CN114377128B

CN114377128B - Preparation and anti-tumor application of composite hydrogel co-loaded with near-infrared photothermal agent and immune drug

Info

Publication number: CN114377128B
Application number: CN202210031622.5A
Authority: CN
Inventors: 李娟�; 刘艳迪; 颜军; 韩宇阳
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2022-01-12
Filing date: 2022-01-12
Publication date: 2023-01-06
Anticipated expiration: 2042-01-12
Also published as: CN114377128A

Abstract

The invention relates to preparation and anti-tumor application of a composite hydrogel co-loaded with a near infrared photothermal agent and an immune drug. The main functional components of the hydrogel are preferably gellan gum composite hydrogel which is loaded with reduced molybdenum-based polyoxometalate clusters and an immune agonist R848. The reduced molybdenum-based polyoxometallate prepared by the method has the performance of quick degradation under the condition of physiological buffer solution and stability in deionized water solution. In addition, the composite hydrogel prepared by the invention has injectability and thermal hysteresis type sol-gel transition behavior. Under the irradiation of near infrared light, the prepared composite hydrogel has excellent photo-thermal conversion property, can rapidly ablate in-situ tumor after laser irradiation, and prevents tumor recurrence and metastasis through the synergistic treatment of immune agonist. The composite hydrogel treated by the combination of the co-load reduced molybdenum-based polyoxometallate and the immune agonist R848 has good safety and curative effect in the aspect of tumor resistance.

Description

Preparation and anti-tumor application of composite hydrogel co-loaded with near-infrared photothermal agent and immune drug

Technical Field

The invention relates to preparation and anti-tumor application of a composite hydrogel co-loaded with a near-infrared photothermal reagent molybdenum-based polyoxometalate cluster and an immune agonist, and belongs to the fields of pharmacy and oncology.

Background

Photothermal therapy (PTT for short) is a minimally invasive methodTumor ablation technology is a therapeutic method which uses Photothermal conversion reagents (PTAs for short) to absorb Near-infrared (NIR) light and converts the light into heat to kill tumor cells. PTAs (such as gold nanoparticles and graphene) widely reported at present are difficult to biodegrade, and have safety problems caused by in vivo accumulation { biochem.biophysis.rep.2021, 26,100991; chi, chem, lett, 2017,28 (4), 691-702; bioMed Res.Int.2021,2021,5518999; small 2013,9 (9-10), 1492-1503}. Thus, some newly emerging biodegradable PTAs (such as black phosphorus nanoplates) are of great interest in cancer treatment, but these materials aggregate themselves easily and degrade rapidly in aqueous solution (about 2 weeks), which makes them difficult to store for long periods { Biomaterials 2020,236,119770; j.am.chem.soc.2015,137 (35), 11376-11382}. In addition, in a single photothermal antitumor therapy mode, due to the limited penetration depth of laser, the photothermal effect is generally only for small-volume tumors: (<100mm ³ ) Is effective. To improve the therapeutic effect, current research is gradually shifting the focus from monotherapy to combination therapy. Among emerging combination therapy modalities, photothermal/immune combination therapy is receiving attention because of its advantages of efficiently eliminating primary tumors and inhibiting distant metastasis of lesions.

Polyoxometalates (POM) is a transition metal oxide cluster, and is an ideal PTA material due to the characteristics of simple synthesis, low cost, precise size, water solubility, easy oxidation reduction, adjustable structure and the like. Shi Jianlin group developed firstly a hyperthermia method of tumor specific targeting of molybdenum-based POM clusters and triggered by NIR, and the reductive substance in the tumor microenvironment activated the conversion of Mo (VI) to Mo (V) in POM, so that it had excellent near infrared absorption properties and corresponding photothermal conversion properties j.am.chem.soc.2016,138 (26), 8156-8164. However, the research is carried out by injection, POM passively targets tumor parts after systemic circulation, and laser power density required in photothermal therapy is as high as 1.5W/cm ² . It was able to eliminate well the orthotopic tumor in the early stages of treatment, but lung metastasis was observed in the mouse model 16 days after treatment. Of interest, the report describes POM clustersIt is very stable in Phosphate Buffered Saline (PBS) at pH 7.4 and the spectral absorption does not change substantially after one year of storage. The high stability of the POM cluster under physiological pH conditions is not beneficial to liver and kidney metabolism, and the safety problems such as excessive accumulation and the like are easily caused in vivo.

