CN105521496B - A kind of preparation method of the injection aquagel of chemical bonding anticancer drug - Google Patents

Abstract

The invention discloses a method for preparing an injectable hydrogel chemically bonded with anticancer drugs. The first component is hyperbranched polyamidoamine grafted with anticancer drugs, and oxidized sodium alginate is used as the second component. Components, the two components form -C=N-bonds as cross-linking sites, and an injectable hydrogel capable of sustaining release of anticancer drugs in vivo is obtained. In the present invention, the drug is combined with the hydrogel component by chemical bonds, which prolongs the release period of the drug; when the hydrogel is degraded, the drug is still combined with the macromolecular substance, prolonging its circulation time in the living body. Correspondingly, the therapeutic effect of the medicine is improved, so that the frequency of medication can be reduced, and the toxic and side effects can be alleviated. The hydrogel has very good application prospects in the fields of biomedicine and tissue engineering.

Description

Preparation method of an injectable hydrogel chemically bonded with anticancer drugs

1. Technical field

The invention relates to a method for preparing an injectable hydrogel, in particular to a method for preparing an injectable hydrogel chemically bonded with anticancer drugs, and belongs to the technical field of injectable hydrogel.

2. Background technology

Hydrogel is a kind of soft material with 3D porous structure, mostly physical crosslinking (such as hydrogen bond, hydrophilic-hydrophobic interaction, ionic interaction, etc.) or chemical crosslinking (such as acyl bond, disulfide bond, imine bond, etc.) Keys, etc.), has the characteristics of high water content, high elasticity, softness and so on. Therefore, hydrogels have important application value in biomedicine, drug delivery, tissue engineering and so on. Research on hydrogels in recent years has mainly focused on injectable hydrogels formed in situ. The components of injectable hydrogels are flowable liquids that can gel in situ after mixing, so they can be implanted in the body without invasive surgery, enabling them to be used as tissue engineering materials. Another feature of injectable hydrogel is that drugs, proteins, cells, etc. can be mixed with component liquids in advance, so that it can be used in the field of drug release. Injectable hydrogels are very suitable for clinical treatment.

Polyamidoamine with branched structure is (HPAMAM) a kind of dendritic macromolecule with specific three-dimensional structure and molecular size. HPAMAM has the characteristics of highly branched structure and highly controllable configuration. HPAMAM is easy to accommodate drugs inside the molecule. Due to the low molecular weight and good fluidity of HPAMMA, it has attractive application prospects in biomedicine and targeted delivery. There are a large number of active functional groups on the periphery of HPAMAM molecules, which can react with other substances to form injectable gels. At present, some gel systems based on branched polyamidoamines have been reported in the literature.

Hongbin Zhang et al. used hyperbranched polymers combined with anticancer drugs to obtain a sustained-release system that can release drugs in the human body. However, in this system, the drug and the polymer are only physically blended, and there is no chemical combination with the gel component. (J. Mater. Chem., 2011, 21, 13530)

Siquan Zhu et al. combined hyperbranched polymers with drugs to obtain a hydrogel system that can control the release of various drugs. However, the combination of drug and polymer in this system is a physical combination. (Biomaterials, 2010, 31, 5445-5454.)

Raghavendra S. Navath et al. used PEG and HPAMAM to obtain an injectable drug-releasing hydrogel for the treatment of reproductive system infections. However, the combination of drug and hydrogel in this system is also physical blending. (Mol. Pharmaceuticals, 2011, 8, 1209-1223).

Wei-Qing Liu et al. used ATRP technology to obtain a pH-responsive drug-releasing hyperbranched polymer hydrogel. The combination of the hydrogel and the drug is a physical combination. (Mater. Sci. Eng. C 2012, 32, 953-960.)

Since the drug in the above reports is physically combined with branched polyamidoamine or hydrogel based on branched polyamidoamine, the binding force between the drug and branched polyamidoamine is not strong, so the release rate of the drug is relatively low. Faster, and the duration of the drug effect is relatively short. This may cause disadvantages such as high frequency of medication and relatively large toxic and side effects of the drug.

3. Contents of the invention

The present invention aims to provide a method for preparing an injectable hydrogel chemically bonded with anticancer drugs, so as to solve the problems of fast drug release rate and short duration of drug effect in the branched polyamidoamine injectable hydrogel system that physically combines drugs. The disadvantage is to prolong the drug release period, prolong the circulation time of the drug in the organism, reduce the frequency of medication, and reduce the purpose of toxic and side effects. The hydrogel of the invention can be well applied in the fields of biomedicine, tissue engineering and the like.

