CN104650194A - Peptide dentritic macromolecular drug and preparation method and application thereof - Google Patents

Abstract

The invention discloses a peptide dendrimer medicine and its preparation method and application, belonging to the field of biomedicine. The medicine comprises a peptide dendrimer skeleton and a terminal functional group. The biological activity of the drug is ensured by the terminal functional group, and a large number of terminal functional groups can be grafted on the periphery of the peptide dendrimer, so that the terminal can be effectively amplified through the amplification effect of the dendrimer. Biological effects of functional groups. The drug has a precise molecular structure, abundant surface functional groups, good water solubility, and also has biological characteristics such as globulin and easy uptake by cells, which enriches the biological characteristics of peptide dendrimers and promotes Peptide drug development. The preparation method has a simple process, and can accurately control the structure of the peptide dendrimer drug by controlling the preparation of each raw material.

Description

A kind of peptide dendrimer drug and its preparation method and application

technical field

The invention belongs to the field of biomedicine, and in particular relates to a therapeutic peptide dendrimer anticancer drug.

technical background

With the increasingly serious problems of environmental pollution and food safety, the incidence of major diseases such as malignant tumors has increased significantly, and the treatment of diseases has attracted widespread attention. At present, chemotherapy based on chemical drugs is one of the most effective methods of disease treatment; drug molecules act on tumor cells to achieve the purpose of disease treatment. However, the development and application of new drugs has always been a major problem in cancer treatment research, but the development of new drugs is an important strategy to improve the efficacy of drugs, reduce side effects, and achieve the reversal of multidrug resistance.

China is the largest drug production and export country in the world, but the production of generic drugs accounts for more than 97%. In order to realize the goal of developing from a big country in medicine to a powerful country in medicine, it is imperative to strengthen the research and development of innovative drugs. The state promotes the development of "major new drug creation" through various policies and scientific and technological projects, encourages the research and development of innovative drugs, and guides the industrialization of new drugs. So far, inventing new drugs with independent intellectual property rights is still a major challenge for my country's drug research and development. The discovery of new targets and the molecular design of new drugs are important breakthroughs in the development of new drugs.

In recent years, the research and development of natural product extraction, prodrugs and metallodrugs have been fruitful in cancer treatment. Strikingly, the advantages of peptide drugs based on natural amino acids in anti-tumor therapy have been highlighted, such as their specific targets, high-efficiency biological activity, and low side effects. Researchers are trying to obtain highly effective antitumor drugs by designing and constructing peptide drugs with different amino acid sequences and molecular structures. At present, the major challenges facing peptide drugs are high production costs, difficult synthesis and purification, poor water solubility, and low cellular uptake; therefore, the development of a class of peptide drugs with easy synthesis and high efficiency is of great significance to the development of new drugs .

Contents of the invention

Based on the characteristics of precise structure, multifunctionality, biological activity and biomimetic function of peptide dendrimers, peptide dendrimers have the potential to become a new type of peptide drugs. The development of a therapeutic dendrimer anti-tumor drug not only expands the development of new peptide drugs from the perspective of molecular structure, but also improves the research of dendrimer in the field of biomedicine. The content of the present invention is not only different from the previous functional development of carrier-type dendrimers for drug loading, but also different from the previous development of linear peptide drugs. It is a novel peptide drug development in terms of structure and composition. On this basis, the present invention provides a therapeutic peptide dendrimer anticancer drug, which effectively amplifies the biological functions of the bioactive groups in amino acids by using the amplification effect of peptide dendrimers, thereby realizing the purpose of treatment.

The present invention is realized through the following technical solutions:

A therapeutic peptide dendrimer drug comprises a peptide dendrimer skeleton and a terminal functional group. The biological activity of the drug is ensured by the terminal functional group, and a large number of terminal functional groups can be grafted on the periphery of the peptide dendrimer, so that the terminal can be effectively amplified through the amplification effect of the dendrimer. Biological effects of functional groups.

