3 rd generation polyamidoamine dendrimer (G3. NH)2) Reacting in solution, dialyzing, and freeze-drying to obtain Ad-G3.NH2；
(2) Activating beta-cyclodextrin (beta-CD) under the condition of solution, and adding into 5 th generation polyamidoamine dendrimer (G5. NH)2) Reacting in solution, dialyzing, and freeze-drying to obtain G5.NH2-CD；
(3) Under the condition of solution, target polypeptide RGD reacts with polyethylene glycol (Mal-PEG-COOH), and the RGD-PEG-COOH is obtained after dialysis and freeze-drying;
(4) reacting a transmembrane polypeptide-Dermorphin (DER) with polyethylene glycol (Mal-PEG-COOH) under the condition of a solution, dialyzing, and freeze-drying to obtain DER-PEG-COOH;
(5) activating 2-pyridine-2-carboxylic acid (Pyr) in solution, and adding into Ad-G3.NH2Reacting in solution, dialyzing, and freeze-drying to obtain Ad-G3.NH2-Pyr；
(6) Under the condition of solution, RGD-PEG-COOH is taken to be activated and then added into Ad-G3.NH2Reacting in solution, dialyzing, and freeze-drying to obtain Ad-G3.NH2-PEG-RGD；
(7) Under the condition of solution, after DER-PEG-COOH is taken to be activated, Ad-G3.NH is added2Reacting in solution, dialyzing, and freeze-drying to obtain Ad-G3.NH2-PEG-DER；
(8) Under the condition of solution, adding Ad-G3.NH2-Pyr、Ad-G3.NH2-PEG-RGD and Ad-G3.NH2Mixing the-PEG-DER uniformly, and adding the mixture to G5.NH2Reacting in a CD solution, dropwise adding triethylamine and an acetic anhydride solution, uniformly mixing, dialyzing, and freeze-drying to obtain M-CSTD.NHAc;
(9) and ultrasonically dispersing M-CSTD.NHAc and copper chloride under the condition of solution to obtain the target product multifunctional core-shell dendrimer copper complex.
Further, in the step (1), the molar ratio of the adamantane acetic acid to the 3 rd generation polyamidoamine dendrimer is 1-1.5: 1. The environment of the solution is DMSO (namely dimethyl sulfoxide) solution. Meanwhile, adamantane acetic acid can be activated using a DMSO-dissolved EDC · HCl and NHS solution. Preferably, Ad-COOH, EDC & HCl, NHS and G3.NH2The molar ratio of (a) to (b) is 1-1.5: 10:10: 1.
Further, in the step (1), the reaction temperature is room temperature, and the reaction time is 2-4 d; the activation temperature can also be room temperature, and the activation time is 2-4 h.
Further, in the step (2), the molar ratio of the beta-cyclodextrin to the 5 th generation polyamidoamine dendrimer is 25-30: 1. Here, the solution condition may be a DMSO (i.e., dimethyl sulfoxide) solution,meanwhile, β -CD can be activated with CDI solution dissolved in DMSO. Preferably, beta-CD, CDI and G5.NH2The molar ratio of (A) to (B) is 25-30: 250: 1.
Further, in the step (2), the reaction temperature is room temperature, and the reaction time is 58-62 hours; the activation temperature is 30 ℃, and the activation time is 5-7 h.
Further, in the step (3), the molar ratio of the targeting polypeptide RGD to the polyethylene glycol is 0.8-1.2: 1, preferably 1: 1. Here, the solution condition may be a DMSO (i.e., dimethyl sulfoxide) solution.
Further, in the step (3), the reaction temperature is room temperature, and the reaction time is 20-24 hours.
Further, in the step (4), the molar ratio of the cell-penetrating polypeptide-enkephalin to the polyethylene glycol is 1.6-2.4: 1, and preferably 2: 1. Here, the solution condition may be a DMSO (i.e., dimethyl sulfoxide) solution.
Further, in the step (4), the reaction temperature is room temperature, and the reaction time is 20-24 hours.
Further, in the step (5), 2-picolinic acid and Ad-G3.NH2The molar ratio of (A) is 45-55: 1, preferably 50: 1. In addition, in the step (5), the reaction temperature is preferably room temperature, and the reaction time is preferably 2-4 d. Here, the solution condition may be a DMSO (i.e., dimethyl sulfoxide) solution. Meanwhile, 2-picolinic acid can be activated by EDC & HCl, corresponding to Pyr, EDC & HCl and Ad-G3.NH2In a molar ratio of 50:100: 1.
Further, in step (6), RGD-PEG-COOH and Ad-G3.NH2The molar ratio of (a) to (b) is 8 to 12:1, preferably 10: 1. Here, the solution condition may be a DMSO (i.e., dimethyl sulfoxide) solution. Meanwhile, RGD-PEG-COOH can be activated with EDC. HCl and NHS solution, preferably RGD-PEG-COOH, EDC. HCl, NHS and Ad-G3.NH2In a molar ratio of 10:50:50: 1.
Further, in the step (6), the reaction temperature is room temperature, and the reaction time is 2-4 d.
Further, in step (7), DER-PEG-COOH and Ad-G3.NH2The molar ratio of (a) to (b) is 8 to 12:1, preferably 10: 1. Here, the solution condition may be DMSO (i.e., bisMethyl sulfoxide) solution. Meanwhile, DER-PEG-COOH can be activated with EDC. HCl and NHS solution, preferably DER-PEG-COOH, EDC. HCl, NHS and Ad-G3.NH2In a molar ratio of 10:50:50: 1.
Further, in the step (7), the reaction temperature is room temperature, and the reaction time is 2-4 d.
Further, in the step (8), the addition amount of each material satisfies: the molar ratio of G5 to G3 is 1: 12-18, preferably 1: 15. Wherein, Ad-G3.NH2-Pyr、Ad-G3.NH2-PEG-RGD and Ad-G3.NH2The PEG-DER molar ratio is preferably 1:1:1, and the molar ratio of the total molar amount of G5 and G3 to the molar amount of acetic anhydride and triethylamine is 1 (10-100) to 10-100.
Further, in the step (8), adding into G5.NH2The reaction temperature in the CD solution is room temperature, and the reaction time is 20-24 hours; and dropwise adding triethylamine and acetic anhydride solution, and mixing at room temperature for 20-24 h.
Further, the dialysis in the steps (1), (3), (4) and (5) is carried out for 3 days by using a cellulose dialysis membrane with the molecular weight cutoff of 1000 in ultrapure water 2L multiplied by 3;
the dialysis in the steps (2), (6) and (7) is to dialyze the cellulose dialysis membrane with the molecular weight cutoff of 5000 in ultrapure water 2L multiplied by 3 for 3 days;
the dialysis in the step (8) was carried out for 3 days by using a cellulose dialysis membrane having a molecular weight cut-off of 50000 in ultrapure water 2 L.times.3.
Further, in the step (9), the molar ratio of M-CSTD.NHAc to copper chloride is 1 (100-140), and the ultrasonic time is 2-4 min. Here, the solution condition is normal saline.
The second technical scheme of the invention provides a multifunctional core-shell dendrimer copper complex which is prepared by the preparation method.
The third technical scheme of the invention provides application of the multifunctional core-shell dendrimer copper complex in preparation of tumor MR imaging and chemodynamics treatment reagents. The macromolecular copper complex has diagnosis and treatment integrated performance, and is used for in-vitro anti-glioma research and tumor MR imaging and chemodynamics treatment research of an in-situ glioma mouse model through tail vein injection.
The invention utilizes the shell component dendrimer at the tail end of the amino group to respectively modify the picorphin, the RGD peptide and the pyridine, and forms the core-shell dendrimer with the core component dendrimer through supermolecule self-assembly and acetylation treatment, thereby complexing copper ions to construct a multifunctional nano compound for the diagnosis and treatment integration of in-situ brain glioma. The multifunctional core-shell dendrimer copper complex is used for realizing the penetration of BBB, targets glioma, obtains good imaging effect and improves the survival rate of glioma mice. Through rational design and accurate synthesis of the dendrimer nano material, the invention overcomes some defects of application of the dendrimer, integrates two separated processes/functions of clinical diagnosis and treatment at present into one nano material, and constructs a diagnosis and treatment integrated nano platform which can pass through a blood brain barrier and target tumors to carry out tumor MR imaging and chemical kinetic treatment by using an acetylated multifunctional core-shell dendrimer copper complex.
