CN109679093B

CN109679093B - Compound, preparation method and application thereof

Info

Publication number: CN109679093B
Application number: CN201910002904.0A
Authority: CN
Inventors: 王国营; 师冰洋
Original assignee: Henan University
Current assignee: Henan University
Priority date: 2019-01-02
Filing date: 2019-01-02
Publication date: 2021-03-30
Anticipated expiration: 2039-01-02
Also published as: CN109679093A

Abstract

The application relates to the field of compounds, in particular to a compound, and a preparation method and application thereof. A compound is a dendrimer, and at least one terminal group of the dendrimer is a Schiff base structure. Due to the specificity of the structure and the molecular framework of the dendrimer of which one or more end groups are Schiff base structures, the compound can fluoresce and has the characteristic of fluorescent color development. The compound has fluorescence tracking performance, and the application field of the dendrimer is increased.

Description

Compound, preparation method and application thereof

Technical Field

The application relates to the field of compounds, in particular to a compound, and a preparation method and application thereof.

Background

Dendrimer refers to an organic molecule having a branched structure, such as porphyrin dendrimer, aryl ether dendrimer, PAMAM dendrimer, and the like. Dendritic molecules have the same size, controllable surface functional groups and good chemical stability, and are good materials for preparing LB single-layer films, self-assembled single-layer films (SAMs), casting films, colloids and nanoclusters. The use of dendrimers in the prior art is limited and needs to be improved.

Disclosure of Invention

The embodiments of the present application aim to provide a compound, a preparation method and applications thereof, which aim to improve the problem that the use of the existing dendrimer is limited, and provide a new class of dendrimers.

In a first aspect, the present application provides a compound, wherein the compound is a dendrimer, and at least one terminal group of the dendrimer is a schiff base structure.

Due to the specificity of the structure and the molecular framework of the dendrimer of which one or more end groups are Schiff base structures, the compound can fluoresce and has the characteristic of fluorescent color development. The compound has fluorescence tracking performance, and the application field of the dendrimer is increased. When the compound is used for LB monolayer films, self-assembled monolayer films (SAMs), cast films, colloids, and nanoclusters, the above substances can be traced and identified.

According to the compound provided by the application, the end group is a Schiff base structure, so that the further modification, hydrophilicity and hydrophobicity and dispersity of the dendrimer cannot be influenced, and the stability of the dendrimer can be improved;

in some embodiments of the first aspect of the present application, the dendrimer comprises a polyethyleneimine or a polyamidoamine.

Polyamidoamines are provided whose end groups comprise Schiff base structures, which enable them to emit fluorescence, which can be detected. The maximum excitation wavelength of the compound is 485 nm, the maximum emission wavelength is 507 nm, and high-brightness green fluorescence is displayed under a fluorescence microscope.

In some embodiments of the first aspect of the present application, the dendrimer comprises polyamidoamine having a molecular weight of 25000 and 35000.

When the polyamidoamine with the molecular weight of 25000-35000 is used as a drug carrier, the carrying, transportation and release of the drug are facilitated.

In a second aspect, the present application provides a method for preparing a compound, the method mainly comprising the steps of:

reacting the dendrimer of which the end group has an amino group with glyoxal.

Reacting the dendrimer of which the end group has amino with glyoxal, and reacting one or more amino groups of the end group with glyoxal to obtain one or more Schiff base structures; the obtained compound can fluoresce, has the characteristic of fluorescence color development, has the performance of fluorescence tracking, and increases the application field of the dendrimer.

The method provided by the embodiment does not influence the further modification of the dendrimer and the hydrophilicity and hydrophobicity of the dendrimer, thereby influencing the dispersibility of the dendrimer; and the preparation method has lower cost.

In some embodiments of the second aspect of the present application, the dendrimer comprises a polyethyleneimine or a polyamidoamine.

Preferably, the dendrimer comprises polyamidoamine; preferably, the polyamidoamines have a molecular weight of 25000-35000.

The toxicity of the polyamidoamine is mainly caused by a large number of positively charged amino groups, and the positive charge on the surface of the polyamidoamine can destroy the structure of a cell membrane, thereby causing apoptosis. The amido on the surface of the polyamide amine reacts with glyoxal to generate a Schiff base structure, so that the generated product has the chemical fluorescence property and simultaneously reduces the toxicity of the polyamide amine.

