CN111607101A

CN111607101A - Dendritic macromolecule with active oxygen responsiveness and preparation method and application thereof

Info

Publication number: CN111607101A
Application number: CN202010606730.1A
Authority: CN
Inventors: 丁寅; 胡子涛
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2020-06-29
Filing date: 2020-06-29
Publication date: 2020-09-01

Abstract

The invention provides a polymer nano-carrier for drug delivery, a dendritic macromolecular carrier with Reactive Oxygen Species (ROS) and Glutathione (GSH) responsiveness, a preparation method and biological application thereof. The method is characterized in that: dendrimers comprising 1, 2-bis (2-aminoethoxy) ethane as the core molecule of the dendrimer, oligo (ethylene glycol) -containing (OEG/PEG) molecules to construct the dendrimer and provide a thermosensitive and hydrophilic scaffold, and ROS-responsive (peroxyoxalate) block copolymer segments in the hydrophobic arms.

Description

Dendritic macromolecule with active oxygen responsiveness and preparation method and application thereof

Technical Field

The invention relates to a dendrimer with Reactive Oxygen Species (ROS) responsiveness, a preparation method and application thereof, and belongs to the field of polymer nano-drug carriers.

Background

The nano-carrier is used for loading micromolecular chemotherapeutic drugs, proteins and genes, so that the toxic and side effects of the drugs can be reduced, and the nano-carrier plays an important role in tumor treatment. The distribution of the nano-drug carrier in the tumor directly influences the treatment effect of the drug loaded on the nano-drug carrier. However, the unique physiopathological features of tumors lead to the inability of the vector to penetrate deeply into the tumor, preventing its uniform distribution.

In addition, after the nano-drug is taken by tumor cells, the drug is not fully released, and the effect of cancer treatment is also greatly limited. In fact, different stimuli may be present simultaneously in the tumor cells, and different stimuli may also be present in different regions of the tumor. A cancer cell may have different levels of stimulation at different stages of development. In order to improve the bioavailability of drugs, the design of multi-responsive carriers has become necessary. Despite the great development of various stimuli-responsive block copolymers, challenges remain. First, an ideal controlled release should allow for a rapid release but not an explosive release. In most reported copolymer systems, the stimulus-sensitive units are located on the crosslinking units, or side chains, and the number on the main chain is limited. Thus, the component structure of the drug delivery system cannot be completely destroyed by environmental stimuli, resulting in a slow but incomplete drug release. At the same time, high Drug Loading (DLC) and high Drug Loading Efficiency (DLE) are also often necessary. Therefore, if additional groups for drug interaction are provided during the design of the stimuli-sensitive segment, the levels of DLC and DLE in the carriers can be greatly improved.

Reactive Oxygen Species (ROS) are internal stimuli with the sensitivity to tumor heterogeneity, resulting from the difference in reactive oxygen species between the mildly oxidized extracellular space of the tumor and the oxidatively increased intracellular space. ROS are highly reactive ions and free radicals, and play a crucial role in regulating protein functions, producing various hormones, regulating cell signals, regulating inflammation, and eliminating pathogens. An increase in ROS levels triggers the risk of cellular DNA mutation, which is closely associated with the proliferation of many cancer cells. Much research on ROS-responsive carrier systems currently focuses mainly on polymeric nanoparticles, hydrogels, inorganic nanoparticles, and activatable prodrugs.

Polymeric nanoparticles have been recognized as effective carriers for imaging and therapeutic drug delivery. By controlling the hydrophilic-lipophilic balance of the segmented copolymer and the self-assembly in a solution state, the physical and chemical properties of colloidal particles, such as the shape, the size, the surface charge and the like, can be conveniently regulated and controlled, so that the distribution of the colloidal particles in a body is influenced. Although several types of stimuli responsive to factors such as pH, temperature, light, etc., enzymatic or bioreductive environmental responses, etc., have been extensively explored in polymer nanocarrier applications, the incorporation of ROS responsive units into polymer nanoslabs has resulted in a regulated drug release, which has not been of interest until recently, as more and more evidence suggests that many pathogenic processes are implicated in ROS. The success of the development of ROS-responsive drug delivery systems depends largely on the choice of effective ROS-sensitive materials. The different ROS-responsive linkages are generally achieved by degradation or solubility switching mechanisms (hydrophobic to hydrophilic transitions) of the ROS-responsive linkages in the carrier at the time of triggering drug release. ROS-responsive polymer micellar systems that have been developed to date, classified according to the type of ROS-responsive unit incorporated into the polymer nanoparticle, are mainly: organometallic chalcogen (selenium or tellurium) and organometallic boron linkage, arylboronic acid esters, sulfur-containing linkages such as thioethers, ketals; aryl oxalates, ferrocenes, and the like. The type of linkage, sensitivity and its location in the carrier system, greatly influence the kinetics of drug release.

It is well established that the tumor penetration capacity of nanomaterials increases with decreasing particle size. Therefore, low nanosized dendrimers are likely to confer good tumor tissue penetration capability to the carrier. In addition, dendrimers have many advantageous properties, such as monodisperse and highly branched structures, spherical shape, the presence of internal cavities, and abundant peripheral groups, making dendrimers very promising nanocarriers. The oligo (ethylene glycol) (OEG)/PEG molecule can be used for constructing a dendrimer, providing a temperature-sensitive and hydrophilic skeleton, and promoting the application of the dendrimer in biomedicine.

