CN109276537B

CN109276537B - Composite hydrogel co-loaded with vascular disrupting agent and near-infrared photothermal response nanoparticles, and preparation and application thereof

Info

Publication number: CN109276537B
Application number: CN201811064878.6A
Authority: CN
Inventors: 李娟�; 梁育晴; 郝义俊; 孙晓毅; 刘又年
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2018-09-12
Filing date: 2018-09-12
Publication date: 2020-11-10
Anticipated expiration: 2038-09-12
Also published as: CN109276537A

Abstract

The invention relates to an injectable composite hydrogel co-loaded with a vascular blocking agent and near-infrared photothermal response nanoparticles and an anti-tumor application. The composite hydrogel comprises a hydrogel substrate material, and a blood vessel blocking agent and near-infrared photo-thermal response nanoparticles which are loaded in the hydrogel substrate material. The nano composite hydrogel prepared by the invention has the sol-gel transformation behavior of injectability and thermal hysteresis. Under the irradiation of near infrared light, the hybrid hydrogel disclosed by the invention has very excellent photo-thermal properties, and the curative effect of the vascular blocking agent is improved by enhancing the permeability of tumor blood vessels. The hybrid hydrogel treated by combining the vascular disrupting agent and the photo-thermal has good safety and curative effect in the aspect of tumor resistance.

Description

Composite hydrogel co-loaded with vascular disrupting agent and near-infrared photothermal response nanoparticles, and preparation and application thereof

Technical Field

The invention relates to an injectable hydrogel co-loaded with a vascular blocking agent and near-infrared photothermal response nanoparticles and an anti-tumor application, and belongs to the fields of pharmaceutics and oncology.

Background

The tumor tissue is composed of parenchyma and stroma, and the tumor parenchyma is tumor cells, is a main component of the tumor and has tissue source specificity. The tumor stroma plays a role in supporting and nourishing the tumor parenchyma, and has no specificity, including connective tissue, blood vessels, lymphatic vessels, and the like.

Vascular disrupting agents (VDAs for short) are a class of antineoplastic drugs that selectively damage tumor-associated blood vessels. The medicine can selectively destroy tumor-related blood vessels, block the supply of nutrients and oxygen to tumor tissues, and cause the death of secondary tumor cells, thereby achieving the aim of targeted therapy of tumors. However, single vascular blocker therapy often results in tumor treatment failure and tumor recurrence due to poor water solubility, short half-life, low bioavailability, insufficient permeability, etc. of vascular inhibitors (e.g., combretastatin a4, etc.).

Near-infrared photothermal therapy is a recently developed anti-tumor physical method, and by utilizing the advantages of long wavelength, strong penetrability and the like of near-infrared light (NIR), tumor tissues can be ablated quickly and locally at fixed points at fixed time and fixed points, and infrared thermal imaging, photoacoustic imaging and the like can be performed according to the relation between the intensity of infrared emission and the temperature of an object. Commonly used photothermal conversion materials include carbon-based nanomaterials (e.g., graphene, etc.), nano-semiconductor materials (e.g., copper sulfide, bismuth selenide, etc.), noble metal nanoparticles (e.g., gold, platinum, etc.). Prussian blue is Fe²⁺And Fe³⁺The hexacyanoferrate with mixed valence has simple preparation method and mild conditions, can be clinically used for treating thallium radiation poisoning, and has good biological safety and metabolic pathway in human bodies. Researches find that the Prussian blue nano particles have near infrared photothermal effect and magnetic resonance imaging enhancement function. Single photothermal therapy is generally only applied to small-volume tumors (< 100 mm) when resisting tumors³) Effective and easily causes adverse phenomena such as thermal burn, pain and the like in the treatment process.

At present, the hydrogels with near infrared light response reported in research are mainly a class of hydrogels containing precious metal nanoparticles (e.g. platinum nanoparticles, gold nanoparticles) with photothermal properties { Biomaterials 2016, 104, 129-; adv.funct.mater.2018, 28(21), 1801000}, and focuses on combination therapy with both photothermal and chemotherapy. Chemotherapy is known to have significant side effects and to be susceptible to drug resistance. No literature report on the realization of photothermal and vascular disrupting agent combination therapy by loading a vascular disrupting agent and near-infrared photothermal response nanoparticles on a hydrogel carrier is found.

Disclosure of Invention

The invention aims to provide a composite hydrogel co-loaded with a vascular disrupting agent and near-infrared photothermal response nanoparticles.

The second purpose of the invention is to provide a preparation method of the composite hydrogel.

The third purpose of the invention is to provide an application of the composite hydrogel in preparing antitumor drugs.

