CN111821436A - Targeted penetrating type nano diagnosis and treatment compound for in-situ tumor oxygen generation and sensitization photodynamic curative effect and construction method thereof - Google Patents

Targeted penetrating type nano diagnosis and treatment compound for in-situ tumor oxygen generation and sensitization photodynamic curative effect and construction method thereof Download PDF

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CN111821436A
CN111821436A CN202010771037.XA CN202010771037A CN111821436A CN 111821436 A CN111821436 A CN 111821436A CN 202010771037 A CN202010771037 A CN 202010771037A CN 111821436 A CN111821436 A CN 111821436A
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tumor
oxygen
targeted
tlyp
treatment compound
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吴春惠
张映雪
谢正鑫
王易坤
刘贻尧
杨红
李亭亭
秦翔
李顺
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University of Electronic Science and Technology of China
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Abstract

The invention provides a targeting penetrating type nano diagnosis and treatment compound for tumor in-situ oxygen generation and sensitization photodynamic curative effect and a preparation method thereof, wherein the nano diagnosis and treatment compound comprises three parts: based on graphene oxideUnderlying GO-MnO2The tumor targeting homing peptide comprises a composite oxygen-producing carrier, a tumor targeting homing penetrating peptide connected with the carrier, and a photosensitizer carried on the carrier. The nano diagnosis and treatment compound has the functions of tumor targeted deep penetration, tumor microenvironment responsive in-situ oxygen production and MRI/fluorescence imaging, and can greatly improve the curative effect of photodynamic therapy.

Description

Targeted penetrating type nano diagnosis and treatment compound for in-situ tumor oxygen generation and sensitization photodynamic curative effect and construction method thereof
Technical Field
The invention belongs to the technical field of medical materials, and particularly relates to a targeted penetrating type nano diagnosis and treatment compound for tumor in-situ oxygen generation and sensitization photodynamic curative effect and a preparation method thereof.
Background
Photodynamic therapy (PDT) is a novel noninvasive tumor therapy, and the basic principle is that a photosensitizer accumulated in target cells or tissues is excited by laser with a proper wavelength to perform photochemical reaction, so that the photosensitizer reacts with oxygen molecules to form Reactive Oxygen Species (ROS), thereby generating cytotoxicity to kill tumor cells and achieving the purpose of resisting tumors. Compared with other methods such as chemotherapy, the photodynamic therapy has the advantages of lower toxic and side effects, higher selectivity, minimally invasive high efficiency and the like. Since the 70's of the 20 th century, in clinical applications, PDT has been extensively studied for the treatment of various types of solid tumors and other diseases.
However, further clinical applications of PDT are still limited by a number of troublesome problems. From the principle of the common PD tii type photochemical reaction, it is known that a photosensitizer, oxygen concentration and laser are the most important three elements thereof. Oxygen is one of the most important factors determining ROS production, however, as PDT proceeds, the concentration of oxygen is gradually consumed, eventually inhibiting ROS production instead. In addition to this self-limiting factor, it is well known that the microenvironment inside solid tumors is in a hypoxic state; in most tumors, the percent hypoxia increased from 10% to 30%, with oxygen pressures below 15mm Hg. Therefore, tumor cells regulate a series of tumor growth and metabolic behaviors through hypoxia response regulatory factor (HIF-1), such as promoting expression of glucose transporter (GLUT), promoting glycolysis, and down-regulating the rate of mitochondrial oxidative phosphorylation. Most importantly, hypoxia greatly limits the yield of cytotoxic ROS in PDT, so that cells with strong invasiveness of tumor hypoxia centers cannot be effectively killed, treatment resistance is caused, and tumor drug resistance is caused. In response to this problem, various strategies for alleviating tumor hypoxia have been proposed, such as hyperbaric oxygen injection, microbubble-carried oxygen, reaction of metal with hydrogen peroxide to generate oxygen in situ, perfluorocarbon or hemoglobin-carried oxygen delivery, etc. However, these methods still cannot effectively solve the problem that the PDT treatment effect of tumor is limited by tumor hypoxia, and some methods have the problems of low oxygen carrying efficiency, complex operation, and possibly causing other biological toxicity, so that there is still an urgent need to develop a novel technology to solve this problem.
Currently, MnO2Due to the unique chemical reaction activity, degradability, high biocompatibility and other excellent properties, the nano material has attracted great interest of researchers. (1) Nano MnO2And H2O2Reacting, in particular, with endogenous H in intratumoral acidic environments2O2Reaction, consumption of H+And oxygen is released, in-situ oxygenation in the tumor is directly realized, and the anaerobic state is improved. The results of the study revealed that MnO2NPs are introduced into the nano-drugs, so that the curative effects of radiotherapy, chemotherapy, PDT and the like are greatly improved. (2) Nano MnO2And H2O2Self-decomposed to Mn during the reaction2+，Mn2+Is easily and rapidly discharged by organism, thereby avoiding nano MnO2Aggregation in vivo results in toxicity, and excellent biocompatibility is shown. More importantly, Mn2+It has 5 spin-parallel unpaired electrons and is an excellent Magnetic Resonance Imaging (MRI) contrast agent. (3) Nano MnO2Can also react with glutathione with higher concentration in tumor cells, and can improve the oxidation reduction stress state in tumors. Thus, nano MnO2The MRI probe is an excellent multifunctional material which responds to multiple biochemical characteristics of a tumor microenvironment and specifically generates oxygen in tumor tissues and releases the MRI imaging probe. In various MnO2In the nanomaterial, MnO2NPs have the obvious advantages of simple and rapid synthesis, cheap raw materials, large specific surface area, controllable size, easy compounding with other nano materials and the like, but MnO with smaller particle size2NPs have low drug loading efficiency of photosensitizers, are particularly difficult to compound with hydrophobic photosensitizers, and still need other carrier materials.