Compared with the prior art, the invention has the following advantages:
(1) the method is simple, easy to operate and separate, low in cost, and has good development prospect, and the raw materials are commercialized;
(2) the multifunctional core-shell dendrimer copper complex nano platform prepared by the invention has good water solubility, can pass through a blood brain barrier, has inhibitory effect on in-situ glioma cells, has a certain r1 relaxation rate, can be used for chemical dynamics treatment and MR imaging of in-situ glioma, and has diagnosis and treatment integrated effect.
Drawings
FIG. 1 is a schematic diagram of the synthesis and application of the multifunctional core-shell dendrimer copper complex prepared by the present invention.
FIG. 2 is the G3.NH prepared in example 12-Ad(a)、G5.NH2-CD(b)、RGD-PEG-COOH(c)、DER-PEG-COOH(d)、RGD-PEG-G3.NH2-Ad(e)、DER-PEG-G3.NH2-Ad(f)、Pyr-G3.NH2Ad (G) and G5.NMR spectrum of NHAc-CD/Ad-G3.NHAc-PEG-DER/PEG-RGD/Pyr (i.e., M-CSTD. NHAc) (h).
Fig. 3 is a graph (a) and a fit graph (b) of UV-Vis and an absorbance analysis graph (c) of M-cstd. nhac prepared in example 1 titrated with copper ions at different ratios.
FIG. 4 is a TEM image of M-CSTD. NHAc/Cu (II) prepared in example 1.
Fig. 5 is a comparison graph of surface potential and hydrodynamic diameter of some nanomaterials prepared in example 1 and comparative example.
FIG. 6 is an infrared spectrum (a) and an ultraviolet spectrum (b) demonstrating that M-CSTD. NHAc/Cu (II) and copper chloride prepared in example 1 can undergo a Fenton-like reaction.
Fig. 7 is a cell activity graph of C6 cells treated with different nanomaterials for 24 hours in example 6 (a), C6 cells treated with copper and copper complexes (b), and bned.3 cells treated with copper and copper complexes for 24 hours (C).
FIG. 8a shows the treatment of alpha in example 7 with different concentrations of copper and copper complex M-CSTD. NHAc/Cu (II)vβ3Graph of copper ion content 4 hours after C6 cells with high/low integrin receptor expression
FIGS. 8b and 8C are graphs of fluorescence intensity in C6 cells detected by flow-assay after in vitro construction of a blood-brain barrier model and treatment of a molecular layer of bEnd.3 cells with FITC-labeled multifunctional modified nucleocapsid dendrimer and its control material for 6 hours in example 8.
FIG. 9 is a flow chart of apoptosis after 24 hours of treatment of C6 cells with different concentrations of copper and copper complex M-CSTD. NHAc/Cu (II) in example 10.
FIG. 10 is a graph showing the glutathione content (a) and the glutathione peroxidase content (b) in C6 cells after treating the cells with different concentrations of copper and copper complex for 24 hours in example 10.
FIG. 11 is a fluorescence image of confocal microscope showing the active oxygen content in C6 cells treated with different concentrations of copper and copper complex M-CSTD. NHAc/Cu (II) in example 11 for 6 hours.
FIG. 12 shows T of copper and copper complex M-CSTD. NHAc/Cu (II) of example 121A linear relation graph (a) of the inverse of the relaxation time along with the change of the Cu concentration, an MR imaging graph (b) of the mouse in-situ brain glioma and a corresponding graph (c) of the change of the signal-to-noise ratio of a tumor part.
Fig. 13 shows the antitumor effect of M-cstd. nhac/Cu (ii) in vivo in saline solution (100mL, [ Cu ] ═ 8mM) prepared in example 1 by tail vein injection in example 13. (a) The change of body weight of the mice is shown in the figure, and the survival rate of the mice is shown in the figure.
FIG. 14 shows G5.NH prepared in comparative example 1 and comparative example 22-CD/G3.NH2Nuclear magnetic resonance hydrogen spectra of-Ad (a), g5.nhac-CD/Ad-g3.nhac (b) and g5.nhac-CD/Ad-g3.nhac-mPEG/pyr (c).
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.
In particular, the endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In the following examples, G3.NH2、G5.NH2The raw materials such as the target polypeptide RGD, the membrane-penetrating polypeptide DER and the like are all commercial products. Wherein, G3.NH2、G5.NH2From Dendritech corporation (Midland, MI); the targeting polypeptide RGD was purchased from Sigma-Aldrich (st. louis, MO) and has the sequence Arginine-glycine-aspartic acid (Arginine-glycine-aspartic acid); the membrane-penetrating polypeptide DER is synthesized by Nanjing peptide biotechnology limited and has the sequence as follows: tyrosine-alanine-phenylalanine-glycine-tyrosine-proline-serine-cysteine (Tyr-DAla-Phe-Gly-Tyr-Pro-Ser-Cys).
And the rest of the raw materials or treatment techniques which are not specifically described indicate that the raw materials or the treatment techniques are all conventional commercial raw materials or conventional treatment techniques in the field.
The invention uses NMR spectrum (1H NMR), ultraviolet visible absorption spectrum (UV-Vis), Zeta potential and dynamic light scattering analysis (DLS), Transmission Electron Microscope (TEM), Fourier infrared spectrum and other means characterize the prepared multifunctional nano platform (M-CSTD. NHAc); testing the biocompatibility of the M-CSTD.NHAc and related materials by using a CCK-8 method, and evaluating the cytotoxicity of the M-CSTD.NHAc copper complex (M-CSTD.NHAc/Cu (II)) and related comparative materials; then, evaluating the targeting performance of M-CSTD.NHAc/Cu (II) to the brain glioma by using an inductively coupled plasma spectrum generator, and constructing an in-vitro blood brain barrier model to evaluate the blood brain barrier crossing performance of the M-CSTD.NHAc; testing the influence of the copper ions and the copper complex on the apoptosis of the glioma cells; analyzing and comparing the capacity of the copper ions and the copper complexes for consuming glutathione and related indexes in glioma cells, and simultaneously determining the influence of the copper complexes on the content of active oxygen in glioma cells; in addition, the relaxivity of copper and the copper complex thereof is measured, tumor MR imaging and chemical kinetics treatment experiments of a rat orthotopic brain glioma model are carried out, the in vivo MR imaging and treatment effects of the prepared multifunctional core-shell dendrimer copper complex nano platform are considered, and fig. 1 is a schematic diagram of the synthesis and application of the nano platform prepared by the invention.
The specific test results are as follows:
(1)1h NMR test results
At G3.NH2In the NMR spectrum of Ad (see FIG. 2a), 2.2-3.4ppm is G3.NH21.6-1.9ppm are characteristic proton peaks of Ad, and each of the G3.NH is calculated from the ratio of their integrated areas21.1 Ad molecules are ligated; at G5.NH2In the NMR spectrum of-CD (see FIG. 2b), 2.2-3.4ppm is G5.NH23.4-4.0ppm and 5.0ppm are characteristic proton peaks of beta-CD, and each G5.NH is calculated according to the ratio of their integral areas213.9 CD molecules are linked; in the NMR spectrum of RGD-PEG-COOH (see FIG. 2c), 3.4-3.6ppm are the methylene proton peaks of PEG, 71-7.3ppm are proton peaks on the benzene ring of the RGD polypeptide, and 0.7 RGD molecules are connected to each PEG according to the ratio of the integral areas of the proton peaks; in the nuclear magnetic resonance hydrogen spectrogram of DER-PEG-COOH (see figure 2d), 3.4-3.6ppm are methylene proton peaks of PEG, 7.1-7.3ppm are proton peaks on benzene ring of DER polypeptide, and according to the ratio of their integrated areas, each PEG is connected with 0.8 DER molecules; in RGD-PEG-G3.NH2In the NMR spectrum of Ad (see FIG. 2e), each G3.NH can be calculated from the ratio of the integral areas of their characteristic peaks2Ad is linked with 3.4 RGD-PEG molecules; in DER-PEG-G3.NH2In the NMR spectrum of Ad (see FIG. 2f), each G3.NH can be calculated from the ratio of the integral areas of their characteristic peaks2Ad to which 3.4 DER-PEG molecules are attached; in Pyr-G3.NH2In the NMR spectrum of Ad (see FIG. 2g), 7.5, 8 and 8.5ppm are characteristic proton peaks on the pyridine ring, and each of the peaks is calculated as G3.NH2Ad is linked to 4 Pyr molecules; in the NMR spectrum of G5.NHAc-CD/Ad-G3.NHAc-PEG-RGD/PEG-DER/Pyr (i.e., M-CSTD. NHAc) (see FIG. 2h), 6.6 functionalized Ad-G3.NHAc molecules were attached to each G5.NHAc-CD according to the ratio of the integrated areas of their characteristic peaks, and in addition, a methyl proton peak of acetyl group was also present at 1.9ppm, indicating that the remaining amino groups on the surface of the final material M-CSTD. NHAc were acetylated.