In some embodiments of the second aspect of the present application, the dendrimer is reacted with glyoxal and the reaction product is modified with polyethylene glycol.

The end group of the compound molecule can be modified by adopting a product obtained after polyethylene glycol modification reaction, so that the steric hindrance of the dendrimer is increased, the biocompatibility of the dendrimer is improved, and the stability of the macromolecule is further improved.

In some embodiments of the second aspect of the present application, the above includes:

mixing the dendrimer solution with glyoxal, reacting for a preset time, and removing substances with the molecular weight less than 10000; the pH of the dendrimer solution is 9.5-10.5.

In a third aspect, the present application provides a use of a compound provided in the first aspect of the present application in fluorescence development.

Preferably, the application of the compound in a fluorescent probe or the application of the compound in preparing a fluorescent nano material.

The compound has fluorescence property, good compatibility and unique hydrodynamic property. Can be applied to fluorescent agents, fluorescent dye preparation, fluorescent powder preparation, cell imaging materials, fluorescent nano materials preparation, fluorescent probes, fluorescent kits and the like. And the fluorescent dye has no toxicity, high chemical stability, safe and environment-friendly fluorescent color development and low cost.

In a fourth aspect, the present application provides a compound as provided in the first aspect of the present application for use in a pharmaceutical delivery vehicle.

The compound can be used as a drug transport carrier, a cavity in the compound can carry drugs and slowly release the drugs, and the fluorescence property of the compound can facilitate observation of the drug transport route and the released curative effect.

In some embodiments of the fourth aspect of the present application, the use of a compound provided above in the first aspect of the present application as a carrier for doxorubicin.

The compound has excellent fluorescence tracking performance and drug transport performance, and has the functions of drug transport and signal tracking. And the compound has acid sensitivity, and is favorable for improving the killing capacity of the compound on tumor cells.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.

FIG. 1 is a nuclear magnetic resonance spectrum provided in test example 1 of the present application;

FIG. 2 is a transmission electron microscope photograph of G5, F-G5, F-G5-PEG in experimental example 2 of the present application;

FIG. 3 is a graph showing a distribution of particle sizes of G5, F-G5, and F-G5-PEG in test example 2 of the present application;

FIG. 4 is a fluorescence spectrum of F-G5 in test example 3 of the present application;

FIG. 5 is a graph showing fluorescence intensity curves of F-G0-F-G5 in test example 3 of the present application;

FIG. 6 shows fluorescence stability of F-G5 under different pH conditions in experimental example 3 of the present application;

FIG. 7 shows a fluorescence spectrum of a PEGylated amine product in Experimental example 4 of the present application;

FIG. 8 shows the surface charge changes of G5, F-G5, F-G5-PEG in test example 5 of the present application;

FIG. 9 shows the cell viability of melanoma cells according to G5, F-G5, F-G5-PEG in test example 5 of the present application;

FIG. 10 is a graph showing the cell survival rates of human vascular epidermal cells by G5, F-G5, F-G5-PEG in test example 5 of the present application;

FIG. 11 shows the results of cellular uptake of F-G5-PEG in melanoma cells over time in Experimental example 6 of the present application;

FIG. 12 shows the drug release profiles of F-G5-PEG/DOX and Free doxorubicin (Free DOX) in Experimental example 7 of the present application under different pH conditions;

FIG. 13 shows confocal fluorescence microscopy images of F-G5-PEG/DOX, G5/DOX, DOX. HCl of experimental example 8 of the present application after 4 hours of melanoma cell culture;

FIG. 14 shows the results of the cell viability of F-G5-PEG/DOX, G5/DOX, DOX. HCl in Experimental example 8 of the present application after 48 hours of culture of melanoma cells.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The compounds of the examples of the present application, methods for their preparation and their use are described in detail below.

A compound is a dendrimer, and at least one terminal group of the dendrimer is a Schiff base structure.