As an alternative, in the above-mentioned peptide dendrimer drug, the terminal functional group is an amino acid with biological activity. The biological functions of amino acids and the interaction characteristics of biomacromolecules are effectively utilized, and the amino acid residues can improve the physicochemical properties (such as solubility) of drugs, so as to facilitate the therapeutic effect in vivo. Amino acids are used not only as terminal functional groups but also as branching units of the peptide dendrimer skeleton, which makes the structure of peptide dendrimers more compact and precise, and has the characteristics of easy synthesis, easy purification, and good water solubility.

As an alternative, in the above-mentioned peptide dendrimer drug, the terminal functional group contains an indole ring. The indole ring can interact with nucleic acid, utilize the amplification effect of dendrimers and the supramolecular action of nucleic acid, interfere with the cell cycle, and realize its anti-tumor activity.

As an alternative, in the above-mentioned peptide dendrimers, the terminal functional group is tryptophan. Using the indole ring in tryptophan as an active group, using the indole ring of tryptophan to interact with nucleic acid supramolecules, causing cell apoptosis, thereby realizing the treatment of tumors; amino acid residues can enhance the water solubility of drugs; Amino acids also serve as part of the branching units of the peptide dendrimer backbone.

As an alternative, in the above-mentioned peptide dendrimer drug, the peptide dendrimer is a first-generation or second-generation or third-generation or fourth-generation peptide dendrimer with amino acids as branching units.

As an alternative, in the above peptide dendrimer drug, the branching units in the peptide dendrimer skeleton are lysine, serine, glutamic acid, arginine, histidine, threonine At least one of amino acid, tyrosine, aspartic acid, tryptophan.

As an alternative, in the above peptide dendrimer drug, the peptide dendrimer is fan-shaped or spherical. Peptide dendrimers are used to render the drug specific to the globulin-like structure.

As an alternative, in the above-mentioned peptide dendrimer drug, the structural formula of the peptide dendrimer is as follows:

Among them, Rm represents the core molecule of the spherical molecule, m represents the functionality of the core molecule, and the functionality of the core molecule can be 3, 4, 6 or 8; K is the amino acid branching unit, G 1.0 and G 2.0 represent the first generation and Second-generation peptide dendrimers; n represents terminal functional groups.

As an alternative, in the above-mentioned peptide dendrimers, the core molecule is One of.

The present invention also provides a method for preparing the peptide dendrimer drug, which is characterized in that it comprises the following steps:

(1) preparing peptide dendrimers;

(2) Grafting terminal functional groups on the periphery of peptide dendrimers.

As an alternative, the preparation of the peptide dendrimers in step (1) may adopt a divergent method or a convergent method or a combined divergent-convergent method.

As an alternative, the preparation method of the peptide dendrimer described in step (1) is specifically:

a) Protect the functional group of the amino acid: protect the amino acid according to the difference of the surface functional group of the core molecule of the peptide dendrimer to be prepared, if the surface functional group of the core molecule is an amino group, then protect the amino group of the amino acid, such as the surface functional group of the core molecule If it is a hydroxyl or carboxyl group, the carboxyl group of the amino acid is protected;

b) Preparation of a first-generation dendrimer: Weigh branched cores (functionality n, n>1), amino acids (1.5n equivalents) containing protective groups, condensing agents (1.5n equivalents), catalysts (1.5 n equivalents) and organic bases (4n equivalents), under the condition of 0 ℃ nitrogen protection, add a solvent to carry out dehydration condensation reaction; then react at room temperature, after the reaction is over, the resulting solution is washed, dried, concentrated under reduced pressure, in an organic solvent The first-generation peptide dendrimers with protective groups were obtained by precipitation and separation in medium;

c) Deprotection: Accurately weigh the first-generation peptide dendrimer, deprotection reagent (20n equivalent), add solvent to dissolve, react at room temperature under nitrogen protection for 4 hours, remove protection, concentrate under reduced pressure, and extract or Precipitate to obtain the first generation of peptide dendrimers;

d) Preparation of second-generation dendrimers: Weigh the first-generation peptide dendrimers (functionality 2n), amino acids (3n equivalents) containing protective groups, condensing agents (3n equivalents), catalysts ( 3n equivalent) and organic base (8n equivalent), under the condition of nitrogen protection at 0 ℃, add a solvent for dehydration condensation reaction; then react at room temperature, after the reaction, the resulting solution is washed, dried, concentrated under reduced pressure, in an organic solvent The second-generation peptide dendrimers with protective groups are obtained by precipitation in medium; the third and fourth-generation dendrimers can be obtained by repeating the above deprotection and condensation reaction steps.