The invention aims to synthesize a novel degradable monodisperse nonionic OEG/PEG-ROS response dendritic block polymer nano-drug carrier, and researches the tumor drug loading capacity of the carrier and the tumor microenvironment response behavior of the composite drug-loaded colloidal particles. Dendrimers up to the fourth generation were synthesized using cu (i) catalyzed azide-alkyne cycloaddition (CuAAC) click polymerization and amidation coupling reactions. A redox (ROS/GSH) double sensitive chain segment is designed and synthesized on the hydrophobic part of the dendritic polymer, for example, a disulfide bond, organometallic sulfur group (selenium or tellurium), thioether, peroxyoxalate and other repeating units are introduced into the hydrophobic chain segment of the polymer. A large number of triazole groups are formed on the main chain of the polymer to serve as hydrogen bond donors and acceptors, so that the loading capacity of the antitumor drug is increased. Acid-labile carboxylic acid amide groups are modified at the tail ends of the dendritic block polymers, so that the colloidal particle surface charge reversal characteristic of the dependence of the tumor microenvironment on pH is obtained.

Disclosure of Invention

The purpose of the invention is: synthesizing a novel degradable monodisperse nonionic OEG/PEG-ROS response dendritic block polymer nano-drug carrier, and providing a preparation method and application thereof.

The technical scheme adopted by the invention is as follows:

a multi-responsive dendrimer containing ROS response is characterized in that the dendrimer comprises a dendrimer system and a molecular chain segment with tumor microenvironment response (ROS, GSH and pH response), a synthetic route of the dendrimer is designed by taking diamino tri (ethylene glycol) as a core molecule (an initial molecule), a monomer with four alkyne groups is used as a branch unit to react with the core molecule to generate 0.5 generation (G0.5), and the branch unit is terminated by the alkyne groups; the hydrophobic chain segment which is rich in organometallic sulfur group selenium or tellurium, thioether, peroxyoxalate, disulfide bond and the like and has ROS or GSH sensitive groups is connected to the G0.5 generation molecule by a Cu (I) catalyzed alkyne-azide 1, 3-dipolar cycloaddition (CuAAC) click polymerization method, and a monomer with a tert-Butyloxycarbonyl (BOC) protected amino group is connected through the CuAAC reaction to generate a generation 1 (G1) dendrimer; cutting Boc protection and repeating the process, so as to synthesize successive generations of dendritic macromolecules G1.5, G2, G2.5 and G3 until G3.5 generation dendritic macromolecules, further grafting O, O' -bis (2-aminoethyl) octaethylene glycol protected by Boc monomer (providing a thermosensitive and hydrophilic scaffold for the dendritic macromolecules), and synthesizing generation 4 dendritic macromolecule G4; the synthesized dendrimer is terminated by alkynyl groups if the generation is half, and terminated by Boc-protected amino groups if the generation is full, thereby greatly facilitating functionalization.

The above ROS-responsive dendrimer, wherein said dendrimer comprises Reactive Oxygen Species (ROS) -sensitive groups, having the structure:

a peroxyoxalate ester.

A method for preparing ROS responsive dendrimer comprises the following steps:

step 1, synthesizing 0.5 generation dendrimer G0.5 with four alkynyl groups

Reacting di (propyne-2-oxymethyl) -propionyl chloride with di (2-aminoethyl) glycol in the presence of triethylamine to obtain

The reaction formula is as follows:

step 2, obtaining a generation 1 dendrimer G1 with ROS response groups as hydrophobic chain segments and amino end groups protected by 4 tert-butoxycarbonyl (Boc) through CuAAC cycloaddition reaction;

(a) g0.5 and a compound 3 (oxalic acid di (3-azidopropyl) ester) react in the presence of copper sulfate pentahydrate to obtain a product A,

(b) reacting the product A with a compound 4 (diacetylene cystine), N, N, N ', N ', N ' -Pentamethyldiethylenetriamine (PMDETA) and cuprous chloride to obtain a product B,

(c) and reacting the product B with a compound 5 (azido-Boc protected o, o' -bis (2-aminoethyl) diethylene glycol) in a mixed solution of tetrahydrofuran and water under the action of copper sulfate pentahydrate and sodium ascorbate to obtain G1.

Step 3, after the Boc protecting group is cleaved, repeating the above process, and synthesizing the G1.5, G2, G2.5, G3 or G3.5 generation dendrimer:

step 4, grafting oligo (ethylene glycol) (OEG/PEG) molecules through CuAAC reaction, namely, O' -bis (2-aminoethyl) octadecyl glycol protected by Boc monomer as a hydrophilic chain segment to synthesize the 4 th generation dendritic macromolecule G4

Step 5, preparation of Doxorubicin (DOX) -loaded ROS response dendrimer drug-loaded nanoparticles

A polymer sample of 0.6mg of G4 was placed in a 10mL white serum glass bottle and dissolved in 1mL DMSO, and the mixture was stirred uniformly, after complete dissolution, a DOX (4mg/mL, 30. mu.L) solution was added, and 2mL of a phosphate buffer solution (PB, 10mmol/L, pH 7.4) was slowly added dropwise with a syringe pump at a rate of 30. mu.L/min, and after completion of the addition, stirring was continued for 1 hour. The solution was transferred to a dialysis bag (2000DaMWCO) and dialyzed against PB (10mmol/L, pH 7.4) for 6h, changing the dialysate every 2 h. And after the dialysis is finished, obtaining DOX polymer wrapped nanoparticle solution with volume of 5mL and concentration of 0.12 mg/mL.