The dendrimer is a novel highly branched, symmetrical and radial functional macromolecule and consists of three parts, namely an inner core (Initiator core), a plurality of inner branched functional groups (Interior) and an outer surface group (Exterior). Its many unique properties: the structure is regular, and the molecular structure is accurate; the relative molecular mass can be controlled; has a large number of surface functional groups; a high degree of geometric symmetry; the spherical molecules are extruded outwards and loose inwards, and cavities exist in the molecules and can be adjusted. Is a good material for preparing LB single-layer films, self-assembly single-layer films (SAMs), casting films, colloids and nanoclusters.

Schiff bases are mainly organic compounds containing an imine or azomethine characteristic group (-RC ═ N-), and are usually formed by condensation of an amine and an active carbonyl group. In the examples of the present application, at least one end group of the dendrimer is a Schiff base structure, meaning that at least one end group of the dendrimer is (H)₃C－CH＝N－)。

The inventor finds out in the experimental process that due to the specificity of the structure and the molecular framework of the dendrimer of which one or more end groups are Schiff base structures, the compound can fluoresce and has the characteristic of fluorescent color development.

Compared with the dendrimer obtained by connecting groups with chemiluminescence on the surface, the compound provided by the embodiment of the application has at least the following advantages:

1. directly connecting groups with chemical fluorescence to the surface of the dendrimer, wherein the groups with chemical fluorescence can change the surface property of the material and influence the further modification of the dendrimer; the compounds provided in the examples of the present application do not have this drawback.

2. Directly connecting groups with chemical fluorescence to the surfaces of the dendrimers, wherein the groups with chemical fluorescence can change the hydrophilicity and hydrophobicity of the dendrimers, so that the dispersibility of the dendrimers is influenced; the compounds provided in the examples of the present application do not have this drawback.

In some embodiments of the present application, the dendrimer comprises a polyethyleneimine or a polyamidoamine.

Further, the molecular weight of the dendrimer may be 700-40000; further, the molecular weight of the dendrimer may be 800-30000; in the examples of the present application, the molecular weight of the dendrimer is not limited.

In some embodiments of the present application, for example, the dendrimer may be a polyethyleneimine having a molecular weight of 800, a polyethyleneimine having a molecular weight of 15000, or a polyethyleneimine having a molecular weight of 25000; further, the dendrimer can be polyamidoamine with molecular weight of 500-350000, further, the dendrimer can be polyamidoamine with molecular weight of 25000-35000; for example, polyamidoamines having a molecular weight of 517, 1430, 3256, 6909, 14215, 28826.

In other embodiments of the present application, the dendrimer is not limited to polyethyleneimine and polyamidoamine, and may be ferrocenyl dendrimer, porphyrin-based dendrimer, or the like.

Polyamidoamine (PAMAM) dendrimers are one of the most extensively and deeply studied dendrimers today, and share common characteristics of dendrimers, and also have their unique properties: good compatibility, low solution viscosity, unique hydrodynamic properties and easy modification. And the external amino terminal machine enables the macromolecule to substitute positive charge, thereby being widely applied to a gene delivery system. The polyamidoamine dendrimer shows wide application prospects in the biological fields of gene vectors, nano composite materials, drug transportation and the like.

However, polyamidoamines also have their limitations, for example, the absence of spontaneous detectable signals. In this example, a polyamidoamine is provided whose end groups comprise Schiff base structures, so that it can emit fluorescence and be detected. The maximum excitation wavelength of the compound is 485 nm, the maximum emission wavelength is 507 nm, and high-brightness green fluorescence is displayed under a fluorescence microscope. For convenience of description, polyamidoamine having a schiff base structure as a terminal group is named FPAMAM in examples of the present application.

The number of schiff base structures may be 1, 2, 3, 4, 6, 8, 64, 90, 128, etc.

The compound provided by the embodiment of the application can fluoresce due to the space structure of the dendrimer and the Schiff base structure of at least one end group, and can be tracked and identified when the compound is used for LB single-layer films, self-assembled single-layer films (SAMs), casting films, colloids and nanoclusters.

The embodiment of the application also provides a preparation method of the compound, which mainly comprises the following steps: reacting the dendrimer of which the end group has an amino group with glyoxal.