As an optional way, in the step (2), the terminal functional group is grafted to the periphery of the peptide dendrimer through the condensation reaction of the amino group and the carboxyl group.

As an alternative, when the terminal functional group of the peptide dendrimer drug is a biologically active amino acid (such as tryptophan), directly use the biologically active amino acid as a peptide tree The branching unit of the outermost layer pair of the macromolecular skeleton can be used to prepare higher-generation dendrimers, which can achieve the purpose of grafting terminal functional groups on the periphery of the peptide dendrimers in step (2). .

The present invention also provides an application of the above-mentioned peptide dendrimers in the preparation of antitumor drugs. The peptide dendrimer drug of the present invention is used as an anticancer drug and has a good inhibitory effect on tumors.

All features disclosed in this specification, or steps in all methods or processes disclosed, may be combined in any manner, except for mutually exclusive features and/or steps.

Beneficial effects of the present invention:

The present invention proposes a new therapeutic peptide dendrimer anticancer drug, which successfully solves the problems of poor water solubility, poor transmembrane permeability and high production cost of peptide drugs, and obtains easy synthesis, good water solubility and anticancer drug. Peptide dendrimer anticancer drug with high tumor activity.

The peptide dendritic macromolecular anticancer drug of the present invention has precise molecular structure, rich surface functional groups, and also has biological characteristics such as globoids and easy uptake by tumor cells, which enriches the peptide dendritic macromolecular The biology should and promote the development of peptide drugs.

In an optional mode of the present invention, tryptophan is used as the terminal functionalized amino acid, and the amplification effect of dendrimers can be used to make the indole ring of tryptophan interact with nucleic acid to develop peptide dendrimers. Biological functions of drugs.

The preparation method of the present invention has a simple process, and can accurately control the structure and function of the peptide drug by controlling the preparation of each raw material.

Description of drawings

Fig. 1 is a schematic diagram of the working principle of the peptide dendrimer drug of the present invention.

Fig. 2 is a synthetic route diagram of the peptide dendrimer drug described in Example 2 of the present invention.

Fig. 3 is the H NMR spectrum of the peptide dendrimer drug described in Example 2 of the present invention.

Fig. 4 is the mass spectrum of the peptide dendrimer drug described in Example 2 of the present invention.

Fig. 5 is a reversed-phase high performance liquid chromatogram of the peptide dendrimer drug described in Example 2 of the present invention.

Fig. 6 is an agarose gel electrophoresis of the interaction between the peptide dendrimer drug described in Example 3 of the present invention and DNA.

Fig. 7 is the fluorescence emission spectrum of the interaction between the peptide dendrimer drug described in Example 3 of the present invention and DNA.

Fig. 8 is a transmission electron micrograph of the interaction between the peptide dendrimer drug described in Example 3 of the present invention and DNA.

Fig. 9 is the ultraviolet-visible spectrum of the interaction between the peptide dendrimer drug described in Example 3 of the present invention and DNA.

Fig. 10 is the hydrogen spectrum of the interaction between the peptide dendrimer drug described in Example 3 of the present invention and DNA.

Fig. 11 is an infrared spectrum of the interaction between the peptide dendrimer drug and DNA in Example 3 of the present invention.

Fig. 12 is the cell survival rate of different tumor cells of the peptide dendrimer drug described in Example 4 of the present invention.

Fig. 13 is a laser confocal picture of the interaction between the peptide dendrimer drug described in Example 4 of the present invention and DNA.

Fig. 14 is a laser confocal image of the cellular uptake of the peptide dendrimer drug described in Example 4 of the present invention.

Fig. 15 is the cell cycle distribution of the interaction between the peptide dendrimer drug described in Example 4 of the present invention and DNA.

Figure 16 shows the changes in tumor volume and body weight of BABL/c mice treated for 21 days in Example 5 of the present invention.

Fig. 17 is a photo of immunohistochemical section of tumor tissue and analysis of semi-quantitative data after 21 days of treatment of BABL/c mice in Example 5 of the present invention.