The invention has the beneficial effects that: 1) the invention provides a preparation method of a multi-response type dendrimer with ROS response inside, which is successfully used for drug delivery and realizes tumor treatment. 2) The dendritic macromolecule selects the biocompatible monomer and the chain segment to participate in the reaction, and the target molecule has an accurate structure, so that the polymer nano carrier has good biological safety and stable pharmacokinetics. 3) The dendrimer is synthesized through CuAAC polymerization and amidation reaction, and has mild reaction, high efficiency and biological friendliness. 4) The dendrimer can be prepared into dendrimers with different tumor microenvironment responses by introducing and designing different chemical bonds, so that the stability of the particles in the circulating process and the release of tumor microenvironment response type drugs are realized. 5) The dendrimer provided by the invention provides a novel dendrimer with ROS response, and the dendrimer has wider biomedical application by utilizing the characteristics of tumor high-expression active oxygen free Radicals (ROS).

Drawings

FIG. 1 is a diagram of the G4 dendrimer structure;

FIG. 2 is a TEM image of a G4 dendrimer;

FIG. 3 is the average particle size of the G2, G3, and G4 molecules;

fig. 4 shows ROS response properties of G4 nanoparticles: at 50mmol/L H₂O₂Emission spectra of the lower NR fluorescent probes as a function of time;

fig. 5 shows the pH response property of G4 nanoparticles: emission spectrum of NR fluorescent probe with time at pH 5;

FIG. 6 shows the molecular weight distribution at 50mmol/L H₂O₂(ii) DOX release profile over time;

fig. 7 is a release profile of DOX over time at pH 5.

Detailed Description

The present invention is further described below in conjunction with examples, which are to be understood as being illustrative only and in no way limiting. The starting materials mentioned in the examples below were selected for analytical purification and are all commercially available.

Examples

1. Synthesis of Generation 0.5 dendrimer G0.5 with four alkynyl groups

a. Synthesis of Compound 1:

sodium hydride (1.5g, 0.0376mol) was washed several times with dry petroleum ether and suspended in 20ml of anhydrous DMF. To the resulting suspension was added a solution of 2, 2-bis (hydroxymethyl) propionic acid (2g, 0.0148mol) in 5ml anhydrous DMF under ice-water bath conditions. The resulting mixture was stirred for 0.5 h under an ice-water bath and a solution of propargyl bromide (80 wt% in toluene, 3.31ml, 0.0307mol) was added slowly via syringe. The resulting mixture was slowly warmed to room temperature and stirred for 5 hours. After quenching the excess sodium hydride with chilled deionized water, a large amount of deionized water was added. The aqueous phase is washed 3 times with 20ml of Dichloromethane (DCM) and then acidified to pH 2 with 1M HCl solution. The crude product was extracted from the aqueous solution by DCM and Na was used₂SO₄Drying and column chromatography using chloroform/petroleum ether/acetic acid eluent (3/7/0.05, vol.) gave compound 1 as a pale yellow oil.

b. Synthesis of Compound 2:

to a solution of compound 1(1g, 4.76mmol) in 20ml of anhydrous DCM was added oxalyl chloride (10ml, 0.118 mol). The resulting mixture was stirred at reflux for 1 hour. The solvent was removed under reduced pressure to give the compound 2, bis (propyne-2-oxymethyl) -propionyl chloride.

c. Synthesis of G0.5 generation dendrimers

Compound 2 was dissolved in 5ml of dry DCM solvent, and diaminotriethylene glycol solution [ 1g, 6.748 mmol ] was slowly added and dissolved in anhydrous DCM containing 4.8 ml (0.033 mol) of triethylamine]Stirring at 0 ℃. The resulting solution was warmed to room temperature and slowly stirred for 5 hours, after which the reaction mixture was washed 3 times with 100ml of DCM, 30ml of water, Na₂SO₄After drying, the solvent is evaporated off. The crude product is further purified by column chromatography using DCM/MeOH eluent(from 99/1 to 95/5 by volume) gave compound G0.5 as a pale yellow viscous oil (3.3G, 92% yield).

G0.51H NMR spectra were assigned as follows:

1H NMR(500MHz，CDCl3)：7.07(br s，2H)，4.17(t，J＝2.5Hz，8H)，3.66-3.62(m，12H)，3.56(t，J＝5.0Hz，4H)，3.45(q，J＝5.0Hz，4H)，2.47(t，J＝2.5Hz，4H)，1.19(s，6H).

2. generation 1 dendrimer G1 with ROS-responsive groups as hydrophobic segments and 4 tert-butoxycarbonyl (Boc) protected amino end groups was obtained by CuAAC cycloaddition.

a. Synthesis of Compound 3

First, 3-chloro-1-propanol (3g, 0.032mol), sodium azide (5g, 0.079mol) and sodium hydroxide (0.2g, 5mmol) were added in proportion to 50ml of distilled water. After stirring at 50 ℃ for 24 hours, the product was extracted with diethyl ether. The combined organic extracts were washed with saturated sodium chloride (NaCl) and MgSO₄And (5) drying. The volatiles were evaporated to give a colorless oil (3-azido-1-propanol).

