CN114601936B - Tumor-targeted near infrared light response nitric oxide nano generator, preparation method and application thereof - Google Patents

Tumor-targeted near infrared light response nitric oxide nano generator, preparation method and application thereof Download PDF

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CN114601936B
CN114601936B CN202210314512.XA CN202210314512A CN114601936B CN 114601936 B CN114601936 B CN 114601936B CN 202210314512 A CN202210314512 A CN 202210314512A CN 114601936 B CN114601936 B CN 114601936B
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peg
nitric oxide
ucnp
tumor
oxide nano
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CN114601936A (en
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王育才
蒋为
郭子萱
汪沁
董旺
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University of Science and Technology of China USTC
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    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
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    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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    • B82—NANOTECHNOLOGY
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    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Abstract

The invention discloses a near infrared light response nitric oxide nano generator for targeting tumor, which is a core-shell structure; the invention also provides a preparation method of the nitric oxide nano generator, which is characterized in that hydrophobic oleic acid modified UCNP is synthesized by a solvothermal method, then a ligand exchange method is utilized to exchange oleic acid ligand on the surface of the UCNP by carboxyl-PEG-acrylamide to obtain PEG modified hydrophilic UCNP@PEG, finally the hydrophilic UCNP@PEG is resuspended, a functional monomer, a cross-linking agent and an initiator are added to generate in-situ free radical polymerization, and an MMP responsive gel layer formed on the surface of a kernel is taken as a shell; the invention also provides application of the nitric oxide nano generator, which is used for preparing medicines for treating tumors and resisting bacteria. The invention is suitable for preparing the nitric oxide nano generator, and the prepared nitric oxide nano generator is further applied to the biological fields of tumor, antibacterial treatment and the like.