The injectable hydrogel is a hydrogel which can be implanted into a human body in an injection mode, and has the advantages of small wound, simple and convenient operation, long in-vivo retention half-life period and the like. The invention designs an injectable hydrogel platform for co-loading a near-infrared photothermal reagent molybdenum-based POM cluster and an immune agonist, and the injectable hydrogel platform is used for photothermal/immune combination therapy. By means of intratumoral injection, the composite hydrogel can directly act on tumor parts to realize local tumor photothermal treatment and gradually release the entrapped immunotherapy medicament. On one hand, the POM cluster generates local high temperature (more than 42 ℃) to ablate most tumor cells under the irradiation of near infrared; on the other hand, the immune drug kills residual and escaped tumor cells by activating the patient's own immune system, thereby preventing the recurrence and metastasis of tumors. By strategically loading the two active ingredients together in an injectable hydrogel, the composite hydrogel achieves maximum efficacy in cancer combination therapy while minimizing systemic toxicity. More importantly, the POM cluster aqueous solution still has stable photo-thermal conversion performance after being stored for a long time, and has controllable biodegradability under the condition of buffer solutions with different pH values. The POM cluster photothermal conversion material with storage stability and controllable in-vivo degradability is not reported.

Disclosure of Invention

The invention aims to provide a composite hydrogel co-loaded with a near infrared photothermal reagent molybdenum-based POM cluster and an immune agonist.

The second purpose of the invention is to provide a pH-controllable degradable reduced POM cluster and a preparation method thereof.

The third purpose of the invention is to provide the application of the injectable composite hydrogel in antitumor therapy.

A composite hydrogel co-loaded with a near infrared photothermal reagent molybdenum-based POM cluster and an immune agonist comprises a hydrogel matrix material, the near infrared photothermal reagent molybdenum-based POM cluster loaded in the hydrogel matrix material and the immune agonist. The composite hydrogel is based on hydrogel matrix materials, and is loaded with near infrared photothermal reagent molybdenum-based POM clusters and an immune agonist. The invention innovatively discovers that the molybdenum-based POM cluster and the immune agonist which are near infrared photothermal reagents are loaded on the hydrogel, so that the purpose of obvious synergy is achieved, the anti-tumor activity of each other can be improved, and the anti-tumor effect is obviously improved. The composite hydrogel co-loaded with the near-infrared photothermal reagent molybdenum-based POM cluster and the immune agonist has a better anti-tumor effect through photothermal/immune combination treatment, can ablate nearly all in-situ tumors and inhibit the lung metastasis of the tumor far end.

Preferably, the near infrared photothermal agent is a reduced POM cluster, and the central metal ion includes at least one of molybdenum and tungsten.

Further preferably, the near-infrared photothermal reagent is a reduced molybdenum-based POM cluster comprising Keggin type PMo ₁₂ And Dawson type P ₂ Mo ₁₈ Two kinds.

The preparation of the reduction state POM of the invention is that firstly a reducing agent reacts with the oxidation state POM, and then the pH value of the solution is adjusted to 6-8 by alkali after the reduction reaction is finished. The reducing agent includes glutathione, ascorbic acid and the like. During the reduction process, colorless POM in oxidation state is gradually changed into blue POM in reduction state, and the spectral absorption range of POM in reduction state is 650nm-900 nm.

The reduced molybdenum-based POM shows different stabilities in buffer solutions with different pH values, so that the POM cluster has good storage stability and rapid degradability under physiological conditions.

The reduced molybdenum-based POM has good photo-thermal conversion efficiency and photo-thermal stability under the irradiation of near infrared light.

The hydrogel matrix material is a natural polysaccharide with the highest critical solution temperature, and has a thermal hysteresis type sol-gel transformation behavior, so that in photo-thermal treatment application, the composite hydrogel is not melted in laser irradiation, but still exists in a gel state at a tumor part, and relatively high local concentration and optimal treatment effect are kept at the tumor part.

Preferably, the hydrogel matrix material is a temperature-sensitive hydrogel with the highest critical solution temperature; preferably, the hydrogel matrix material is injectable. The hydrogel matrix is preferably at least one of Gellan Gum (GG), agar, carrageenan, and xanthan gum. The preferable matrix material is more favorable for improving the performance of the composite hydrogel, and further improves the local drug concentration and biocompatibility of the material after injection. Still more preferably, the hydrogel base material is gellan gum; most preferably low acyl gellan gum. The hydrogel provided by the invention is made of materials with good safety.