A solution of oxalyl chloride (0.8mL, 9.454mmol) in 30mL of dichloromethane was added dropwise to a well-stirred ice-cooled solution of 3-azido-1-propanol (1.00g, 9.9mmol), TEA (5mL, 36mmol) and DMAP (1.222g, 10mmol) in 20mL of dichloromethane. The reaction was stirred vigorously under argon at room temperature for 15 hours. After filtration, the filtrate was washed with brine 4 times, over MgSO₄Drying and evaporating the solvent to obtain the oily substance which is oxalic acid di (3-azidopropyl) ester with the yield of 71.3 percent.

b. Synthesis of Compound 4

A stirred solution of BOC-cystine (1g, 2.26mmol) and TEA (1.8ml, 0.013mol) in 100ml of anhydrous DCM was cooled to give the dideoxy cystine derivative. Propargylamine (0.8ml, 0.0125mol) was added in portions in an ice bath. The mixture was kept at 0 ℃ and a solution of BOP reagent (2.3g, 5.20mmol) in 7ml of dichloromethane was added and reacted for 20 minutes under argon. After 1 hour in ice bath, the reaction was kept at room temperature for 12 hours. The methylene chloride was removed by distillation under reduced pressure. The crude product is dissolved in 100ml of ethyl acetate and the mixture is taken up with saturated potassium hydrogen sulfate (KHSO)₄) Saturated sodium dicarbonate (NaHCO)₃) The solution and saturated sodium chloride (NaCl) were washed sequentially. Followed by magnesium sulfate (MgSO)₄) Drying and evaporating to remove the solvent to obtain the compound 4 of the bis-alkynylated cystine.

c. Synthesis of Compound 5

a) Functional group protection of o, o' -bis (2-aminoethyl) diethylene glycol: protecting according to the difference of the surface functional groups of the core molecules of the dendrimer to be prepared, if the surface functional groups of the core molecules are amino groups, protecting the amino groups.

b) Imidazole-1-sulfonylazide hydrochloride (0.3g, 1.4319mmol) was added to a solution of Boc protected o, o' -bis (2-aminoethyl) diethylene glycol (0.5g, 2.0145mmol), potassium carbonate (0.35g, 2.53mmol) and copper (II) sulfate pentahydrate (2mmg, 0.0080mmol) in 10ml of methanol after completion of the reaction monitored by TLC, the mixture was concentrated, 30ml of dichloromethane were added, the resulting mixture was washed with water (3 × 20ml), washed with Na₂SO₄After drying the solvent was evaporated off. The crude product was further purified by column chromatography using DCM/methanol (92/8, vol/vol) eluent to afford compound 5, azido-Boc protected o, o' -bis (2-aminoethyl) diethylene glycol as a colorless oil.

d. Synthesis of G1 generation dendrimer

G0.5(1G, 1.879mmol), compound 3(1G, 5.10mmol), and copper (II) sulfate pentahydrate (100mg, 0.401mmol) were dispersed in 30ml of anhydrous THF, and the reaction mixture was stirred under argon for 1 hour. Polycyclic addition reaction under argon atmosphere gave product A, which was deoxygenated by dissolving A (0.7575g, 0.5mmol), Compound 4(0.257g, 0.5mmol), PMDETA (0.02g, 0.115mmol) and CuCl (0.01g, 0.101mmol) in 5mL of anhydrous DMF at 30 deg.C for 12 h. After the reaction was complete the reaction mixture was precipitated in cold ether and the precipitate was washed with copious amounts of water to give the hydrophobic block B.

Product B described above, compound 5(2.0g, 7.3mmol) and copper (II) sulfate pentahydrate (100mg, 0.401mmol) were dispersed in 40ml of anhydrous THF. To the resulting mixture was added dropwise, under argon, a solution of sodium ascorbate (100mg, 0.505mmol) in 40ml of deionized water. The reaction mixture was stirred under argon for 1 hour. After removal of most of the THF under reduced pressure, the crude product was extracted with DCM and then Na₂SO₄Dried and further purified by column chromatography with DCM/methanol (98/2, vol) eluent to give G1 as a pale yellow viscous oil.

G1¹The H NMR spectrum is attributed as follows:

¹H NMR(500MHz，CDCl₃)：4.4(t，2H)、3.46(t，2H)、2.02(m，2H)1.48(s，18H)、2.18(s，2H)、2.94(m，4H)，4.08(m，4H)，4.92(br s，2H)、5.53(d，2H)、8.07(br s，2H)。

3. after the Boc protecting group is cleaved, the above procedure is repeated, and dendrimers of G1.5, G2, G2.5, G3, G3.5 generation can be synthesized.

Trifluoroacetic acid (TFA) (3.8ml, 0.051mol) was added to a solution of G1(1G, 0.215mmol) in 2.8ml DCM under ice bath conditions. The resulting mixture was stirred at room temperature for 3 hours. Removal of the solvent under reduced pressure gave deprotected G1.