Detailed ways:

The above-mentioned content of the present invention will be further described in detail below through the examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. Any modification made without departing from the spirit and principles of the present invention, as well as equivalent replacements or improvements made according to ordinary technical knowledge and conventional means in the field shall be included in the protection scope of the present invention.

Embodiment 1: the preparation of peptide dendrimer

a) Protect the functional group of the amino acid: protect the amino acid according to the difference of the surface functional group of the core molecule of the peptide dendrimer to be prepared. If the surface functional group of the core molecule is an amino group or a hydroxyl group, protect the amino group of the amino acid, such as the core molecule If the surface functional group is a carboxyl group, the carboxyl group of the amino acid is protected;

b) Preparation of a first-generation dendrimer: Weigh branched cores (functionality n, n>1), amino acids (1.5n equivalents) containing protective groups, condensing agents (1.5n equivalents), catalysts (1.5 n equivalents) and an organic base (4n equivalents), at 0°C, under nitrogen protection conditions, add a solvent for dehydration condensation reaction; then react at room temperature, after the reaction, the resulting solution is washed and dried, concentrated under reduced pressure, and organic solvent Precipitate to obtain the first generation of peptide dendrimers with protective groups;

c) Deprotection: Accurately weigh the first-generation peptide dendrimer, deprotection reagent (20n equivalent), add solvent to dissolve, react at room temperature under nitrogen protection for 4 hours, remove protection, concentrate under reduced pressure, and extract or Precipitate to obtain the first generation of peptide dendrimers;

d) Preparation of second-generation dendrimers: Weigh the first-generation peptide dendrimers (functionality 2n), amino acids (3n equivalents) containing protective groups, condensing agents (3n equivalents), catalysts ( 3n equivalent) and organic base (8n equivalent), at 0°C, under the condition of nitrogen protection, add a solvent for dehydration condensation reaction; then react at room temperature, after the reaction, the resulting solution is washed, dried, concentrated under reduced pressure, organic solvent Precipitate to obtain the second-generation peptide dendrimers with protective groups;

By repeating the above deprotection and condensation reaction steps, the third and fourth generation dendrimers can be obtained.

select separately One of them is used as a branched core, and one or more of lysine, arginine, histidine, glutamic acid or aspartic acid, and tryptophan are used as branching units to prepare a series of One to four generations of peptide dendrimers. Its structural formula is shown in formula 1.

The condensing agent, catalyst, organic base, and solvent described in this embodiment can be selected from various condensing agents, catalysts, organic bases, and solvents known in the prior art for the dehydration condensation of carboxyl and amino groups.

Embodiment 2: A specific synthetic example of a peptide dendrimer drug (the synthetic route is shown in Figure 2)

Synthesis of a new generation of peptide dendrimers (G1-Poss-Lys)

Add 30 mL of concentrated hydrochloric acid dropwise to 350 mL of methanol at 50°C, continue to raise the temperature to 90°C, slowly add 15 mL of 3-aminopropyltriethoxysilane dropwise, and react in a closed state for 24 hours, tetrahydrofuran precipitates to obtain octamer (3 - Aminopropyl)silsesquioxane hydrochloride (Poss.HCl). ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 8.14 (s, 2H), 3.61-3.59 (t, 2H), 1.69 (m, 2H), 0.75-0.72 (t, 2H).

Weigh 2.0g cage-type octamer (3-aminopropyl) silsesquioxane hydrochloride (Poss HCl), 8.0g O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) and 2.5 Add 1-g 1-hydroxybenzotriazole (HOBT) into a 100mL single-neck flask, add 5.0g Boc-lys(Boc)-OH into a 50mL constant pressure dropping funnel, vacuumize and fill with nitrogen. Add about 40 mL of distilled DMF solvent with a syringe, add 3 mL of N,N-diisopropylethylamine (DIPEA) under stirring in an ice bath, continue stirring for half an hour, remove the ice bath, and react at room temperature for 48 h. After the solvent was removed under reduced pressure, chloroform was added for dissolution, followed by washing with saturated NaHCO ₃ , NaHSO ₄ , and NaCl. After drying with anhydrous MgSO ₄ , the solvent was spun off and recrystallized in acetonitrile to obtain G1-Poss-Lys-Boc as a white powdery solid. ¹ H NMR (600 MHz, DMSO-d ₆ )δ7.76(s,1H),6.73(s,2H),3.83(d,1H),3.02-2.86(d,4H),1.46-1.19(m, J=7.0 Hz, 26H), 1.37(s, 6H), 0.55(s, 2H).

