CN108969766B

CN108969766B - Photosensitive composite material with tumor microenvironment response oxygenation performance and preparation method thereof

Info

Publication number: CN108969766B
Application number: CN201810798006.6A
Authority: CN
Inventors: 刘健哲; 郑明彬
Original assignee: Shenzhen Huachuang Biomedical Technology Co ltd
Current assignee: Shenzhen Huachuang Biomedical Technology Co ltd
Priority date: 2018-07-19
Filing date: 2018-07-19
Publication date: 2021-09-10
Anticipated expiration: 2038-07-19
Also published as: CN108969766A

Abstract

The invention relates to a photosensitive composite material with tumor microenvironment response oxygenation performance and a preparation method thereof. The composite material takes manganese dioxide particles as a carrier to load ICG, the manganese dioxide particles are of a hollow porous particle structure, the particle size is 65-75 nm, and the thickness of a shell of each hollow porous particle is 7-9 nm. The preparation method comprises the steps of preparing silver nanospheres, preparing hollow manganese dioxide nanoparticles and loading ICG. The technical scheme provided by the invention has the advantages that the prepared composite material has uniform particle size, the temperature is rapidly increased under the irradiation of near-infrared light with the wavelength of 808nm, the highest temperature can reach 50 ℃ after 10min, the composite material has good photo-thermal conversion performance, and an in-vitro simulation tumor microenvironment response oxygen release experiment shows that the prepared H-MnO is₂The nano particles release oxygen rapidly, the oxygen increasing effect is good, and the high-sensitivity tumor microenvironment responds to the oxygen releasing characteristic and the high-loading ICG photosensitizer is beneficial to generating a high-efficiency photodynamic effect.

Description

Photosensitive composite material with tumor microenvironment response oxygenation performance and preparation method thereof

Technical Field

The invention belongs to the technical field of nano photosensitizers, and particularly relates to a photosensitive composite material with tumor microenvironment response oxygenation performance and a preparation method thereof.

Background

In recent years, cancer has become a leading problem to the health of humans. The traditional cancer treatment methods mainly comprise operations, radiotherapy and chemotherapy, but the treatment methods have the side effects of large trauma, systemic toxicity, damage to normal cells and tissues and the like. With the continuous development of tumor treatment means, the non-invasive and space-time controllable photodynamic therapy (PDT) and photothermal therapy (PTT) as novel therapies provide a new approach for the clinical treatment of tumors. PDT is carried out by irradiating tumor part with specific light source, acting on photosensitizer enriched in tumor part, and inducing peripheral O after photosensitizer absorbs light energy₂Converted into singlet oxygen and other Reactive Oxygen Species (ROS) which are toxic to the tumor cells, thereby achieving the effect of killing the tumor cells. The PTT principle is that the surface of the photosensitizer has a plasma resonance absorption effect to convert light energy into heat energy under the irradiation of near infrared light, so that the temperature of a tumor part is raised. When the temperature at the tumor site is higher than 42 ℃, the tumor cells die due to factors such as DNA synthesis inhibition or protein denaturation. Compared with the traditional treatment means, the PDT and PTT treatment means have the advantages of low toxicity, strong targeting property, no toxic or side effect, strong universality and the like, and are officially approved by relevant health departments of the countries such as the United states, France, Germany, UK, Japan, Netherlands, Canada and the like to be applied to the treatment of various malignant tumors (such as lung cancer, skin cancer, bladder cancer, esophageal cancer, cervical cancer and the like)). However, oxygen consumption in the PDT treatment process can aggravate local tumor hypoxia, and the tumor hypoxia microenvironment can not only promote tumor growth, invasion and metastasis, but also greatly reduce the PDT treatment effect. And the tumor hypoxia can further induce tumor cells to excessively express hypoxia induction factors, promote the rapid formation of tumor neovascularization and induce the recurrence, invasion and metastasis of tumors.

