CN108126198B - W18O49-tirapazamine composite nano particle and preparation method and application thereof - Google Patents

Abstract

The invention discloses a W₁₈O₄₉-tirapazamine composite nanoparticles, which take PEG-PCL as a carrier and are internally loaded with W₁₈O₄₉And tirapazamine; w₁₈O₄₉And tirapazamine drug loading of 2.99% and 1.29%, respectively; the composite nano particles are in a dispersed spherical structure, and the particle size is 60-100 nm. The invention can improve the treatment effect of TPZ.

Description

W18O49-tirapazamine composite nano particle and preparation method and application thereof

Technical Field

The invention relates to a W with good tumor treatment effect₁₈O₄₉-tirapazamine composite nanoparticles.

Background

Tumor hypoxia has been one of the major causes of failure in solid tumor chemoradiotherapy (chemoradiotherapy resistance). Solid tumors contain 10% -50% of hypoxic cells, and the tolerance of the hypoxic cells to radiation and chemotherapeutic drugs is 2.5-3 times stronger than that of aerobic cells, so that the hypoxic cells become one of the important factors that tumors are difficult to cure and easy to relapse and transfer.

The action mechanism of the TPZ is that under the hypoxic condition, a high-activity TPZ free radical can obtain a hydrogen atom from DNA through the mediation of topoisomerase II, so that single/double-strand break and chromosome destruction are caused, and then cell death is caused. And its efficiency is reduced under the normal oxygen condition.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide composite particles capable of improving the treatment effect of TPZ.

In order to achieve the above object, the present invention provides a W₁₈O₄₉-tirapazamine composite nanoparticles, said composite nanoparticles having PL (polyethylene glycol-polycaprolactone) as carrier, on which W is loaded₁₈O₄₉And tirapazamine; w₁₈O₄₉And tirapazamine drug loading of 2.99% and 1.29%, respectively; the composite nano particles are in a dispersed spherical structure, and the particle size is 60-100 nm.

The invention also provides a preparation method of the composite nano particle, which comprises the following steps:

(1) dissolving 50mg of PEG-PCL (polyethylene glycol-polycaprolactone, PL for short) in 5-10mL of dichloromethane to prepare a solution a;

(2) adding 50-100mL of pure water into the solution a to obtain a solution b;

(3) adding 5mg-10mg of W into the solution b₁₈O₄₉Stirring for 10-20 min to obtain solution c;

(4) adding 2-4mg of tirapazamine into the solution c, and stirring for 10-20 minutes to obtain a solution d;

(5) carrying out ultrasonic treatment on the solution d for 30-50 minutes;

(6) then centrifuging for 10-20 minutes under the condition of 14000 r/min;

(7) and (4) adding deionized water into the precipitate obtained in the step (6) for washing, repeating the step (6) for centrifuging, and repeating the washing and centrifuging for three times to obtain the composite nano particles.

The invention also provides application of the composite nano particle in preparing a medicament for treating tumors, in particular application of the composite nano particle in preparing a medicament for combining chemotherapy and photothermal therapy.

Compared with the prior art, the invention has the following advantages:

the composite nanoparticle of the present invention utilizes W₁₈O₄₉Consuming oxygen inside the tumor (after illumination, forming electron-hole pairs which can react with water and oxygen in the air to generate oxygen anions (O) with ultrahigh activity₂-) and hydroxyl radicals, which have strong oxidizing properties and are capable of reacting directly with most of the organic substances adsorbed on the surface of the semiconductor catalyst and oxidatively degrading the organic substances to the final product of CO₂And H₂O) to induce tumor hypoxia, so that the tumor hypoxia area is enlarged, and the action effect of tirapazamine in a hypoxic area is optimal, thereby further enhancing the therapeutic effect of tirapazamine chemotherapy.

In addition, W₁₈O₄₉Has good photothermal conversion effect and can be used for photothermal treatment.