The hydrogel provided by the invention has the characteristics of simple preparation method and simple treatment operation. The three selected materials do not have strong complexation, so that the composite hydrogel can be obtained by directly mixing. When in treatment, only one intratumoral injection and one near infrared illumination are needed to completely cure the tumor and inhibit the tumor distal metastasis. In addition, the reduced POM cluster is gradually decomposed into low-toxicity soluble molybdate in a physiological environment, so that accumulation in the body is avoided.

The preferred composite hydrogel is a gellan gum-based composite hydrogel (R848/POM @ GG) containing a TLR7/8 agonist Rasimimod (Resiquimod, R848 for short) and a controllably degradable near-infrared photothermal agent molybdenum-based POM cluster.

Researches find that the preferable reduced POM, the immune agonist R848 and the hydrogel matrix material have better synergistic treatment effect, and the biological safety and the tumor treatment effect of the composite hydrogel can be further improved.

The preparation method of the R848/POM @ GG composite hydrogel comprises the following steps: and heating and dissolving the gellan gum with water, cooling to 50 ℃, quickly adding the reduced POM and R848 sample solution, fully mixing, and further cooling to room temperature to obtain the gellan gum composite hydrogel system jointly loaded with the reduced POM and R848.

In the R848/POM @ GG composite hydrogel prepared by the invention, the mass percentage concentration of the gellan gum is 0.5-5%, the mass concentration of the POM is 0.01-10.0 mg/mL, and the R848 concentration is 50-500 mug/mL.

The most preferable technical scheme of the invention comprises that in the mixed aqueous solution, the mass percentage concentration of the gellan gum is 2%, the mass concentration of the POM is 300 mug/mL, and the concentration of the R848 is 150 mug/mL.

Compared with the existing photothermal conversion reagent, the molybdenum-based POM in the controllable degradation reduction state has better biological safety and higher photothermal conversion efficiency. The novelty is that the reduced molybdenum-based POM cluster has adjustable degradation performance and rapid degradation performance under the condition of physiological pH buffer solution. Meanwhile, the reduced POM has better storage stability in the deionized water solution and better clinical transformation value. Moreover, the prepared R848/POM @ GG composite hydrogel can not only effectively remove in-situ tumor, but also prevent tumor recurrence and metastasis, and has a better photothermal/immune combined anti-tumor treatment effect.

Drawings

FIG. 1. Absorption spectra of POM in oxidized and reduced states of example 2

FIG. 2. Example 2 photo-thermal temperature rise curves of two reduced POMs at different laser powers and different sample concentrations

FIG. 3. Example 3 degradation of two reduced POMs under different pH buffer conditions

FIG. 4. Example 3 spectral absorption and cytotoxicity of two reduced POMs after 12h storage under different solution conditions

FIG. 5 temperature rising rheological curve of POM @ GG composite hydrogel in example 4

FIG. 6 shear thinning Properties of POM @ GG composite hydrogel in example 4

FIG. 7 strain scan curve of POM @ GG composite hydrogel in example 4

FIG. 8 is the alternate strain cycle scan curve of the composite hydrogel of POM @ GG in example 4

FIG. 9 photo-thermal temperature rise curves of POM @ GG composite hydrogel in example 4 in different storage periods

FIG. 10. Example 6 in vivo degradation of reduced POM and POM @ GG composite hydrogel

FIG. 11 is a graph showing the antitumor effect of the composite hydrogel of example 7 after 15 days of treatment

FIG. 12H & E staining of Lung tissue 15 days after treatment with composite hydrogel of example 7

Detailed Description

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

EXAMPLE 1 preparation of two reduced POMs

Weighing 15mg of oxidation state H ₃ [PMo ₁₂ O ₄₀ ]The crystals were dissolved in 5mL of deionized water. Adding a certain amount of glutathione (GSH, 5 mM), mixing, placing the above solution in water bath at 37 deg.C for reacting for 4h, adjusting pH of the solution to 7.4 to obtain Keggin type reduced POM (PMo for short) ₁₂ 。

Weighing 15mg of the oxidized form (NH) ₄ ) ₆ [P ₂ Mo ₁₈ O ₆₂ ]·14.2H ₂ O crystal, and dissolving it in 5mL of deionized water. Adding a certain amount of glutathione (GSH, 5 mM), mixing, placing the above solution in water bath at 37 deg.C for reacting for 4h, adjusting pH of the solution to 7.4 to obtain Dawson type reduced POM (P for short) ₂ Mo ₁₈ 。