(2) UV-Vis Spectroscopy test results
Referring to FIG. 3, FIG. 3a is a graph of the UV-Vis spectra of copper ions and acetylated dendrimers at different molar ratios (60: 1, 80: 1, 100:1, 120: 1, 140: 1, 160: 1). And simultaneously, nonlinear fitting is carried out on the absorbance of the maximum absorption peak at 602nm and the proportion of the copper ions to the acetylated dendrimer (see figure 3b), so that 110-130 moles of copper ions can be complexed by each mole of acetylated core-shell dendrimer. Finally, the copper ions and the multifunctional acetylated core-shell dendrimer are mixed in a proportion of 120: 1 molar ratio for subsequent experiments.
By measuring CuCl2M-CSTD. NHAc, M-CSTD. NHAc/Cu (II) (see FIG. 3c),the results show that the absorption peak of the aqueous solution of copper chloride is 814nm, the absorption peak of the aqueous solution of M-CSTD.NHAc/Cu (II) is 602nm, and that there is no absorption peak at both positions of M-CSTD.NHAc. This indicates that the position of the absorption peak is affected by the change in the environment surrounding the copper complex due to the functional modification. Meanwhile, the disappearance of the absorption peak of the copper ions in the UV-Vis spectrum of the M-CSTD.NHAc/Cu (II) complex indicates that no free copper ions exist in the solution, and after the M-CSTD.NHAc is complexed with 120 molar equivalents of the copper ions, the copper ions are completely in a complexing state.
(3) TEM and Zeta potential and hydrodynamic diameter test results
The TEM test was used to determine the actual size of the final material copper complex M-CSTD. NHAc/Cu (II) (see FIG. 4), and finally the average size of the final material copper complex M-CSTD. NHAc/Cu (II) was found to be 27.89 + -1.54 nm.
Hydrodynamic diameter test results (see fig. 5b) show that the hydrodynamic diameter (410.3nm) of the M-cstd. nhac subjected to multifunctional modification is obviously increased compared with the hydrodynamic diameter (207.3nm) of the core-shell dendrimer cstd. nhac not subjected to functional modification, but slightly shows a downward trend after complexing copper ions, and is reduced from 410.3nm to 365.6 nm.
The Zeta potential measurement result (see fig. 5a) shows that the surface potential of the multifunctional acetyl core-shell dendrimer M-CSTD. NHAc is +20.1mV, which is slightly higher than that of the acetylated core-shell dendrimer CSTD. NHAc (the potential is +18.4mV) which is not subjected to functional modification under the same condition, but is slightly lower than that of the acetylated core-shell dendrimer (21.2mV) after copper complexing, which may be the reason for influencing the surface potential of the nano platform after copper ion complexing.
(4) Fenton-like reaction test results
The ability of copper and copper complexes to undergo fenton-like reactions was tested using fourier infrared spectroscopy (fig. 6) which shows that copper and copper complexes are capable of reducing reduced Glutathione (GSH) to oxidized glutathione (GSSG) and Uv-vis spectroscopy (fig. 6b) which demonstrates that copper and copper complexes are in the presence of GSH and hydrogen peroxide (H) in the presence of GSH2O2) The reaction can generate hydroxyl radicals, degrading Methylene Blue (MB).
(5) Results of cytotoxicity test
Referring to fig. 7a, compared with the saline control group, M-cstd-nhac and its related control material cstd-nhac-mPEG/Pyr have no obvious cytotoxicity to glioma C6 cells in the tested concentration range, and the cell survival rate is above 90%, indicating that M-cstd-nhac has no tumor cell inhibition per se before complexing copper. After complexing copper ions (see fig. 7b), the cell survival rate of each concentration of the copper complex of the M-CSTD.NHAc/Cu (II) and related control materials is obviously reduced compared with that of the M-CSTD.NHAc and related control materials, which indicates that the copper ions have certain cytotoxicity, namely the copper complexes are helpful for enhancing the tumor cell inhibition of the materials. Meanwhile, copper and copper complex M-CSTD.NHAc/Cu (II) were selected to test their cytostatic property against endothelial cells (bEnd.3) by the CCK-8 method (see FIG. 7C), copper ions alone and M-CSTD.NHAc/Cu (II) were found to be less cytotoxic to bEnd.3 cells than C6 cells in the tested concentration range, and it was found that copper ion concentration was in the concentration range of 0-100. mu.M, M-CSTD.NHAc/Cu (II) was not significantly cytotoxic to bEnd.3 cells, and cell survival rate was above 90%.
(6) Inductively coupled plasma spectroscopy test results
Fig. 8a shows the amount of phagocytosis of copper by C6 cells after treatment with different concentrations of copper and copper complex to evaluate the specific targeting effect of M-cstd. Using normal saline group as blank control, respectively mixing copper and copper complex with alphavβ3The C6 cells with high and low expression of integrin receptor were co-cultured for 4 hours, and then the phagocytosis amount of nanoparticles by C6 cells was tested by inductively coupled plasma spectroscopy. The results show that the amount of phagocytosis of copper and copper complexes by cells increases with increasing concentration of Cu ions, and that alpha co-cultured with copper complexes in the concentration range studiedvβ3The phagocytosis amount of C6 cells with high integrin receptor expression (without RGD blocking) to copper is obviously higher than that of alphavβ3C6 cells with low integrin receptor expression (blocked by RGD) demonstrated that modification of the targeting molecule RGD in the material allows specific targeting of M-cstd. nhac/cu (ii) to C6 cells.
(7) Flow cytometer test results
Fig. 8b and 8C show the ability of FITC-labeled multifunctional modified nucleocapsid dendrimer and its control material to be phagocytosed by C6 cells across the mend.3 cell monolayer. A normal saline group is used as a blank control, multifunctional modified core-shell dendritic macromolecules and a control material thereof are subjected to fluorescence labeling by FITC, then bEnd.3 cells are cultured in an upper chamber of a transwell plate, and C6 cells are cultured in a lower layer of the transwell plate to construct an in vitro blood brain barrier model. When the upper cell is fully paved in the whole chamber and the resistance of the cell membrane reaches 200 omega m (the in vitro blood brain barrier model is successfully constructed), the upper culture medium is changed into a culture medium containing FITC labeled multifunctional modified core-shell dendrimer or a contrast material thereof for culture for 6h, the C6 cells in the lower chamber are collected, and the fluorescence intensity of the cells is detected by using a flow cytometer. The results show that FITC-labeled multifunctional modified nucleocapsid dendrimer has the ability to cross the mend.3 cell monolayer and be phagocytized by C6 cells, while the control material (without modification by the transmembrane polypeptide DER) is not much different from the normal saline group, demonstrating the ability to cross the blood brain barrier.
FIG. 9 shows the effect on apoptosis after treatment with different concentrations of copper and copper complexes. Under the same concentration of the experiment, C6 cells treated by copper and M-CSTD, NHAc/Cu (II) all show apoptosis, and the apoptosis degree is increased along with the increase of the copper concentration in the range of the research concentration, and the cell apoptosis degree shows copper concentration dependence.
(8) Intracellular glutathione level and related index test results
FIG. 10 shows the change in intracellular glutathione levels and the levels of glutathione peroxidase (GPx), a relevant index, after treatment of C6 cells with different concentrations of copper and copper complexes. At the same concentration in the experiment, the levels of glutathione and GPx in C6 cells treated by copper and M-CSTD.NHAc/Cu (II) are not greatly different and are reduced along with the increase of the copper concentration, thereby indicating that M-CSTD.NHAc/Cu (II) has the same capacity as the single copper ion and can consume the glutathione in vivo.
(9) Results of cellular active oxygen content test
C6 cells were cultured in vitro, and C6 cells were treated with copper complex M-cstd. nhac/cu (ii), and the change in the active oxygen content of C6 cells at different concentrations at the same time was observed (see fig. 11). Confocal microscopy results show that C6 cell active oxygen content increases with increasing copper concentration in the material after copper complex treatment, in concert with decreasing glutathione levels.