The combination of chemotherapy and photothermal therapy can improve the tumor treatment effect, and the killing rate is higher, thereby reducing the risk of tumor recurrence and metastasis.

The composite nano particle (PL-W) prepared by the invention₁₈O₄₉TPZ) and irradiating the tumor site with laser light at 808nm to release TPZ from the nanoparticles while W is simultaneously present₁₈O₄₉Consuming oxygen to generate active oxygen, aggravating hypoxic area of tumor, and improving chemotherapy effect of TPZ. Combines chemotherapy and photothermal therapy, and improves the tumor treatment effect.

Drawings

FIG. 1 is a scanning electron micrograph of a composite nanoparticle of the present invention;

FIG. 2 is a UV spectrum of a composite nanoparticle of the present invention;

FIG. 3 is a graph showing the temperature change under photothermal effect of the composite nanoparticle of the present invention;

FIG. 4 is a graph showing the release effect of tirapazamine in the composite nanoparticle of the present invention with time;

FIG. 5 is a graph comparing the effect of different concentrations of composite nanoparticles on cell viability in the absence of laser irradiation in accordance with the present invention;

FIG. 6 is a graph comparing the effect of different concentrations of composite nanoparticles on cell viability under laser irradiation in accordance with the present invention;

FIG. 7 is a graph comparing the results of ROS/hypoxia assays in HeLa cells;

in FIG. 7, Undated is the saline group, PL-W₁₈O₄₉Is via PL-W₁₈O₄₉Nanoparticle treated and laser irradiation free set, PL-W₁₈O₄₉TPZ is PL-W₁₈O₄₉TPZ nanoparticle treatment and laser irradiation free set, PL-W₁₈O₄₉+ Laser is via PL-W₁₈O₄₉NPs treatment and laser irradiation set, PL-W₁₈O₄₉-TPZ + Laser is PL-W₁₈O₄₉-TPZ nanoparticle treatment and laser irradiation group;

FIG. 8 is a comparative plot of in vivo Micro-PET imaging after injection of composite nanoparticles of the present invention;

FIG. 9 is a graph comparing the change in tumor volume after different drug treatments;

FIG. 10 is a graph comparing the change in body weight of mice after different drug treatments;

FIG. 11 is a graph comparing HE and TUNEL staining of tumor tissue after treatment with different drugs;

FIG. 12 is a graph comparing the distribution of drugs in vivo;

FIG. 13 is a comparison of pathological results of important organs treated with different drugs;

FIG. 14 is the biochemical index chart of the blood after the composite nano particle treatment of the present invention.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

Preparation of example 1

The preparation of the composite nano-particles of the invention is carried out by adopting the following steps:

(1) dissolving 50mg of PEG-PCL (polyethylene glycol-polycaprolactone) in dichloromethane (5-10mL) to obtain a solution a;

(2) adding 50-100mL of pure water into the solution a to obtain a solution b;

(3) adding W into the solution b₁₈O₄₉(5mg-10mg), and stirring for 10-20 minutes by adopting a magnetic stirrer, wherein the rotating speed of a magnetic rotor is 600 revolutions per minute, so as to obtain a solution c;

(4) adding tirapazamine TPZ (2-4mg) into the solution c, and stirring for 10-20 minutes by adopting a magnetic stirrer, wherein the rotating speed of a magnetic rotor is 600 revolutions per minute to obtain a solution d;

(5) taking the solution d, and carrying out ultrasonic treatment for 30-50 minutes;

(6) centrifuging (14000 rpm) for 10-20 min;

(7) adding 20-50mL of deionized water into the precipitate, washing, and centrifuging (the same centrifugation conditions as the step (6));

(8) repeating the step (6) and the step (7) for three times, and finally centrifuging to obtain a precipitate, namely the composite nano particle.

Product characterization example 2

The composite nanoparticle (PL-W) prepared in example 1 was used₁₈O₄₉TPZ NPs) were characterized:

1. PL-W observation by scanning Electron microscope₁₈O₄₉-TPZ NPs nanoparticles

As shown in FIG. 1, it can be seen that the composite nanoparticles have a dispersed spherical structure, and the particle diameter is 60 to 100nm (average 80 nm).