Example 2 test of photothermal conversion Performance of two reduced POMs

The maximum absorption wavelength of the reduced POM prepared by the invention is about 710nm (figure 1), and the reduced POM is suitable for being used as a near infrared photothermal reagent. 1.0mL of reduced POM clusters (PMo) with different concentrations ₁₂ And P ₂ Mo ₁₈ ) Solution by near infrared laser (808nm, 0.3W/cm) ² ) Irradiating for 10min. During which the temperature change of the sample was tracked with an infrared thermal imaging camera and the real-time temperature was recorded every 30 seconds. Under the stimulation of near infrared light of 808nm, the reduced POM prepared by the invention has excellent photo-thermal property and photo-thermal stability, and the temperature change is in direct proportion to the concentration of POM clusters, the irradiation time and the power of the near infrared light (figure 2).

Example 3 degradation experiments of two reduced POMs at different pH conditions

To reduce PMo in its reduced state ₁₂ And P ₂ Mo ₁₈ The solution was diluted to 300. Mu.g/mL with PBS buffer solutions of different pH. The samples were then placed in a 37 ℃ water bath and observed for color change at various time points.

The reduced POM cluster prepared by the invention is rapidly degraded in PBS buffer solution, and the degradation rate is accelerated along with the increase of the pH value of the buffer solution, which shows that the material can be rapidly degraded into colorless molybdate after 12 hours under the physiological condition of pH 7.4 (figure 3). Moreover, the reduced POM itself has good storage stability in water, and the absorption is basically unchanged after 12 hours. Cytotoxicity experiments showed that the degraded product was less cytotoxic than the reduced POM (fig. 4).

Example 4 Loading of reduced POM (in P) ₂ Mo ₁₈ Example) composite hydrogel

1.2g of gellan gum was weighed and dissolved in 40mL of deionized water, and the solution was dissolved by magnetic stirring at 90 ℃. After the sample is completely dissolved, cooling to 50 ℃, adding a certain amount of reduced POM to prepare the controllable degradation P ₂ Mo ₁₈ The photothermal hydrogel of (1), hereinafter abbreviated as POM @ GG hydrogel. The final concentrations of the components in the hydrogel were 300. Mu.g/mL POM and 2% gellan gum, respectively, and the samples were stored in a refrigerator at 4 ℃.

The temperature rise rheological curve of the composite hydrogel (POM @ GG) prepared by the invention does not have a gel-sol transition intersection point between 25 ℃ and 80 ℃, and the thermal hysteresis property of the composite hydrogel is illustrated (figure 5). The composite hydrogel prepared by the invention has good injectability and self-recovery property. The shear-thinning properties of the composite hydrogels were characterized by rheometry, the gel viscosity gradually decreased with increasing shear rate, and the transition was reversible (fig. 6). In addition, the storage modulus of the composite hydrogel gradually decreased with increasing strain, and when the strain was greater than about 27.9%, G' < G ", exhibited fluid properties (fig. 7). When the strain was set at 100%, the storage modulus G 'of the composite hydrogel dropped significantly (G' < G "), indicating that the gel was broken, and when the strain was 0.1%, the system immediately gelled again, the modulus returned to near the original level, and this process could be cycled many times, indicating that the composite hydrogel had very good self-healing properties (fig. 8).

The POM @ GG composite hydrogel is 0.3W/cm ² Exhibits excellent photothermal conversion efficiency (63.1%) and good photothermal stability at a safe power density of (2) (fig. 9).

Example 5 Co-Loading of reduced POM (with P) ₂ Mo ₁₈ For example) and R848

1.2g of gellan gum was weighed and dissolved in 40mL of deionized water, and the solution was dissolved by magnetic stirring at 90 ℃. Cooling to 50 deg.C after the sample is completely dissolved, adding a certain amount of reduced POM (P) ₂ Mo ₁₈ ) And preparing the POM @ GG hydrogel. When loading R848, firstly dissolving R848 in a small amount of DMSO solution, and then adding the R848 solution into the gel to obtain the co-loaded reduction state P ₂ Mo ₁₈ And R848, hereinafter referred to as R848/POM @ GG hydrogel. The final concentrations of the components in the hydrogel were 300. Mu.g/mL POM, 150. Mu.g/mL R848, and 2% gellan gum, respectively, and the samples were stored in a 4 ℃ refrigerator.

