CN113304280A - Rare earth up-conversion composite nano material for treating tumor - Google Patents

Rare earth up-conversion composite nano material for treating tumor Download PDF

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CN113304280A
CN113304280A CN202110519982.5A CN202110519982A CN113304280A CN 113304280 A CN113304280 A CN 113304280A CN 202110519982 A CN202110519982 A CN 202110519982A CN 113304280 A CN113304280 A CN 113304280A
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mno
tumor
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兰建明
陈敬华
李春艳
吴芳
罗登旺
韩罗丹
方垚
沈毅萍
余春晓
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Fujian Medical University
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Abstract

The invention discloses a rare earth up-conversion composite nano material for treating tumors, and constructs a near-infrared remote light-controlled up-conversion photodynamic therapy and tumor microenvironment triggerable chemical kinetics cooperative therapy platform to realize the treatment of hypoxic tumorsCan be used for treating diseases. The UCNPs with high efficiency and stable luminescence can convert 980 nm NIR light into 540nm green light, so that HB of a photosensitizer after red shift of an absorption spectrum is excited to carry out a PDT process. Endogenous acidic H in the tumor microenvironment2O2Hydrolyzed MnO2Can realize O2The self-sufficiency of the PDT can overcome the tumor hypoxia obstacle and improve the oxygen-dependent treatment efficiency of the PDT. Mn produced by hydrolysis2+Can mediate Fenton-like reaction to generate virulent OH, and cause oxidative stress of tumor cells. MnO2Can also consume the intracellular antioxidant GSH, reduce OH clearance and improve CDT efficiency. The platform combines the tumor microenvironment response, PDT and CDT for cooperative treatment, improves the treatment efficiency and realizes the effective treatment of the hypoxic tumor.

Description

Rare earth up-conversion composite nano material for treating tumor
Technical Field
The invention relates to a phospholipid-coated up-conversion nano material (UCNPs) loaded with photosensitizer Hypocrellin B (HB), and MnO is grown in situ on PEG on the surface of the UCNPs through O-Mn coordination2A tumor photodynamic/chemokinetic (PDT/CDT) synergistic treatment platform is constructed. Is a new tumor treatment technology integrating nanotechnology, optical technology and chemical action, and belongs to the field of modern medical minimally invasive or non-invasive treatment combining interdisciplinary disciplines.
The present invention relates to the above-mentioned conversion nanomaterial loaded with photosensitizer HB and MnO2The composite nano material directly growing on the surface of UCNPs in situ serves as an ideal TME (tumor microenvironment response therapy) response type nano treatment platform and is used for effectively treating hypoxic tumors in a body.
Background
In recent years, optomechanicalForce therapy (PDT) has received a great deal of attention in the treatment of clinical malignancies. PDT involves irradiation of a Photosensitizer (PS) enriched in the tumor site with light of a specific wavelength to introduce O2Converted into cytotoxic Reactive Oxygen Species (ROS), which can cause irreversible damage to proteins or DNA, thereby inducing apoptosis in tumor cells. Compared with the traditional operation, chemotherapy and radiotherapy, PDT has the advantages of small invasion, low toxicity, high selectivity, high efficiency, good curative effect and the like, and has great development prospect in the field of tumor treatment. However, there are still a number of unavoidable factors that limit the clinical utility of PDT.
First, the selection of a suitable photosensitizer is advantageous for obtaining efficient PDT results. However, it is difficult to balance the penetration depth of conventional photosensitizers with the efficiency of PDT. The hypocrellin is reported to have antiviral, antitumor and other effects, and shows great medicinal potential in preventing and treating diseases such as cancer. The photosensitive activity and phototherapy effect of the photosensitizer Hypocrellin B (HB) are more prominent, and the photosensitizer hypocrellin B is an ideal PS for PDT due to the advantages of strong phototoxicity, fast metabolism, high quantum yield of triplet oxygen and singlet oxygen, small damage to normal tissues and the like. However, the poor biocompatibility and penetration depth of visible light as excitation light limits its application in PDT. Coincidentally, rare earth doped upconversion nanoparticles (UCNPs) can convert near infrared light into ultraviolet or visible light, and their mediated photodynamic therapy shows a great effect in increasing tissue penetration depth of light to combat deep tumors, which makes PDT a critical step forward. After the liposome is coated, the absorption peak of HB generates a larger red shift, and the high matching with the emission spectrum of UCNPs is realized, so that the fluorescence quantum transfer efficiency is improved, and the high-efficiency PDT effect is generated. The PDT process is realized by exciting UCNPs by near infrared light, and deep tumor treatment can be realized due to the fact that the penetration depth of the near infrared light in biological tissues is deep.
Second, extreme hypoxia to the Tumor Microenvironment (TME), due to O2The high dependence of (a) limits the development of PDT. In addition, O consumed during PDT2May further aggravate the tumorHypoxia, reduced efficacy of PDT, and tumor progression. An effective strategy is needed to increase the oxygen concentration in the anoxic zone. Hyperbaric oxygen therapy of pure O breathed in a pressurized chamber2Have been used to overcome hypoxic environments, but the side effects of hyperoxic convulsions and barotrauma caused by excess oxygen in normal tissues have limited their widespread use. Various nanomaterials (e.g. perfluorocarbon, CaO)2Etc.) have also been used to selectively alleviate cancer hypoxia, but suffer from poor biocompatibility, O2Short generation effect and the like.