On the other hand, the hypoxic state inside the tumor often increases from the tumor margin to the interior of the tumor, solving the problem of tumor hypoxia, and at the same time, the problem of how to deliver the photosensitizer and the oxygen-generating carrier to the hypoxic center of the tumor needs to be considered. Although existing research suggests that nano-drugs can enhance the permeability and retention effect (namely EPR effect) of tumor tissues, photosensitizers (>100nm) are passively targeted to be delivered to tumors; however, due to pathological obstacles such as disordered vascular systems inside the tumor, high interstitial pressure, dense extracellular matrix and the like, the nano photosensitive drug is only gathered around the tumor vessels, is difficult to effectively diffuse in tissues inside the tumor, and cannot reach hypoxic regions, so that the curative effect of the nano photosensitive drug is greatly limited. At present, in order to increase the tumor tissue permeability of nano-drugs, strategies such as modifying tumor homing penetrating peptide on the surface of a nano-carrier or utilizing responsive molecules (such as matrix metalloproteinase and weak acidity) in tumor microenvironment to enable the size of nanoparticles to be disintegrated or gradually reduced in tumor are gradually reported. However, these methods do not simultaneously achieve the problem of improving hypoxia inside the tumor.
Therefore, considering the complicated microenvironment inside the tumor and the gradual deepening of the tumor deep hypoxia, how to improve the PDT therapeutic effect of the tumor from the combined angle of the relaxation of the tumor internal hypoxia and the targeted permeation inside the tumor becomes a problem to be solved by those skilled in the art. Therefore, the construction of the nano-carrier-based targeted penetration type nano diagnosis and treatment system with the tumor in-situ oxygen generation and sensitization photodynamic curative effect has important significance for tumor treatment.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a targeted penetration type nano diagnosis and treatment compound with tumor in-situ oxygen generation and sensitization photodynamic curative effects and a preparation method thereof.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a targeting penetrating nano diagnosis and treatment compound for in-situ generating oxygen and sensitizing photodynamic therapy effect of tumor comprises GO-MnO with graphene oxide as a substrate2The tumor targeting homing penetration peptide is connected with the composite oxygen-generating carrier in a chemical covalent connection mode, and the photosensitizer is connected with the composite oxygen-generating carrier in a pi-pi accumulation effect, a hydrophobic effect, a hydrogen bond or an electrostatic effect;
wherein, GO-MnO taking graphene oxide as substrate2The composite oxygen-generating carrier is formed by in-situ growth of MnO on the surface of graphene oxide through modification of aminated cations2And (4) preparing the nano particles.
Further, the tumor targeting homing penetrating peptide is LyP-1, F3, iRGD or tLyP-1, preferably tLyP-1.
Further, the photosensitizer is an organic fluorescent dye capable of generating singlet oxygen and capable of being excited by near infrared light (wavelength of 620-1100 nm), specifically chlorins, porphyrins, phthalocyanines, rhodamines, methylene blues, cyanines (such as ICG, IR820 and IR825) and the like, and preferably chlorins (such as Ce 6).
The preparation method of the tumor in-situ oxygen generation sensitization photodynamic therapy targeted penetrating type nano diagnosis and treatment compound comprises the following steps:
(1) synthesis of GO-MnO2Composite oxygen-generating carrier
Carrying out self-assembly or amide reaction on GO and an aminated polycation polymer to prepare a GO-polycation compound, adding a potassium permanganate solution into the GO-polycation compound, and reacting at room temperature for 12-36h to prepare the GO-polycation compound; the mass ratio of polycationic polymer to GO is 0.25: 1-2: 1; the mass ratio of GO to potassium permanganate is 1:8-12, preferably 1: 8.4;
(2) preparation of targeting penetrating oxygen-producing nano composite carrier
Synthesizing FITC-labeled thiolated tumor targeting homing penetrating peptide, and then adding GO-MnO2Mixing the composite oxygen-producing carrier, maleimide polyethylene glycol succinimide acetate and FITC marked sulfhydrylation tumor targeted homing penetrating peptide according to the weight ratio of 1:0.1-1:0.1-1, and reacting at room temperature for 20-28h to obtain the product;
(3) preparation of targeting penetrating nano diagnosis and treatment compound
The photosensitizer and the targeted penetrating oxygen-producing nano composite carrier are self-assembled to prepare the targeted penetrating nano diagnosis and treatment composite.
Further, the aminated polycationic polymer is poly (allylamine hydrochloride) (PAH), polylysine or polyethyleneimine, preferably PAH, with a molecular weight in the range of 15-50.0kDa, preferably 15000, in daltons.
Further, the mass ratio of the aminated polycationic polymer to GO is 1: 1.
Further, the reaction concentration of potassium permanganate is 10-20mM, the reaction concentration of potassium permanganate is preferably 13.3mM, and the reaction time is preferably 24 h.
Further, GO-MnO2The weight ratio of the composite oxygen-generating carrier, the maleimide polyethylene glycol succinimide acetate and the FITC-labeled thiolated tLyP-1 is 1:0.18: 0.18.
Further, the mass ratio of the photosensitizer to the targeted penetration type oxygen production nano composite carrier is 0.1-4: 1.
further, the mass ratio of the photosensitizer to the targeted penetration type oxygen production nano composite carrier is 1: 1.