Oily compound 2(0.8g, 3.47mmol) was dissolved in 2ml of anhydrous DCM. To the resulting solution was slowly added, with stirring in an ice bath, deprotected G1(0.215mmol) dissolved in 10ml of anhydrous DCM containing TEA (0.5ml, 3.61mmol)And (4) liquid. The resulting solution was slowly warmed to room temperature and stirred for 3 hours. After this time, the reaction mixture was diluted with 50ml of DCM, washed 3 times with 20ml of water and Na₂SO₄Drying and evaporating. The crude product was further purified by column chromatography with DCM/methanol eluent (98/2 vol) to give G1.5 as a pale yellow viscous oil.

G1.5(2.5G, 0.448mmol), compound 3(1.8G, 9.18mmol) and copper (II) sulfate pentahydrate (130mg, 0.521mmol) were dispersed in 152040ml of anhydrous THF. The reaction mixture was stirred under argon for 2.5 hours. The polycyclic addition reaction is carried out under argon. A solution of the above product, Compound 4(1g, 1.95mmol), PMDETA (0.1g, 0.577mmol) and CuCl (0.03g, 0.303mmol) in 20mL anhydrous DMF was deoxygenated at 60 ℃ for 12 h. At the end of the polymerization, a degassed solution of compound 5 was added to a Schlenk tube to achieve a di-end group on the polymer. Precipitation in cold ether and washing of the precipitate with copious amounts of water gave the product (hydrophobic block).

The above product, Compound 5(2.3g, 8.40mmol) and copper (II) sulfate pentahydrate (130mg, 0.521mmol) were dispersed in 40ml of anhydrous THF, to the resulting mixture was added dropwise a solution of sodium ascorbate (180mg, 0.909mmol) in 35ml of deionized water under argon, the reaction mixture was stirred under argon for 2 hours, after removal of most of the THF under reduced pressure, the crude product was extracted with 3 × 60ml of DCM, extracted with Na₂SO₄Dried and further purified by column chromatography with DCM/methanol (290/10, vol) eluent to give G2 as a pale yellow viscous oil.

The above steps are repeated to synthesize G2.5, G3 and G3.5.

4. Oligomeric (ethylene glycol) (OEG/PEG) molecules are grafted through CuAAC reaction, namely O, O' -bis (2-aminoethyl) octadecyl glycol protected by Boc monomers is used as a hydrophilic chain segment, and the generation 4 dendritic macromolecule G4 is synthesized.

Synthesis of Boc monomer protected O, O' -bis (2-aminoethyl) octanediol according to the published procedure

Imidazole-1-sulfonylazide hydrochloride (200mg, 0.954mmol) was added to a solution of compound 4(2g, 0.021mmol), potassium carbonate (0.2g, 1.45mmol) and copper (II) pentahydrate (1.2mg, 0.00481mmol) in 3ml of methanol after completion of the reaction monitored by TLC, the mixture was concentrated, 40ml of DCM was added and the resulting mixture was washed with water (3 × 20ml), Na₂SO₄Dried and evaporated. The crude product was further purified by column chromatography with DCM/MeOH eluent (99/1, vol.) to afford Boc-PEG-N₃As a white solid.

Mixing G3.5, Boc-PEG-N₃And (0.4g, 0.391mmol) and copper (II) sulfate pentahydrate (4mg, 0.016mmol) in 2ml of anhydrous THF. To the resulting mixture was added dropwise a solution of sodium ascorbate (8mg, 0.040mmol) in 2ml of deionized water under argon. The reaction mixture was stirred under argon for 1 hour. After removal of most of the THF under reduced pressure, the crude product was extracted 3 times with 15ml of DCM and Na₂SO₄Dried and further purified by column chromatography with DCM/methanol (13/1, vol.) eluent to give G4 as a pale yellow viscous oil.

G4¹The H NMR spectrum is attributed as follows:

1H NMR(500MHz，CDCl3)：4.4(t，2H)、3.46(t，2H)、2.02(m，2H)1.48(s，18H)、2.18(s，2H)、2.94(m，4H)，4.08(m，4H)，4.92(br s，2H)、5.53(d，2H)、8.07(br s，2H)5.01(br s，1H)，3.68(t，J＝5.0Hz，2H)，3.65(s，4H)，3.55(t，J＝5.0Hz，2H)，3.40(t，J＝5.0Hz，2H)，3.32(q，J＝5.0Hz，2H)，1.45(s，9H).7.80(s，60H)，7.40(br s，30H)，5.12(br s，32H)，4.63(s，120H)，4.56(s，120H)，3.89(s，120H)，3.58-3.41(m，548H)，3.30(q，J＝5.0Hz，64H)，1.43(s，288H)，1.14(s，90H).

5. preparation of G4 nanoparticle suspension

Taking a polymer sample, namely 0.5mg of G4 molecule, dissolving the polymer sample in a 10mL white serum glass bottle and 1mL DMSO, uniformly stirring, slowly dropwise adding 2mL phosphoric acid buffer solution (PB, 10mmol/L, pH 7.4) by using a syringe pump, setting the dropwise adding speed to be 30 mu L/min, and continuously stirring for 1h after the dropwise adding is finished. The solution was transferred to dialysis bags (2000Da MWCO) and dialyzed against PB (10mmol/L, pH 7.4) for 6h, changing dialysate every 2 h. After the dialysis, a volume of 5mL of G4 polymer nanoparticle suspension with a concentration of 0.10mg/mL was obtained.