2. The tungsten concentration was measured using an inductively coupled plasma mass spectrometer (ICP-MS, NexION 300D, Perkin-Elmer Corporation, USA).

PL-W₁₈O₄₉The drug loading in TPZ NPs is 2.99% (W)₁₈O₄₉) And 1.29% (TPZ).

3. TPZ, PEG-PCL NPs, W using Shimadzu UV-3100 Spectrophotometer₁₈O₄₉And PL-W₁₈O₄₉TPZ was subjected to uv absorption spectroscopy scan.

As shown in FIG. 2, the UV characterization results indicate that the UV light intensity is within PL-W₁₈O₄₉The ultraviolet spectrum of-TPZ NPs has W₁₈O₄₉And TPZ, proving W₁₈O₄₉And TPZ successful wrapping to PL-W₁₈O₄₉TPZ nanoparticles.

4. Photothermal effects were studied using a 808nm diode laser (LEO photonics co. In order to prevent evaporation of water, a quartz cell was covered with tin foil, and PL-W was studied₁₈O₄₉Temperature change of TPZ nanoparticles under 808nm laser irradiation.

As shown in FIG. 3, laser irradiation at 808nm in vitro for 10 minutes, PL-W₁₈O₄₉The temperature of the TPZ nanoparticles rose from 25.4 ℃ to 63.6 ℃ demonstrating PL-W₁₈O₄₉TPZ nanoparticles have photothermal properties.

Tirapazamine Release Effect example 3

For PL-W₁₈O₄₉The TPZ nanoparticles were irradiated with laser light at 808nm, PL-W after irradiation with laser light at 808nm as shown in FIG. 4₁₈O₄₉TPZ nanoparticles were significantly released, reaching 80% after 24, whereas only 20% of TPZ was released without laser irradiation. Description of PL to W₁₈O₄₉And TPZ has good wrapping effect and can release drugs under the excitation of laser.

In vitro cytotoxicity assay example 4

Detection of PL-TPZ-W by MTT method₁₈O₄₉Cytotoxicity of the nanoparticles.

HeLa cells were seeded in 96-well plates (10)⁴Individual cells/well) and incubated for 12 h. Each hole is added with nanoparticles PL-TPZ, PL-W with different concentrations₁₈O₄₉Nanoparticles or PL-W₁₈O₄₉Medium of TPZ nanoparticles, with or without laser irradiation at 808nm for 24h, five replicates were performed for all concentration groups. MTT solution was added and incubated for 4 h. DMSO was added and the absorbance at 490nm was measured for each well using a microplate reader.

The preparation method of the PL-TPZ nano particles comprises the following steps:

(1) dissolving 50mg of PEG-PCL (polyethylene glycol-polycaprolactone) in dichloromethane (5-10mL) to obtain a solution a;

(2) adding 50-100mL of pure water into the solution a to obtain a solution b;

(3) adding tirapazamine TPZ (2-4mg) into the solution b, and stirring for 10-20 minutes by adopting a magnetic stirrer at the rotating speed of a magnetic rotor of 600 revolutions per minute to obtain a solution c;

(4) taking the solution c, and carrying out ultrasonic treatment for 30-50 minutes;

(5) centrifuging (14000 rpm) for 10-20 min;

(6) adding 20-50mL of deionized water into the precipitate, washing, and centrifuging (the centrifugation condition is the same as the step (5));

(7) repeating the step (5) and the step (6) for three times, and finally centrifuging to obtain a precipitate, namely the composite nano particle.