The present invention utilizes the reactivity of the terminal functional group of the branched polyamidoamine to react with the drug to form a chemically bonded macromolecular drug carrier; then the chemically bonded macromolecular drug carrier is combined with oxidized sodium alginate to form an injectable Hydrogel system. Since the drug and the hydrogel are combined by chemical bonds, the release of the drug needs to undergo hydrolysis of the chemical bond, and accordingly, the drug release period is prolonged. In addition, since the drug is bound to the macromolecule, the drug will always stay in the organism until the macromolecule is completely degraded by the organism, prolonging its circulation time in the organism. Thereby, the dosage of medication can be reduced, and the toxic and side effects of the medicine can be reduced.

The preparation method of the injectable hydrogel chemically bonded with anticancer drugs of the present invention is based on hyperbranched polyamidoamine grafted with anticancer drugs (such as camptothecin, doxorubicin, 5-fluorouracil, paclitaxel, etc.) One component, with oxidized sodium alginate as the second component, and the two components formed -C=N- bonds as cross-linking sites, resulting in an injectable hydrogel capable of sustained release of anticancer drugs in vivo , including the following steps:

(1) Dissolve the anticancer drug and methyl bromoacetate of equimolar amount in methanol, react at room temperature for 2-4h in the presence of a catalyst, and obtain an intermediate product—an anticancer drug grafted with methyl bromoacetate; The product and hyperbranched polyamidoamine were dissolved in methanol at the same time, heated to reflux temperature, and reacted for 4-6 hours. After the reaction was completed, the reaction solution was rotary evaporated at 45°C to remove the solvent, and then precipitated in ice acetone for 1-2 times, at room temperature Under vacuum drying, obtain the hyperbranched polyamide amine of anticancer drug grafting;

The anticancer drug is camptothecin, doxorubicin, 5-fluorouracil or paclitaxel, etc.;

The catalyst is triethylamine or sodium methoxide; the added amount of the catalyst is 5-15% of the mass of the anticancer drug.

The mass ratio of hyperbranched polyamidoamine to intermediate product is 30:1;

The preparation of the hyperbranched polyamidoamine is a conventional method, specifically taking 6.146g methylenebisacrylamide and 2.584g N-aminoethylpiperazine as monomers in 60mL of methanol/water mixed solvent (V _methanol : V (Chem. Commun. _, 2007, 2587–2589).

(2) Dissolve the hyperbranched polyamidoamine grafted with anticancer drugs in deionized water, which is recorded as solution A; dissolve the oxidized sodium alginate in deionized water, which is recorded as solution B; After being injected into the living body, the injectable hydrogel can be obtained in situ.

The concentration of hyperbranched polyamidoamine grafted with anticancer drugs in solution A is 50-350 mg/mL;

The concentration of oxidized sodium alginate in solution B is 50-250 mg/mL;

When injected into the living body, the volume ratio of solution A to solution B is 3:1 to 1:3.

The oxidized sodium alginate is obtained by reacting 0.173 g/mL sodium periodate as an oxidizing agent and 0.2 g/mL sodium alginate at a volume ratio of 1:1 at room temperature for 6 hours.

The present invention has following characteristics:

1. Compared with other branched polyamidoamine hydrogel systems, the hydrogel provided by the present invention is combined with the drug by chemical bonds, and the release of the drug depends on the hydrolysis of the chemical bond, thus prolonging the drug release period.

2. Compared with other branched polyamidoamine hydrogel systems, in the hydrogel provided by the present invention, the drug and hyperbranched polyamidoamine are chemically bonded. Before the hyperbranched polyamidoamine is completely degraded by the organism, the drug will always stay in the organism, prolonging the circulation time of the drug in the organism.

3. Compared with other branched polyamidoamine hydrogel systems, the technology provided by the present invention can reduce the frequency of medication, thereby reducing the toxic and side effects of drugs.

4. This injectable hydrogel with long-term drug release ability is expected to be applied in biomedicine, tissue engineering and other fields.

4. Description of drawings

Figure 1 is a schematic diagram of the reaction process of synthesizing hyperbranched polyamidoamine grafted with 5-fluorouracil as a model anticancer drug.