A composite hydrogel co-loaded with a vascular blocking agent and near-infrared photo-thermal response nanoparticles comprises a hydrogel substrate material, the vascular blocking agent loaded in the hydrogel substrate material and the near-infrared photo-thermal response nanoparticles.

The composite hydrogel is a nano composite hydrogel based on a hydrogel substrate material and containing near-infrared photo-thermal response nano particles and a vascular blocking agent. The invention innovatively discovers that loading the vascular disrupting agent and the near-infrared photothermal response nanoparticles on the hydrogel has the obvious synergistic effect, can improve the anti-tumor activity of each other and obviously improve the anti-tumor effect, and the inventor also discovers that loading the vascular disrupting agent and the near-infrared photothermal response nanoparticles on the hydrogel together can avoid the burst release of the vascular disrupting agent and realize the long-acting effect through slow release. The results of in vivo antitumor experiments in mice show that compared with the single use of VDAs/hydrogel and PB/hydrogel, the experimental group treated by the composite hydrogel has better antitumor effect, and can almost completely remove tumors.

The near-infrared photothermal response nanoparticles of the present invention require a material having good biocompatibility, higher photothermal conversion efficiency, and better photothermal stability.

Preferably, the near-infrared photothermal response nanoparticles are at least one of mesoporous Prussian blue nanoparticles (PB), platinum and metal chalcogenide.

Further preferably, the near-infrared photothermal response nanoparticles are mesoporous prussian blue nanoparticles. Researches find that the preferable PB, the blood vessel blocking agent and the hydrogel substrate material have better synergistic effect, and the synergistic effect can be further improved.

Preferably, the particle size of the near-infrared photo-thermal response nanoparticles is 20-200 nm.

Preferably, the near-infrared photothermal response nanoparticles are mesoporous Prussian blue nanoparticles with the particle size distribution of 20-200 nm and the maximum absorption wavelength of about 720 nm.

The Prussian blue nano particles are prepared by a polyvinylpyrrolidone (PVP) reduction dispersion method, a sodium citrate modification precipitation method, a hydrothermal etching method and the like.

Preferably, the vascular disrupting agent is at least one of combretastatin a4(CA4), combretastatin a4 water-soluble phosphate prodrug CA4P, DMXAA and TZT-1027; preferably CA 4. The inventor researches and discovers that the preferable CA4 and the composite hydrogel of the near-infrared photothermal response nanoparticles have a better synergistic effect, and the anti-tumor and slow-release effects can be further improved.

The hydrogel matrix material is a natural polysaccharide with the highest critical solution temperature (UCST), has a sol-gel transition behavior with heat hysteresis, and can be cooperated with other components in the composite hydrogel to ensure that the thermal effect generated by the illumination of nano particles in the composite hydrogel prevents the burst release of the vascular disrupting agent, so that the vascular disrupting agent keeps relatively high local concentration and treatment effect at a tumor part.

Preferably, the hydrogel substrate material is temperature-sensitive hydrogel; preferably, the hydrogel matrix material is injectable.

The hydrogel base material is more preferably at least one of gellan gum and agar. The preferable substrate material is more beneficial to improving the performance of the composite hydrogel, and further improving the in-situ treatment capability and biocompatibility of the material.

Still more preferably, the hydrogel base material is gellan gum; most preferably low acyl gellan gum.

The research of the invention finds that under the cooperation of the innovative components, the composite hydrogel can be endowed with good anti-tumor effect, and the research further finds that the cooperative performance can be further improved by controlling the proportional relation among the components.

Preferably, in the composite hydrogel, the mass percentage concentration of the hydrogel substrate material is 0.5-5%, the mass concentration of the near-infrared photothermal response nanoparticles is 0.01-1.0 mg/mL, and the concentration of the vascular disrupting agent is 1.0-10.0 mg/mL.

Further preferably, in the composite hydrogel, the hydrogel substrate material is gellan gum, the near-infrared photothermal response nanoparticles are PB with the particle size of 20-200 nm, and the vascular blocking agent is CA 4; wherein the mass percentage concentration of the gellan gum is 0.5-5%, the mass concentration of PB is 0.05-1 mg/mL, and the concentration of the vascular disrupting agent is 1-10 mg/mL.

The invention discloses a preparation method of the composite hydrogel, which is to mix a vascular blocking agent, near-infrared photo-thermal response nano particles, a hydrogel matrix material and water to prepare the composite hydrogel.

According to the preparation method, the vascular blocking agent, the near-infrared photo-thermal response nano particles and the matrix material are gelatinized in water to prepare the composite hydrogel.