Studies have shown that tumor cells have a unique metabolic pattern that is different from normal cells. On the one hand, solid tumors produce large amounts of lactic acid due to the upregulation of glycolytic metabolism during tumorigenesis, resulting in an acidic tumor microenvironment with a significant decrease in pH. On the other hand, malignant cells produce excessive H2O2Resulting in H in TME2O2The levels were significantly elevated. In addition, studies have shown that reduced Glutathione (GSH) levels in tumor tissues are at least 4-fold higher than in normal tissues. In conclusion, the solid tumor TME has obvious GSH and H2O2、H+High content. In recent years, MnO2The nano structure is used as a unique TME-response type nano therapeutic and diagnostic material, and has attracted great interest. Due to pH/redox characteristics, exfoliated MnO2The nanostructure can be bound by acidic H in solid tumors2O2Reduction to Mn2+Meanwhile, a large amount of oxygen is generated, and the treatment effect of PDT (photodynamic therapy) dependent on oxygen on hypoxic tumors can be obviously improved. In addition, MnO2Highly reactive hydroxyl radicals (. OH) can be generated by Fenton-like reactions and have proven useful in chemokinetic therapy (CDT). In addition, GSH acts as an intracellular antioxidant for PDT production1O2OH generated by CDT has strong scavenging effect, thereby greatly increasing the resistance of cancer cells to oxidative stress and reducing the curative effect of PDT and CDT. While passing through MnO2Reducing intracellular GSH level is an important means for PDT/CDT nano-drugs to avoid tumor drug resistance and improve the curative effect of tumor treatment.
Inspired by the above research, the research method uses MnO2Directly grows on the surface of UCNPs in situ, and is used as an ideal TME (tumor microenvironment response therapy) response type nano treatment platform. The principle is that the multifunctional nano platform adopts amphiphilic copolymer DSPE-PEG2000The method is characterized in that hydrophobic interaction is combined with oleic acid ligands on the surfaces of UCNPs to realize that phospholipid wraps up conversion nanoparticles, a photosensitizer HB is assembled through a compact hydrophobic layer, and finally MnO grows in situ through O-Mn coordination on PEG on the surfaces of the UCNPs2Nanosheets. The platform is an ideal design integrating tumor microenvironment response and PDT/CDT, can fully play the role of up-converting each component in the nano composite material, and realizes the effective treatment of tumors.
Disclosure of Invention
The invention aims to construct a near-infrared remote light-controlled up-conversion photodynamic therapy and tumor microenvironment triggerable chemical kinetics cooperative therapy platform, improve the treatment efficiency through tumor microenvironment response and PDT/CDT cooperative therapy, and realize effective treatment on hypoxic tumors.
In order to achieve the purpose, the invention adopts the following technical scheme: an up-conversion nanocomposite material capable of serving as a tumor microenvironment responsive (TME) nanotherapeutic platform, comprising: the device is an ideal design integrating tumor microenvironment response, photodynamic therapy and chemodynamic therapy; the multifunctional nano platform adopts amphiphilic copolymer DSPE-PEG2000The method is characterized in that hydrophobic interaction is combined with oleic acid ligands on the surfaces of UCNPs to realize that phospholipid wraps up conversion nanoparticles, a photosensitizer HB is assembled through a compact hydrophobic layer, and finally MnO grows in situ through O-Mn coordination on PEG on the surfaces of the UCNPs2The nano-sheet can fully play the role of each component in the up-conversion nano-composite material, and realize the effective treatment of tumors.
The preparation method of the up-conversion nano composite material used as the tumor treatment platform is characterized by comprising the following steps: the method comprises the following steps:
1) NaYF is prepared according to a thermal decomposition method4Yb, Er nanocrystals (UCNPs) prepared by thin film hydration methodDSPE-PEG2000Coated NaYF4Nano particles UCNPs @ DSPE-PEG of Yb, Er nano crystal2000（UCNPs@DP）；
2) Loading of photosensitizer HB: each of the brown vials was charged with 1 mL of UCNPs @ DSPE-PEG synthesized in step 12000(UCNPs @ DP) and 10. mu.L of DMF solutions of different concentrations of HB selected from 0, 0.5, 1, 5, 10, 20, 30, 40 mM were stirred for 24 h in the dark to obtain UCNPs @ DSPE-PEG2000@HB（UCNPs@DPB）；
3）UCNPs@DSPE-PEG2000@HB-MnO2（UCNPs@DPB-MnO2) The preparation of (1): mu.L of the UCNPs @ DPB aqueous solution obtained in step 2) was added to a 1.5 mL EP tube, 320. mu.L of deionized water was added, and then 40. mu.L of a 50 mM MES solution and 40. mu.L of 4 mM KMnO were added4Adding the solution into tubes in sequence, and performing ultrasonic treatment at room temperature for 30 min; finally, the precipitate was collected by centrifugation at 14500 rpm for 10 min, washed 3 times with deionized water, and dispersed in 100. mu.L of deionized water to obtain UCNPs @ DSPE-PEG2000@HB-MnO2（UCNPs@DPB-MnO2) For further use;
4) the killing effect of the up-conversion nano composite material on tumor cells: near infrared spectroscopy experiments show that 980 nm laser has no adverse effect on HeLa cells, and cancer cells incubated with UCNPs @ DPB are inhibited in various concentration ranges under 980 nm laser irradiation, which indicates that PDT plays a role in killing HeLa cells, compared with UCNPs @ DPB, UCNPs @ DPB-MnO2Has more obvious inhibiting effect on HeLa cells due to the coated MnO2Degradation takes place, on the one hand, O is produced2Improving the oxygen-dependent treatment efficiency of PDT; on the other hand, Mn is produced2+And mediates the Fenton-like reaction to generate virulent OH, so that the oxidative stress of the tumor cells is caused, and the effective treatment of the tumor cells is realized.