6. Transmission Electron microscopy analysis (TEM)

Taking a G4 nano particle solution (20 mu L, 0.10mg/mL) on a copper net containing a carbon supporting film, sucking excess solution by using filter paper after 1min, dripping 20 mu L of uranyl acetate for dyeing, sucking excess dyeing agent by using filter paper after 1min, washing by using 20 mu L of ultrapure water, naturally airing, and observing by using a transmission electron microscope after sample preparation is finished, wherein the result is shown in figure 2.

7. Dynamic light scattering particle size analysis (DLS)

G2, G3 and G4 nanoparticle solutions (0.5mL and 0.10mg/mL) were added to a 1mL quartz cell, and the mixture was measured in a dynamic laser light scattering apparatus at a reflection angle of 173 and a temperature of 25 ℃, and the results are shown in FIG. 3, wherein the particle size was 15-20 nm.

8. Nile Red (NR) fluorescence assay

Mixing NR in ethanol (10. mu.L, 1.0 × 10)^-3mol/L) was added to 5mL of the polymer nanoparticle solution (0.10mg/mL, 10mmol/L pH 7.4PB) and stirred overnight at 25 ℃ in the dark. Adding acetic acid buffer solution with pH 5 into the partial solution, adjusting to corresponding pH, and adding different amounts of H₂O₂Adding into the partial solution, and adjusting to H₂O₂＝50mmol/L、H₂O₂The emission spectra at different times NR were recorded by fluorescence spectroscopy at room temperature under 100mmol/L with an excitation wavelength of 545nm and an emission wavelength range of 560-700 nm. The results are shown in FIGS. 4-5.

9. Encapsulation and release test of G4 loaded antitumor drug doxorubicin DOX

A polymer sample of 0.6mg of G4 was placed in a 10mL white serum glass bottle and dissolved in 1mL DMSO, and the mixture was stirred uniformly, after complete dissolution, a DOX (4mg/mL, 30. mu.L) solution was added, and 2mL of a phosphate buffer solution (PB, 10mmol/L, pH 7.4) was slowly added dropwise with a syringe pump at a rate of 30. mu.L/min, and after completion of the addition, stirring was continued for 1 hour. The solution was transferred to a dialysis bag (2000DaMWCO) and dialyzed against PB (10mmol/L, pH 7.4) for 6h, changing the dialysate every 2 h. And after the dialysis is finished, obtaining DOX polymer wrapped nanoparticle solution with volume of 5mL and concentration of 0.12 mg/mL. Adding acetic acid buffer solution with pH 5 to the partial solution, adjusting to corresponding pH, and adding H₂O₂Adding into the partial solution, and adjusting to H₂O₂Absorbance at 485nm was recorded at different times of DOX with a uv spectrophotometer at room temperature as 50 mmol/L. The results are shown in FIGS. 6-7, at acidic pH (pH 5) or ROS (H)₂O₂50mmol/L), the release of DOX increased 8-fold over that under physiological conditions.

Claims

1. A ROS-responsive dendrimer characterized by: the dendrimer comprises a dendrimer system and a molecule chain segment with biological response, diaminotri (ethylene glycol) is used as a core molecule of the dendrimer, oligo (ethylene glycol) (OEG/PEG) containing molecules are used for constructing the dendrimer and providing a thermosensitive and hydrophilic scaffold, cystine of natural amino acid with a disubstituted group is selected as a starting material for synthesizing a dehydrative monomer, and the dendrimer with ROS responsiveness in the first generation to the fourth generation is synthesized by using a Cu (I) -catalyzed alkyne-azide 1, 3-dipolar cycloaddition (CuAAC) reaction. The synthesized dendrimer is terminated with an alkynyl group in the half generation and with an amino group protected with Boc in the full generation.

2. The ROS-responsive dendrimer according to claim 1, wherein the dendrimer is a first through fourth generation dendrimer.

3. The ROS-responsive dendrimer according to claim 1, wherein said dendrimer comprises a Reactive Oxygen Species (ROS) -responsive sensing group having the structure:

a peroxyoxalate ester.

4. A method of preparing the ROS-responsive dendrimer of claim 1, comprising the steps of:

step 1, synthesizing 0.5 generation dendrimer G0.5 with four alkynyl groups.