Further, the dendrimer having amino groups at the terminal is reacted with glyoxal, and one or more of the amino groups at the terminal are reacted with glyoxalCarrying out a reaction to obtain one or more Schiff base structures; at least one end group of the dendrimer is (H)₃C－CH＝N－)。

Due to the specificity of the structure and the molecular framework of the dendrimer of which one or more end groups are Schiff base structures, the compound can fluoresce and has the characteristic of fluorescent color development.

According to the preparation method of the compound provided by the embodiment of the application, the ethylene glycol changes the amino end group to obtain the dendrimer of which the end group is of the Schiff base structure, and the product obtained by the preparation method has chemical fluorescence property; compared with the preparation method of connecting the group with chemical fluorescence through the surface, the method has at least the following advantages:

1. the attachment of a group with chemiluminescence affects further modification of the dendrimer, which is not the case with the methods provided in this example.

2. The attachment of a group with chemiluminescence affects the hydrophilicity and hydrophobicity of the dendrimer, and thus the dispersibility thereof, which is not the case with the method provided in this example.

3. The cost of the method of attaching groups with chemiluminescence is large.

4. The method for connecting the group with the chemiluminescence is complex in the process of removing the group with the chemiluminescence, the group with the chemiluminescence has great influence on the use of a final product, and the method provided by the embodiment does not have the defect.

In the examples of the present application, the number of amino groups of the dendrimer having an amino group at the terminal is not limited, and may be, for example, 1, 2, 4, 8, 64, 128, etc.; accordingly, during the reaction, the number of schiff base structures generated by the reaction may be less than or equal to the number of amino groups.

Further, in some embodiments of the present application, the dendrimer includes a polyethyleneimine or a polyamidoamine. In other embodiments of the present application, the dendrimer is not limited to polyethyleneimine and polyamidoamine, and may be ferrocenyl dendrimer, porphyrin-based dendrimer, or the like.

The polyamidoamine has toxicity, and the toxicity of the polyamidoamine is mainly caused by a large number of positively charged amino groups, and the positive charge on the surface of the polyamidoamine can destroy the structure of a cell membrane, thereby causing apoptosis.

In some embodiments of the present application, the amino groups on the surface of the polyamidoamine react with glyoxal to form schiff base structures, which provide the resulting product with chemiluminescent properties while reducing the toxicity of the polyamidoamine.

In some embodiments of the present application, the above compounds are prepared by a process comprising: after the reaction of the dendrimer with glyoxal, the product of the reaction is modified with polyethylene glycol.

The product obtained after the polyethylene glycol modification reaction is adopted, the molecular end group of the compound can be modified, the steric hindrance of the dendrimer is enhanced, the biocompatibility of the dendrimer is improved, and the stability of the macromolecule is further improved.

In embodiments of the present application, a method of preparing a compound may comprise:

Dissolving dendrimer in water, adjusting pH of dendrimer solution to 9.5-10.5, such as pH 10, mixing dendrimer solution with glyoxal, reacting in dark for a predetermined time (such as 8-14 hr), and removing substance with molecular weight less than 10000 (such as placing the reacted mixture in a dialysis bag with molecular weight cut-off of 10000).

In other embodiments of the present application, the step of removing substances having a molecular weight less than 10000 may be performed by selecting a molecular weight to be removed according to a molecular weight of the product.

The application also provides application of the compound.

In light of the above, the inventors have found that the above compound has chemical fluorescence properties, and therefore, the above compound can be used for fluorescence development, including but not limited to preparation of fluorescent agents, preparation of fluorescent dyes, preparation of fluorescent powders, application of cell imaging materials, preparation of fluorescent nanomaterials, preparation of fluorescent probes, fluorescent kits, and the like.

The compound has the advantages of fluorescence property, no toxicity, high chemical stability, safety in fluorescence color development, environmental protection and low cost.

The application also provides application of the compound.

As mentioned above, the compound has the advantages of chemical fluorescence property, good compatibility and unique hydrodynamic property.

Therefore, the compound can be used as a drug delivery carrier, the cavity in the compound can carry the drug and slowly release the drug, and the fluorescence property of the compound can facilitate the observation of the drug delivery route and the released curative effect.

For example, in some embodiments of the present application, the above compounds may be used to carry doxorubicin. In other words, the above compounds may act as carriers for doxorubicin.