Fig. 2 is the formation process of the injectable hydrogel and the photo of the hydrogel. The left picture shows that the sodium alginate solution is injected into the hyperbranched polyamide solution to form a gel rapidly; the right picture shows the large gel formed after the sodium alginate solution and the hyperbranched polyamide amine solution are uniformly mixed.

Figure 3 is the degradation process of the drug-loaded hydrogel. The abscissa is time/day, and the ordinate is the mass ratio of the degraded hydrogel to the original hydrogel.

Fig. 4 is the drug release curve of the hyperbranched polyamidoamine/oxidized sodium alginate injectable hydrogel grafted with anticancer drugs in the example. (The abscissa is time/hour; the ordinate is cumulative release rate/%).

Fig. 5 is the drug release curve within 12 hours of the hyperbranched polyamidoamine/oxidized sodium alginate injectable hydrogel grafted with anticancer drugs in the example. (The abscissa is time/hour; the ordinate is cumulative release rate/%).

5. Specific implementation

The injectable hydrogel chemically bonded with anticancer drugs of the present invention will be described in detail below in conjunction with the examples.

Example 1:

1. Take 5 mmol of 5-fluorouracil and methyl bromoacetate, dissolve them in 5 mL of methanol, add 0.1 g of sodium methoxide, and react at room temperature for 2 hours to obtain 5-fluorouracil grafted with methyl bromoacetate; take 3 g of hyperbranched polyamidoamine and Add 0.1g of methyl acrylate-modified 5-fluorouracil into 10mL of methanol solution, stir evenly, heat to 75°C for reflux reaction for 4h, rotate the reaction solution at 45°C, and then precipitate twice with 10 times the volume of ice acetone, The precipitate was placed in a vacuum oven and dried at room temperature for 12 hours to obtain hyperbranched polyamidoamine grafted with 5-fluorouracil.

2. Dissolve 0.15g of hyperbranched polyamidoamine grafted with 5-fluorouracil in 1mL of deionized water, and record it as solution A; dissolve 0.10g of oxidized sodium alginate in 1mL of deionized water, and record it as solution B; Mix A and solution B at a volume ratio of 1:3, shake and mix evenly at room temperature, and an injectable hydrogel can be obtained within 1 min.

3. Hydrogel drug release test in vitro

The injectable hydrogel prepared in step 2 was placed in a semipermeable membrane with a dialysis molecular weight of 6000 (MWCO 6 KD), soaked in 6 mL of PBS buffer solution with a pH value of 7.4, and incubated at 37°C. Add the same amount of the same fresh PBS buffer solution after taking 4mL culture solution each time, and the sampling time is 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, and then take a sample every 24h. The hydrogel without drug loading was used as a blank reference, and samples were taken at the same time. Absorbance was measured with a UV spectrophotometer. The drug release concentration was determined by a standard curve. As shown in Figure 3, it can be seen that the burst release effect of the drug is small, the sustained release time is long, and the metabolism in vitro is slow.

Example 2:

1. Take 5 mmol each of camptothecin and methyl bromoacetate, dissolve them in 5 mL of methanol, add 0.1 g of triethylamine, and react at room temperature for 3 hours to obtain camptothecin grafted with methyl bromoacetate; then take 3 g of hyperbranched polyamide Add amine and 0.1g camptothecin with methyl bromoacetate to 10mL of methanol solution, stir evenly, place in an oil bath at 70°C and heat to reflux for 5h, rotate the reaction solution at 45°C, and then 10 times the volume of ice acetone was precipitated twice, and the precipitate was placed in a vacuum drying oven and dried at room temperature for 12 hours to obtain hyperbranched polyamidoamine grafted with camptothecin.

2. Dissolve 0.15g camptothecin-grafted hyperbranched polyamidoamine in 1mL deionized water, which is called solution A; dissolve 0.20g oxidized sodium alginate in 1mL deionized water, which is called solution B; Mix A and solution B at a volume ratio of 2:3, shake and mix evenly at room temperature, and an injectable hydrogel can be obtained within 1 min.