The targeted penetrating type nano diagnosis and treatment compound for the in-situ oxygen generation and sensitization photodynamic curative effect of the tumor and the preparation method thereof provided by the invention have the following beneficial effects:
the targeted penetrating type nano diagnosis and treatment compound for the in-situ oxygen generation and sensitization photodynamic curative effect of the tumor mainly comprises three parts: GO-MnO with graphene oxide as substrate2The tumor targeting homing peptide comprises a composite oxygen-producing carrier, a tumor targeting homing penetrating peptide connected with the carrier, and a photosensitizer carried on the carrier. GO-MnO with graphene oxide as substrate2The particle size of the graphene oxide in the composite oxygen-generating carrier is 150-500 nm, the surface of the graphene oxide is provided with carboxylic acid residues, hydroxyl and epoxy groups, and the graphene oxide can be functionalized by chemical modification, namely small-size MnO can be grown on the surface of the graphene oxide in situ2The nano particles, and the two-dimensional plane structure and the aromatic structure of the nano particles can carry various medicines, such as photosensitizer, through pi-pi accumulation effect, hydrophobic force effect, hydrogen bond, electrostatic effect and other modes. The tumor targeting homing penetration peptide is a small molecule peptide with unique tumor tissue targeting and penetration capacity, the action mechanism of the tumor targeting homing penetration peptide is mainly a C-end Rule (C-end Rule, CendR) approach, namely, the free C end of the homing penetration peptide must contain (R/K) XX (R/K) motifs which are hidden in a peptide segment; when bound to Receptors (such as integrins) on the surface of tumor cells, the peptides are proteolytically cleaved to expose the cryptic CendR motif, which in turn specifically binds to the b1 domain of Neuropilin Receptors (NRPs) on the surface of target cells, triggering the cells to undergo a specific endocytosis/transcellular transport, transporting the "cargo" carried by the homing peptides deep into the tumor tissue. The photosensitizer is an organic fluorescent dye which can be excited by near infrared light (with the wavelength of 620-1100 nm) and can generate singlet oxygen, and when the photosensitizer is combined on the nano diagnosis and treatment compound, the photosensitizer can be excited to participate in oxygen reaction to generate cytotoxic singlet oxygen. In conclusion, the plane structure and the large specific surface area of GO are utilized, and MnO is grown on the surface of GO in situ through modification of amination cations2The nanoparticle is simultaneously modified with a targeting homing penetrating peptide, and the targeting homing penetrating peptide can be specifically combined with a b1 structural domain of Neuropilin Receptors (NRPs) on the cell surface to trigger cells to generate a special endocytosis/transcellular transport function so as to penetrate into the interior of a three-dimensional ellipsoid and further targetTumor hypoxic center, tumor microenvironment and intracellular H thereof2O2And MnO with MnO2The nano particles react to generate oxygen, so that an anoxic microenvironment deep in the tumor is relieved; then GO through GO-MnO2The pi-pi conjugation and the hydrophobic effect carry the photosensitizer efficiently, the photosensitive compound drug is penetrated through the depth of the tumor in a targeted way, and the photosensitizer is excited to react with oxygen to generate cytotoxic singlet oxygen, so that the hypoxia state in the depth of the tumor can be relieved better, and the curative effect of photodynamic therapy is improved greatly.
The nano composite prepared by the invention also has the functions of tumor microenvironment responsive in-situ oxygen generation and MRI imaging, and specifically comprises the following steps: the nano-composite of the invention utilizes H of the tumor microenvironment2O2Can be in situ with MnO2The nano particles react to generate oxygen and release Mn simultaneously2+The improvement of the anoxic state in the deep part of the tumor can be realized, so that the photodynamic therapy effect is improved, the dosage is reduced, and the toxic and side effects are obviously reduced; at the same time, Mn is used2+Is T1An MRI imaging probe for MRI imaging to detect the targeted accumulation of the nano diagnosis and treatment system in the tumor; meanwhile, the photosensitizer can also be used as a fluorescent probe to carry out fluorescence imaging, the distribution and metabolism conditions of the nano diagnosis and treatment system in each tissue and tumor of a mouse are detected, the photodynamic therapy guided by magnetic resonance/fluorescence imaging can be realized, and the photodynamic therapy efficiency of the tumor is greatly improved. In addition, MnO may be avoided2The nanoparticles aggregate in vivo to generate toxicity, and show excellent biocompatibility.
Based on the advantages, the nano-composite prepared by the invention can be used for treating various solid tumors including but not limited to skin cancer, malignant melanoma, nasopharyngeal carcinoma, esophageal cancer, gastric cancer, liver cancer, breast cancer, laryngeal cancer, thyroid cancer, tongue cancer, prostatic cancer, penile cancer, testicular tumor, vaginal malignant tumor and vulval malignant tumor appearing on the surface or inside of organs. The treatment can be carried out by intratumoral direct administration or intravenous administration.
Drawings
FIG. 1 is GO-MnO2Ultraviolet of oxygen generating composite carrierVisible absorption spectrum and Zeta potential results.
FIG. 2 is GO-MnO2And (5) transmission electron microscope characterization results of the oxygen-generating composite carrier.
FIG. 3 shows Zeta potential, violet-visible absorption spectrum, fluorescence spectrum and drug loading and encapsulation efficiency of tLyP-1 of GM @ tLyP-1 vector.
FIG. 4 is a schematic structural diagram of GM @ tLyP-1/Ce 6.
FIG. 5 is a graph showing UV-visible absorption spectrum, fluorescence spectrum, Zeta potential and hydrated particle size of GM @ tLyP-1/Ce 6.
FIG. 6 shows the oxygen generating performance and the active oxygen performance characterization results of GM @ tLyP-1/Ce 6.
FIG. 7 is in vitro magnetic resonance imaging T of GM @ tLyP-1/Ce61And (5) imaging performance characterization results.