(10) Magnetic Resonance (MR) analysis results
Preparing 1mL of aqueous solution of copper and copper complex M-CSTD-NHAc/Cu (II) with Cu concentrations of 0.2, 0.4, 0.8, 1.6 and 3.2mM respectively, and determining T of the material under different Cu concentrations by a magnetic resonance imaging analyzer1Relaxation effect (see fig. 12 a). R of Cu (II) and M-CSTD. NHAc/Cu (II) is calculated1Values of 0.8679 and 0.7331mM, respectively-1s-1. Similar r is maintained after complexation of Cu ion and M-CSTD. NHAc1The values indicate that M-CSTD. NHAc/Cu (II) can be used as T in the diagnosis of MR molecular imaging1A positive contrast agent.
In vivo construction of in situ brain glioma models in rats, M-CSTD. NHAc/Cu (II) saline solution (100mL, [ Cu ] was injected via tail vein]8mM) to evaluate the effect of MR imaging at the tumor site. The mice tumor sites were progressively enhanced in MR signal after injection compared to the pre-injection blank group (fig. 12b), indicating that M-cstd. nhac/cu (ii) can enrich and display MR signal at the tumor sites with blood flow. Meanwhile, the results of MR signal-to-noise ratio quantitative analysis (FIG. 12c) show that the SNR of the tumor site reaches the maximum value after the injection of M-CSTD. NHAc/Cu (II) for 45min, and the results show that M-CSTD. NHAc/Cu (II) can be used as T1Contrast agents are used for enhanced in vivo tumor MR imaging diagnosis.
(11) Evaluation of in vivo antitumor Effect
An orthotopic brain glioma model was constructed in mice treated every 3 days by tail vein injection of a physiological saline solution of M-cstd. nhac/Cu (ii) (100mL, [ Cu ] ═ 8mM), and the chemokinetic treatment effect was evaluated by recording the weight change (see fig. 13a) and survival rate (see fig. 13b) of the mice. After chemokinetic treatment, the body weight change of the M-CSTD. NHAc/Cu (II) group mice is smaller than that of the blank normal saline group, and the survival rate is higher than that of the blank group, for example, the survival rate of the blank group mice is 0 at the modeling time of 28 days, while the survival rate of the treatment group mice is still maintained at 50%. This shows that the multifunctional core-shell dendrimer M-CSTD. NHAc/Cu (II) prepared by the invention has good in vivo anti-tumor activity.
The present invention will be described in detail with reference to specific examples.
Example 1
A preparation method of a multifunctional core-shell dendrimer copper complex nano platform is shown in figure 1, and specifically comprises the following steps:
(1) 4.22mg of Ad-COOH, 41.62mg of EDC & HCl and 21.98mg of NHS were weighed out and dissolved in 5mL of DMSO solution, respectively, and then the EDC & HCl and NHS solutions were added dropwise to the Ad-COOH solution and stirred at room temperature for 3 hours. Then 100mg of G3.NH are weighed out2Dissolved in 5mL of DMSO solution, the activated Ad-COOH solution was added dropwise to G3.NH2Continuing the reaction in the solution for 3 days, transferring the obtained product into dialysis bag with molecular weight cutoff of 1000, dialyzing in distilled water for three days (2L × 3), and freeze drying to obtain dry Ad-G3.NH2And storing at-20 ℃ for later use.
(2) 43.64mg of beta-CD and 62.34mg of CDI were weighed out and dissolved in 5mL of DMSO solution, and the CDI solution was added dropwise to the beta-CD solution and stirred at room temperature for 6 hours. 40mg of G5.NH are weighed2Dissolved in 5mL of DMSO solution, and the resulting activated beta-CD solution was added dropwise to G5.NH2The reaction was continued for 60 hours, and the obtained product was transferred to a dialysis bag having a molecular weight cut-off of 5000, dialyzed in distilled water for three days (2 L.times.3), and then subjected to freeze-drying treatment to obtain dry G5-CD, which was stored at-20 ℃ for further use.
(3) Respectively weighing 10mg RGD and 29mg Mal-PEG-COOH, respectively dissolving in 5mL DMSO solution, then dropwise adding the RGD solution into polyethylene glycol (Mal-PEG-COOH) solution dissolved in DMSO, reacting for 24h, transferring the obtained product into a dialysis bag with cut-off molecular weight of 1000, dialyzing in distilled water for three days (2L x 3), freeze-drying, and finally obtaining dry RGD-PEG-COOH, and storing at-20 ℃ for later use.
(4) 26.36mg of DER and 29mg of Mal-PEG-COOH were weighed out and dissolved in 5mL of DMSO solution, respectively, the DER solution was added dropwise to the polyethylene glycol (Mal-PEG-COOH) solution dissolved in DMSO likewise for 24 hours, the resulting product was transferred into a dialysis bag having a molecular weight cut-off of 1000, dialyzed against distilled water for three days (2L. times.3), and then subjected to freeze-drying treatment to obtain dried DER-PEG-COOH, which was stored at-20 ℃ until use.
(5) 4.44mg Pyr, 13.85mg EDC & HCl, 5.16mg G3.NH were weighed out separately2Respectively dissolving Ad in 5mL DMSO solutions, adding EDC & HCl solution dropwise into Pyr solution, stirring at room temperature for 0.5h, and adding the obtained activated Pyr solution into the same DMSO-dissolved Ad-G3.NH2Reacting in solution for 3 days, transferring the obtained product into dialysis bag with molecular weight cutoff of 1000, dialyzing in distilled water for three days (2L × 3), and freeze drying to obtain dry Ad-G3.NH2-Pyr, -20 ℃ for storage.
(6) 16.95mg of RGD-PEG-COOH, 6.94mg of EDC. HCl and 3.77mg of NHS obtained in step (3) were weighed out and dissolved in 5mL of DMSO, respectively, and then the EDC. HCl and NHS solutions were added dropwise to the RGD-PEG-COOH solution and stirred at room temperature for 3 hours. Then 5.16mg of G3.NH are weighed out2-Ad in 5mL DMSO solution, and the resulting activated RGD-PEG-COOH solution was added dropwise to G3.NH2In Ad solution, the reaction is continued for 3 days, the product obtained is transferred into a dialysis bag with a molecular weight cut-off of 5000, dialyzed in distilled water for three days (2 L.times.3), and then subjected to freeze-drying treatment to obtain dry Ad-G3.NH2-PEG-RGD, -storing at-20 ℃ for later use.
(7) 18.86mg of DER-PEG-COOH obtained in the step (4), 6.94mg of EDC. HCl and 3.77mg of NHS were weighed out and dissolved in 5mL of DMSO, respectively, and then EDC. HCl and NHS solutions were added dropwise to the DER-PEG-COOH solution and stirred at room temperature for 3 hours. Then 5.16mg of G3.NH are weighed out2-Ad in 5mL DMSO solution, and the resulting activated DER-PEG-COOH solution was added dropwise to G3.NH2In Ad solution, the reaction is continued for 3 days, the product obtained is transferred into a dialysis bag with a molecular weight cut-off of 5000, dialyzed in distilled water for three days (2 L.times.3), and then frozenDrying to obtain dry Ad-G3.NH2-PEG-DER, -storing at 20 ℃ for future use.
(8) 2.75mg, 5.63mg and 5.96mg of each of the materials obtained in the above steps (5), (6) and (7) were dissolved in 5mL of DMSO, and mixed with stirring at room temperature, and then 2.93mg of G5.NH was weighed2-CD is dissolved in 5mL DMSO solution, and the mixed solution is added dropwise to G5.NH2Reacting in a-CD solution for 24 hours, adding triethylamine (250 mu L), uniformly mixing, dropwise adding an acetic anhydride solution (150 mu L) after 30 minutes, continuously stirring at room temperature for 24 hours, transferring the obtained product into a dialysis bag with the molecular weight cutoff of 50000, dialyzing in distilled water for three days (2L multiplied by 3), and then carrying out freeze drying treatment to finally obtain the dried multifunctional core-shell tree-shaped macromolecule G5.NHAc-CD/Ad-G3.NHAc-PEG-DER/PEG-RGD/Pyr, namely M-CSTD.NHAc, and storing at-20 ℃ for later use.
(9) Weighing the material M-CSTD.NHAc obtained in the step (8), dissolving the material M-CSTD.NHAc in normal saline, adding a proper amount of copper chloride solid (the molar ratio of the M-CSTD.NHAc to the copper chloride is 1:120), and then carrying out ultrasonic dispersion to obtain the normal saline solution of the multifunctional core-shell tree-like macromolecule copper complex M-CSTD.NHAc/Cu (II).