Description

Tumor-targeted near infrared light response nitric oxide nano generator, preparation method and application thereof
Technical Field
The invention belongs to the technical field of medicines, relates to a nanometer delivery system, and in particular relates to a near infrared light response nitric oxide nanometer generator for targeting tumors, a preparation method and application thereof.
Background
Nitric Oxide (NO) is one of the important gas signaling molecules in the human body, has various important physiological roles and potential pharmacological activities in the body, and plays an important role in various vital activities such as vascular smooth muscle relaxation, platelet adhesion, inflammation and immune response, neurotransmission and the like. Recent studies have shown that NO also shows potential application prospects in tumor treatment. Studies show that NO can increase the sensitivity (about 1000 times) of tumor cells to chemotherapeutic drugs, and meanwhile, NO combined with radiotherapy, chemotherapy and photodynamic therapy also has good sensitization effect, so that the drug resistance of tumors to various drugs can be reversed. In addition, the NO molecules can inhibit the expression of P-type glycoprotein of drug-resistant cancer cells, so that the enrichment of intracellular chemotherapeutics is effectively improved, and the killing effect is enhanced; NO can also promote the normalization of tumor blood vessels, promote the intratumoral osmotic pressure of tumor-associated macrophages and T cells, and inhibit the expression of PD-L1 on the surface of tumor cells.
At present, the delivery modes of NO in basic and preclinical studies are mainly divided into four types: 1. directly inhaling NO gas; 2. NO donor small molecules (e.g. azoglycol olefins NONOates, nitrosothiols RSNO, nitrobenzene PhNO) 2 Metal nitrosyl compounds M-NO, organic nitrates and organic nitrites); 3. nanocarriers deliver NO (including liposomes, silica nanoparticles, quantum dots, titania, carbon materials); 4. photocatalysis produces endogenous NO. The direct inhalation of low-concentration NO has the problems of poor dosage control, low targeting, easy NO poisoning and the like due to different NO tolerance degrees of patients, and the NO gas is unstable and is easily oxidized into nitrogen dioxide with stronger toxicity, so that the clinical conversion of the nitrogen dioxide is limited. Although the NO donor small molecule can directly act on tissues to release NO, the concentration control is relatively easy, the common NO donor small molecule usually lacks tumor tissue targeting, and serious toxic and side effects are easy to cause, so that the clinical use effect of the NO donor small molecule is limited to a great extent. The nanometer delivery system can effectively regulate and control the in-vivo process of the medicine, improve the distribution and enrichment of the medicine in tumor tissues, improve the cell uptake and release behaviors of the medicine, realize attenuation and synergy, and realize high-efficiency NO in-vivo delivery by utilizing the nanometer delivery system. A series of nano-carriers (e.g. liposomes, silica nanoparticles, quantum dots, titania, carbon materials, bovine serum albumin nanoparticles, metal-organic frameworks, prussian blue particles, etc.) with NO donors bound by surface have been designed for in vivo NO delivery, significantly improving the NO enrichment within the tumor. Although NO delivery platforms have broader application prospects in biomedicine than NO donor small molecules, each NO delivery system has its own advantages and disadvantages. For example: the NO delivery platform begins to release NO when the NO delivery platform does not reach the focus part in the process of delivering NO. Similarly, NO delivery platforms loaded with NO donors also result in uncontrolled release of NO due to instability of the carrier. And these nanocarrier materials still rely on the release efficiency of NO itself. Furthermore, the in vivo safety of inorganic materials to which the related systems mainly relate is also an important issue for future clinical transformations not to be negligible.
Strategies for triggering NO release by ultraviolet-visible light (UV-vis) have potential for NO toxicity and phototoxicity, and poor tissue penetration by ultraviolet light, which limits the further use of most NO donors in vivo. Thus, developing a more controlled, efficient, nano-delivery system that catalyzes the in situ generation of NO is a significant challenge in the field of NO delivery.
Disclosure of Invention
The invention aims to provide a near infrared light response nitric oxide nano generator for targeting tumors, which effectively realizes tumor treatment by targeting tumor tissues through Matrix Metalloproteinase (MMP) responsiveness and simultaneously reduces the effect of systemic toxicity.
Another object of the present invention is to provide a method for preparing the above-mentioned tumor-targeting near infrared light responsive nitric oxide nano generator.
It is still another object of the present invention to provide an application of the above-mentioned tumor-targeting near infrared light responsive nitric oxide nano generator.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a near infrared light responsive nitric oxide nano-generator for targeting tumors, which is a core-shell structure.