The above-described up-conversion nanocomposite (UCNPs @ DPB-MnO)2) Preparing;
1) determination of photosensitizer HB loading and stability investigation: loading HB with different concentrations by using UCNPs @ DP, and keeping the color unchanged and basically achieving saturation when the concentration reaches 200 mu M; further examining the loading capacity, in the DMF solution, when the concentration of HB is 200 μ M, the loading capacity is saturated to 15 μ g/mg; placing the compound for 24 h, centrifuging to obtain supernatant, and testing absorption spectrum; the supernatant after the compound is dispersed in deionized water, PBS, ethanol, chloroform, DMEM and RPMI-1640 for centrifugation is subjected to absorption spectrum measurement, and only a small amount of leakage occurs in the ethanol, because the ethanol can destroy the phospholipid structure, and no leakage of HB is found in other solutions; HB loaded in the phospholipid layer can be completely extracted by using dichloromethane; the phospholipid layer has stable property, and can well load photosensitizer for photodynamic therapy of tumor;
2) testing of singlet oxygen in vitro: generated by using chemical probe 1, 3-diphenyl isobenzofuran (DPBF) to up-conversion nano composite material under 980 nm laser irradiation1O2Carrying out detection; at 980 nm, the laser irradiation of HB and UCNPs @ DP respectively alone is absent1O2After the photosensitizer HB is generated and loaded, the attenuation degree of DPBF is gradually increased along with the increase of the concentration of the compound and the extension of the irradiation time, so that the photosensitizer HB can be efficiently generated1O2The compound lays a foundation for improving the next photodynamic therapy effect;
3) testing of up-conversion nanocomposite-mediated Fenton-like reaction: to verify Mn2+The mediated Fenton-like reaction will contain 10. mu.g/mL MB, 8 mM H2O2And 0.5 mM MnCl 225 mM NaHCO3The solution was allowed to stand at 37 ℃ for 30 min, monitored by the change in absorbance at 665 nm for OH-induced MB degradation;
4) OH scavenging by GSH: 25 mM NaHCO3Solution containing 10. mu.g/mL MB, 8 mM H2O2GSH at various concentrations selected from 0, 1, 5 and 10 mM and 0.5 mM MnCl2Or UCNPs @ DPB-MnO2OH-induced MB degradation was monitored by absorbance change at 665 nm, standing at 37 ℃ for 30 min.
The up-conversion nano composite material is characterized in that: using amphiphilic copolymers DSPE-PEG2000By hydrophobic interaction with each otherActing to combine with oleic acid ligand on the surface of UCNPs, and realizing phospholipid coated up-conversion nanoparticles; HB is used as a photosensitizer, UCNPs can convert near infrared light into 540nm green light, and the green light is used as a mediated carrier in PDT therapy, so that the photosensitizer HB is excited to carry out PDT; finally, in-situ growth of MnO on PEG on the surface of UCNPs through O-Mn coordination2Nanosheets; endogenous acidic H in the tumor microenvironment2O2Hydrolyzed MnO2Can realize O2The self-sufficiency of the PDT can overcome the tumor hypoxia obstacle and improve the oxygen-dependent treatment efficiency of the PDT; mn produced by hydrolysis2+Can mediate Fenton-like reaction to generate virulent OH, so as to cause oxidative stress of tumor cells; MnO2Can consume the intracellular antioxidant GSH, reduce OH elimination and improve CDT efficiency; MTT experiment shows that the concentration of the up-conversion nano composite material is within 200 mug/mL, the cell survival rate can reach more than 90 percent, and UCNPs @ DPB-MnO2Little or no toxicity to non-cancerous cells; UCNPs @ DPB-MnO under 980 nm laser processing2+ the number of killer cells in the NIR treated group was the greatest; the up-conversion nano composite material has good cell compatibility and shows good treatment effect on tumor cells.
Specifically, the invention designs and constructs a near-infrared remote light-controlled up-conversion photodynamic therapy and tumor microenvironment triggerable chemical kinetics cooperative therapy platform for effectively treating hypoxic tumors. The strategy is as follows: firstly, amphiphilic copolymer DSPE-PEG is adopted2000The phospholipid coated up-conversion nanoparticles are realized by combining hydrophobic interaction with oleic acid ligands on the surface of UCNPs. Secondly, the photosensitizer HB is assembled through the compact hydrophobic layer, UCNPs which are efficient and stable in luminescence can convert 980 nm NIR light into 540nm green strongest fluorescence, and therefore the photosensitizer HB after red shift of an absorption spectrum is excited by 540nm green light to perform PDT. Meanwhile, MnO is grown in situ by coordination of O-Mn in PEG on the surface of UCNPs2Nanosheets, MnO2Can hydrolyze endogenous acidic H in the tumor microenvironment2O2Thereby realizing O2The self-sufficiency of the PDT can overcome the tumor hypoxia obstacle and improve the oxygen-dependent treatment efficiency of the PDT. Mn produced by hydrolysis2+Mediating Fenton-like reactionsThe generation of virulent OH causes the oxidative stress of tumor cells. MnO2Can also consume the intracellular antioxidant GSH, reduce OH clearance and improve CDT efficiency. The platform combines the tumor microenvironment response, PDT and CDT for cooperative treatment, improves the treatment efficiency and realizes the effective treatment of the hypoxic tumor.