FIG. 8 shows the results of in vitro cytotoxicity, targeted endocytosis and penetration assay of GM @ tLyP-1/Ce 6.
FIG. 9 shows the in vitro targeting oxygen production enhancing photodynamic anti-cancer effect of GM @ tLyP-1/Ce 6.
FIG. 10 is the results of improved tumor hypoxia in GM @ tLyP-1/Ce 6.
FIG. 11 shows the results of in vivo MRI/FL bimodal imaging of GM @ tLyP-1/Ce 6.
FIG. 12 is the results of GM @ tLyP-1/Ce6 enhancing ROS in vivo.
FIG. 13 shows the in vivo oxygen production enhanced photodynamic anti-tumor results of GM @ tLyP-1/Ce 6.
Detailed Description
Example 1
A targeting penetrating nano diagnosis and treatment compound for in-situ generating oxygen and sensitizing photodynamic therapy effect of tumor comprises GO-MnO with graphene oxide as a substrate2A composite oxygen-generating type carrier is prepared,
tumor targeting homing penetrating peptide connected with the composite oxygen-generating carrier in a chemical covalent connection mode, and photosensitizer connected with the composite oxygen-generating carrier in a pi-pi accumulation effect, a hydrophobic effect, a hydrogen bond or an electrostatic effect, and the preparation process comprises the following steps:
first step, synthesis of GO-MnO2Composite oxygen-generating carrier
1mL of poly(allylamine hydrochloride) (PAH) solution (with the concentration of 1mg/mL) is added with 2mL of graphene oxide solution (with the concentration of 0.5mg/mL) dropwise under the condition of magnetic stirring, and the reaction is carried out overnight to obtain GO-PAH compound; adding 1mL of potassium permanganate solution (8.4mg/mL) into the GO-PAH compound dropwise, stirring at room temperature overnight, centrifuging the solution at high speed (15000rpm,15min), discarding the supernatant, washing the precipitate with deionized water for three times, adding 0.1mL of deionized water to constant volume to obtain GO-MnO2An oxygen-generating composite carrier.
For the GO-MnO prepared2The oxygen-producing composite carrier is detected as follows:
1. the results of ultraviolet-visible absorption spectrum detection and Zeta potential detection are shown in figure 1. Wherein, fig. 1a is the ultraviolet-visible absorption spectrum detection result, and fig. 1b is the Zeta potential detection result.
From FIG. 1a, GO-MnO2Having MnO in oxygen-generating composite carrier (abbreviated as GM)2Typical UV-visible absorption peaks (300-400 nm); from FIG. 1b, GO-MnO2The Zeta potential of the oxygen-producing composite carrier is reduced from +47.3mV of GO-PAH to +10.8 mV.
2. Transmission electron microscopy characterization
Characterization by transmission electron microscopy, as shown in fig. 2, wherein (a) in fig. 2 is a Transmission Electron Microscopy (TEM) characterization image of GO; (b) TEM image as GM; (c) EDS elemental analysis chart for GM; (d) STEM Mapping element distribution map of GM.
From FIG. 2, GO-MnO2A clear black nanoparticle appears on the TEM image of (A) and confirmed to be MnO by EDS elemental analysis2And (4) NPs. Further proves GO-MnO through STEM Mapping element distribution diagram2The upper C element, the Mn element and the N element are uniformly distributed and almost have no free state, and the GO-MnO is proved2Successful synthesis of oxygen-generating composite carriers.
Secondly, synthesizing a targeting penetrating oxygen-producing nano composite carrier GM @ tLyP-1
0.1mL of GM solution (GM solution concentration 1mg/mL, GM mass 0.1mg) was diluted to 5.0mL with PBS buffer, and 18. mu.g of bifunctional PEG (NHS-PEG-MAL, maleimide polyethylene glycol succinimidyl acetate) and 18. mu.g of FITC-labeled thiolated tLyP-1 were weighed, quickly added to the reaction flask, and stirred at room temperature overnight. After the reaction, the reaction mixture was centrifuged at high speed (15000rpm,15min), and the supernatant was discarded, followed by repeated washing three times to remove unbound NHS-PEG-MAL and tLyP-1. Adding 2mL of deionized water to a constant volume to obtain a GM @ tLyP-1 carrier solution, and storing at 4 ℃.
The GM @ tLyP-1 carrier is characterized by Zeta potential, a purple light-visible absorption spectrum and a fluorescence spectrum, and the characterization result is shown in figure 3. FIG. 3 (a) shows the Zeta potentials of GO, GM and GM @ tLyP-1; (b) is a graph of the ultraviolet-visible absorption spectra of GM, GM @ tLyP-1 and tLyP-1; (c) is a fluorescence spectrum diagram of GM @ tLyP-1 and tLyP-1; (d) the drug loading rate and the encapsulation rate of the tLyP-1 are shown.
Through detecting the Zeta potentials of GO, GM and GM @ tLyP-1, the Zeta potential is further increased to +23mV after the tLyP-1 is connected; while the successful modification of the tLyP-1 is proved by the results of the characteristic peak of the tLyP-1 marked by FITC appearing at 480nm in the ultraviolet-visible absorption spectrum and the characteristic peak of the tLyP-1 marked by FITC appearing at about 518nm in the fluorescence spectrum; the drug loading of tLyP-1 when fully encapsulated was 18% as measured by UV-Vis absorption spectroscopy.