Deprotection

Add 1.0 g of G1-Poss-Lys-Boc white powder to 6 mL of trifluoroacetic acid (TFA), and react at room temperature for 6 h. The TFA was spun off under reduced pressure, pumped dry with an oil pump, added anhydrous ether and stirred, and a white precipitate was formed to obtain the compound G1-Poss-Lys. The product was directly used in the next reaction without purification.

Synthesis of Peptide Dendrimers (G2-Poss-Trp)

Repeat the synthetic steps of G1-Poss-Lys-Boc, and replace the cage octapoly (3-aminopropyl) silsesquioxane hydrochloride with a first-generation peptide dendrimer (G1-POSS-Lys). Boc-lys(Boc)-OH is replaced by Boc-Trp-OH to synthesize G2-Poss-Trp-Boc. ¹ H NMR (600MHz,DMSO-d ₆ )δ10.76(s,2H),8.32(s,3H),7.84-6.66(m,10H),4.24-4.16(t,3H),3.02(m,8H ), 1.27-1.11(s,26H), 0.55(s,2H).

Deprotection and purification

Add 1.0 g of white powder of G2-Poss-Trp-Boc to 2 mL of trifluoroacetic acid, and react at room temperature for 6 h. The TFA was spun off under reduced pressure, drained with an oil pump, and anhydrous ether was added to stir to produce a white precipitate to obtain the compound G2-Poss-Trp. The obtained G2 peptide dendrimers were dissolved in deionized water, dialyzed for 3 days, and freeze-dried for later use to obtain peptide dendrimers surrounded by tryptophan (G2-Poss-Trp).

The NMR detection (see Figure 3) shows that the first and second generation peptide dendrimers were successfully prepared by this method (Figure 3 is the NMR spectrum of G2-Poss-Trp-Boc). In order to further confirm the synthesized compound, the molecular weight of the obtained compound was also determined using a mass spectrometer (Q-TOF premier from Waters, USA). The results also confirmed that the obtained product was the target compound (Figure 4 is the mass spectrum of G2-Poss-Trp). The purity of the peptide dendrimer drug was further evaluated by high performance liquid chromatography to reach 99.9% (see FIG. 5 ).

Embodiment 3: to the peptide dendrimer drug prepared in embodiment 2, it is carried out the detection of following aspects:

(1) Use gel electrophoresis retardation experiments to observe the ability of different amounts of peptide dendrimer drugs to interact with DNA. - EGFPC1 plasmid DNA (including 200ng DNA) was compounded at room temperature for 30 minutes, and the W/P ratio refers to the molar ratio of tryptophan (W) in the peptide drug to the phosphate group (P) in the DNA. The complex was subjected to 1% agarose gel electrophoresis at 100mV for 45min, stained with ethidium bromide, and observed under a 254nm ultraviolet light. The results in Figure 6 show that when the molar ratio of peptide dendrimers to p-EGFPC1 plasmid is greater than 10 :1, it can effectively interact with the p-EGFPC1 plasmid and block its movement.

(2) At the same time, the fluorescence spectrum further indicated that there was a supramolecular interaction between peptide dendrimer drugs (TRPDs) and p-EGFPC1 plasmid. First, according to different W/P ratios (from 20:1 to 1:1), peptide dendrimer drugs (concentration: 5 μg/mL) were compounded with DNA at room temperature for 30 min, and peptides were detected by F-7000 fluorescence spectrophotometer Fluorescence emission spectrum changes of dendrimer drugs. As shown in Figure 7, at a certain concentration of peptide dendrimers (5 μg/mL), as the amount of DNA continues to increase, peptide dendrimers interact with DNA, resulting in tryptophan molecules The characteristic absorption peak at 365nm was quenched, and the formation of aggregates between peptide dendrimers and DNA led to the enhancement of the absorption peak at 578nm of scattered light.