The compound has excellent fluorescence tracking performance and drug transport performance, and has the functions of drug transport and signal tracking. The compound has acid sensitivity, and is favorable for improving the killing capacity of the compound on tumor cells. Can be used as adriamycin carrier to improve the killing ability to tumor cells.

The features and properties of the present application are described in further detail below with reference to examples.

Example 1

This example provides six compounds, prepared primarily by the following steps:

dissolving 10 mg of polyamidoamine in 3 ml of aqueous solution, adjusting the pH value of the solution to 10, then dropwise adding excessive glyoxal, reacting and stirring for 12 hours in a dark place, and changing the solution into reddish brown after the reaction. After the reaction, the solution was placed in dialysis bags of different cut-off molecular weights and dialyzed with 2L of deionized water for 24 hours. The dialyzed solution is lyophilized into powder by a lyophilizer and can be stored for a long time.

In this example, 6 polyamidoamines of different molecular weights and different amino numbers were prepared and the products were designated F-G0, F-G1, F-G2, F-G3, F-G4, F-G5, respectively; accordingly, the polyamidoamine with a molecular weight of 28826 and a number of amino groups of 128 was designated as G5. The molecular weight, the number of amino groups and the product designation of the starting polyamidoamine are given in table 1.

Table 1 example 1 raw material and product items of the preparation method of each compound

Polyamidoamine molecular weight	Number of amino groups	Nomenclature of Compounds
			517	4	F-G0
1430	8	F-G1
			3256	16	F-G2
6909	32	F-G3
			14215	64	F-G4
28826	128	F-G5

In this example, the lyophilized F-G5 is dissolved in PBS (phosphate buffered saline) solution with ph7.2, 10 times molar amount of carboxyl activated polyethylene glycol (NHS-PEG) is added, the mixed solution is reacted for 4 hours, after the reaction is finished, the reaction solution is placed in a dialysis bag with 10K molecular weight cut-off, phosphate and unreacted PEG are removed by dialysis, and after dialysis, the reaction solution is lyophilized into powder to obtain polyethylene glycol PEG modified autofluorescence polyamidoamine.

The F-G0, F-G1, F-G2, F-G3, F-G4 and F-G5 provided by the embodiment can be used for manufacturing fluorescent agents, fluorescent dyes, fluorescent powder, cell imaging materials, fluorescent nano materials, fluorescent probes, fluorescent kits, fluorescent probes, drug carriers and the like. Furthermore, the compound can be used as a carrier of the adriamycin.

Example 2

This example provides three compounds, prepared primarily by the following steps:

respectively dissolving 10 mg of branched polyethylene glycol amine with different molecular weights in 3 ml of aqueous solution, adjusting the pH value of the solution to 9.5, then dropwise adding excessive glyoxal, reacting and stirring for 12 hours in a dark place, and changing the solution into reddish brown after the reaction. After the reaction is finished, the solution is placed in a dialysis bag with the corresponding molecular weight cut-off, and is dialyzed for 24 hours by using 2L of deionized water to obtain a product.

The branched polyethylene glycol amines have mass average molecular weights of 800, 15000, and 25000, respectively.

The compound provided by the embodiment can be used for preparing fluorescent agents, fluorescent dyes, fluorescent powder, cell imaging materials, fluorescent nano materials, fluorescent probes, fluorescent kits, fluorescent probes, drug carriers and the like.

Example 3

This example provides a compound prepared by essentially the following steps:

dissolving 10 mg of polyamidoamine with the molecular weight of 25000 in 3 ml of aqueous solution, adjusting the pH value of the solution to 9.5, then dropwise adding excessive glyoxal, and stirring for 12 hours in a dark reaction, wherein the solution becomes reddish brown after the reaction. After the reaction, the solution was placed in dialysis bags of different cut-off molecular weights and dialyzed with 2L of deionized water for 24 hours. The dialyzed solution is lyophilized into powder by a lyophilizer and can be stored for a long time.