The constructed near-infrared remote light-controlled up-conversion photodynamic therapy and tumor microenvironment triggerable chemical kinetics cooperative therapy platform is used for the effective treatment of the hypoxic tumor, and comprises the following steps:
1) NaYF is prepared according to a high-temperature cracking method4Yb, Er nanocrystals (UCNPs) and DSPE-PEG prepared by thin film hydration method2000Coated NaYF4Nano particles of Yb, Er nanocrystals.
2) Loading of photosensitizer HB: loading of photosensitizer HB: 1 mL each of UCNPs @ DSPE-PEG was added to a brown vial2000(abbreviated as UCNPs @ DP) and 10. mu.L of DMF solutions of different concentrations of HB (0, 0.5, 1, 5, 10, 20, 30, 40 mM) were stirred for 24 h in the absence of light.
3）MnO2Compounding: UCNPs @ DSPE-PEG2000@HB-MnO2(abbreviated as UCNPs @ DPB-MnO)2) And (4) preparing. Add 100. mu.L of UCNPs @ DPB in water to a 1.5 mL EP tube, add 320. mu.L of deionized water, then add 40. mu.L of MES solution (50 mM) and 40. mu.L of KMnO4Solutions (4 mM) were added to the tubes in sequence and sonicated at room temperature for 30 min. Finally, the precipitate was collected by centrifugation at 14500 rpm for 10 min, washed 3 times with deionized water, and dispersed in 100 μ L of deionized water for further use.
4) Testing of composite mediated Fenton-like reaction: mn2+Validation of mediated Fenton-like reaction generation OH: MB is a dye that is degraded by the. OH group and is selected as an indicator of the formation of the. OH group. First, to verify Mn2+The mediated Fenton-like reaction will contain 10. mu.g/mL MB, 8 mM H2O2And 0.5 mM MnCl 225 mM NaHCO3The solution was left at 37 ℃ for 30 min, monitored by the change in absorbance at 665 nm. OH scavenging by GSH: 25 mM NaHCO3Solution containing 10. mu.g/mL MB, 8 mM H2O2Different concentrations of GSH (0, 1, 5 and 10 mM) and 0.5 mM MnCl2Or UCNPs @ DPB-MnO2OH-induced MB degradation was monitored by absorbance change at 665 nm, standing at 37 ℃ for 30 min.
5) Cell survival rate by MTT assay: the survival rates of normal cells and tumor cells treated with CDT were first examined separately as control groups. Secondly, the survival rate of tumor cells after PDT and CDT combined treatment is detected, HeLa cells with good growth condition are adopted, and 100 mu L of the HeLa cells per well contain 1.0X 104Cell density of individual cells plated in 96-well plates at 37 ℃ with 5% CO2The cell culture box is used for culturing for 24 hours. UCNPs @ DPB and UCNPs @ DPB-MnO at different concentrations (0, 25, 50, 100, 200, 400 mug/mL) were added to the corresponding wells of the experimental group2Incubate for 1 h, wash 3 times with PBS. Then using 980 nm excitation light (2W/cm)2) Irradiating for 20 min. After that, the culture medium was replaced with fresh medium and cultured for another 24 hours, the culture solution was aspirated, PBS was washed 1 time, 100. mu.L of MTT (1 mg/mL) was added to each well, followed by incubation at 37 ℃ for 4 hours, the culture solution was carefully aspirated and 150. mu.L of DMSO was added, shaking was performed on a shaker at a low speed for 10 minutes, absorbance at 490 nm was measured by a microplate reader, and the cell survival rate was calculated.
6) Photodynamic/chemokinetic treatment of upconverting composite nanomaterials: BALB/C mice were cultured according to standard procedures and injected with appropriate amounts of 4T1 cells on their right hind limb dorsum after two weeks of observation. When tumors grew to a usable size, mice were randomly assigned to 5 groups for light experiments, respectively salene, NIR, HB, UCNPs @ DPB and UCNPs @ DPB + NIR, with 5 mice per group. Injecting 60 μ L of sample solution by in-situ injection, irradiating tumor part with 2W 980 nm exciter for 25min, stopping irradiation every 5min for 1min, and setting the vertical distance from fiber head to tumor part at 1.5 cm. The treatment period lasted 14 days and the body weight of the mice was recorded as well as the volume of solid tumors.
7) Histopathological analysis
H & E staining: mice were fixed with 4% paraformaldehyde after removal of tumors and major organs. The samples were then embedded, sectioned and stained. Histopathological changes were observed and evaluated under an inverted microscope.
Tunel experiment: tumors from mice were paraffin embedded, sectioned and stained, observed under a fluorescent microscope and evaluated for histopathological changes.
The invention successfully constructs a near-infrared remote light-controlled up-conversion photodynamic therapy and chemical kinetics cooperative therapy platform which can be triggered by a tumor microenvironment. The UCNPs with high efficiency and stable luminescence can convert 980 nm NIR light into 540nm green light, and the 540nm green light excites photosensitizer HB to carry out PDT process. Endogenous acidic H in the tumor microenvironment2O2Hydrolyzed MnO2Can realize O2The self-sufficiency of the PDT can overcome the tumor hypoxia obstacle and improve the oxygen-dependent treatment efficiency of the PDT. Mn produced by hydrolysis2+Can mediate Fenton-like reaction to generate virulent OH, and cause oxidative stress of tumor cells. MnO2Can also consume the intracellular antioxidant GSH, reduce OH clearance and improve CDT efficiency. The tumor cell targeting property (EPR) enables the tumor cells to have higher selectivity on the up-conversion composite material, avoids the damage to normal cells, and obviously reduces the side effect. The platform combines the tumor microenvironment response and the PDT/CDT cooperative treatment, improves the treatment efficiency and realizes the effective treatment of the hypoxic tumor.