Preferably, the preparation method comprises the steps of adding a hydrogel matrix material into a dispersion liquid in which the near-infrared photothermal response nanoparticles are dispersed, heating for dissolving, cooling to a temperature range where the drug is not inactivated, adding the vascular disrupting agent drug, and cooling to room temperature.

Still more preferably, the preparation method comprises: and (2) after the gellan gum is heated and dissolved by water, the gellan gum is cooled to a temperature range which does not cause inactivation of the drug, PB and the vascular disrupting agent drug are added, and then the mixed aqueous solution is cooled to room temperature, so that the gellan gum nano composite hydrogel system containing PB nano particles and the vascular disrupting agent is obtained.

The composite hydrogel has the characteristics of simple preparation method and simple treatment operation. The selected three materials are mixed to obtain the nano composite hydrogel. When in treatment, the tumor can be completely cured only by one intratumoral injection and one or a plurality of times of near infrared illumination.

The invention also provides the application of the composite hydrogel for preparing anti-tumor medicaments;

preferably, the medicine is used for preparing the tumor in situ treatment;

further preferably, the compound is used for preparing the medicine for resisting malignant sarcoma.

Most preferably, the use is characterized by being used for preparing an anti-tumor injection preparation.

The invention also provides an anti-tumor injection preparation which comprises the composite hydrogel.

Advantageous effects

The composite hydrogel has a high-efficiency anti-tumor effect through the cooperation of the components. When near infrared light irradiates, the nano composite hydrogel generates a heat effect, so that not only can the tumor permeability be enhanced, but also the interaction between molecules of a hydrogel substrate material (such as gellan gum) can be weakened, thereby enhancing the gel network fluidity, and the vascular blocking agent is gradually diffused to the peripheral area of an injection part, so that the comprehensive collapse of tumor blood vessels and the tumor cell necrosis can be caused. Because of the thermal hysteresis effect of the gellan gum hydrogel, the gel cannot be completely destroyed in the heating process, and the coated substance can be kept near tumor tissues, so that the action time of the medicine is better prolonged, and the purposes of in-situ and long-acting administration are achieved.

In addition, the invention can well solve the technical problems of poor water solubility, short half-life period, low bioavailability, insufficient permeability and the like of the vascular disrupting agent through the synergy of the components; moreover, the near-infrared photothermal response nanoparticles can be matched with the hydrogel substrate material to regulate and control the release and tissue permeability of effective components, so that the aim of remarkably improving the anti-tumor effect is fulfilled.

The nano composite hydrogel is not simply combined, and through the synergy of the components, the composite hydrogel has the sol-gel transformation behavior of heat hysteresis, the heat effect generated under illumination can not cause the burst release of the vascular blocking agent, and the vascular blocking agent can keep relatively high local concentration and treatment effect at a tumor part; moreover, the composite hydrogel disclosed by the invention has injectability, improves the permeability of tumor blood vessels, can realize the aim of in-situ efficient treatment, and effectively ensures the good safety and curative effect of the composite hydrogel in the aspect of tumor resistance.

Drawings

FIG. 1 example 4 rheological Properties of nanocomposite hydrogels during warming and Cooling

FIG. 2 shear thinning Properties of example 4 nanocomposite hydrogels

FIG. 3 Strain Scan Curve of nanocomposite hydrogel of example 4

FIG. 4 Strain cycling Scan Curve of the 3 nanocomposite hydrogel of example 4

FIG. 5 absorption spectra of nanocomposite hydrogel of example 4

FIG. 6 photo-thermal property of example 4 nanocomposite hydrogel

FIG. 7 light controlled Release Profile of EXAMPLE 6 nanocomposite hydrogels

FIG. 8 is a graph showing the antitumor effects of the nanocomposite hydrogel of example 7

FIG. 9 is a quantitative analysis of the anti-tumor effect of the nanocomposite hydrogel of example 7

Detailed Description

The following examples are intended to illustrate the invention without further limiting it.

Example 1.

Prussian blue nano particle prepared by polyvinylpyrrolidone (PVP) reduction dispersion method

15g of polyvinylpyrrolidone (PVP) and 664mg of hematite (K) were weighed out₃[Fe(CN)₆]) Adding 200mL of 0.01M HCl, stirring at room temperature for 30min, transferring into a constant-temperature oil bath kettle at 80 ℃ for reaction for 20h, cooling to room temperature after the reaction is finished, centrifuging at high speed for 20min, removing supernatant, washing with secondary water to be neutral, and freeze-drying for later use.