Example 2
5-10mg of each of the materials prepared in example 1 was dissolved in 500. mu.L of deuterated water and subjected to hydrogen spectroscopy using a Bruker400MHz NMR spectrometer, the results of which are shown in FIG. 2 at G3.NH2In the NMR spectrum of Ad (FIG. 2a), 2.2-3.4ppm is G3.NH21.6-1.9ppm are characteristic proton peaks of Ad, and each of the G3.NH is calculated from the ratio of their integrated areas21.1 Ad molecules are ligated; at G5.NH2NMR spectrum of-CD (FIG. 2b), 2.2-3.4ppm is G5.NH23.4-4.0ppm and 5.0ppm are characteristic proton peaks of beta-CD, and each G5.NH is calculated according to the ratio of their integral areas213.9 CD molecules are linked; in the nuclear magnetic resonance hydrogen spectrogram of RGD-PEG-COOH (figure 2c), 3.4-3.6ppm are methylene proton peaks of PEG, 7.1-7.3ppm are proton peaks on benzene ring of RGD polypeptide, and according to the ratio of their integral areas, each PEG is connected with 0.7 RGD molecules; in DER-PEIn the NMR spectrum of G-COOH (FIG. 2d), 3.4-3.6ppm are methylene proton peaks of PEG, 7.1-7.3ppm are proton peaks on benzene ring of DER polypeptide, and according to their ratio of integral area, each PEG is connected with 0.8 DER molecules; in RGD-PEG-G3.NH2In the NMR spectrum of Ad (FIG. 2e), each G3.NH can be calculated from the ratio of the integral areas of their characteristic peaks2Ad is linked with 3.4 RGD-PEG molecules; in DER-PEG-G3.NH2In the NMR spectrum of Ad (FIG. 2f), 3.4 DER-PEG molecules were linked to each G3.NH2-Ad, as calculated from the ratio of the integrated areas of their characteristic peaks; in Pyr-G3.NH2In the nuclear magnetic resonance hydrogen spectrum of Ad (see the attached figure 2g of the specification), 7.5, 8 and 8.5ppm are characteristic proton peaks on a pyridine ring, and each G3.NH can be calculated according to the ratio of the integral areas of the characteristic peaks2Ad is linked to 4 Pyr molecules; in the NMR spectrum of G5.NHAc-CD/Ad-G3.NHAc-PEG-RGD/PEG-DER/Pyr (i.e. M-CSTD. NHAc) (FIG. 2h), 6.6 functionalized Ad-G3.NHAc molecules were attached to each G5.NHAc-CD according to the ratio of the integrated areas of their characteristic peaks, and in addition, a methyl proton peak of acetyl group was also present at 1.9ppm, indicating that the remaining amino groups on the surface of the final material M-CSTD. NHAc were acetylated.
Example 3
M-CSTD. NHAc prepared in example 1 was titrated for copper ions at different molar ratios by UV-Vis assay by first preparing M-CSTD. NHAc solution, then adding it in parallel to M-CSTD. NHAc solution according to different molar ratios of copper ions to M-CSTD. NHAc (60: 1, 80: 1, 100:1, 120: 1, 140: 1, 160: 1) and finally diluting the mixture to 700. mu.L with water. Detection by uv-vis spectroscopy and fitting was performed to check for saturation binding of cu (ii). The result is shown in fig. 3, the maximum absorption peak of M-cstd.nhac/cu (ii) is at 602nm (fig. 3a), the absorbance at 602nm of the maximum absorption peak is nonlinearly fitted with the ratio of copper ions to M-cstd.nhac to obtain 110-130 moles of copper ions per mole of M-cstd.nhac (fig. 3b), and then the ratio of copper ions to M-cstd.nhac is 120: 1 molar ratio of copper chloride (CuCl)2) The UV-Vis spectra of M-CSTD. NHAc, M-CSTD. NHAc/Cu (II) (FIG. 3c) show thatCuCl2The absorption peak of the aqueous solution was 814nm, the absorption peak of the M-CSTD. NHAc/Cu (II) aqueous solution was 602nm, and there was no absorption peak at both of these positions. This indicates that the position of the absorption peak is affected by the change in the environment surrounding the copper complex due to the functional modification. Meanwhile, the disappearance of the copper absorption peak at 814nm in the UV-Vis spectrum of the M-CSTD.NHAc/Cu (II) complex indicates that no free copper ions exist in the solution, and after the M-CSTD.NHAc is complexed with 120 molar equivalents of copper ions, the copper ions are completely in a complexing state.
Example 4
Formulation example 1 the resulting M-CSTD. NHAc/Cu (II) solution (1mg/mL) was sized by TEM and the final copper complex M-CSTD. NHAc/Cu (II) showed an average size of 27.89 + -1.54 nm as shown in FIG. 4. Subsequently, an aqueous solution (2mg/mL) of the corresponding material was formulated for determination of hydrodynamic diameter and surface potential by means of a Malvern laser particle sizer (Malvern, μm K, 633nm laser). Hydrodynamic diameter test results (fig. 5a) show that the hydrodynamic diameter (410.3nm) of the M-cstd-nhac subjected to multifunctional modification is obviously increased compared with that of the core-shell dendrimer cstd-nhac (207.3nm) not subjected to functional modification, but the M-cstd-nhac/cu (ii) after copper ion complexation shows a slight decrease trend, which is decreased from 410.3nm to 365.6 nm. The Zeta potential measurement result (figure 5b) shows that the surface potential of the M-CSTD.NHAc is +20.1mV, which is slightly higher than that of acetyl core-shell dendrimer CSTD.NHAc (the potential is +18.4mV) which is not subjected to functional modification under the same condition, but is slightly lower than that of M-CSTD.NHAc/Cu (II) (21.2mV) after copper complexing, which is probably the reason for influencing the surface potential of the nano platform after copper ion complexing.
Example 5
According to a molar ratio (copper chloride to M-cstd. nhac) of 120: 1 preparing M-CSTD.NHAc/Cu (II) compound solution and keeping the concentration of Cu (II) in the solution at 10mM, mixing with GSH with the same concentration of 10mM in an equal volume, freeze-drying the mixture, and characterizing by a Fourier infrared spectrometer, wherein the results are shown in FIG. 6a and are similar to the Fourier infrared spectrum of commercial GSSG, and the M-CSTD.NHAc/Cu (II) compound + GSH group and the copper chloride + GSH group are 2553cm-1No corresponding peak appears at any position (representing sulfydryl and-SH),indicating that the presence of copper ions oxidized the-SH groups of GSH to generate GSSG.
Separately, 10. mu.g/mL Methylene Blue (MB) solution and 10mM H solution were prepared2O2Solutions, 10mM GSH solution and 10mM Cu (II) in M-CSTD. NHAc/Cu (II) complex were mixed in equal volumes in different combinations and OH-induced MB degradation was measured after 30 minutes using Uv-vis absorbance measurements. The results are shown in FIG. 6b, similar to copper chloride, M-CSTD. NHAc/Cu (II) complex in the presence of GSH and hydrogen peroxide (H)2O2) The reaction can generate hydroxyl radicals, degrading Methylene Blue (MB).
Example 6
The cytotoxicity of M-CSTD. NHAc and its control material, copper and copper complex M-CSTD. NHAc/Cu (II) prepared in example 1 was examined using C6 cells or bEnd.3 cells as model cells. Cells were seeded in a 96-well plate at a density of 10000 cells/well and cultured in 100. mu.L DMEM medium supplemented with 100U/mL penicillin, 100U/mL streptomycin and 10% FBS overnight at 37 ℃ at a carbon dioxide concentration of 5%. The medium was then replaced with 100. mu.L of DMEM containing the material at different concentrations and incubation continued for 24 h. The culture was then poured off, and 100. mu.L of DMEM medium solution containing 10% CCK-8 was added to continue the culture for 2 hours. And (3) testing the light absorption value under the wavelength of 450nm by using a multifunctional microplate reader. The results are shown in fig. 7, compared with the normal saline control group, the M-cstd-nhac and its related control material cstd-nhac-mPEG/Pyr have no obvious cytotoxicity to the glioma C6 cells in the tested concentration range, and the cell survival rate is above 90% (fig. 7a), which indicates that the M-cstd-nhac has no tumor cell inhibition property before complexing copper. After complexing copper ions, the cell survival rate of the copper complex of M-CSTD.NHAc/Cu (II) and related control materials at each concentration is obviously reduced compared with that of the copper complex of M-CSTD.NHAc and related control materials (figure 7b), which indicates that the copper ions have certain cytotoxicity, namely the copper complexes are helpful for enhancing the tumor cell inhibition of the materials. Meanwhile, copper and copper complex M-CSTD.NHAc/Cu (II) were selected to test their cytostatic property on endothelial cells (bEnd.3) by the CCK-8 method (FIG. 7C), copper ions alone and M-CSTD.NHAc/Cu (II) were found to have lower cytotoxicity on bEnd.3 cells than on C6 cells in the tested concentration range, and it was found that the copper ion concentration was in the concentration range of 0-100. mu.M, M-CSTD.NHAc/Cu (II) had no significant cytotoxicity on bEnd.3 cells, and the cell survival rate was above 90%.