(3) In order to further explore the interaction between the peptide dendrimer drug and nucleic acid, first, the nanoscale of the peptide dendrimer drug nucleic acid aggregate was characterized by transmission electron microscopy (TEM). Peptide dendrimers (concentration: 100 μg/mL) were compounded with DNA at a W/P of 10 at room temperature for 30 min and then freeze-dried. The aggregates formed between peptide dendrimers and DNA were observed by TEM. As shown in Figure 8, when the peptide dendrimer drug concentration is 100 μg/mL and the W/P molar ratio is 10, the peptide dendrimer drug and DNA form aggregates of about 60 nm.

(4) Secondly, the UV-Vis absorption spectrum indicated that there was a π-interaction between the peptide dendrimers and DNA. First, according to different W/P ratios (from 5:1 to 30:1), the peptide dendrimer drug (concentration: 10 μg/mL) was compounded with DNA at room temperature for 30 min, and the peptide dendrimer drug without DNA complexed (TRPDs) as a control, the UV-Vis spectrum changes of peptide dendrimer drugs were detected by UV-Vis spectrophotometer to study the π-interaction between peptide dendrimer drugs and DNA. As shown in Figure 9, under a certain concentration of peptide dendrimer drug (10 μg/mL), with the continuous increase of DNA amount, due to the tryptophan indole ring of peptide dendrimer and DNA There is a π-interaction, which broadens and increases the peak of the ultraviolet absorption of tryptophan at 250-300nm.

(5) In addition, the peptide dendrimer drug and DNA were compounded at room temperature for 30 min at different W/P (such as 100:1 and 25:1), freeze-dried, and the π ratio between the peptide dendrimer drug and DNA was studied by NMR. -interaction. As shown in Figure 10, the NMR results show that there is a π-interaction between the tryptophan indole ring in the peptide dendrimer drug and DNA. Compared with the NMR spectrum of the peptide dendrimer drug, the With the increase of DNA, the chemical shift of the proton of the indole ring obviously moves to the high field, and the peak shape becomes wider at the same time.

(6) Finally, compound peptide dendrimers and DNA with different W/P (such as 10:1 and 1:1) at room temperature for 30 minutes, and freeze-dry. We can see from infrared spectroscopy (Figure 11) that peptides Dendrimer drugs can interact with DNA, resulting in the displacement of guanine stretching vibration from 1698cm ^-1 to 1675cm ^-1 , and the displacement of PO ₂ symmetric stretching vibration from 1088cm ^-1 to 1016cm ^-1 .

Embodiment 4: biological evaluation

In vitro toxicity test of peptide dendrimers by CCK-8 method.

Select a variety of tumor cells in the active growth phase (select 7 tumor cell lines such as 4T1, HepG2, HeLa, MCF-7, MCF-7/ADR, SKOV3 and SKOV3/ADR) and inoculate them in 96-well plates, and culture them for 24 hours Afterwards, the peptide dendrimer drug prepared in Example 2 was added, and divided into 4 groups, the administration concentrations were 10ug/mL, 50ug/mL, 70ug/mL, 100ug/mL, and each experimental group was set to 6 parallel samples, and set the following control groups at the same time: replace the peptide dendrimers with tryptophan, first-generation lysine, tryptophan/first-generation lysine mixture, and second-generation lysine, and the rest of the operations same. After continuing to cultivate for a certain period of time, rinse with PBS buffer (pH 7.4) and replace with a new medium, add CCK-8, and continue to cultivate in the dark for 2 hours, measure the absorbance at 490nm with a microplate reader, and calculate the cell survival rate. The experimental results are shown in Figure 12. The peptide dendrimers have highly effective anti-tumor activity against various tumor cells, and the anti-tumor activity is enhanced with the increase of the concentration. The experimental results of the control group showed that tryptophan, first-generation lysine, tryptophan/first-generation lysine mixture and second-generation lysine had good biocompatibility.

Fluorescence changes in cells in vitro.