PL-W₁₈O₄₉The preparation method of the nano-particles comprises the following steps:

(1) dissolving 50mg of PEG-PCL (polyethylene glycol-polycaprolactone) in dichloromethane (5-10mL) to obtain a solution a;

(2) adding 50-100mL of pure water into the solution a to obtain a solution b;

(3) adding W into the solution b₁₈O₄₉(5mg-10mg), and stirring for 10-20 minutes by adopting a magnetic stirrer, wherein the rotating speed of a magnetic rotor is 600 revolutions per minute, so as to obtain a solution c;

(4) taking the solution c, and carrying out ultrasonic treatment for 30-50 minutes;

(5) centrifuging (14000 rpm) for 10-20 min;

(6) adding 20-50mL of deionized water into the precipitate, washing, and centrifuging (the centrifugation condition is the same as the step (5));

(7) repeating the step (5) and the step (6) for three times, and finally centrifuging to obtain a precipitate, namely the composite nano particle.

Mixing PL-W₁₈After co-culturing 24O 49-TPZ NPs with HeLa cells, the cells all maintained normal cell viability in the absence of laser 808 irradiation, as shown in FIG. 5.

After laser 808 irradiation, cell viability decreased as the nanoparticle concentration increased. As shown in fig. 6.

Detection of reactive oxygen species/Hypoxia (ROS/Hypoxia) in HeLa cells example 5

The level of reactive oxygen species/hypoxia in HeLa cells was detected using an oxidative stress/hypoxia detection kit.

Inoculating HeLa cells into a confocal culture dish, and culturing, wherein 5 groups are selected, namely a physiological saline water group, PL-W₁₈O₄₉Nanoparticle laser-free irradiation group, PL-W₁₈O₄₉TPZ nanoparticle laserless irradiation group, PL-W₁₈O₄₉Nano self-care auxiliary laser irradiation group and PL-W₁₈O₄₉TPZ nanoparticle assisted laser irradiation group.

Adding hypoxia/oxidative stress detection mixture after 4 hr, culturing for 30min, washing with PBS, and laser (1W/cm) at 808nm²) Irradiating for 5 min. PBS was washed three times and fluorescence imaging was performed with confocal laser scanning microscopy (CLSM, LSM 710, Zeiss). The excitation wavelength of the hypoxia fluorescence signal is 561nm, and the emission wavelength is 600 nm. The excitation wavelength of ROS is 488nm, and the emission wavelength is 520 nm.

After 808nm laser irradiation, PL-W was measured as shown in FIG. 7₁₈O₄₉And PL-W₁₈O₄₉The TPZ groups all produce ROS (reactive oxygen species) and Hypoxia (hypoxic species). Description of W₁₈O₄₉The tumor cells can generate hypoxic after laser irradiation, so that 10% -50% of hypoxic cells in tumors in animal experiments are subjected to administration (PL-W)₁₈O₄₉TPZ NPs), laser irradiation, so that the entire tumor produced hypoxic oxygen provides data support.

In vivo Micro-PET imaging example 6

Physiological saline containing 18F-MISO (75. mu. Ci/mouse) was injected into nude mice via tail vein to evaluate hypoxia after treatment with different treatments.

Scans were performed 1h after injection using Inveon PET/CT system (Siemens, Malvern, Pa.) Micro-PET.

FIG. 8 is a comparison of 4 different layers of PET imaging after administration to mice in tumor models, the left mouse in each layer being injected with PL-W via the tail vein₁₈O₄₉TPZ nanoparticles, right mice injected with saline as a control. After laser irradiation, the left side (PL-W) can be seen through the figure₁₈O₄₉TPZ NPs) mice had a much larger hypoxic zone than the right (saline) mice. Further proves (PL-W)₁₈O₄₉TPZ nanoparticles) can increase the proportion of tumor hypoxia upon laser irradiation.

Antitumor Effect animal experiment example 7

When the tumor volume reaches 500mm³In this case, HeLa cell-induced tumor model mice were randomly divided into 4 groups of 8 mice each (i.e., Saline, PL-W in physiological Saline group)₁₈O₄₉Nanoparticle laser irradiation group PL-W₁₈O₄₉+ Laser, PL-TPZ nano particle and Laser irradiation group PL-TPZ + Laser and PL-W₁₈O₄₉-TPZ nanoparticle plus laser irradiation group PL-W₁₈O₄₉-TPZ+Laser)。

PL-W8 h after intravenous injection₁₈O₄₉+ Laser set, PL-TPZ + Laser set and PL-W₁₈O₄₉The group of-TPZ + Laser was irradiated with a 808nm Laser (1W/cm)²) And 8 min. Body weight and tumor volume were recorded.