Example 7
Selecting murine glioma C6 cells as model cells, and evaluating the targeting effect of the prepared M-CSTD-NHAc/Cu (II) nanoparticles by detecting the phagocytosis amount of the cells to the copper complex M-CSTD-NHAc/Cu (II) nanoparticles. First, CSTD.NHAc-mPEG/Pyr/Cu (II) and M-CSTD.NHAc/Cu (II) solutions with different copper concentrations were prepared in sterile physiological saline, and C6 cells were seeded in 12-well plates at a density of 20 ten thousand cells/well. After overnight culture, a group of C6 cells were co-cultured for 3 hours with 2. mu.M RGD addition (defined as RGD blocked C6 cells, i.e.. alpha.)vβ3C6 cells with low integrin receptor expression), and the other two groups of C6 cells co-cultured without RGD were defined as αvβ3C6 cells highly expressing integrin receptor. Then, the DMEM medium solution added with the prepared nanoparticle solution and the alpha solution are respectively addedvβ3C6 cells with high and low integrin receptor expression were co-cultured at 37 ℃ for 4 hours with saline as a blank. Then digesting and centrifuging, collecting cells, digesting with aqua regia, and detecting the phagocytosis amount of the copper element by an inductively coupled plasma spectrometer. As a result, as shown in FIG. 8a, the amount of phagocytosis of copper and copper complexes by cells gradually increased with the increase of the Cu ion concentration, and α co-cultured with copper complexes in the range of concentration to be investigatedvβ3The phagocytosis amount of C6 cells with high integrin receptor expression (without RGD blocking) to copper is obviously higher than that of alphavβ3C6 cells with low integrin receptor expression (blocked by RGD) demonstrated that modification of the targeting molecule RGD in the material allows specific targeting of M-cstd. nhac/cu (ii) to C6 cells.
Example 8
Establishing an in vitro BBB model, and carrying out in-vitro BBB model on the bEnd.3 cells at 2 multiplied by 105The individual cells were seeded in the upper chamber (pore size: 0.4 μm). The medium was changed every 2 days. After 6 days, C6 cells were seeded in the lower chamber. After 24 hours, the cells were passed through a cell membrane resistance meter (Millicel-RES, Millipore, USA)) Transendothelial electrical resistance (TEER) was tested to confirm barrier integrity. When the TEER exceeds 200 omega/cm2Were used for further experiments. FITC-labeled multifunctional modified nucleocapsid dendritic macromolecules and control materials thereof are added into an upper chamber culture medium at the concentration of 1mg/mL, after 6h of incubation, C6 cells in a lower chamber are washed by PBS and digested and collected in 1mL of PBS buffer, and then the fluorescence intensity (green fluorescence) of FITC in the cells is determined by a BD Calibur flow cytometer (BD company, USA) so as to evaluate the capacity of the multifunctional nucleocapsid dendritic macromolecules to cross the blood brain barrier in vitro. As shown in FIGS. 8b-C, FITC-labeled multifunctional nucleocapsid dendrimer was able to cross bEnd.3 cell monolayer and phagocytose by C6 cells, while the control material (without modification by DER) was not as different from the saline group, demonstrating no ability to cross the blood brain barrier.
Example 9
Each hole is 2 multiplied by 105C6 cells were plated in 12-well plates, and after overnight incubation, the medium was decanted and incubated for 24 hours with various concentrations of copper and copper complex M-CSTD. NHAc/Cu (II). Then pouring out the culture medium, washing for 3 times by PBS, digesting the cells by pancreatin, transferring to a 15ml centrifuge tube, centrifuging for 5 minutes at 1000 revolutions, removing the supernatant, adding 1ml of PBS, blowing uniformly, placing into an ice box, staining by an Annexin V/PI apoptosis detection kit according to the instruction, and detecting by a flow cytometer. As shown in fig. 9, at the same concentration in the experiment, C6 cells treated with copper and M-cstd. nhac/cu (ii) all showed apoptosis, and the degree of apoptosis increased with the increase of copper concentration in the concentration range studied, showing dependence on copper concentration.
Example 10
Each hole is 2 multiplied by 105C6 cells were plated in 12-well plates, and after overnight incubation, the medium was decanted and incubated for 24 hours with various concentrations of copper chloride and copper complex M-CSTD. NHAc/Cu (II). Then pouring out the culture medium, washing for 3 times by PBS, digesting the cells by pancreatin, transferring to a 15mL centrifuge tube, centrifuging for 5 minutes at 1000r/min to remove the supernatant, adding a proper lysate to lyse the cells, and determining the total protein according to the Byunnan BCA protein kit and the specification thereofAnd (3) measuring the content of glutathione in the cell according to the Biyun day total glutathione detection kit and the instruction thereof, and measuring the content of glutathione peroxidase in the cell according to the Biyun day glutathione peroxidase detection kit and the instruction thereof. As shown in FIG. 10, the levels of glutathione and GPx in C6 cells treated with copper and M-CSTD. NHAc/Cu (II) were not much different at the same concentrations as in the test, but decreased with the increase in copper concentration, indicating that M-CSTD. NHAc/Cu (II) has the same capacity as copper ions alone and consumes glutathione in the cells.
Example 11
Each hole is 2 multiplied by 105C6 cells were seeded in 12-well plates, and after overnight culture, the medium was decanted and incubated for 4 hours with various concentrations of the copper complex M-CSTD. NHAc/Cu (II). After that, the medium was poured off, washed 3 times with PBS, the supernatant was removed, and an appropriate volume of active oxygen probe (DCFH-DA) was added at a final concentration of 10. mu.M. The volume added is preferably sufficient to cover the cells, and usually, for one well of a six-well plate, not less than 1ml of diluted DCFH-DA is added. Incubate at 37 ℃ for 20 minutes in a cell incubator. Cells were washed three times with serum-free cell culture medium to remove DCFH-DA well without entering the cells. Thereafter, observation and photographing were performed with a confocal microscope. As a result, as shown in FIG. 11, the active oxygen content of C6 cells increased with the increase of copper concentration in the material after the copper complex treatment, which corresponded to the decrease of glutathione level.
Example 12
Firstly, 1mL of aqueous solution of copper and copper complex M-CSTD.NHAc/Cu (II) with Cu concentration of 0.2, 0.4, 0.8, 1.6 and 3.2mM is prepared respectively, and T of the material under different Cu concentration is determined by a magnetic resonance imaging analyzer1Relaxation effect, r of Cu and M-CSTD. NHAc/Cu (II) is calculated1Values, results are shown in FIG. 12a for r for Cu (II) and M-CSTD. NHAc/Cu (II)1Values of 0.8679 and 0.7331mM, respectively-1s-1，r1The values are similar, and the M-CSTD. NHAc/Cu (II) can be used as a T1 positive contrast agent in the MR molecular imaging diagnosis.
Then, a mouse body was constructedAn intra-orthotopic brain glioma model (all animal experiments were performed strictly according to the animal protection Association standards), i.e. 4-6 week female ICR mice for experiments were purchased from Shanghai Si Laike laboratory animal center (China, Shanghai). Intracranial injection (bregma moved 2mm to the left and then up 1mm and drilled 3mm deep) by mice at 1X 106C6 cell/mouse dose injection tumor cells in mice in vivo construction of in situ glioma model, 7 days later through the tail vein injection containing copper complexes M-CSTD. NHAc/Cu (II) physiological saline solution (100mL, [ Cu [)]8mM) to evaluate the effect of MR imaging at the tumor site (see fig. 12). Wherein, the scanning parameters of the nuclear magnetic resonance imager are set as follows: the scan sequence parameters are: the repetition Time (TR) is 300ms, the echo Time (TE) is 8.6ms, the thickness is 0.5mm, the acquisition matrix (matrix) is 256mm × 256, the field of view (FOV) is 2.5 × 2.5cm2, and the Flip Angle (FA) is 180 °. The results are shown in FIG. 12b, and the MR signal at the tumor site of the mice was gradually increased after injection compared to the blank group before injection, indicating that M-CSTD. NHAc/Cu (II) can be enriched at the tumor site with blood circulation and show MR signal. Meanwhile, the results of MR signal-to-noise ratio quantitative analysis (FIG. 12c) show that the SNR of the tumor site reaches the maximum value after the injection of M-CSTD. NHAc/Cu (II) for 45min, and the results show that M-CSTD. NHAc/Cu (II) can be used as T1Contrast agents are used for enhanced in vivo tumor MR imaging diagnosis.