The invention has the advantages that:
(1) HB as the second generation photosensitizer has great potential, has the advantages of strong phototoxicity, rapid metabolism, high quantum yield of singlet oxygen and the like, and can effectively generate active oxygen and kill cancer cells. The UCNPs can convert near infrared light into visible light or ultraviolet light, and HB and the UCNPs can be indirectly excited by the near infrared light in deep tumors after being combined, thereby showing obvious effect on increasing tissue penetration depth of light to resist the deep tumors. Overcomes the defects of poor biocompatibility, poor penetration depth and the like of other methods, improves the specificity and reduces the side effect;
(2) using amphiphilic copolymers DSPE-PEG2000By passingHydrophobic interaction is combined with oleic acid ligands on the surfaces of UCNPs to realize that phospholipid coats up-conversion nanoparticles, and photosensitizer HB is assembled through a compact hydrophobic layer, so that the absorption peak of HB is red-shifted by 105nm and is matched with the emission peak of Er-doped UCNPs at 540 nm. Fully utilizes the up-conversion nano material NaYF4: the strongest upconversion fluorescence of Yb/Er at 540nm greatly improves the PDT energy transfer efficiency.
(3) Endogenous acidic H in the tumor microenvironment2O2Hydrolyzed MnO2Can realize O2The self-sufficiency of the PDT can overcome the tumor hypoxia obstacle and improve the oxygen-dependent treatment efficiency of the PDT.
（4）MnO2Can also consume the intracellular antioxidant GSH, reduce OH clearance and improve CDT efficiency. The tumor cell targeting property (EPR) enables the tumor cells to have higher selectivity on the up-conversion composite material, avoids the damage to normal cells, and obviously reduces the side effect.
Drawings
FIG. 1 is a standard curve diagram of the concentration of HB versus its fluorescence intensity, in which A is the fluorescence spectrum of different concentrations of HB dissolved in DMF solution; b is a standard graph of the concentration of HB versus its fluorescence intensity.
FIG. 2 is a UV-VIS absorption spectrum of UCNPs, an upconversion emission spectrum and UV-VIS absorption spectra before and after loading of HB on UCNPs @ DP.
Fig. 3 is a comparison graph of the experiments, in which: (A) UV pattern of HB; (B) a UV map of UCNPs @ DP; (C-G) UV plots of UCNPs @ DPB at different concentrations; (H) DPBF and different materials show the time course of their absorbance at 415 nm under 980 nm excitation light.
FIG. 4 shows UCNPs @ DP-MnO2And 100 mu M H2O2Graph of in vitro oxygen production over time after reaction in acidic solution (pH = 5.5) (blue line).
FIG. 5 is a graph of UCNPs @ DP-MnO treatment with GSH at various concentrations2Post-spectrum, in which: (A) is an absorption spectrum; (B) is an up-conversion emission spectrogram.
FIG. 6 is a graph depicting the uptake of the upconversion nanocomposite mediated Fenton-like reactionSpectrogram, in the figure: (A) respectively with H2O2、Mn2+、H2O2+Mn2+、H2O2+Mn2++HCO3 -Absorption spectra after MB treatment (inset: sample photo); (B) at different concentrations (1, 2, 4, 8, 10 mM) of H2O2Absorption spectra after treatment of MB (inset: sample photo), control group: MB, experimental group: MB + Mn2++HCO3 -+H2O2(ii) a (C) Absorption spectra after glutathione treatment of MB at various concentrations (1, 5, 10 mM) (inset: sample photograph) for control group: MB, experimental group: MB + Mn2++HCO3 -+H2O2+ GSH; (D) absorption spectra after glutathione treatment of MB at various concentrations (1, 5, 10 mM) (inset: sample photograph), control: MB, experimental group: MB + UCNPs @ DP-MnO2+HCO3 -+H2O2+GSH。
FIG. 7 shows the cell viability of HeLa cells incubated with different concentrations of material under different conditions.
FIG. 8 is fluorescence imaging of HeLa cells stained with PI after treatment in 5 different ways.
Fig. 9 is a schematic representation of a platform for photodynamic/chemokinetic treatment based on upconverting nanocomposites, wherein: a is an up-conversion nano composite material synthesis scheme; b is an anti-tumor mechanism diagram of the up-conversion nano composite material.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples.