After 12 days, mice were sacrificed, tumors collected, weighed, hematoxylin-eosin (H & E) stained and TUNEL apoptotic staining assay, observed under fluorescent microscope (IX71, Olympus).

PL-W as shown in FIGS. 9 and 10₁₈O₄₉The TPZ + Laser group is passed through PL-W₁₈O₄₉TPZ NPs treatment and after laser irradiation, tumor volume remained almost unchanged, while PL-W₁₈O₄₉The + Laser group, PL-TPZ + Laser group apply TPZ or W separately₁₈O₄₉After laser irradiation, the tumor volume increased to different degrees. PL-W was found by HE and TUNEL staining of tumor tissue₁₈O₄₉The TPZ nanoparticles had good tumor treatment effect, as shown in fig. 11.

And to PL-W₁₈O₄₉PL-W is found in the TPZ + Laser group and Saline group according to the distribution of the drugs in vivo and the pathological study of important organs₁₈O₄₉TPZ NPs are metabolized primarily in the liver, as shown in FIGS. 12 and 13.

Blood and Biochemical analysis example 8

Through PL-W₁₈O₄₉TPZ nanoparticles hypoxia-activated chemotherapy and photothermal therapy, on days 1 and 7, respectivelyAnd mice blood was collected on day 21 for blood and biochemical examination.

The main observation indexes include liver function indexes: alanine Aminotransferase (ALT), total bilirubin levels (TBIL) and Total Protein (TP).

Renal function indexes: blood urea Creatinine (CRE) and nitrogen (BUN) levels.

The blood indicators mainly include white blood cell count (WBC), red blood cell count (RBC), lymphocyte count, monocyte count (MON) and neutrophil count (NEU), Hemoglobin (HB) content, and influence on liver and kidney functions and blood after administration is explored.

As shown in FIG. 14, through PL-W₁₈O₄₉After the TPZ nano-particles are injected into the vein, the biochemical indexes of the blood are not changed.

PL-W will be described with reference to FIGS. 12 to 14₁₈O₄₉The TPZ nanoparticles have good biocompatibility.

Claims (3)

1. W₁₈O₄₉-tirapazamine composite nanoparticles characterized by: the composite nano particle takes PEG-PCL as a carrier, and W is loaded in the composite nano particle₁₈O₄₉And tirapazamine; the W is₁₈O₄₉And tirapazamine drug loading of 2.99% and 1.29%, respectively; the composite nano particles are in a dispersed spherical structure, and the particle size is 60-100 nm;

the composite nano particle is prepared by the following method:

(1) dissolving 50mg of PEG-PCL in 5-10mL of dichloromethane to prepare a solution a;

(2) adding 50-100mL of pure water into the solution a to obtain a solution b;

(3) adding 5mg-10mg of W into the solution b₁₈O₄₉Stirring for 10-20 min to obtain solution c;

(4) adding 2-4mg of tirapazamine into the solution c, and stirring for 10-20 minutes to obtain a solution d;

(5) carrying out ultrasonic treatment on the solution d for 30-50 minutes;

(6) then centrifuging for 10-20 minutes under the condition of 14000 r/min;

(7) and (4) adding deionized water into the precipitate obtained in the step (6) for washing, repeating the step (6) for centrifuging, and repeating the washing and centrifuging for three times to obtain the composite nano particles.

2. The use of the composite nanoparticle of claim 1 for the preparation of a medicament for the treatment of tumors.

3. Use of the composite nanoparticle according to claim 1 for the preparation of a combination drug for chemotherapy and photothermal therapy.

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