Indocyanine green is a photosensitizer approved by the U.S. Food and Drug Administration (FDA) for clinical use, has characteristic absorption in the near infrared region with high tissue penetration, and produces good PDT and PTT therapeutic effects under NIR irradiation. However, ICG when used for PDT and PTT therapy results in hypoxia at the tumor site, thereby reducing PDT treatment efficacy and promoting tumor growth and metastasis.

Therefore, the development of a photosensitive and renaturated material which can effectively increase oxygen in the tumor microenvironment is continued to realize the treatment effect of PDT or PTT.

Disclosure of Invention

The invention provides a photosensitive composite material with tumor microenvironment response oxygenation performance and a preparation method thereof, which are used for solving the problem that hypoxia hinders the treatment effect in the PDT or PTT process.

In order to solve the technical problems, the technical scheme of the invention is as follows: the photosensitive composite material with the tumor microenvironment response oxygenation performance is beneficial to generating an efficient photodynamic effect, manganese dioxide particles are used as carriers to load ICG (indocyanine green), the manganese dioxide particles are of hollow porous particle structures, the particle size is 65-75 nm, and the thickness of a shell of each hollow porous particle is 7-9 nm.

The invention also provides a preparation method of the photosensitive composite material with the tumor microenvironment response oxygenation performance, which comprises the following steps:

step one, preparing silver nanospheres: with silver trifluoroacetate (CF)₃COOAg) as raw material and polyalcohol as reducing agent to prepare silver nanospheres;

step two, preparing hollow manganese dioxide nano particles: preparing hollow manganese dioxide nanoparticles by taking the silver nanospheres obtained in the step one as templates, potassium permanganate as a raw material and silver as a reducing agent;

step three, loading of ICG: and (3) placing the hollow manganese dioxide nanoparticles obtained in the second step into an indocyanine green (ICG) -Phosphate Buffer Solution (PBS) solution, and ultrasonically loading a sufficient amount of ICG on the inner surface and/or the outer surface of the hollow manganese dioxide nanoparticles through electrostatic adsorption.

Using MnO₂In the tumor microenvironment (acidic/H-rich)₂O₂) Oxygen released by redox reaction with hydrogen peroxide improves tumor hypoxia microenvironment and improves tumor treatment effect; the generated manganese ions can be discharged out of the body through the kidney, and the nano-carrier is safe and efficient.

Optionally, the step one specifically includes sequentially mixing a sodium hydrosulfide (NaSH) -glycol solution, a hydrochloric acid-glycol solution, a polyvinylpyrrolidone (PVP) -glycol solution, and CF₃Adding the COOAg-glycol solution into an ethylene glycol reaction system, keeping the reaction temperature at 140 ℃ and 160 ℃ for reaction for 30-60min to obtain silver nanospheres, performing post-treatment on the separated silver nanospheres, and finally dispersing the silver nanospheres in water for later use.

Optionally, the concentration of the NaSH-glycol solution is 3mM, the concentration of the hydrochloric acid-glycol solution is 3mM, the concentration of the PVP-glycol solution is 20mg/mL, and the CF is₃The concentration of the COOAg-glycol solution was 282 mM.

Optionally, the volume ratio of each component in the first step is as follows:

optionally, the post-treatment in the step one specifically comprises washing with acetone, ethanol and ultrapure water for 1 time respectively, and centrifuging at 8500rpm for 10min to remove the supernatant.

Optionally, the second step specifically comprises adding potassium permanganate (KMnO) to the silver nanospheres dispersed in water₄) Adding polyallylamine hydrochloride (PAH) aqueous solution, stirring for 10-15min, and centrifuging to obtain H-MnO dispersed in water₂And (5) standby.

Optionally, the KMnO₄The concentration of the water solution is 8mg/mL, the concentration of the PAH water solution is 50mg/mL, and KMnO₄The volume ratio of the aqueous solution to the PAH aqueous solution was 1: 1.

Optionally, the centrifugal washing in the second step is specifically washing three times in ultrapure water, the centrifugation rate is 12000rpm, and the time duration is 10min each time.