Example 4

This example provides a compound prepared by essentially the following steps:

dissolving 10 mg of polyamide amine with molecular weight of 35000 in 3 ml of water solution, adjusting the pH value of the solution to 10.5, then dropwise adding excessive glyoxal, reacting and stirring for 12 hours in a dark place, and changing the solution into reddish brown after the reaction. After the reaction, the solution was placed in dialysis bags of different cut-off molecular weights and dialyzed with 2L of deionized water for 24 hours. The dialyzed solution is lyophilized into powder by a lyophilizer and can be stored for a long time.

Test example 1

The chemical structure of polyamidoamine having 28826 amino groups and 128 molecular weight (designated G5 in this example for convenience of description), which is one of the raw materials of example 1, M-SCM-2000, product F-G5 and F-G5-PEG was determined by hydrogen spectroscopy using a nuclear magnetic resonance spectrometer.

The NMR spectrum is shown in FIG. 1. In FIG. 1, a represents NHS-PEG2000-CH₃Nuclear magnetic resonance spectrum of (a); b represents the nuclear magnetic resonance spectrum of F-G5-PEG; c represents the nuclear magnetic resonance spectrum of F-G5; d represents the NMR spectrum of G5. The figure circles a plurality of characteristic peaks, and the oval circles in fig. 1 have no specific meaning.

As can be seen from fig. 1: the specific peaks of F-G5 at 1.98 and 8.37ppm were from methyl and carbon-nitrogen double bond structures, demonstrating the successful introduction of Schiff base structures on the PAMAM surface. The characteristic peak of F-G5-PEG at 3.63ppm is from a carbon-carbon single bond structure, and the successful bonding of PEG on the PAMAM surface is proved.

Test example 2

Respectively taking polyamidoamine (G5) with the molecular weight of 28826 and the amino number of 128, F-G5 and F-G5-PEG, dissolving a small amount of the polyamidoamine, the F-G5 and the F-G5-PEG in an aqueous solution, then dropwise adding the solution to a special copper net for a projection electron microscope, carrying out negative dyeing on a sample by using 2% phosphotungstic acid, drying the sample, and then placing the dried sample in the projection electron microscope to determine the physical appearance and the.

The transmission electron microscope pictures of G5, F-G5 and F-G5-PEG are shown in FIG. 2. The colors and states of G5, F-G5 and F-G5-PEG are shown in the upper right border of FIG. 2, G5 is a colorless liquid, F-G5 is a brownish yellow liquid, and F-G5-PEG is a brownish yellow liquid. The particle size distribution of G5, F-G5, and F-G5-PEG is shown in FIG. 3.

As can be seen from fig. 2 and 3: the projection electron microscope shows that F-G5 and F-G5-PEG both have good dispersibility and the particle size distribution is 25 to 35 nanometers.

Test example 3

A certain amount of F-G5 was dissolved in PBS solution, and the Excitation (Excitation) and Emission (Emission) spectra were measured using a fluorescence photometer.

The fluorescence spectrum of F-G5 is shown in FIG. 4. As can be seen from FIG. 4, the maximum excitation wavelength of F-G5 is 485 nm and the maximum emission wavelength is 507 nm.

The same mass concentration of F-G0-F-G5 was dissolved in PBS solution and the fluorescence Intensity was measured using a fluorescence photometer (fluorescence Intensity).

The fluorescence intensity curves for F-G0-F-G5 are shown in FIG. 5. As can be seen from FIG. 5, the fluorescence intensities of F-G5 and F-G4 are close to and almost coincident with each other, and the fluorescence intensities of F-G5 and F-G4 are all higher than those of F-G3, F-G42, F-G1 and F-G0. Indicating that the substances F-G3, F-G42, F-G1 and F-G0 all have the chemical fluorescence property.

A certain amount of F-G5 is dissolved in PBS solutions with different pH values, and the fluorescence intensity of the F-PAMAM under different pH conditions is measured.

The fluorescence stability of F-G5 under different pH conditions is shown in FIG. 6. As can be seen from FIG. 6, the fluorescence intensities under different pH conditions show that the fluorescence intensity of F-G5 does not vary with pH.

Test example 4

Taking the compound prepared from the polyethylene glycol amine with the mass-average molecular weight of 800 in the example 2; dissolved in PBS solution, and the Excitation (Excitation) and Emission (Emission) spectra were measured using a fluorescence photometer.