Take tumor cells, add medium to dilute, inoculate in a glass-bottom dish at a fixed number per well, and add peptide dendrimer drug (blue fluorescence) at a concentration of 10 μg/mL after the cells adhere to the wall. . After the cells were cultured for 2 hours, rinsed twice with PBS (pH 7.4), Hoechst 33342 labeled DNA (blue fluorescence), rinsed twice with PBS (pH 7.4), then added PBS, and observed in vitro cells with confocal laser Fluorescence changes of peptide dendrimers. The experimental results are shown in Figure 13. In the case of excitation at 405nm, not only can fluorescence be emitted in the low band (400nm to 500nm) (a and b in Figure 13), but also in the higher band (550nm to 800nm) (in Figure 13 c and d) Fluorescence emission, indicating that the fluorescence emitted in the low band is produced by peptide dendrimers, and the fluorescence emitted in the high band is due to the interaction between peptide dendrimer drugs and intracellular DNA, resulting in an increase in the intensity of scattered light. This phenomenon It is consistent with the change of fluorescence spectrum of peptide dendrimer drug and DNA complex in vitro, indicating that the peptide drug can indeed interact with intracellular DNA.

In vitro cell uptake experiments.

Tumor cells were taken, diluted with culture medium, and inoculated in a glass-bottomed dish at a fixed number per well. After the cells adhered to the wall, FITC-labeled peptide dendrimers were added at a concentration of 10 μg/mL (green fluorescence). After the cells were cultured for several time points, rinsed twice with PBS (pH 7.4), Hoechst 33342 labeled DNA (blue fluorescence), rinsed twice with PBS (pH 7.4), then added PBS, and observed in vitro with laser confocal Intracellular distribution and changes of peptide dendrimers uptake by cells. The results of laser confocal experiments ( FIG. 14 ) show that peptide dendrimers can effectively enter cells and interact with intracellular nucleic acid substances to achieve their anti-tumor effects.

Apoptosis detection.

Take tumor cells, add medium to dilute, inoculate in six-well plate according to the density of fixed number per well, after the cells adhere to the wall, add peptide dendrimers with a concentration of 10 μg/mL, and cultivate for 24 hours , collect the cells, fix with 70% ethanol for 24 hours, centrifuge, rinse with PBS once, stain the cells with PI for 30 minutes, centrifuge, rinse with PBS once, resuspend the cells with PBS, observe the effect of peptide drugs on the cell cycle by flow cytometry Influence. The experimental results are shown in Figure 15. The peptide dendrimer drug can arrest the cell cycle in the sub-G1 phase, while tryptophan, first-generation lysine, tryptophan/first-generation lysine mixture, and second-generation lysine Amino acids did not cause changes in the cell cycle. Peptide dendrimers can interact with nucleic acid supramolecules to affect the cell cycle and eventually lead to cell apoptosis.

Embodiment 5: antitumor experiment in animal body.

The 4T1 tumor model was established in 4-week-old BABL/c mice (about 20-25g). When the tumor grew to 100cm ³ , the mice were randomly divided into groups, and the peptide dendrimer drug and the control group G2-Lys were configured as At a certain concentration, each mouse was injected intramuscularly, 100 μL for each mouse, administered once every two days through the tail vein, and administered four times in total. At the same time, the tumor size and mouse weight were recorded every two days, and the observation period was 21 days. . After the experiment, the mice were dissected to prepare slices of important tissues and organs. The experimental results are shown in Figures 16, 17 and 18, Figure 16A is the arrangement of the in vivo anti-tumor experiment, Figure 16B is the change in the relative volume of the tumor within 21 days of the observation period, and Figure 16C is the change in the relative weight of the tumor during the 21 days of the observation period, Figure 17A is tumor section immunohistochemistry (H&E), CD-31, Ki-67 and TUNEL staining, Figure 17B is the semi-quantitative analysis of tumor tissue section CD-31 staining positive cells, Figure 17C is tumor tissue section Ki-67 staining positive Semi-quantitative analysis of cells, Figure 17D is a semi-quantitative analysis of TUNEL-positive cells in tumor tissue sections. The peptide dendrimer drug can effectively inhibit tumor growth, new blood vessels, tumor proliferation, and induce apoptosis of tumor cells. At the same time, the mice are in good health and have not caused lesions in other organs.