Thirdly, preparing a targeted penetrating nano diagnosis and treatment compound GM @ tLyP-1/Ce6
2mL of GM @ tLyP-1 carrier solution (0.5mg/mL) was added with 1mL of Ce6 solution (1mg/mL) under low-speed magnetic stirring, stirred at room temperature overnight for reaction, centrifuged at high speed (15000rpm,15min), the supernatant was discarded, and washing was repeated three times to remove unbound Ce 6. Adding 2mL of deionized water for constant volume to obtain GM @ tLyP-1/Ce6 nano diagnosis and treatment compound solution, and storing at 4 ℃.
The schematic diagram of the prepared GM @ tLyP-1/Ce6 nano diagnosis and treatment compound is shown in FIG. 4. Then, synthesizing and characterizing the GM @ tLyP-1/Ce6 nano diagnosis and treatment compound by an ultraviolet-visible absorption spectrum, a fluorescence spectrum and a particle size analyzer, wherein the result is shown in figure 5; wherein, (a) is the ultraviolet-visible spectrum of each nano system; (b) the fluorescence spectra of GM @ tLyP-1/Ce6 and Ce 6; (c) zeta potential results for each nanosystem; and (d) is a hydrodynamic diameter and physical diagram of each nano system (an inset, 1 is GO, 2 is GM, 3 is GM @ tLyP-1, and 4 is GM @ tLyP-1/Ce 6).
As shown in FIG. 5a, the final targeting penetrating type nano diagnosis and treatment compound GM @ tLyP-1/Ce6 has a characteristic absorption peak of manganese dioxide near 350nm and characteristic absorption peaks of Ce6 near 404nm and 660nm, and successful synthesis of GM @ tLyP-1/Ce6 is proved; as shown in FIG. 5b, the fluorescence spectrum results show that GM @ tLyP-1/Ce6 shows significant Ce6 fluorescence quenching, which is caused by Ce6 supported on GO, further demonstrating the successful loading of Ce 6. Zeta potential and hydrodynamic diameter measurements were performed by a particle sizer, as shown in FIG. 5c, after loading Ce6, the potential of GM @ tLyP-1/Ce6 changed from +23mV to-26 mV of GM @ tLyP-1, and the hydrodynamic diameter also gradually increased; further, the drug loading rate of GM @ tLyP-1/Ce6 calculated by spectroscopy can reach up to 45%. The above results further confirm the successful synthesis of GM @ tLyP-1/Ce 6.
Example 2 GM @ tLyP-1/Ce6 in vitro oxygen production, singlet oxygen yield, and MRI imaging Performance testing
The performance of the targeted penetrating nano diagnosis and treatment compound prepared by the invention is evaluated mainly by the following method:
1. characterization of oxygen production Properties
An equal amount of 100mg of GM @ tLyP-1/Ce6 solution was reacted with 100. mu.M hydrogen peroxide solution at pH 6.5 and 7.4, respectively, and a blank control was set. The oxygen content of the solution was measured every 10 seconds using a JPBJ-608 portable dissolved oxygen analyzer.
As shown in fig. 6a, at H2O2Solution (100. mu.M) + GM @ tLyP-1/Ce6 group, can rapidly trigger the reaction to produce O2And the oxygen generation rate is remarkably increased along with the increase of acidity, which proves that the GM @ tLyP-1/Ce6 nano-composite has good oxygen generation effect.
2. Characterization of active oxygen Properties
Detecting the singlet oxygen yield of the photosensitizer by adopting a singlet oxygen probe (SOSG), wherein when no singlet oxygen exists, the fluorescence of chromophore is quenched by electron transfer in the molecule of the SOSG; in the presence of singlet oxygen, SOSG forms endoperoxides, which result in a change in electron transfer and a restoration of chromophore fluorescence (530 nm). Therefore, the increase in fluorescence intensity of SOSG at 530nm directly reflects the singlet oxygen production.
In 0.5mL GM @ tLyP-1/Ce6 solution (equiv. [ Ce6 ]]Adding 0.5 μ L of a singlet oxygen fluorescent probe SOSG stock solution (final concentration of SOSG is 2.5 μ M), mixing well, and using a 660nm laser (power is 2W/cm)2) After exciting the solution (0, 2, 4, 6, 8, 10, 12min), the fluorescence emission spectrum at 530nm was detected.
As shown in FIG. 6b, the SOSG signal of the GM @ tLyP-1/Ce6 nanocomposite was gradually increased with the increase of the light irradiation time, which proves that GM @ tLyP-1 has good singlet oxygen yield. However, compared with free Ce6, the fluorescent intensity of SOSG under the same conditions is slightly weaker due to energy transfer between the graphene carrier and Ce6, but can be further used for photodynamic therapy.
3. Detection of in vitro magnetic resonance imaging function
GM @ tLyP-1/Ce6 nanocomposite solutions of different Mn concentrations were fixed in order and placed in a magnetic resonance imager, parameters were adjusted (T1 test: IR sequence, SFO1 ═ 22.106MHz, P1 ═ 16.20 μ s, P2 ═ 32.80 μ s, SW ═ 200KHz, RG1 ═ 20, DRGl ═ 3, PRG ═ 3, TW ═ 25000ms, NTI ═ 30, NS ═ 2), and magnetic resonance imaging images of the samples were examined.
As shown in fig. 7 (each set of bars in fig. 7b represents pH 7.4, pH 6.5 and pH 5.0, respectively, from left to right), it can be observed that as pH decreases and Mn content increases, T increases1The signal becomes clearly brighter. Thus, under acidic conditions, T1The signal is enhanced along with the increase of the Mn concentration, and GM @ tLyP-1/Ce6 has obvious T1-MRI contrast effect.
Example 3 GM @ tLyP-1/Ce6 in vitro cytotoxicity, Targeted endocytosis and penetration experiments
GM @ tLyP-1/Ce6, mouse breast cancer cell 4T1, prepared by the preparation method provided in example 1, was purchased from ATCC and 4T1 cells were cultured using RPMI 1640 medium.