The fluorescence spectrum is shown in FIG. 7. As can be seen from FIG. 7, the compound prepared from the PEGylamine with the mass-average molecular weight of 800 has fluorescence property.

Test example 5

F-G5 and F-G5-PEG prepared in example 1 and two cell lines (human brain blood vessel cell wall cells and melanoma cells) are respectively incubated for 48 hours, and the cytotoxicity is tested, wherein the concentration ranges of F-G5 and F-G5-PEG are 0-100 micrograms/ml. And a market pin G5 was used as a control.

The surface charge changes of G5, F-G5 and F-G5-PEG are shown in figure 8, and the cell survival rates (viatility) of G5, F-G5 and F-G5-PEG to melanoma cells at different concentrations are shown in figure 9; the cell viability of G5, F-G5, and F-G5-PEG on human vascular epidermal cells at different concentrations is shown in FIG. 10.

From FIGS. 8-10, it can be seen that the survival rate of melanoma cells and human vascular epidermal cells is very low and the cytotoxicity is very high when the concentration of G5 is higher than 25 μ G/ml; the survival rates of melanoma cells and human vascular epidermal cells are high in the tested concentration range of F-G5 and F-G5-PEG, F-G5 and F-G5-PEG hardly show any cytotoxicity, and F-G5 and F-G5-PEG are high in biosafety. The reduction of F-G5 cytotoxicity is related to the reduction of surface charge, as shown in FIG. 8, the absolute value of surface charge (Zeta-potential) of F-G5, F-G5-PEG is obviously reduced compared with that of G5, thereby being beneficial to reducing the cytotoxicity brought by the material per se.

Test example 6

F-G5-PEG and melanoma cells were incubated for 2 hours, 4 hours, and 24 hours, and the uptake of material and cellular imaging effect were tested using a fluorescence microscope after cell treatment. The control group consisted of melanoma cells only and F-G5-PEG only material.

The results of the cellular uptake of F-G5-PEG into melanoma cells over time are shown in FIG. 11, wherein in FIG. 11, the number represents the images of melanoma cells alone under 2-hour, 4-hour and 24-hour fluorescence microscopy, respectively; merge represents images under fluorescent microscope of F-G5-PEG and melanoma cells incubated for 2 hours, 4 hours, and 24 hours. F-G5-PEG represents images under a fluorescent microscope incubated for 2 hours, 4 hours, 24 hours with F-G5-PEG alone.

As can be seen in fig. 11: the green fluorescence intensity became stronger with time, and after 24 hours, a strong green fluorescence foci were shown in the cells, indicating that more material was taken up by the cells with time. The fluorescence microscope shows clear and bright green fluorescence, and the material has excellent biological imaging capacity.

Test example 7

Uniformly stirring the adriamycin and the F-G5-PEG in a mass ratio of 1: 10. And after the drug is loaded, the drug is centrifuged to remove the loaded free adriamycin aggregate, and the supernatant is the adriamycin-loaded F-G5-PEG (named F-G5-PEG/DOX). Dissolving centrifugally collected adriamycin into a methanol solution, determining the amount of precipitated adriamycin by measuring ultraviolet absorbance, and subtracting centrifuged medicine from the initial total medicine amount to obtain the total medicine amount carried.

F-G5-PEG (F-G5-PEG/DOX) loaded with adriamycin is dissolved in 2 ml of phosphate buffer solution and placed in a dialysis bag with 10K molecular weight cut-off, the dialysis bag is put into the phosphate buffer solution with different pH values, and the point is taken every specific time to test the drug release speed.

The encapsulation efficiency is equal to the amount of the dendrimer encapsulated drug/the total amount of the drug added multiplied by 100 percent

The encapsulation efficiency of the mixed system is 85-95%.

The drug release profiles of F-G5-PEG/DOX and Free doxorubicin (Free DOX) at different pH environments are shown in FIG. 12. As can be seen in FIG. 12, the release of the free doxorubicin hydrochloride drug was rapid, with almost 100% being released within 2 hours. Compared with the prior art, the F-PAMAM/DOX system drug is released rapidly and slowly in the first 2 hours, so that the effect of controlled release of the drug is achieved. In addition, the drug release speed of the F-G5-PEG/DOX is related to the pH value of the solution, and the drug release speed is higher when the solution is acidic, which shows that the F-PAMAM/adriamycin system has acid sensitivity and is beneficial to improving the killing capability of the F-PAMAM/adriamycin system on tumor cells.