Optionally, step three specifically comprises separating H-MnO from water₂Adding ICG-PBS solution, ultrasonically dispersing to load ICG on the surface of manganese dioxide nanoparticles by electrostatic adsorption, standing for 20-40min to obtain precipitate as photosensitive composite material (H-MnO) with tumor microenvironment response oxygenation performance₂@ ICG) which was dispersed in PBS and stored until use.

Alternatively, the concentration of the ICG-PBS solution is 15. mu.g/mL.

The technical scheme provided by the invention adopts a potassium permanganate reduction method to prepare H-MnO with a hollow porous structure and uniform particle size₂The thickness of the shell of the nano particle is about 8nm, the temperature is rapidly raised under the irradiation of near infrared light with the wavelength of 808nm, the highest temperature can reach 50 ℃ after 10min, the nano particle has good photo-thermal conversion performance, and the experiment of simulating the tumor microenvironment response oxygen release in vitro shows that the prepared H-MnO has good photo-thermal conversion performance₂The nano-particles release oxygen rapidly, the oxygenation effect is good, the high-sensitivity tumor microenvironment responds to the oxygen release characteristic and the high-loading ICG photosensitizer is favorable for generating high-efficiency photodynamic effect, and in addition, Mn released by the photosensitizer²⁺Can be used for guiding biological imaging, can modify targeting molecules on the surface of the nano photosensitizer, provides a new way for visualization and accurate treatment of tumors, and has wide application prospect in the future.

Drawings

FIG. 1 is a schematic diagram of the preparation process of the photosensitive composite material with tumor microenvironment response oxygenation capability of the invention;

FIG. 2 is TEM image of nanoparticles, wherein FIG. 2a is TEM image of silver nanospheres prepared in example 1, and FIG. 2b is H-MnO prepared in example 1₂Transmission electron microscopy of nanoparticles;

FIG. 3 is a scheme showing a scheme of preparation in example 1Prepared silver nanosphere and H-MnO₂A particle size map of the nanoparticles;

FIG. 4 is a silver nanosphere, H-MnO prepared in example 1₂Nanoparticles and H-MnO₂Zeta potential diagram of @ ICG;

FIG. 5 is UV-VISIBLE ABSORPTION SPECTRUM, wherein FIG. 5a is UV-VISIBLE ABSORPTION SPECTRUM of Ag nanospheres prepared in example 1, and FIG. 5b is ICG and H-MnO prepared in example 1₂Ultraviolet-visible absorption spectrum of @ ICG;

FIG. 6 shows PBS, ICG and H-MnO prepared in example 1₂@ ICG photothermal conversion curve under 808 laser irradiation;

FIG. 7 is H-MnO prepared in example 1₂An oxygen release map simulating the tumor microenvironment in vitro.

Detailed Description

For the convenience of understanding, the photosensitive composite material with oxygen increasing capability in response to tumor microenvironment and the preparation method thereof are described in the following with reference to the examples, which should be understood as merely illustrative and not limiting the scope of the present invention.

The chemical reagents used in the experiment are analytically pure, and the water is ultrapure water, and the method mainly comprises the following steps: sodium hydrosulfide (NaSH), hydrochloric acid, silver trifluoroacetate (CF)₃COOAg), polyvinylpyrrolidone (PVP), ethylene glycol, acetone, anhydrous ethanol, indocyanine green (ICG), potassium permanganate, polyallylamine hydrochloride (PAH), Phosphate Buffer Solution (PBS), tris (4, 7-biphenyl-1, 10-phenanthroline) ruthenium dichloride (Ru (dpp)₃Cl₂)。

The instrument used in the experiment mainly comprises: ultrasonic cleaning apparatus, UV-visible spectrophotometer (Perkinelmer Lambda25), nanometer particle size potentiometer (Malvern Zetasizer Nano ZS90, UK), transmission electron microscope (TEM, Tecnai G2F20S-Twin, USA), 808 laser (PSU-III-LED, China, New vinpoch industries electro-optical technology Co., Ltd.), thermocouple (Omega, China), high speed refrigerated centrifuge (Sigma 3-18KS, Germany), fluorescence spectrometer (Edinburgh FLS920, UK)

As shown in fig. 1, the present invention prepares silver nanospheres by a sulfide-assisted polyol reduction method; then the silver nanospheres are used as templates,preparing H-MnO2 nano-particles with hollow porous structures by adopting a potassium permanganate reduction method; then loading a photosensitizer indocyanine green (ICG) in H-MnO₂In the nano particles, the ICG-loaded nano photosensitizer H-MnO with tumor oxygenation performance is obtained₂@ ICG. Take a specific implementation as an example.