Example 3:

1. Take 4 mmol of paclitaxel and methyl bromoacetate, dissolve them in 10 mL of methanol, add 0.1 g of triethylamine, and react at room temperature for 2 hours to obtain paclitaxel grafted with methyl bromoacetate; then take 3 g of hyperbranched polyamidoamine and 0.1 g Paclitaxel with methyl bromoacetate was added to 10 mL of methanol solution, stirred evenly, placed in an oil bath at 80°C and heated to reflux for 4.5 hours, the reaction solution was rotary evaporated at 45°C, and then mixed with 10 times the volume of ice acetone Precipitate once, and place the precipitate in a vacuum oven to dry at room temperature for 12 hours to obtain paclitaxel-grafted hyperbranched polyamidoamine.

2. Dissolve 0.20g of paclitaxel-grafted hyperbranched polyamidoamine in 1mL of deionized water, and record it as solution A; dissolve 0.10g of oxidized sodium alginate in 1mL of deionized water, and record it as solution B; Solution B was mixed at a volume ratio of 1:1, shaken and mixed evenly at room temperature, and an injectable hydrogel could be obtained within 1 min.

Example 4:

1. Take 6 mmol each of doxorubicin and methyl bromoacetate, dissolve them in 5 mL of methanol, add 0.1 g of triethylamine, and react at room temperature for 4 hours to obtain doxorubicin grafted with methyl bromoacetate; then take 3 g of hyperbranched polyamide Add amine and 0.1g of doxorubicin with methyl bromoacetate to 10mL of methanol solution, stir evenly, place in an oil bath at 75°C and heat to reflux for 6h, and rotate the reaction solution at 45°C, then use Precipitate once with 10 times the volume of ice acetone, and place the precipitate in a vacuum drying oven to dry at room temperature for 12 hours to obtain hyperbranched polyamidoamine grafted with doxorubicin.

2. Dissolve 0.20g of doxorubicin-grafted hyperbranched polyamidoamine in 1mL of deionized water, and record it as solution A; dissolve 0.15g of oxidized sodium alginate in 1mL of deionized water, and record it as solution B; Mix A and solution B at a volume ratio of 3:2, shake and mix evenly at room temperature, and an injectable hydrogel can be obtained within 1 min.

Claims (7)

1. a kind of preparation method of the injection aquagel of chemical bonding anticancer drug, it is characterised in that：It is to be grafted anticancer The over-branched polyamidoamine of drug is the first component, and using the sodium alginate of oxidation as the second component, two kinds of components pass through formation- C=N- keys have obtained the injection aquagel for capableing of sustained release anticancer drug in vivo as crosslink sites；Including walking as follows Suddenly：

(1) anticancer drug of equimolar amounts and methyl bromoacetate are dissolved in methyl alcohol, is reacted at room temperature in the presence of a catalyst 2-4h obtains intermediate product；Intermediate product and over-branched polyamidoamine are dissolved in methanol simultaneously, are heated to reflux temperature, 4-6h is reacted, solvent is evaporated off in 45 DEG C of backspins in reaction solution after reaction, is then precipitated 1-2 times in ice acetone, room temperature Lower vacuum drying obtains the over-branched polyamidoamine of anticancer drug grafting；

(2) in deionized water by the over-branched polyamidoamine dissolving of anticancer drug grafting, it is denoted as solution A；By the seaweed of oxidation Sour sodium dissolving in deionized water, is denoted as solution B；Solution A is vibrated at room temperature with solution B and is uniformly mixed, in 1min Obtain injection aquagel；

The anticancer drug includes camptothecine, adriamycin, 5 FU 5 fluorouracil or taxol.

2. preparation method according to claim 1, it is characterised in that：

The catalyst is triethylamine or sodium methoxide；The additive amount of catalyst is the 5-15% of anticancer drug quality.

3. preparation method according to claim 1, it is characterised in that：

The mass ratio of over-branched polyamidoamine and intermediate product is 30：1.

4. preparation method according to claim 1, it is characterised in that：

A concentration of 50-350mg/mL for the over-branched polyamidoamine that anticancer drug is grafted in solution A.

5. preparation method according to claim 1, it is characterised in that：

A concentration of 50-250mg/mL of the sodium alginate aoxidized in solution B.

6. according to the preparation method described in claim 1,4 or 5, it is characterised in that：

The volume ratio of solution A and solution B is 3 when being injected in organism:1 to 1:3.

7. preparation method according to claim 1, it is characterised in that：

It is oxidant and the sodium alginate of 0.2g/mL with body that the sodium alginate of the oxidation, which is using the sodium metaperiodate of 0.173g/mL, Product ratio 1:1, react what 6h was obtained at ambient temperature.