Test example 8

Uniformly stirring the adriamycin and the F-G5-PEG in a mass ratio of 1: 10. And after the drug is loaded, the drug is centrifuged to remove the loaded free adriamycin aggregate, and the supernatant is the adriamycin-loaded F-G5-PEG (named F-G5-PEG/DOX).

F-G5-PEG (F-G5-PEG/DOX) loaded with adriamycin, G5 molecules (G5/DOX) loaded with adriamycin, free adriamycin (DOX) and adriamycin hydrochloride (DOX. HCl) are respectively incubated with the tumor cells for 4 hours, and the uptake capacity of the tumor cells is tested by a confocal microscope and a flow cytometer after the cells are washed by buffer solution.

F-G5-PEG (F-G5-PEG/DOX) loaded with adriamycin, G5 molecules (G5/DOX) loaded with adriamycin, free adriamycin (DOX) and adriamycin hydrochloride (DOX. HCl) are respectively incubated with the tumor cells for 48 hours, the concentration of the adriamycin is set to be 0-5 micrograms/ml, and the survival rate of the cells is tested to evaluate the capability of the cells to kill the tumor cells.

Confocal fluorescence microscopy pictures of F-G5-PEG/DOX, G5/DOX, DOX. HCl after 4 hours of melanoma cell culture are shown in FIG. 13.

Fig. 13 shows microscope pictures of 16 experimental groups; in fig. 13:

row (row) of hcl represents doxorubicin hydrochloride-treated cells alone; the row with dox (tea) represents cells treated with hydrophobic doxorubicin alone; the row in which G5/DOX is located represents G5 loaded doxorubicin-treated cells; the row where F-G5-PEG/DOX is located represents FG5-PEG/DOX treated cells.

The column (vertical) in which nucleous (blue) is located represents the DAPI stained nuclei, and signals are collected by the blue channel of the fluorescence microscope; the column where carrier (Green) is located is a green fluorescence channel of a fluorescence microscope, and signals of F-G5 are collected; the column in which DOX (Red) is located is the fluorescence microscope red fluorescence channel, the signal of the ingested doxorubicin is collected, and the column in which Merge is located represents the superposition of the three colors.

The results of cell viability of F-G5-PEG/DOX, G5/DOX, DOX. HCl after 48 hours of melanoma cell culture are shown in FIG. 14.

As can be seen from FIG. 13 and FIG. 14, F-G5-PEG/DOX significantly improved the uptake and killing ability of tumor cells compared to free Doxorubicin (DOX).

The F-G5-PEG loaded adriamycin can kill tumor cells, and the F-G5-PEG loaded adriamycin has larger killing capacity on the tumor cells when the concentration of the tumor cells is higher.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A compound, wherein said compound is a dendrimer and at least one end group of said dendrimer is a schiff base structure; the dendrimer comprises a polyamidoamine.

2. The compound of claim 1 wherein the polyamidoamine has a molecular weight of 25000-35000.

3. A preparation method of a compound is characterized by mainly comprising the following steps:

reacting dendrimer with amino at the end group with glyoxal; the dendrimer comprises a polyamidoamine.

4. The method of claim 3, wherein the polyamidoamine has a molecular weight of 25000-35000.

5. The method of claim 3 or 4, wherein the reaction of the dendrimer with the glyoxal is followed by modifying the reaction product with polyethylene glycol.

6. A process for the preparation of a compound according to claim 3 or 4, comprising:

mixing the dendrimer solution with the glyoxal, reacting for a preset time, and removing substances with the molecular weight less than 10000; the pH of the dendrimer solution is 9.5-10.5.

7. Use of a compound according to claim 1 or 2 for fluorescence development.

8. The use according to claim 7, wherein the use of the compound is in a fluorescent probe or in the preparation of a fluorescent nanomaterial.

9. Use of a compound according to claim 1 or 2 in a drug delivery vehicle.

10. Use according to claim 9, characterized in that the compound is used as a carrier for doxorubicin.