Example 1H-MnO₂Preparation of @ ICG

The method comprises the following steps: preparing silver nanospheres: the oil bath was heated to 150 ℃ and 10mL of ethylene glycol was added to a 50mL round bottom flask and heated with stirring. After 10min, the ethylene glycol reached 150 ℃ and 0.12mL of a 3mM NaSH-ethylene glycol solution was injected immediately. After 2min, 1mL of a 3mM HCl-ethylene glycol solution and 2.5mL of a 20mg/mL PVP-ethylene glycol solution were added. After 2min, 0.8mL of 282mM CF was added₃COOAg-glycol solution. Then, the temperature of the reaction solution was maintained at 150 ℃ and the reaction was carried out for 45 min. The obtained silver nanospheres are washed by acetone, ethanol and ultrapure water for 1 time respectively, then centrifuged at 8500rpm for 10min, the supernatant is removed, and finally the silver nanospheres are dispersed in 10mL of ultrapure water for standby.

Step two: preparing hollow manganese dioxide nanoparticles: diluting 0.5mL of silver nanosphere to 2.5mL, and magnetically stirring at room temperature; then 100. mu.L KMnO with concentration of 8mg/mL was added₄Magnetically stirring the aqueous solution for 5 min; then 100. mu.L PAH with a concentration of 50mg/ml was added and stirred for 10 min. Washing the precipitate with ultrapure water for three times (12000rpm, 10min) by centrifugation to obtain H-MnO₂Dispersed in 10mL of ultrapure water for use.

Step three: load of ICG: 3mL of H-MnO was taken₂Centrifuging, removing supernatant to obtain precipitate, adding 3mL ICG-PBS solution with concentration of 15 μ g/mL into the precipitate, and ultrasonically dispersing to load ICG on manganese dioxide nanoparticles by electrostatic adsorption. Standing for 30min, centrifuging to remove supernatant, and dispersing precipitate in 3mL PBS buffer solution to obtain H-MnO₂@ ICG composite.

Example 2 physicochemical Property testing

2.1 Experimental methods

The obtained H-MnO₂The @ ICG nanoparticles were diluted with ultrapure water and thenThe sample is dripped on a 200-mesh ultrathin carbon film copper net, naturally dried, and finally the morphology and the particle size are characterized by a transmission electron microscope (TEM, Tecnai G2F20S-Twin, USA).

The particle size and surface Zeta potential of the nanoparticles were determined by means of a dynamic laser scattering instrument (DLS, Malvern Zetasizer Nano ZS 90); the absorption spectrum of the nanoparticles was determined by a UV-Vis spectrophotometer (Perkinelmer Lambda 25).

2.2 analysis of results:

as can be seen from the TEM image of FIG. 2a, the prepared silver nanospheres are mostly spherical, and a small part is elliptical, and have uniform size and 45 + -5 nm particle size. In the TEM image of FIG. 2b, H-MnO₂The nano particles are hollow spheres, the particle size of the nano particles is increased, the particle size is 70nm +/-5 nm, and the shell thickness is about 8 nm.

As shown in FIG. 3, the average core particle diameter of the synthesized silver nanosphere measured by DLS particle sizer is 59.18nm, and H-MnO of hollow porous structure₂The average core particle size of the nanoparticles was 85.80nm, which is consistent with TEM characterization.

As shown in FIG. 4, the average Zeta potential of the silver nanospheres measured by the DLS particle size analyzer is-13.7 mV, and the H-MnO is₂The average Zeta potential of the nano-particles is 36.6mV, and H-MnO of the ICG with negative charge is loaded₂The average Zeta potential of the @ ICG nanoparticles was-33.3 mV. The change in particle size and Zeta potential indicates that the negatively charged photosensitizer ICG was successfully loaded into H-MnO₂On the nanoparticles.