Example 2 preparation of Prussian blue nanoparticles by sodium citrate modification precipitation method

0.10g of sodium citrate is weighed out and added to 20mL of FeCl₃(1mM) to solution, 0.10g of sodium citrate was weighed out and added to 20mL of K₄[Fe(CN)₆]Adding sodium citrate/K into (1mM) aqueous solution, stirring at constant temperature of 60 deg.C₄[Fe(CN)₆]And dropwise adding the mixed solution into the sodium citrate/FeCl 3 mixed solution, cooling to room temperature, and continuously stirring for 30min to obtain the Prussian blue nanoparticles.

Example 3 preparation of mesoporous Prussian blue nanoparticles

Weighing 0.08g of Prussian Blue (PB) nano particles and 125mg of PVP, adding into 80mL of 1.0M HCl, stirring for 30min, transferring into a 100mL reaction kettle, placing in an oven at 140 ℃ for reaction for 4h, centrifuging at high speed for 20min, collecting precipitate, washing with secondary water to be neutral, and freeze-drying for later use.

Example 4 Co-supporting Prussian blue nanoparticles and CA4 nanocomposite hydrogel

Weighing 0.2g of gellan gum (and low acyl of Wako pure industries, Ltd.) and adding into 10mL of 0.1mg/mL PB (example 3) ultrasonic dispersion, mixing uniformly, placing in a constant temperature water bath kettle at 85 ℃, placing the mixed solution in a constant temperature water bath kettle at 50 ℃ for 1h after the gellan gum is fully dissolved, adding 60mg of CA4, mixing uniformly, cooling to room temperature to obtain the nano composite hydrogel coating PB and CA4, and placing the sample in a refrigerator at 4 ℃ for dark storage (the product is marked as CA4+ PB @ gellan hydrogel).

Example 5 Co-supporting Prussian blue nanoparticles and CA4P nanocomposite hydrogel

Weighing 0.2g of gellan gum, adding the gellan gum into 10mL of 0.1mg/mL PB ultrasonic dispersion (example 3), uniformly mixing, placing the mixture in a constant-temperature water bath kettle at 85 ℃, placing the mixture in a constant-temperature water bath kettle at 50 ℃ for 1h after the gellan gum is fully dissolved, adding 30mg of CA4P, fully mixing uniformly, cooling to room temperature to obtain the nano composite hydrogel coated with PB and CA4P, and placing the sample in a refrigerator at 4 ℃ for dark storage (the product is marked as CA4P + PB @ gellan hydrogel).

Example 6 Release test of nanocomposite hydrogel

Two parts of 1.0g of the nanocomposite hydrogel prepared in example 5 were weighed, 2.0mL of PBS buffer solution with pH of 7.4 was added as release media, and the mixture was placed in a shaking table with a constant temperature water bath at 37 ℃ to simulate release. Wherein one sample is applied at 1.0W/cm²After 5min of 808nm laser irradiation (temperature increased to 54 ℃), the laser was turned off, and four cycles of irradiation were repeated. The other part was a control group without laser irradiation. The drug concentration in the release medium is quantified at specific time points and a cumulative drug release kinetic curve is plotted.

Example 7 antitumor assay of nanocomposite hydrogels

After shaving, the mice were fed with 100. mu.L of physiological saline containing 4T1 cells subcutaneously and the back of the mice grew about 150mm after about one week³Size of the tumor. Mice were randomly divided into 6 groups (5 per group): (1) the blank control group was normal saline PBS; (2) PB (example 3) + NIR, 0.1 mg/mLPB; (3) PB @ gellan hydrogel + NIR; (4) CA4, 6 mg/mL; (5) CA4@ gellan hydrogel; (6) CA4+ PB @ gellan hydrogel (example 3) + NIR. The samples of different experimental groups were injected intratumorally into mice, and the volume of the intratumoral injection sample of each mouse was 200 uL. About 2h after injection, 808nm laser irradiation was applied to the tumor sites of each mouse of groups (2), (3) and (6) for 3min at an optical power density of 1W/cm². Tumor volume and mouse body weight were recorded every other day, and after 22 days of treatment, tumor tissue was dissected and collected after mice were sacrificed under ether anesthesia.

The nano-composite hydrogel (CA4+ PB @ gellan hydrogel) prepared by the invention shows different rheological properties in the processes of temperature rise and temperature drop, and the temperature drop curve shows that the sol-gel transition temperature of the nano-composite hydrogel is about 52 ℃, while the temperature rise curve shows that the nano-composite hydrogel does not have the gel-sol transition temperature, which shows the thermal hysteresis property of the nano-composite hydrogel (figure 1).