G0.5 (chemical formula: C)₂₈H₄₀N₂O₈，m/z：532.2785(100％)，533.2818(30.3％)，534.2852(4.4％)，534.2827(1.6％))

Sodium hydride was washed several times with dry petroleum ether and suspended in 20ml of anhydrous DMF. To the resulting suspension was added a solution of 2, 2-bis (hydroxymethyl) propionic acid in 5ml dry DMF under ice water bath conditions. The resulting mixture was stirred for 0.5 hours under an ice-water bath and a solution of propargyl bromide was added slowly via syringe. The resulting mixture was slowly warmed to room temperature and stirred for 5 hours. After quenching the excess sodium hydride with chilled deionized water, a large amount of deionized water was added. The aqueous phase was washed 3 times with 20ml Dichloromethane (DCM) and then acidified to pH 2 with 1M HCl solution. The crude product was extracted from the aqueous solution by DCM and Na was used₂SO₄Drying and column chromatography using chloroform/petroleum ether/acetic acid eluent (3/7/0.05, vol.) gave compound 1 as a pale yellow oil. To a solution of compound 1 in 20ml of anhydrous DCM was added oxalyl chloride. The resulting mixture was stirred at reflux for 1 hour. The solvent was removed under reduced pressure to give the compound 2, bis (propyne-2-oxymethyl) -propionyl chloride. Will combine withDissolve product 2 in 5ml dry DCM solvent, slowly add diaminotriethylene glycol solution, stir at 0 ℃. The resulting solution was warmed to room temperature and slowly stirred for 5 hours, after which the reaction mixture was washed 3 times with 100ml of DCM, 30ml of water, Na₂SO₄After drying, the solvent is evaporated off. The crude product obtained is further purified by column chromatography (from 99/1 to 95/5 by volume) using DCM/methanol eluent to give compound G0.5 as a pale yellow viscous oil.

And 2, obtaining a generation 1 dendrimer G1 with ROS response groups as hydrophobic chain segments and amino end groups protected by 4 tert-butyloxycarbonyl (Boc) groups through CuAAC cycloaddition reaction.

G1 (chemical formula: C)₁₉₂H₃₁₂N₅₈O₆₄S₈The precise mass number: 4710.0708, molecular weight: 4713.4300)

m/z：4712.0775(100.0％)，4711.0742(96.8％)，4713.0809(68.4％)，4710.0708(46.6％)，4714.0733(36.2％)，4713.0700(35.0％)，4714.0842(34.5％)，4715.0767(24.7％)，4713.0745(21.4％)，4712.0712(20.7％)，4712.0666(16.9％)，4714.0779(14.7％)，4714.0818(13.2％)，4715.0876(12.9％)，4713.0784(12.7％)，4716.0800(12.5％)，4711.0678(10.0％)，4715.0851(8.1％)，4715.0703(7.7％)，4714.0670(7.5％)，4713.0769(6.4％)，4715.0813(6.4％)，4712.0735(6.2％)，4712.0750(6.1％)，4716.0691(5.7％)，4715.0658(5.5％)，4716.0737(5.3％)，4716.0776(4.8％)，4717.0834(4.7％)，4715.0742(4.6％)，4714.0803(4.4％)，4717.0725(3.9％)，4713.0636(3.6％)，4711.0702(3.0％)，4717.0809(2.9％)，4714.0624(2.7％)，4716.0909(2.3％)，4714.0708(2.2％)，4715.0836(2.2％)，4717.0771(2.0％)，4715.0727(2.0％)，4714.0693(1.9％)，4718.0758(1.9％)，4714.0716(1.6％)，4713.0682(1.5％)，4714.0739(1.4％)，4713.0706(1.3％)，4716.0761(1.2％)，4717.0661(1.2％)，4716.0628(1.1％)

First, 3-chloro-1-propanol, sodium azide and sodium hydroxide were added in proportion to 50ml of distilled water. After stirring at 50 ℃ for 24 hours, the product was extracted with diethyl ether. The combined organic extracts were washed with saturated sodium chloride (NaCl) and MgSO₄And (5) drying. The volatiles were evaporated to give a colorless oil (3-azido-1-propanol).

A solution of oxalyl chloride in 30ml of dichloromethane was added dropwise to a well-stirred ice-cooled solution of 3-azido-1-propanol, Triethylamine (TEA) and 4-Dimethylaminopyridine (DMAP) in 20ml of dichloromethane. The reaction was stirred vigorously under argon at room temperature for 15 hours. After filtration, the filtrate was washed with brine 4 times, over MgSO₄Drying and evaporating the solvent to obtain the oily substance which is oxalic acid di (3-azidopropyl) ester.

Functional group protection of o, o' -bis (2-aminoethyl) diethylene glycol: protecting according to the difference of the surface functional groups of the core molecules of the dendrimer to be prepared, if the surface functional groups of the core molecules are amino groups, protecting the amino groups.

Imidazole-1-sulfonyl azide hydrochloride was added to Boc protection of o, o' -bis (2-aminoethyl) diethylene glycol, potassium carbonate and copper (II) sulfate pentahydrate in 10ml methanol after completion of the reaction monitored by TLC, the mixture was concentrated, 30ml dichloromethane was added, the resulting mixture was washed with water (3 × 20ml), Na was added₂SO₄After drying the solvent was evaporated off. The crude product was further purified by column chromatography using DCM/methanol (92/8, vol/vol) eluent to afford compound 5, azido-Boc protected o, o' -bis (2-aminoethyl) diethylene glycol as a colorless oil.

Synthesis of G1 generation dendrimer

G0.5, Compound 3, and copper (II) sulfate pentahydrate were dispersed in 30ml of anhydrous THF, and the reaction mixture was stirred under argon for 1 hour. Polycyclic addition reaction was carried out under argon atmosphere to obtain product A, and A, compound 4, N, N, N ', N', N "-Pentamethyldiethylenetriamine (PMDETA) and CuCl in 5mL of anhydrous DMF were subjected to deoxidation process at 30 ℃ for 12 hours. After the reaction was complete the reaction mixture was precipitated in cold ether and the precipitate was washed with copious amounts of water to give the hydrophobic block B.