Example 6 POM (in P) ₂ Mo ₁₈ Example) in vivo degradation experiments

BALB/c female mice (5-6 weeks old) are used as animal models to study the in vivo degradation characteristics of POM clusters. The reduced POM sample solution (P) prepared in example 1 was added ₂

Mo

₁₈ 100 μ L) and the composite hydrogel sample prepared in example 4 (pom @ gg,100 μ L) were injected subcutaneously into the back of mice, the mice were sacrificed at different time points, the skin of the treated area was dissected, and the color change and the retention of the hydrogel sample were observed. The results show that the molybdenum-based POM and POM @ GG prepared by the invention can be rapidly degraded under physiological conditions (figure 10).

Example 7 composite hydrogel (in P) ₂ Mo ₁₈ Example) antitumor assay

After shaving, the mice were fed with 100. Mu.L of physiological saline containing 4T1 breast cancer cells subcutaneously on their backs, and the back of the mice grew about 150mm after about one week ³ Size of tumor. Mice were randomly divided into 5 groups (4 per group): (1) the blank control group is an untreated group; (2) R848@ GG; (3) POM @ GG; (4) POM @ GG with NIR; (5) R848/POM @ GG with NIR. The hydrogel of the experimental group had a gellan gum concentration of 2%, a POM concentration of 300. Mu.g/mL, and an injection dose of R848 of 15. Mu.g/mouse. The samples from different experimental groups were injected intratumorally into mice, each mouse having an intratumoral injection sample volume of 100. Mu.L. After injection, 808nm laser irradiation was applied to the tumor site of each mouse in experimental groups (4) and (5) for 10min at a laser power density of 0.3W/cm ² . Tumor volume and mouse body weight were recorded at intervals, and after 15 days of treatment, mice were sacrificed and lung tissue and tumor tissue were taken for H&And E, dyeing.

Research shows that compared with a single photothermal treatment control group and a single immunotherapy control group, the experimental group R848/POM @ GG with NIR composite hydrogel has photothermal/immune synergistic treatment effect on solid tumors, the in-situ tumor volume inhibition rate reaches 99.3%, and no obvious lung metastasis exists. Dissection after 15 days of treatment revealed that 4 mice in the experimental group (R848/POM @ GG with NIR) had greatly reduced tumor volumes after treatment, and 2 mice had completely disappeared in situ, indicating that the photothermal/immune combination treatment could almost completely ablate in situ tumors in mice (FIG. 11). In addition, compared with the control group, the pulmonary tissue section of the experimental group (R848/POM @ GG with NIR) has uniform alveolar distribution and no obvious cancer cell aggregation growth, which indicates that the experimental group can obviously inhibit the lung metastasis of the tumor (FIG. 12).

Claims

1. The composite hydrogel co-loaded with the near infrared photothermal agent and the immune drug is characterized by comprising a hydrogel matrix material, a reduced molybdenum-based POM cluster loaded in the hydrogel matrix material and the immune drug;

the reduced molybdenum-based POM cluster is Keggin type PMo ₁₂ Or Dawson type P ₂ Mo ₁₈ ，

The immune drug is Rasimotent R848,

the hydrogel matrix material is gellan gum.

2. The composite hydrogel according to claim 1, wherein the mass percentage concentration of the gellan gum is 0.5-5%, the concentration of the reduced molybdenum-based POM cluster is 0.01-10.0 mg/mL, and the concentration of R848 is 50-500 μ g/mL.

3. The preparation method of the composite hydrogel according to claim 1, wherein the reduced molybdenum-based POM cluster and R848 are added into a fully dissolved gellan gum solution, and the solution is naturally cooled to room temperature to obtain the composite hydrogel co-loaded with the reduced molybdenum-based POM cluster and the immune drug R848.

4. The method for preparing the composite hydrogel according to claim 3, wherein the reduced molybdenum-based POM cluster is reacted with the oxidized POM by a reducing agent, and the pH of the solution is adjusted to 6-8 by alkali after the reduction reaction is finished.

5. The method for producing a composite hydrogel according to claim 4, wherein the reducing agent used for producing the reduced molybdenum-based POM cluster is one of glutathione and ascorbic acid.

6. Use of the composite hydrogel of claim 1 for the preparation of an anti-tumor medicament.

7. The use according to claim 6, for the preparation of a medicament for the photothermal/immunological combination therapy of tumors.