1. Cytotoxicity assay of GM @ tLyP-1 vector
4T1 cells in logarithmic growth phase were seeded in 96-well plates (density 1.0X 10)4Per well), cultured for 24 h. The culture medium in the well plate was removed and different concentrations of GM @ tLyP-1 (25)0, 50.0, 100.0, 150.0, 200.0, 250.0 μ g/mL) were added to the well plate (three wells were repeated for each concentration) and incubation was continued for 12 h. The cell culture medium was removed and washed three times with PBS buffer. Add 100. mu.L of prepared CCK-8 working solution to each well and continue incubation for 45 min. Detecting the light absorption value (OD value) at 450nm by a microplate reader, and calculating the cell survival rate according to the formula: survival rate ═ (experimental OD value-blank OD value) - (control OD value-blank OD value).
As shown in FIG. 8a (each column represents 24h and 48h from left to right), the survival rate of 24h and 48h cells after incubation of GM @ tLyP-1 vector with 4T1 cells at different concentrations was 85%, indicating that the vector has no cytotoxicity in a certain range and good biological safety, even if the concentration of the vector is as high as 250. mu.g/mL.
2. Targeted endocytosis capacity of GM @ tLyP-1/Ce6 in two-dimensional cell plane
4T1 cells in logarithmic growth phase were seeded on a confocal culture dish 15mm in diameter (density 6.5X 10)4One/well), three sets were repeated and incubation continued for 24 h. Washed 3 times with PBS buffer, and added with Ce6, GM @ PEG/Ce6, and GM @ tLyP-1/Ce6 nanocomplexes ([ GM ] diluted with cell culture medium]＝200μg/mL，[Ce6]9. mu.g/mL)), and the culture was continued for 4 hours. The supernatant was aspirated off, washed 3 times with PBS buffer, cells were fixed with 4% paraformaldehyde, and aspirated off after 20 min. After washing 3 times with PBS buffer, 1mL of cell nuclear staining solution (DAPI) was added to the petri dish and the dish was stained in the dark at room temperature for 5 min. The DAPI stain was discarded and washed 3 times with PBS buffer. The cells were immersed in 1mL PBS buffer, keeping the bottom of the dish free of clear water. Placing the culture dish under a laser confocal microscope for observation, observing by a 40-time oil scope, and respectively detecting DAPI (lambda)Ex＝405nm)、Ce6(λEx640 nm).
As shown in FIG. 8b, the green fluorescence of the fluorescent probe FITC labeled on tLyP-1 was observed in the GM @ tLyP-1/Ce6 group, and the red fluorescence of Ce6 was the strongest in the GM @ tLyP-1/Ce6 group compared to the control group; the GM @ tLyP-1/Ce6 nano diagnosis and treatment compound synthesized in the example 1 is shown to have a good targeting endocytosis effect.
3. Transmembrane permeability of GM @ tLyP-1/Ce6 nano diagnosis and treatment compound to three-dimensional tumor spheres
Three-dimensional tumor spheres were constructed by the hanging drop method, and 4T1 cells in the logarithmic growth phase were digested at 20. mu.L per drop (density 1X 10)5one/mL) was added dropwise to the lid of the cell culture dish, 1mL of PBS was added to the dish, the lid was quickly closed, and the plate was placed in an incubator at 37 ± 2 ℃ for 3 days in a hanging manner. Gently flick with medium and transpose into low-adhesion cell culture dishes. Respectively adding a nano diagnosis and treatment compound ([ GM ] GM) containing Ce6, GM @ PEG/Ce6 and GM @ tLyP-1/Ce6]＝500μg/mL，[Ce6]22.5 μ g/mL) and endocytosis for 24 h. Transferring to a glass slide, and detecting FITC (lambda) by three-dimensional layer scanning with a confocal laser microscopeEx＝488nm)、 Ce6(λEx640 nm).
As shown in FIG. 8c, the GM @ tLyP-1/Ce6 group penetrated into the interior of the tumor sphere through a distance of about 90 μm compared to the control group. And only a few nanoparticles enter the tumor spheres in the non-targeting group and the free Ce6 group, so that the GM @ tLyP-1/Ce6 nano diagnosis and treatment compound has good targeting penetrating capability.
Example 4 in vitro Targeted enhanced tumor photodynamic therapy Effect of GM @ tLyP-1/Ce6
4T1 cells in logarithmic growth phase were seeded in 96-well plates (density 10)4One/well), cultured for 24 h. The old culture medium was removed, washed 3 times with PBS buffer, and cell culture medium diluted Ce6, GO @ tLyP-1/Ce6, GM @ PEG/Ce6, GM @ tLyP-1/Ce6([ GM @ tLyP-1/Ce6) were combined in the following manner]＝200μg/mL， [Ce6]Not 9 μ g/mL) solution was added separately to incubate cells for 4 h:
a first group: control; second group: control + Laser; third group: h2O2+H+(ii) a And a fourth group: h2O2+H++Laser。
Removing old culture solution after culturing, washing with PBS buffer solution for 3 times, adding new cell culture solution into the well plate, and using laser with wavelength of 660nm and 0.5W/cm2Irradiating each hole for 5min under the condition of power density. After illumination, the culture was continued for 24h, the old culture medium was removed and PB was usedWash 3 times with S buffer. Each well was added with 100.0. mu.L of CCK8 working solution and incubation was continued for 45 min. And (3) placing the cell pore plate in an enzyme labeling instrument, setting the detection wavelength to be 450nm, recording the absorbance (OD value), and calculating the relative cell survival rate.