As can be seen from fig. 5a, the absorption peak of the silver nanosphere is located at 426nm, which is derived from the excitation of the surface plasmon resonance effect or dipole resonance effect of the nanoparticles, and the main peak is obvious and narrow, which indicates that the prepared silver nanosphere has uniform particle size distribution and good dispersibility. As can be seen from FIG. 5b, H-MnO₂The ultraviolet spectrogram of @ ICG can observe the ultraviolet absorption characteristics of manganese dioxide, and the ultraviolet absorption peak of silver nanospheres is not observed, which indicates that the silver nanospheres are successfully replaced by H-MnO₂A nanoparticle; the absorption peak of ICG is near 780nm, compared with ICG, the nano photosensitizer H-MnO₂The @ ICG absorption peak is near 800nm and is red-shifted, indicating that ICG is loaded in H-MnO₂Within the nanoparticle.

EXAMPLE 3 photothermal Effect detection

3.1 Experimental methods

H-MnO₂The in vitro photothermal properties of @ ICG are mainly characterized by detecting the temperature change of the dispersion of the photosensitizers under NIR illumination. The experiment was divided into 3 groups, the first group being PBS solution, the second group being ICG-PBS solution (15. mu.g/mL), the third group being H-MnO₂@ ICG Dispersion (15. mu.g/mL). The 808 laser (PSU-III-LED, china) with a wavelength of 808nm was used as an infrared light source, and the change in temperature of the nano photosensitizer solution under NIR illumination was recorded using a thermocouple (Omega, china) once every 5 seconds until the solution temperature did not change significantly.

3.2 analysis of results:

due to H-MnO₂The absorption peak of @ ICG is in the near infrared region, so that it can produce a remarkable photothermal conversion performance under irradiation of near infrared light. As can be seen from FIG. 6, the temperature of the PBS solution was slowly increased under the 808-minute laser irradiation, and the maximum temperature was only 33 ℃ within 10 min. ICG solution and H-MnO within the first 2min compared to PBS solution₂The temperature of the @ ICG dispersion is rapidly raised, and H-MnO₂@ ICG light 10min, the temperature rose to 53 ℃. In addition, ICG solution with H-MnO₂The photothermal curves of the @ ICG dispersions were substantially the same, and the maximum temperatures were substantially the same, indicating H-MnO₂The nanoparticles have little influence on the photothermal performance of ICG, and the result shows that the nano photosensitizer has good photothermal conversion performance.

Example 4 oxygenation Performance testing

4.1 Experimental methods

This experiment was carried out by using Ru (dpp)₃Cl₂Oxygen probe for detecting nano photosensitizer to simulate tumor microenvironment (acidity/H-rich) in vitro₂O₂) The response oxygenation performance of (2). The experiment was divided into two groups, the first (PBS6.4+ H)₂O₂) Second group (H-MnO)₂+PBS6.4+H₂O₂) The fluorometric excitation/emission wavelengths are: 455nm/610nm, scanning range: 550nm-700nm, record for 10 min.

4.2 analysis of results:

as shown in fig. 7, in vitroMimicking tumor microenvironment (acidic/H-rich)₂O₂) Under the condition, within 10min, H-MnO₂The oxygen release amount of the nano particles is far higher than that of the control group, which shows that H-MnO₂The @ ICG has good response oxygen increasing performance in a tumor microenvironment, can overcome the problem of tumor part oxygen deficiency caused in photodynamic therapy, and improves the treatment effect.