Example 13
A mouse orthotopic brain glioma model was constructed and the chemokinetic treatment effect was evaluated seven days later by tail vein injection of physiological saline solution (100mL, [ Cu ] ═ 8mM) containing copper complex M-cstd. Injections were given every 3 days for a total of 3 administrations. Mice were weighed every 2 days and survival was recorded every two days. The results are shown in fig. 13, after chemokinetic treatment, the body weight change of the M-cstd. nhac/cu (ii) group mice is smaller than that of the blank saline group, and the survival rate is higher than that of the blank group, for example, the survival rate of the blank group mice is 0 at 28 days of modeling, while the survival rate of the treated group mice is still maintained at 50%. This demonstrates that the multifunctional core-shell dendrimer M-CSTD. NHAc/Cu (II) prepared in example 1 has good in vivo anti-tumor activity.
Comparative example 1
The preparation method of the contrast material acetyl-only core-shell dendrimer (G5.NHAc-CD/Ad-G3.NHAc, namely CSTD. NHAc) comprises the following steps:
15.48mg of Ad-G3.NH obtained in example 1 were weighed out2Dissolved in 5mL of DMSO and 5.86mg of G5.NH was weighed2CD was dissolved in 5mL DMSO solution, then Ad-G3.NH2The solution was added dropwise to G5.NH2Reaction in CD solution for 24h, transferring the obtained product to a dialysis bag with molecular weight cut-off of 50000, dialyzing in distilled water for three days (2L X3), and freeze-drying to obtain product G5.NH2-CD/Ad-G3.NH2(CSTD). Then weighing 20mg CSTD to dissolve in DMSO solution, adding triethylamine (250 muL) to mix evenly, adding acetic anhydride solution (150 muL) dropwise after 30 minutes, continuing stirring for 24 hours at room temperature, transferring the obtained product into a dialysis bag with cut-off molecular weight of 1000, dialyzing in distilled water for three days (2L multiplied by 3), then carrying out freeze drying treatment, and finally obtaining the dry contrast material acetyl core-shell dendrimer G5.NHAc-CD/Ad-G3.NHAc, namely CSTD. NHAc, and storing at-20 ℃ for later use. The characterization results are shown in FIGS. 14a and 14b, and each G5.NH can be calculated according to the ratio of the integral areas of their characteristic peaks2CD linked to 12 functional Ad-G3.NH2The molecule (fig. 14a), in addition, a methyl proton peak of acetyl groups also appears at 1.9ppm in fig. 14b, indicating that the remaining amino groups on the surface of the final material cstd. nhac have been acetylated.
Subsequently, the hydrated particle size and potential change of the prepared CSTD.NHAc, the multifunctional modified M-CSTD.NHAc and the M-CSTD.NHAc/Cu after complexing copper ions are compared through potential particle size determination. Hydrodynamic diameter test results (see fig. 5b) show that the hydrodynamic diameter (410.3nm) of the M-cstd. nhac subjected to multifunctional modification is obviously increased compared with the hydrodynamic diameter (207.3nm) of the core-shell dendrimer cstd. nhac not subjected to functional modification, but slightly shows a downward trend after complexing copper ions, and is reduced from 410.3nm to 365.6 nm. The Zeta potential measurement result (see fig. 5a) shows that the surface potential of the multifunctional acetyl core-shell dendrimer M-CSTD. NHAc is +20.1mV, which is slightly higher than that of the acetylated core-shell dendrimer CSTD. NHAc (the potential is +18.4mV) which is not subjected to functional modification under the same condition, but is slightly lower than that of the acetylated core-shell dendrimer (21.2mV) after copper complexing, which may be the reason for influencing the surface potential of the nano platform after copper ion complexing.
Comparative example 2
The control material acetyl nucleocapsid arborescent macromolecule (G5.NHAc-CD/Ad-G3. NHAc-mPEG/Pyr) without target and transmembrane function is prepared by the following steps:
(1) 29mg of mPEG-COOH, 13.88mg of EDC & HCl and 7.54mg of NHS were weighed out and dissolved in 5mL of DMSO, respectively, and then EDC & HCl and NHS solutions were added dropwise to the mPEG-COOH solution and stirred at room temperature for 3 hours. Then 5.16mg of G3.NH are weighed out2Ad dissolved in 5mL DMSO solution and the resulting activated mPEG-COOH solution added dropwise to G3.NH2Reacting in Ad solution for 3 days, transferring the obtained product into a dialysis bag with molecular weight cutoff of 3500, dialyzing in distilled water for three days (2L × 3), and freeze-drying to obtain dry Ad-G3.NH2-mPEG, stored at-20 ℃ until use.
(2) 10.07mg of Ad-G3.NH obtained in (1) were weighed2mPEG and 2.75mg of Ad-G3.NH obtained in example 12Pyr was dissolved in 5mL of DMSO, and mixed with stirring at room temperature, followed by weighing 2.93mg of G5.NH2-CD is dissolved in 5mL DMSO solution, and the mixed solution is added dropwise to G5.NH2Reacting in a CD solution for 24 hours, adding triethylamine (250 mu L), uniformly mixing, adding an acetic anhydride solution (150 mu L) dropwise after 30 minutes, continuing stirring at room temperature for 24 hours, transferring the obtained product into a dialysis bag with the cut-off molecular weight of 50000, dialyzing in distilled water for three days (2L multiplied by 3), and then carrying out freeze drying treatment to obtain a dried control material G5.NHAc-CD/Ad-G3.NHAc-mPEG/Pyr, namely CSTD. The characterization results are shown in FIG. 14c, and according to the ratio of the integrated areas of their characteristic peaks, it can be calculated that 7.1 functionalized Ad-G3.NHAc molecules are connected to each G5.NHAc-CD, and in addition, a methyl proton peak of acetyl group appears at 1.9ppm, which indicates that the residual amino group on the surface of the final material is acetylated.
(3) Dissolving the material CSTD.NHAc-mPEG/Pyr in physiological saline, adding copper chloride solid according to the molar ratio of 1:120, and performing ultrasonic dispersion to obtain a physiological saline solution of the acetylated core-shell dendrimer copper complex CSTD.NHAc-mPEG/Pyr/Cu (II).
Then, selecting murine glioma C6 cells as model cells, and evaluating the targeting superiority of the prepared M-CSTD-NHAc/Cu (II) nanoparticles compared with copper and control materials CSTD-NHAc-mPEG/Pyr/Cu (II) nanoparticles by detecting the phagocytosis amount of the cells to the M-CSTD-NHAc/Cu (II) and the control materials CSTD-NHAc-mPEG/Pyr/Cu (II) nanoparticles. First, CSTD.NHAc-mPEG/Pyr/Cu (II) and M-CSTD.NHAc/Cu (II) solutions with different copper concentrations were prepared in sterile physiological saline, and C6 cells were seeded in 12-well plates at a density of 20 ten thousand cells/well. After overnight culture, a group of C6 cells were co-cultured for 3 hours with 2. mu.M RGD addition (defined as RGD blocked C6 cells, i.e.. alpha.)vβ3C6 cells with low integrin receptor expression), and the other two groups of C6 cells co-cultured without RGD were defined as αvβ3C6 cells highly expressing integrin receptor. Then, the DMEM medium solution added with the prepared nanoparticle solution and the alpha solution are respectively addedvβ3C6 cells with high and low integrin receptor expression were co-cultured at 37 ℃ for 4 hours with saline as a blank. Then digesting and centrifuging, collecting cells, digesting with aqua regia, and detecting the phagocytosis amount of the copper element by an inductively coupled plasma spectrometer. As a result, as shown in FIG. 8a, the amount of phagocytosis of copper and copper complexes by cells gradually increased with the increase of the Cu ion concentration, and α co-cultured with copper complexes in the concentration range studiedvβ3The phagocytosis amount of C6 cells with high integrin receptor expression (without RGD blocking) to copper is obviously higher than that of alphavβ3C6 cells with low integrin receptor expression (blocked by RGD). Under the same cell condition (without RGD blocking), the phagocytosis amount of M-CSTD. NHAc/Cu (II) by the cell is also significantly higher than that of CSTD. NHAc-mPEG/Pyr/Cu (II). These results indicate that modification of the targeting molecule RGD in the material enables specific targeting of M-cstd. nhac/cu (ii) to C6 cells.