As shown in FIG. 9 (each set of columns in FIG. 9a represents Ce6, GM @ PEG/Ce6, GO @ tLyP-1/Ce6, and GM @ tLyP-1/Ce6, respectively, from left to right; each set of columns in FIG. 9b represents control, H, respectively, from left to right2O2+H+、Laser、H2O2+H++ Laser; in the absence of a simulated tumor microenvironment, the survival rate of free Ce6 cells reaches about 80%, and the survival rate of 4T1 cells can be reduced to 40% after the Ce6 is combined on the GM @ tLyP-1 vector, which indicates that the GM @ tLyP-1 can enhance the uptake rate of Ce6 by cells. Under the microenvironment simulating tumor acidity and a large amount of hydrogen peroxide, compared with a control group, the survival rate of cells of the GM @ tLyP-1/Ce6 group is reduced to be within 20%, the photodynamic curative effect is improved by nearly 2 times, and the GM @ tLyP-1/Ce6 nano diagnosis and treatment compound has enhanced PDT treatment potential in tumors.
Example 5 study of in vivo tumor hypoxia amelioration of GM @ tLyP-1/Ce6
Balb/c mice are provided by the experimental animal center of Sichuan university, are female, are 5-6 weeks old and have the weight of 18-22 g.
Cells were expanded in vitro, digested and counted, diluted in sterile saline, and 100. mu.L of saline (1X 10) was injected subcutaneously into the right hind leg of mice74T1 cells), the tumor grows to a volume of about 200mm after 8-14 days3Intravenous injections were performed, divided into 4 groups of 2 per group, and blank groups were intratumorally injected with saline as follows: a first group: control (control group); second group: GO @ tLyP-1/Ce6 (Targeted, oxygen-non-generating group); third group: GM @ PEG/Ce6 (non-targeting, oxygen-producing group); and a fourth group: GM @ tLyP-1/Ce6 (Targeted, oxygen-producing group); tail vein injection: each mouse was 100. mu.L ([ MnO ]2]＝0.045mg/mL，[Ce6]＝0.45mg/mL， [GO]1 mg/mL); after 6 hours of administration, the hypoxic probe pethidine hydrochloride (60mg/kg) was injected intratumorally, and after 90 minutes, the tumors were surgically excised and frozen sections. Tumor sections were mixed with mouse anti-pimozole antibody (1:100 dilution, Hypopyprobe Inc.) andalex 488 conjugated goat anti-mouse secondary antibody (1:200 dilution, Jackson Inc.) was incubated together and nuclei were stained with DAPI, and the results are shown in figure 10. FIG. 10 is a DCFH-DA fluorescence signal plot (sacle bar 50 μm) at the tumor site of mice after injection of each set of nanoparticles.
As can be seen from FIG. 10, the tumor tissues in the control group showed a clear green fluorescence signal of the hypoxia probe, indicating that the interior of the solid tumor itself was in a state of severe hypoxia; while the GM @ tLyP-1/Ce6 treated group had a significantly reduced green fluorescence signal and a high degree of co-localization of the green fluorescence signal with the blue fluorescence signal of DAPI, indicating that GM @ tLyP-1/Ce6 could indeed alleviate the tumor hypoxic state.
Example 6 study of GM @ tLyP-1/Ce6 in vivo MRI/FL
The 4T1 tumor-bearing mouse model was constructed and grouped as in example 5 for 4 groups of 3 mice each, and tail vein dosing was performed: 100 μ L ([ MnO ]2]＝0.045mg/mL，[Ce6]＝0.45mg/mL，[GO]1 mg/mL); after 48h of imaging is finished, the mouse is dissected, fluorescence imaging is carried out on main organs and tumors, and the accumulation condition of the GM @ tLyP-1/Ce6 nano diagnosis and treatment compound is analyzed by detecting the fluorescence of Ce 6.
As shown in fig. 11a, significant mri signal enhancement was detectable at the tumor site relative to the control group. The strongest signal was detected at the tumor site 8h after administration, after which the signal intensity decreased with time, indicating that the GM @ tLyP-1/Ce6 Nanocouch Complex can be used as a T1 contrast agent for magnetic resonance imaging. The group GM @ tLyP-1/Ce6 showed significant distribution at the tumor site 48h after injection (see FIG. 11b), indicating that GM @ tLyP-1/Ce6 indeed enables targeted enrichment of the drug at the tumor site, and has a longer tumor residence time, which is beneficial for the subsequent PDT.
Example 7 GM @ tLyP-1/Ce6 enhanced photodynamic anti-tumor Effect in vivo
1. Detection of ROS production in mice
A tumor-bearing mouse model was constructed as in example 5, and a DCFH-DA solution containing GM @ tLyP-1/Ce6 (intratumoral administration) was injected into the tumor, after which the tumor tissue was collected and frozen and sectioned on a glass slide for fluorescence detection.
As shown in FIG. 12 (bar graphs representing, from left to right, Pre-injection, GO @ tLyP-1/Ce6, GM @ PEG/Ce6, GM @ tLyP-1/Ce6, respectively), there was almost no significant green fluorescence signal at the tumor site due to the inhibition of PDT effect caused by severe hypoxia at the tumor site. In contrast to the control group, the GM @ tLyP-1/Ce6 group showed the strongest green fluorescence signal, representing a large amount of ROS production, and confirmed that the GM @ tLyP-1/Ce6 nanocomposite can indeed enhance PDT effect by generating oxygen.