Example 1:
loading of photosensitizer HB
1) Thermal decomposition method for preparing NaYF4Yb, Er nanocrystals (see patent application No. CN201810591016.2 for a specific preparation method, example 2): 0.8 mmol of rare earth stearate and 28 mmol of NaF are weighed into a 100 mL three-necked flask, and 12 mL of OA (oleic acid) and 8 mL of ODE (octadecene) are added; heating the system to 140 ℃ in argon atmosphere, keeping for 30 min, and dehydrating and degassing; then quickly heating toMaintaining the reaction at 312-314 ℃ for 45 min, cooling to room temperature, centrifuging the obtained product at 11000 rpm for 3 min, removing supernatant, washing the precipitate with ethanol, cyclohexane and distilled water, centrifuging until no organic oily matter and NaF component exist, and drying in vacuum at 60 ℃ to obtain OA-UCNPs for later use;
2）DSPE-PEG2000coating UCNPs: 10mg of OA-UCNPs (NaYF for short) prepared in the step 1) are taken4Yb and Er) are dispersed in chloroform and fully ultrasonically treated to ensure that the Yb and the Er are uniformly dispersed; taking 12.5mg of DSPE-PEG2000(distearoylphosphatidylethanolamine-polyethylene glycol 2000 available from Shanghai Yanyi Biotech Co., Ltd.) was dissolved in chloroform, and dispersed OA-UCNPs (NaYF)4Yb, Er) is dripped into the solution, and the solution is stirred in a light-proof glass vial until the chloroform is naturally volatilized; adding ultrapure water, performing intense ultrasonic treatment for 10 min, placing in a water bath kettle at 80 ℃, and stirring vigorously; centrifuging, removing supernatant, adding ultrapure water, washing, centrifuging, removing supernatant, and freeze drying to obtain UCNPs @ DSPE-PEG2000(UCNPs @ DP for short), dispersed with deionized water to 2.5mg/mL for use.
3) Loading of photosensitizer Hypocrellin B (HB): the brown vials were each charged with 1 mL of 2.5mg/mL of UCNPs @ DP obtained in step 2) (UCNPs @ DSPE-PEG)2000) The DMF solution of (5) and 10. mu.L of DMF solution of HB of different concentrations (0, 0.5, 1, 5, 10, 20, 30, 40 mM) were stirred for 24 h in the dark to obtain UCNPs @ DSPE-PEG2000@ HB (UCNPs @ DPB for short).
3) Determination of HB loading and stability investigation: DMF solutions (5, 10, 20, 30, 40 and 50 mu M) with different concentrations of HB are prepared, an emission spectrum of the HB at 550-800 nm can be measured under the excitation of 470 nm, the maximum absorbance at 620nm is determined, the fluorescence intensity at 620nm is measured, and a standard curve of the fluorescence intensity-concentration is drawn, as shown in FIG. 1. Solutions of UCNPs (UCNPs @ DPB) of each assembled HB were centrifuged, unassembled HB in supernatant DMF, the supernatant was aspirated, the fluorescence intensity at 620nm was measured, and the concentration of free HB in the supernatant was calculated according to a standard curve.
Examination of the stability of HB-loaded phospholipid layer, the complex was left to stand for 24 h, the supernatant was centrifuged and the absorbance spectrum was measured. As shown in FIG. 2, the supernatant after centrifugation of the complex dispersed in deionized water, PBS, ethanol, chloroform, DMEM, RPMI-1640 was subjected to absorption spectroscopy, and only a small leak occurred in ethanol, since ethanol destroyed the phospholipid structure, while no leak of HB was found in other solutions. Therefore, the phospholipid layer has stable property, and can be well loaded with photosensitizer for photodynamic therapy of tumor.
4) Testing of singlet oxygen in vitro: at the same excitation light power density of 980 nm (2W/cm)2) And HB, UCNPs @ DP, and various concentrations of UCNPs @ DPB (25, 50, 100, 200, 400. mu.g/mL) were tested under the test conditions, respectively. As shown in FIG. 3, it can be seen from the attenuation of 1, 3-Diphenylisobenzofuran (DPBF) as a chemical probe that HB and UCNPs @ DP were not irradiated with 980 nm laser alone, respectively, as a control1O2And (3) the product is obtained. After loading the photosensitizer HB, the degree of DPBF fading gradually increases with increasing concentration of the complex and with increasing irradiation time.
Example 2
UCNPs@DPB -MnO2Preparation of composite materials
1）UCNPs@DPB-MnO2（UCNPs@DSPE-PEG2000@HB-MnO2) The preparation of (1): mu.L of 2.5mg/mL aqueous UCNPs @ DPB solution was added to a 1.5 mL EP tube, 320. mu.L deionized water was added, and then 40. mu.L of 2-morpholinoethanesulfonic acid (MES) solution (50 mM) and 40. mu.L of KMnO4Solutions (4 mM) were added to the tubes in sequence and sonicated at room temperature for 30 min. Finally, the precipitate was collected by centrifugation at 14500 rpm for 10 min, washed 3 times with deionized water, and dispersed in 100. mu.L of deionized water for further use.
2) Characterization of the self-oxygen supply capacity of the up-converted nanocomposites: due to the existence of one acidity in solid tumor and the enrichment of H2O2And a microenvironment of2The nano-sheet has pH/oxidation reduction reaction characteristics, and can be substituted by H under the acidic condition in the tumor microenvironment2O2Reduction to Mn2+And generates a large amount of oxygen, and can obviously improve the oxygen-dependent PDT on hypoxic tumorsHas good therapeutic effect. To directly verify that the nanocomposite catalyzes H2O2The ability to convert to dissolved oxygen. The complexes were measured at 100. mu. M H2O2The neutral (pH = 7.4)/acidic (pH = 5.5) solution of (a) produced oxygen, and as a result, as shown in fig. 4, more dissolved oxygen was produced under the weak acidic condition, and oxygen production was fast and continuous, and conformed to the acidic condition in the tumor microenvironment.