The nano composite hydrogel prepared by the invention has good injectability and self-recovery property. The rheometer characterizes the shear-thinning behavior of the hybrid gel, with a gradual decrease in viscosity with increasing shear rate and reversibility (fig. 2). The storage modulus of the hybrid gel gradually decreased with increasing strain, with G' < G "at strains greater than about 6%, showing fluid properties (FIG. 3). The nano composite hydrogel has a great reduction of storage modulus G ' under the action of 300% strain (G ' < G '), which indicates that the gel is destroyed, when the strain is 1%, the system immediately undergoes gelation within 10s, the modulus is recovered to be close to the initial level, and the process can be cycled for a plurality of times, which indicates that the hybrid gel has very good self-healing property (figure 4).

The maximum absorption wavelength of the nanocomposite hydrogel prepared by the present invention is around 720nm (fig. 5). Under the stimulation of near infrared light of 808nm, the nano composite hydrogel of the inventionHas excellent photo-thermal property. The temperature change is proportional to the irradiation time and power of the near infrared light (fig. 6). At 1.0W/cm²When the power irradiation is carried out for 5min and the PB mass concentration is 0.1mg/mL, the temperature can be increased by 50 ℃. Although the nano-composite hydrogel can generate heat effect under the irradiation of near-infrared light of 808nm, the influence of the light on the release speed of the vascular blocking agent is small, the hydrogel does not show burst release after the near-infrared light irradiation, and the nano-composite hydrogel still has the characteristic of slowly releasing the medicine (figure 7).

By establishing subcutaneous tumors in mice, tumor volumes exceed 150mm³In this case, 200. mu.L of hydrogel was injected subcutaneously and irradiated with near-infrared light (808nm, 1.0W/cm)²3 min). The experimental group (CA4+ PB @ gellan hydrogel) was found to have the best antitumor synergy compared to the photothermal treatment alone or the control group treated with the vascular blocking agent. The tumor volume of 5 mice in the experimental group (CA4+ PB @ gellan hydrogel) was greatly reduced after 22 days of treatment, and the tumor tissues of 2 mice completely disappeared, indicating that the tumor of the mice can be completely inhibited or even ablated by the combined treatment of photothermal therapy and vascular blocking agent (FIG. 8).

The change in tumor volume within 22 days after treatment is shown in FIG. 9, and the tumor is essentially completely resected. Compared with a control group, the PB + gellan had better tumor treatment effect compared with the PB alone, and similarly, the CA4+ gellan had better cancer treatment effect compared with the CA4 alone, but the tumors of four groups of mice showed a remarkable growth trend in the later treatment period, and the experimental group treated by the composite hydrogel had better anti-tumor effect.

Claims

1. The composite hydrogel is characterized by comprising a hydrogel substrate material, and a blood vessel blocking agent and near-infrared photo-thermal response nanoparticles which are loaded in the hydrogel substrate material;

the near-infrared photo-thermal response nanoparticles are mesoporous Prussian blue nanoparticles;

the vascular blocking agent is combretastatin A4;

the hydrogel substrate material is gellan gum.

2. The composite hydrogel according to claim 1, wherein the particle size of the near-infrared photothermal response nanoparticles is 20-200 nm.

3. The composite hydrogel of claim 1, wherein the gellan gum is a low acyl gellan gum.

4. The composite hydrogel according to any one of claims 1 to 3, wherein the mass percentage concentration of the hydrogel substrate material in the nanocomposite hydrogel is 0.5% to 5%, the mass concentration of the near-infrared photothermal response nanoparticles is 0.01 to 1.0mg/mL, and the concentration of the vascular disrupting agent is 1.0 to 10.0 mg/mL.

5. A preparation method of the composite hydrogel according to any one of claims 1 to 4, wherein the composite hydrogel is prepared by dissolving and mixing the vascular blocking agent, the near-infrared photothermal response nanoparticles and the hydrogel matrix material with water.

6. The method for preparing a composite hydrogel according to claim 5, wherein the hydrogel matrix material is added to the dispersion in which the near-infrared photothermal response nanoparticles are dispersed, and the hydrogel matrix material is heated and dissolved, then cooled to a temperature range that does not cause inactivation of the drug, and then cooled to room temperature to obtain the hydrogel.

7. Use of the composite hydrogel according to any one of claims 1 to 4 or the composite hydrogel prepared by the preparation method according to claim 5 or 6 for preparing an anti-tumor drug.

8. The use according to claim 7 for the preparation of a medicament for the in situ treatment of tumors.

9. The use according to claim 7, for the preparation of a medicament against malignant sarcoma.

10. An antitumor injection preparation comprising the composite hydrogel according to any one of claims 1 to 4.