2. In vivo enhanced photodynamic anti-tumor effect detection
The mouse 4T1 breast cancer cells are cultured in vitro to construct a xenograft tumor-bearing mouse model, and the xenograft tumor-bearing mouse model is divided into 5 mice in groups at random. Blank groups were injected intravenously with 100. mu.L of saline, GM @ tLyP-1/Ce6, GM @ PEG/Ce6, and GO @ tLyP-1/Ce 6. The injection dosage of Ce6 is 90 mug/piece, the injection dosage of GO is 200 mug/piece, MnO is2The injection dose is 9 mug/body; mice were dehaired, exposing the illuminated area. Using a 660nm near-infrared laser at 0.5W/cm2The laser density of (2) is used for irradiating the tumor part of the mouse for 10 min. The size of the tumor was measured in two dimensions by a vernier caliper every other day, the body weight of the tumor-bearing mice was measured, and the second administration treatment was performed on day 3. The formula for calculating the tumor volume is V ═ WL2And/2 (W represents the major diameter of the tumor and L represents the minor diameter of the tumor). On day 15, mice were sacrificed and tumor masses were isolated and weighed.
As shown in fig. 13, showing the in vivo combined anti-tumor results of example six, the targeted penetration type nanotechnology complex GM @ tLyP-1/Ce6 showed very significant tumor inhibition effect (fig. 13a), minimal final tumor volume and weight (fig. 13b), and no significant change in mouse body weight throughout the treatment process, compared to the GM @ PEG/Ce6 treated group and the GO @ tLyP-1/Ce6 treated group, indicating that GM @ tLyP-1/Ce6 has very good biosafety.

Claims (10)

1. The tumor in-situ oxygen generation sensitization photodynamic therapy targeted penetration type nano diagnosis and treatment compound is characterized by comprising GO-MnO with graphene oxide as a substrate2A composite oxygen generating carrier chemically bonded to the composite oxygen generating carrierTumor targeting homing penetrating peptide connected in a valence connection mode and photosensitizer connected with the composite oxygen-generating carrier through pi-pi accumulation, hydrophobic force action, hydrogen bond or electrostatic action;
wherein, GO-MnO taking graphene oxide as substrate2The composite oxygen-generating carrier is formed by in-situ growth of MnO on the surface of graphene oxide through modification of aminated cations2And (4) preparing the nano particles.
2. The tumor in-situ oxygenation-producing sensitization photodynamic therapy targeted penetration type nano diagnosis and treatment compound as claimed in claim 1, wherein the tumor targeted homing penetration peptide is LyP-1, F3, iRGD or tLyP-1.
3. The tumor in-situ oxygen generation and sensitization targeted penetration type nano diagnosis and treatment compound with photodynamic therapy effect according to claim 1, wherein the photosensitizer is organic fluorescent dye capable of generating singlet oxygen under the excitation of near infrared light.
4. The tumor in-situ oxygen production sensitization photodynamic therapy targeted penetration type nanometer diagnosis and treatment compound as claimed in claim 3, wherein the photosensitizer is chlorins, porphyrins, phthalocyanines, rhodamines, methylene blue or cyanines.
5. The method for preparing the tumor in-situ oxygen production sensitization photodynamic therapy targeted penetration type nano diagnosis and treatment compound of any one of claims 1 to 4 is characterized by comprising the following steps:
(1) synthesis of GO-MnO2Composite oxygen-generating carrier
Carrying out self-assembly or amide reaction on GO and an aminated polycation polymer to prepare a GO-polycation compound, adding a potassium permanganate solution into the GO-polycation compound, and reacting at room temperature for 12-36h to prepare the GO-polycation compound; the mass ratio of polycationic polymer to GO is 0.25: 1-2: 1; the mass ratio of GO to potassium permanganate is 1: 8-12;
(2) preparation of targeting penetrating oxygen-producing nano composite carrier
Synthesizing FITC-labeled thiolated tumor targeting homing penetrating peptide, and then adding GO-MnO2Mixing the composite oxygen-producing carrier, maleimide polyethylene glycol succinimide acetate and FITC marked sulfhydrylation tumor targeted homing penetrating peptide according to the weight ratio of 1:0.1-1:0.1-1, and reacting at room temperature for 20-28h to obtain the product;
(3) preparation of targeting penetrating nano diagnosis and treatment compound
The photosensitizer and the targeted penetrating oxygen-producing nano composite carrier are self-assembled to prepare the targeted penetrating nano diagnosis and treatment composite.
6. The method for preparing the tumor in-situ oxygenation sensitization photodynamic therapy targeted penetration type nano diagnosis and treatment compound according to claim 5, wherein the amination polycation polymer is poly (allylamine hydrochloride), polylysine or polyethyleneimine.
7. The method for preparing the tumor in-situ oxygen production sensitization photodynamic therapy targeted penetration type nano diagnosis and treatment compound according to claim 6, wherein the mass ratio of the aminated polycation polymer to GO is 1: 1.
8. The method for preparing the tumor in-situ oxygen generation sensitization photodynamic therapy targeted penetration type nano diagnosis and treatment compound according to claim 5, wherein GO-MnO is added2The weight ratio of the composite oxygen-generating carrier, the maleimide polyethylene glycol succinimide acetate and the FITC-labeled thiolated tLyP-1 is 1:0.18: 0.18.
9. The method for preparing the tumor in-situ oxygen-generating and sensitizing photodynamic therapy targeted penetrating type nano diagnosis and treatment compound according to claim 5, wherein the mass ratio of the photosensitizer to the targeted penetrating type oxygen-generating nano compound carrier is 0.1-4: 1.
10. the method for preparing the tumor in-situ oxygen-generating and sensitizing photodynamic therapy targeted penetrating type nano diagnosis and treatment compound according to claim 9, wherein the mass ratio of the photosensitizer to the targeted penetrating type oxygen-generating nano compound carrier is 1: 1.
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