Example 6

by As a branching core, tryptophan is used as a branching unit to prepare one to four generations of peptide dendrimers. According to the biobiological evaluation method described in Example 4, the antitumor effect of the obtained first to fourth generation peptide dendrimer drugs was tested. The results showed that the peptide dendrimers of the first to fourth generations all had good water solubility, membrane penetration and anti-tumor effect, and the anti-tumor effect was enhanced with the increase of generations. Since the lumen and end groups of the therapeutic peptide dendrimers have both indole ring structures, it can further enhance its supramolecular interactions with nucleic acid molecules (conjugation, intercalation and electrostatic interactions, etc.), so it can Enhance its anti-tumor effect.

Example 7

select Condensation with a carboxyl-containing indole ring (such as 3-indole propionic acid) to obtain a 0-generation dendritic macromolecular drug with the indole ring as the terminal functional group. select As the branched core, the 1st, 2nd, and 3rd generation dendritic macromolecular frameworks were prepared respectively with amino acids as the branching units, and the amino groups on the periphery of the macromolecular frameworks of each generation were condensed with carboxyl-containing indole rings to obtain 1 and 2 , 3rd generation dendrimer drugs.

Example 8

select Condensation with a carboxyl-containing indole ring (such as 3-indole propionic acid) to obtain a 0-generation dendrimer drug with the indole ring as the terminal functional group. select As the branched core, the 1st, 2nd, and 3rd generation dendritic macromolecular frameworks were prepared respectively with amino acids as the branching units, and the amino groups on the periphery of the macromolecular frameworks of each generation were condensed with carboxyl-containing indole rings to obtain 1 and 2 , 3rd generation dendrimer drugs.

According to the biobiological evaluation method described in Example 4, the anti-tumor effects of the 0-3 generation peptide dendrimer drugs obtained in Examples 7 and 8 were tested respectively. The results of the 0 to 3 generation peptide dendrimer drugs obtained in Examples 7 and 8 are basically the same: as the number of generations increases, the number of peripheral tryptophan residues gradually increases; therefore, the peptide dendrimers and The binding ability of nucleic acid is enhanced, and the anti-tumor ability is gradually improved; with the increase of algebra, the increase of indole residues increases the possibility of the interaction between dendrimers and amino acids, but reduces the water solubility of dendrimers, and its anti-tumor Less effective than peptide dendrimers containing tryptophan residues.

The above is only a preferred embodiment of the present invention, and it is only illustrative of the present invention, rather than restrictive; those of ordinary skill in the art understand that it is carried out within the spirit and scope defined by the claims of the present invention. Many changes, modifications, and even equivalent changes will fall within the protection scope of the present invention.

Claims (10)

1. A peptide dendrimer drug, characterized in that: it comprises a peptide dendrimer skeleton and a terminal functional group. 2. The peptide dendrimer drug according to claim 1, characterized in that, the terminal functional group contains an indole ring. 3 . The peptide dendrimer drug according to claim 1 , wherein the terminal functional group is tryptophan. 4 . 4. The peptide dendrimer drug according to claim 1, wherein the branching unit in the peptide dendrimer is lysine, serine, glutamic acid, arginine, group At least one of amino acid, threonine, tyrosine, aspartic acid, tryptophan. 5. The peptide dendrimer drug according to claim 1, characterized in that the peptide dendrimer is spherical. 6. The peptide dendrimer drug according to claim 5, wherein the structural formula of the peptide dendrimer is as follows: Among them, Rm represents the core molecule of the spherical molecule, m represents the functionality of the core molecule, and the functionality of the core molecule can be 3, 4, 6 or 8; K is the amino acid branching unit, G 1.0 and G 2.0 represent the first generation and Second-generation peptide dendrimers; n represents terminal functional groups. 7. The peptide dendrimer drug according to claim 6, wherein the core molecule is One of. 8. A method for preparing a peptide dendrimer drug according to any one of claims 1-7, comprising the following steps: (1) preparing peptide dendrimers; (2) Grafting terminal functional groups on the periphery of peptide dendrimers. 9 . The preparation method according to claim 8 , characterized in that, in the step (2), the terminal functional group is grafted to the periphery of the peptide dendrimer through the condensation reaction of the amino group and the carboxyl group. 10. The use of a peptide dendrimer drug according to any one of claims 1-7 in the preparation of antitumor drugs.