3) Characterization of the up-conversion nanocomposite-mediated Fenton-like reaction: the solid tumor microenvironment has obvious GSH and H2O2、H+High content. Thus, MnO in up-conversion nanocomposites2The nano sheet can be associated with H in a tumor microenvironment+GSH and H2O2Reaction, then release of Mn2+And mediates the Fenton-like reaction to generate virulent OH, thereby killing tumor cells.
In order to verify whether the Glutathione (GSH) with different concentrations can perform oxidation-reduction reaction on the compound, the absorption spectrum and the UCL emission spectrum of the compound treated by the GSH with different concentrations are experimentally determined. As shown in a in fig. 5, the absorption value decreases as the GSH concentration increases; also, as the concentration of GSH increases, UCL gradually recovers, as shown by B in the figure. Description of MnO2Is easily degraded into Mn in the environment2+。
Mn2+The mediated Fenton-like response effects the CDT of tumor cells by the production of OH. To verify the formation of OH, in bicarbonate (HCO)3 -) Using a Methylene Blue (MB) dye that is degradable by the. OH as an indicator of the formation of the. OH, as shown in a of fig. 6.
With H2O2The increase in concentration, decrease in absorbance of MB, indicates the decrease in the concentration of Mn2+OH is H dependent2O2Concentration, as shown by B in fig. 6. Notably, intracellular GSH acts as a scavenger of OH, limiting the therapeutic efficacy of CDT. Mn after GSH addition, as shown by C in FIG. 62+The mediated Fenton-like reaction has very limited degradation of MB. In contrast, due to the depletion characteristics of GSH(D in FIG. 6), MnO2This scavenging effect can be effectively eliminated. Thus, with Mn alone2+In contrast, MnO in the presence of GSH2The generation of OH can be obviously improved.
Example 3
Construction of photodynamic/chemokinetic treatment platform based on up-conversion composite material
1) MTT detection PDT and CDT combined treatment effect on tumor cells: to investigate the therapeutic effect of PDT in combination with CDT on tumor cells, UCNPs @ DPB and UCNPs @ DPB-MnO were compared under 980 nm laser irradiation2The anticancer effect of (1). As shown in FIG. 7, the cell survival rate of the NIR treated group is close to 95%, which indicates that 980 nm laser has no adverse effect on HeLa cells, the cancer cells incubated with UCNPs @ DPB are obviously inhibited in each concentration range under 980 nm laser irradiation, which indicates that PDT plays a role in killing HeLa cells, and compared with UCNPs @ DPB, UCNPs @ DPB-MnO2HeLa cells were more inhibited and cell death by fluorescence microscopy is shown in FIG. 8 due to the coated MnO2Degradation takes place, on the one hand, O is produced2Improving the oxygen-dependent treatment efficiency of PDT; on the other hand, Mn is produced2+The Fenton-like reaction is mediated, toxic OH is generated, and oxidative stress of tumor cells is caused. Therefore, combining PDT with CDT is more beneficial for tumor treatment, as shown schematically in FIG. 9.
2) Photodynamic/chemokinetic treatment of upconverting composite nanomaterials: BALB/C mice were cultured according to standard procedures and injected with appropriate amounts of 4T1 cells on their right hind limb dorsum after two weeks of observation. When tumors grew to a usable size, mice were randomly assigned to 5 groups for light experiments, respectively salene, NIR, HB, UCNPs @ DPB and UCNPs @ DPB + NIR, with 5 mice per group. Injecting 60 μ L of sample solution by in-situ injection, irradiating tumor part with 2W 980 nm exciter for 25min, stopping irradiation every 5min for 1min, and setting the vertical distance from fiber head to tumor part at 1.5 cm. Treatment time the body weight of the mice and the volume of solid tumors were continuously recorded.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims (4)

1. An up-conversion nanocomposite material capable of serving as a tumor microenvironment responsive (TME) nanotherapeutic platform, comprising: the device is an ideal design integrating tumor microenvironment response, photodynamic therapy and chemodynamic therapy; the multifunctional nano platform adopts amphiphilic copolymer DSPE-PEG2000The method is characterized in that hydrophobic interaction is combined with oleic acid ligands on the surfaces of UCNPs to realize that phospholipid wraps up conversion nanoparticles, a photosensitizer HB is assembled through a compact hydrophobic layer, and finally MnO grows in situ through O-Mn coordination on PEG on the surfaces of the UCNPs2The nano-sheet can fully play the role of each component in the up-conversion nano-composite material, and realize the effective treatment of tumors.