Then, an in vitro BBB model was established, and bEnd.3 cells were cultured at 2X 105The individual cells were seeded in the upper chamber (pore size: 0.4 μm). The medium was changed every 2 days. After 6 days, C6 cells were seededIn the lower chamber. The integrity of the barrier was confirmed after 24 hours by measuring the transendothelial resistance (TEER) by a cell membrane resistance meter (milligel-RES, millipore, usa). When the TEER exceeds 200 omega/cm2Were used for further experiments. FITC-labeled multifunctional modified nucleocapsid dendritic macromolecules and control materials thereof are added into an upper chamber culture medium at the concentration of 1mg/mL, after 6h of incubation, C6 cells in a lower chamber are washed by PBS and digested and collected in 1mL of PBS buffer, and then the fluorescence intensity (green fluorescence) of FITC in the cells is determined by a BD Calibur flow cytometer (BD company, USA) so as to evaluate the capacity of the multifunctional nucleocapsid dendritic macromolecules to cross the blood brain barrier in vitro. As shown in FIGS. 8b-C, FITC-labeled multifunctional nucleocapsid dendrimer has the ability to cross bEnd.3 cell monolayer and be phagocytized by C6 cells, while the control material (FITC-CSTD. NHAc-mPEG/Pyr, i.e., without modification by DER) is not much different from the normal saline group, demonstrating the ability to cross the blood brain barrier.
In the above example 1, the addition amount of each raw material used may be arbitrarily adjusted within the following range (i.e., arbitrarily adjusted to an end value or any intermediate value thereof):
in the step (1), the molar ratio of the adamantane acetic acid to the 3 rd generation polyamidoamine dendrimer is 1-1.5: 1;
in the step (2), the molar ratio of the beta-cyclodextrin to the 5 th generation polyamidoamine dendrimer is 25-30: 1;
in the step (3), the molar ratio of the targeting polypeptide RGD to the polyethylene glycol is 0.8-1.2: 1;
in the step (4), the molar ratio of the cell-penetrating polypeptide-enkephalin to the polyethylene glycol is 1.6-2.4: 1;
in step (5), 2-picolinic acid and Ad-G3.NH2The molar ratio of (A) to (B) is 45-55: 1, the reaction temperature is room temperature, and the reaction time is 2-4 d;
in step (6), RGD-PEG-COOH and Ad-G3.NH2The molar ratio of (A) to (B) is 8-12: 1, the reaction temperature is room temperature, and the reaction time is 2-4 d;
in step (7), DER-PEG-COOH and Ad-G3.NH2The molar ratio of (A) to (B) is 8-12: 1, the reaction temperature is room temperature, and the reaction time is 2-4 d;
in step (8)And the addition amount of each material meets the following requirements: the molar ratio of G5 to G3 is 1: 12-18, and the molar ratio of the total molar amount of G5 and G3 to acetic anhydride and triethylamine is 1 (10-100) to (10-100); adding into G5.NH2The reaction temperature in the CD solution is room temperature, and the reaction time is 20-24 hours; and dropwise adding triethylamine and acetic anhydride solution, and mixing at room temperature for 20-24 h.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims (10)

1. A preparation method of a multifunctional core-shell dendrimer copper complex is characterized by comprising the following steps:
(1) under the condition of solution, the adamantane acetic acid is taken for activation, and then the activated adamantane acetic acid is added into the 3 rd generation polyamidoamine dendrimer solution for reaction, and Ad-G3.NH is obtained after dialysis and freeze-drying2；
(2) Under the condition of solution, activating beta-cyclodextrin, adding the activated beta-cyclodextrin into 5 th generation polyamidoamine dendrimer solution for reaction, dialyzing and freeze-drying to obtain G5.NH2-CD；
(3) Under the condition of solution, target polypeptide RGD reacts with polyethylene glycol, and RGD-PEG-COOH is obtained after dialysis and freeze-drying;
(4) under the condition of solution, reacting the transmembrane polypeptide-enkephalin with polyethylene glycol, dialyzing, and freeze-drying to obtain DER-PEG-COOH;
(5) under the condition of solution, 2-picolinic acid is taken out for activation and then added into Ad-G3.NH2Reacting in solution, dialyzing, and freeze-drying to obtain Ad-G3.NH2-Pyr；
(6) Under the condition of solution, RGD-PEG-COOH is taken to be activated and then added into Ad-G3.NH2Reacting in solution, dialyzing, and freeze-drying to obtain Ad-G3.NH2-PEG-RGD；
(7) Under the condition of solution, after DER-PEG-COOH is taken to be activated, Ad-G3.NH is added2Reacting in solution, dialyzing, and freeze-drying to obtain Ad-G3.NH2-PEG-DER；
(8) Under the condition of solution, adding Ad-G3.NH2-Pyr、Ad-G3.NH2-PEG-RGD and Ad-G3.NH2Mixing the-PEG-DER uniformly, and adding the mixture to G5.NH2Reacting in a CD solution, dropwise adding triethylamine and an acetic anhydride solution, uniformly mixing, dialyzing, and freeze-drying to obtain M-CSTD.NHAc;
(9) and ultrasonically dispersing M-CSTD.NHAc and copper chloride under the condition of solution to obtain the target product multifunctional core-shell dendrimer copper complex.
2. The method for preparing the multifunctional core-shell dendrimer copper complex according to claim 1, wherein in the step (1), the molar ratio of the adamantane acetic acid to the 3 rd generation polyamidoamine dendrimer is 1-1.5: 1;
the reaction temperature is room temperature, and the reaction time is 2-4 d.
3. The preparation method of the multifunctional core-shell dendrimer copper complex according to claim 1, wherein in the step (2), the molar ratio of the beta-cyclodextrin to the 5 th-generation polyamidoamine dendrimer is 25-30: 1;
the reaction temperature is room temperature, and the reaction time is 58-62 h.
4. The preparation method of the multifunctional core-shell dendrimer copper complex according to claim 1, wherein in the step (3), the molar ratio of the targeting polypeptide RGD to the polyethylene glycol is 0.8-1.2: 1;
the reaction temperature is room temperature, and the reaction time is 20-24 h.
5. The preparation method of the multifunctional core-shell dendrimer copper complex according to claim 1, wherein in the step (4), the molar ratio of the cell-penetrating polypeptide-enkephalin to the polyethylene glycol is 1.6-2.4: 1;
the reaction temperature is room temperature, and the reaction time is 20-24 h.
6. The method for preparing a multifunctional core-shell dendrimer copper complex according to claim 1, wherein in step (5), 2-picolinic acid and Ad-G3.NH2The molar ratio of (A) to (B) is 45-55: 1, the reaction temperature is room temperature, and the reaction time is 2-4 d;
in step (6), RGD-PEG-COOH and Ad-G3.NH2The molar ratio of (A) to (B) is 8-12: 1, the reaction temperature is room temperature, and the reaction time is 2-4 d;
in step (7), DER-PEG-COOH and Ad-G3.NH2The molar ratio of (A) to (B) is 8-12: 1, the reaction temperature is room temperature, and the reaction time is 2-4 d.
7. The preparation method of the multifunctional core-shell dendrimer copper complex according to claim 1, wherein in the step (8), the addition amount of each material satisfies: the molar ratio of G5 to G3 is 1: 12-18, and the molar ratio of the total molar amount of G5 and G3 to acetic anhydride and triethylamine is 1 (10-100) to (10-100);
adding into G5.NH2The reaction temperature in the CD solution is room temperature, and the reaction time is 20-24 hours;
and dropwise adding triethylamine and acetic anhydride solution, and mixing at room temperature for 20-24 h.
8. The preparation method of the multifunctional core-shell dendrimer copper complex according to claim 1, wherein in the step (9), the molar ratio of M-CSTD.NHAc to copper chloride is 1 (100-140), and the ultrasonic time is 2-4 min.
9. A multifunctional core-shell dendrimer copper complex prepared by the preparation method according to any one of claims 1 to 8.
10. The use of the multifunctional core-shell dendrimer copper complex of claim 9 in the preparation of agents for MR imaging and chemokinetic therapy of tumors.
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