The above product B, compound 5 and copper (II) sulfate pentahydrate were dispersed in 40ml of anhydrous THF. To the resulting mixture was added dropwise a solution of sodium ascorbate in 40ml of deionized water under argon. The reaction mixture was stirred under argon for 1 hour. After removal of most of the THF under reduced pressure, the crude product was extracted with DCM and then Na₂SO₄Dried and further purified by column chromatography with DCM/methanol (98/2, vol) eluent to give G1 as a pale yellow viscous oil.

And 3, after the Boc protecting group is cut, repeating the process to synthesize the G1.5 generation, G2 generation, G2.5 generation, G3 generation and G3.5 generation dendritic macromolecules.

Trifluoroacetic acid (TFA) (3.8ml, 0.051mol) was added to a solution of G1 in 2.8ml DCM under ice bath conditions. The resulting mixture was stirred at room temperature for 3 hours. Removal of the solvent under reduced pressure gave deprotected G1.

The oily compound 2 was dissolved in 2ml of anhydrous DCM. To the resulting solution was slowly added a solution of deprotected G1 in 10ml of TEA in anhydrous DCM with stirring in an ice bath. The resulting solution was slowly warmed to room temperature and stirred for 3 hours. After this time, the reaction mixture was diluted with 50ml of DCM, washed 3 times with 20ml of water and Na₂SO₄Drying and evaporating. The crude product was further purified by column chromatography with DCM/methanol eluent (98/2 vol) to give G1.5 as a pale yellow viscous oil.

G1.5, Compound 3 and copper (II) sulfate pentahydrate were dispersed in 40ml of anhydrous THF. The reaction mixture was stirred under argon for 2.5 hours. The polycyclic addition reaction is carried out under argon. A20 mL anhydrous DMF solution of the above product, Compound 4, PMDETA and CuCl was deoxygenated at 60 ℃ for 12 hours. At the end of the polymerization, a degassed solution of compound 5 was added to a Schlenk tube to achieve a di-end group on the polymer. Precipitation in cold ether and washing of the precipitate with copious amounts of water gave the product (hydrophobic block).

The above product, compound 5 and copper (II) sulfate pentahydrate were dispersed in 40ml of anhydrous THF. To the resulting mixture was added dropwise a solution of sodium ascorbate in 35ml of deionized water under argonThe reaction mixture was stirred under argon for 2 hours after most of the THF was removed under reduced pressure, the crude product was extracted with 3 × 60ml of DCM, over Na₂SO₄Dried and further purified by column chromatography with DCM/methanol (290/10, vol) eluent to give G2 as a pale yellow viscous oil.

The above steps are repeated to synthesize G2.5, G3 and G3.5.

And 4, grafting oligo (ethylene glycol) (OEG/PEG) molecules through CuAAC reaction, namely taking O, O' -bis (2-aminoethyl) octadecyl glycol protected by Boc monomers as a hydrophilic chain segment to synthesize the 4 th generation dendritic macromolecule G4.

Imidazole-1-sulfonylazide hydrochloride was added to a solution of compound 4, potassium carbonate and copper (II) sulfate pentahydrate in 3ml of methanol after completion of the reaction monitored by TLC, the mixture was concentrated, 40ml of DCM was added, the resulting mixture was washed with water (3 × 20ml), Na was added, and₂SO₄dried and evaporated. The crude product was further purified by column chromatography with DCM/MeOH eluent (99/1, vol.) to afford Boc-PEG-N₃As a white solid.

Mixing G3.5, Boc-PEG-N₃And copper (II) sulfate pentahydrate dispersed in 2ml of anhydrous THF. To the resulting mixture was added dropwise a solution of sodium ascorbate in 2ml of deionized water under argon. The reaction mixture was stirred under argon for 1 hour. After removal of most of the THF under reduced pressure, the crude product was extracted 3 times with 15ml of DCM and Na₂SO₄Dried and further purified by column chromatography with DCM/methanol (13/1, vol.) eluent to give G4 as a pale yellow viscous oil.

Molecular structural formula of G4 (chemical formula: C)₁₆₆₂H₂₇₅₈N₅₂₂O₅₂₄8₅₆The precise mass number: 40204.9573, molecular weight: 40232.8360

m/z：40223.9724(100.0％)

Elemental analysis: c, 49.62; h, 6.91; n, 18.17; o, 20.84; s, 4.46).

5. Preparation of composite drug-loaded nanoparticle loaded with antitumor drug doxorubicin DOX

The polymer sample G4 was put into a 10mL white serum glass bottle and dissolved in DMSO, and stirred uniformly, after complete dissolution, DOX solution was added, 2mL phosphoric acid buffer solution (PB, 10mmol/L, pH 7.4) was slowly added dropwise with a syringe pump at a rate of 30. mu.L/min, and after the addition was complete, stirring was continued for 1 h. The solution was transferred to a dialysis bag (2000Da MWCO) and dialyzed against PB (10mmol/L, pH 7.4) for 6h, changing the dialysate every 2 h. And (5) obtaining a DOX polymer wrapped nanoparticle solution after dialysis.