2. A method of preparing the up-conversion nanocomposite as claimed in claim 1 as a platform for tumor therapy, characterized in that: the method comprises the following steps:
1) NaYF is prepared according to a thermal decomposition method4Yb, Er nanocrystals (UCNPs) and DSPE-PEG prepared by thin film hydration method2000Coated NaYF4Nano particles UCNPs @ DSPE-PEG of Yb, Er nano crystal2000（UCNPs@DP）；
2) Loading of photosensitizer HB: each of the brown vials was charged with 1 mL of UCNPs @ DSPE-PEG synthesized in step 12000(UCNPs @ DP) and 10. mu.L of DMF solutions of different concentrations of HB selected from 0, 0.5, 1, 5, 10, 20, 30, 40 mM were stirred for 24 h in the dark to obtain UCNPs @ DSPE-PEG2000@HB（UCNPs@DPB）；
3）UCNPs@DSPE-PEG2000@HB-MnO2（UCNPs@DPB-MnO2) The preparation of (1): mu.L of the UCNPs @ DPB aqueous solution obtained in step 2) was added to a 1.5 mL EP tube, 320. mu.L of deionized water was added, and then 40. mu.L of a 50 mM MES solution and 40. mu.L of 4 mM KMnO were added4The solutions were added sequentially to the tubes and allowed to super-incubate at room temperatureSounding for 30 min; finally, the precipitate was collected by centrifugation at 14500 rpm for 10 min, washed 3 times with deionized water, and dispersed in 100. mu.L of deionized water to obtain UCNPs @ DSPE-PEG2000@HB-MnO2（UCNPs@DPB-MnO2) For further use;
4) the killing effect of the up-conversion nano composite material on tumor cells: near infrared spectroscopy experiments show that 980 nm laser has no adverse effect on HeLa cells, and cancer cells incubated with UCNPs @ DPB are inhibited in various concentration ranges under 980 nm laser irradiation, which indicates that PDT plays a role in killing HeLa cells, compared with UCNPs @ DPB, UCNPs @ DPB-MnO2Has more obvious inhibiting effect on HeLa cells due to the coated MnO2Degradation takes place, on the one hand, O is produced2Improving the oxygen-dependent treatment efficiency of PDT; on the other hand, Mn is produced2+And mediates the Fenton-like reaction to generate virulent OH, so that the oxidative stress of the tumor cells is caused, and the effective treatment of the tumor cells is realized.
3. The method of claim 2, wherein: up-converting nanocomposites (UCNPs @ DPB-MnO)2) Preparing;
1) determination of photosensitizer HB loading and stability investigation: loading HB with different concentrations by using UCNPs @ DP, and keeping the color unchanged and basically achieving saturation when the concentration reaches 200 mu M; further examining the loading capacity, in the DMF solution, when the concentration of HB is 200 μ M, the loading capacity is saturated to 15 μ g/mg; placing the compound for 24 h, centrifuging to obtain supernatant, and testing absorption spectrum; the supernatant after the compound is dispersed in deionized water, PBS, ethanol, chloroform, DMEM and RPMI-1640 for centrifugation is subjected to absorption spectrum measurement, and only a small amount of leakage occurs in the ethanol, because the ethanol can destroy the phospholipid structure, and no leakage of HB is found in other solutions; HB loaded in the phospholipid layer can be completely extracted by using dichloromethane; the phospholipid layer has stable property, and can well load photosensitizer for photodynamic therapy of tumor;
2) testing of singlet oxygen in vitro: by using chemistryGenerated by probe 1, 3-diphenyl isobenzofuran (DPBF) on up-conversion nano composite material under 980 nm laser irradiation1O2Carrying out detection; at 980 nm, the laser irradiation of HB and UCNPs @ DP respectively alone is absent1O2After the photosensitizer HB is generated and loaded, the attenuation degree of DPBF is gradually increased along with the increase of the concentration of the compound and the extension of the irradiation time, so that the photosensitizer HB can be efficiently generated1O2The compound lays a foundation for improving the next photodynamic therapy effect;
3) testing of up-conversion nanocomposite-mediated Fenton-like reaction: to verify Mn2+The mediated Fenton-like reaction will contain 10. mu.g/mL MB, 8 mM H2O2And 0.5 mM MnCl225 mM NaHCO3The solution was allowed to stand at 37 ℃ for 30 min, monitored by the change in absorbance at 665 nm for OH-induced MB degradation;
4) OH scavenging by GSH: 25 mM NaHCO3Solution containing 10. mu.g/mL MB, 8 mM H2O2GSH at various concentrations selected from 0, 1, 5 and 10 mM and 0.5 mM MnCl2Or UCNPs @ DPB-MnO2OH-induced MB degradation was monitored by absorbance change at 665 nm, standing at 37 ℃ for 30 min.
4. The upconverting nanocomposite capable of acting as a tumor microenvironment responsive (TME) nanotherapeutic platform of claim 1 or made by the method of claim 2 or 3, wherein: using amphiphilic copolymers DSPE-PEG2000The phospholipid is coated with the up-conversion nanoparticles by combining hydrophobic interaction with oleic acid ligands on the surface of UCNPs; HB is used as a photosensitizer, UCNPs can convert near infrared light into 540nm green light, and the green light is used as a mediated carrier in PDT therapy, so that the photosensitizer HB is excited to carry out PDT; finally, in-situ growth of MnO on PEG on the surface of UCNPs through O-Mn coordination2Nanosheets; endogenous acidic H in the tumor microenvironment2O2Hydrolyzed MnO2Can realize O2The self-sufficiency of the PDT can overcome the tumor hypoxia obstacle and improve the oxygen-dependent treatment efficiency of the PDT; hydrolysis to produceMn of (2)2+Can mediate Fenton-like reaction to generate virulent OH, so as to cause oxidative stress of tumor cells; MnO2Can consume the intracellular antioxidant GSH, reduce OH elimination and improve CDT efficiency; MTT experiment shows that the concentration of the up-conversion nano composite material is within 200 mug/mL, the cell survival rate can reach more than 90 percent, and UCNPs @ DPB-MnO2Little or no toxicity to non-cancerous cells; UCNPs @ DPB-MnO under 980 nm laser processing2+ the number of killer cells in the NIR treated group was the greatest; the up-conversion nano composite material has good cell compatibility and shows good treatment effect on tumor cells.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115558488A (en) * 2022-10-14 2023-01-03 赣南师范大学 Rare earth composite nano material, preparation method and application thereof, and up-conversion detection method of cysteine

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