Finally, it should be noted that: the above examples are only for illustrating the technical solutions of the present invention, and are not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: it is to be understood that modifications may be made to the technical solutions described in the foregoing embodiments, or some or all of the technical features may be equivalently replaced, and such modifications or replacements may not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The photosensitive composite material with the tumor microenvironment response oxygenation performance is characterized in that manganese dioxide particles are used as carriers to load ICG, the manganese dioxide particles are of hollow porous particle structures, the particle size is 65-75 nm, and the shell thickness of the hollow porous particles is 7-9 nm; the preparation method of the photosensitive composite material with the tumor microenvironment response oxygenation performance comprises the following steps:

step one, preparing silver nanospheres: with CF₃COOAg as raw material and polyalcohol as reducing agent to prepare silver nanospheres, which specifically comprises sequentially mixing NaSH-glycol solution, hydrochloric acid-glycol solution, PVP-glycol solution and CF₃Adding a COOAg-glycol solution into an ethylene glycol reaction system, keeping the reaction temperature at 140-160 ℃ for reaction for 30-60min to obtain silver nanospheres, performing post-treatment on the separated silver nanospheres, dispersing the silver nanospheres in water for later use, wherein the post-treatment specifically comprises washing the silver nanospheres for 1 time by using acetone, ethanol and ultrapure water in sequence, centrifuging the silver nanospheres at 8500rpm for 10min, and removing a supernatant;

step three, loading of ICG: putting the hollow manganese dioxide nano-particles in the second step into an ICG-PBS solution, and carrying out ultrasonic treatment to enable ICG to be loaded on the inner surface and/or the outer surface of the hollow manganese dioxide nano-particles through electrostatic adsorption; specifically, the method comprises separating H-MnO from water₂Then adding ICG-PBS solution, ultrasonic dispersing to load ICG on the surface of manganese dioxide nano-particles by electrostatic adsorption, standing for 20-40min to obtain precipitate H-MnO₂@ ICG, which was stored in PBS dispersed, the concentration of the ICG-PBS solution was 15. mu.g/mL.

2. The preparation method of the photosensitive composite material with the tumor microenvironment response oxygenation capacity of claim 1, which is characterized by comprising the following steps:

step three, loading of ICG: putting the hollow manganese dioxide nano-particles in the second step into an ICG-PBS solution, and carrying out ultrasonic treatment to enable ICG to be loaded on the inner surface and/or the outer surface of the hollow manganese dioxide nano-particles through electrostatic adsorption; specifically, the method comprises separating H-MnO from water₂Then adding ICG-PBS solution, ultrasonic dispersing to load ICG on the surface of manganese dioxide nano-particles by electrostatic adsorption, standing for 20-40min to obtain precipitate H-MnO₂@ ICG, dispersed in PBS for storageFor use, the concentration of the ICG-PBS solution was 15. mu.g/mL.

3. The method for preparing the photosensitive composite material with the tumor microenvironment response oxygenation capacity of claim 2, wherein the concentration of the NaSH-glycol solution is 3mM, the concentration of the hydrochloric acid-glycol solution is 3mM, the concentration of the PVP-glycol solution is 20mg/mL, and the CF is CF₃The concentration of the COOAg-glycol solution was 282 mM.

4. The method for preparing the photosensitive composite material with the tumor microenvironment response oxygenation capacity of claim 2, wherein the second step specifically comprises adding KMnO into silver nanospheres dispersed in water₄Adding PAH aqueous solution into the aqueous solution, stirring for 10-15min, and washing by centrifugation to obtain H-MnO dispersed in water₂And (5) standby.

5. The method for preparing the photosensitive composite material with the tumor microenvironment response oxygenation capacity of claim 4, wherein the KMnO is KMnO₄The concentration of the water solution is 8mg/mL, the concentration of the PAH water solution is 50mg/mL, and KMnO₄The volume ratio of the aqueous solution to the PAH aqueous solution was 1: 1.

6. The method for preparing the photosensitive composite material with the tumor microenvironment response oxygenation capacity of claim 4, wherein the centrifugal cleaning in the second step is specifically three times of cleaning in ultrapure water, the centrifugal rate is 12000rpm, and the time duration of each time is 10 min.

7. The method for preparing the photosensitive composite material with the tumor microenvironment response oxygenation capacity of claim 2, wherein the concentration of the ICG-PBS solution is 15 μ g/mL.