As a limitation, the core-shell structure is characterized in that the shell is a gel layer formed by in-situ free radical polymerization of a functional monomer, a cross-linking agent and an initiator, and the inner core is an up-conversion luminescent rare earth material modified by acrylated polyethylene glycol;
the size of the inner core is 20-60 nm, and the size of the outer shell is 10-20 nm.
As a further limitation, the functional monomers include 2-methacryloxyethyl phosphorylcholine, an acrylated nitric oxide donor, and an acrylamido matrix metalloproteinase-responsive RGD targeting peptide-bonded polyethylene glycol;
the cross-linking agent is N, N-methylene bisacrylamide;
the initiator is ammonium persulfate;
the up-conversion luminescent rare earth material modified by the propenyl polyethylene glycol is UCNP@PEG.
As a further definition, the amino acid sequence of the polypeptide in the acrylamido matrix metalloproteinase-responsive RGD targeting peptide-bound polyethylene glycol is GPLGVRGDG, CPENFFGRGDSG, VPLGVRGDK, PVGLIGRGDK, GGPLGVRGDK or GPQGIWGGCRGDK;
the acrylated nitric oxide donor is an acrylamide modified 4-nitro-3-trifluoromethyl halogenated benzene derivative ANTP, and the structural formula is as follows:
as a further definition, the 2-methacryloyloxyethyl phosphorylcholine, the acrylated nitric oxide donor, the acrylamido matrix metalloproteinase-responsive RGD targeting peptide-bonded polyethylene glycol, and the N, N-methylenebisacrylamide have a molar ratio of 5:5:0.2:1.
as another limitation, the polyethylene glycol has a molecular weight of 500 to 5000.
The invention also provides a preparation method of the tumor-targeted near infrared light response nitric oxide nano generator, which comprises the following steps in sequence:
s1, synthesizing UCNP modified by hydrophobic oleic acid through a solvothermal method;
s2, the UCNP modified by oleic acid is prepared, and a ligand exchange method is utilized to exchange oleic acid ligand on the surface of the UCNP modified by oleic acid by carboxyl-PEG-acrylamide, so that hydrophilic UCNP@PEG with the surface modified by PEG is obtained;
s3, resuspension the hydrophilic UCNP@PEG, adding a functional monomer, a cross-linking agent and an initiator to perform in-situ free radical polymerization, wherein an MMP responsive gel layer formed on the surface of the inner core is taken as an outer shell, and the tumor-targeted near infrared light response nitric oxide nano generator is obtained.
As a limitation, the PEG-modified hydrophilic UCNP (UCNP@PEG) is obtained by exchanging oleic acid ligand on the surface of the UCNP modified by hetero-difunctional carboxyl-PEG-acrylamide by using a ligand exchange method.
As another limitation, in the step S2, the mass ratio of UCNP to carboxyl-PEG-acrylamide is 5:2.
As a third definition, the method for preparing the acrylated nitric oxide donor comprises the following steps:
s1, taking 5-chloro-2-nitro benzotrifluoride and 1, 6-hexamethylenediamine to carry out halogenation reaction to obtain a 4-nitro-3-trifluoromethyl halogenated benzene derivative;
and reacting the amino group of the S2.4-nitro-3-trifluoromethyl halogenated benzene derivative with acryloyl chloride to obtain the acrylamide modified 4-nitro-3-trifluoromethyl halogenated benzene derivative, namely the acrylated nitric oxide donor.
As a fourth limitation, the preparation method of the RGD targeting peptide-bonded polyethylene glycol responsive to the matrix metalloproteinase is as follows:
s1, taking azido polyethylene glycol and alkynyl matrix metalloproteinase response peptide to bond under the catalysis of CuBr, and obtaining RGD targeting peptide-bonded polyethylene glycol PEG-GPLGVRGDG-NH with amino matrix metalloproteinase responsiveness 2 、PEG-CPENFFGRGDSG-NH 2 、PEG-VPLGVRTGDK-NH 2 、PEG-PVGLIGRGDK-NH 2 、PEG-GGPLGVRGDK-NH 2 Or PEG-GPQGIWGGCRGDK-NH 2 ；
S2, reacting the RGD targeting peptide-bonded polyethylene glycol of the amination matrix metalloproteinase with acrylic acid-N-succinimidyl ester in sodium borate buffer solution to obtain the RGD targeting peptide-bonded polyethylene glycol PEG-GPLGVRGDG-Alkene, PEG-CPENFFGRGDSG-Alkene, PEG-VPLGVRTGDK-Alkene, PEG-PVGLIGRGDK-Alkene, PEG-GGPLGVRGDK-Alkene or PEG-GPQGIWGGCRGDK-Alkene of the acrylamide matrix metalloproteinase.
As a further limitation, in the step S1, the bonding is performed by click chemistry;
the concentration of the sodium borate buffer solution is 15-25 mM, and the pH value is 8.0-8.5.
The invention also provides an application of the tumor-targeted near infrared light response nitric oxide nano generator, and the tumor-targeted near infrared light response nitric oxide nano generator is used for preparing medicines for treating tumors and resisting bacteria.
By adopting the technical scheme, compared with the prior art, the invention has the following technical progress:
(1) the acrylated nitric oxide donor used in the invention can release nitric oxide gas with high efficiency under the action of ultraviolet light (365 nm);
(2) the up-conversion luminescent rare earth material (UCNP) used in the invention is a functional material capable of converting photons with low energy into photons with high energy to emit anti-Stokes luminescence, the photon energy absorbed by the material is lower than the photon energy emitted by the material, and the photon energy is excited by 980nm near infrared light, thus ultraviolet light with 365nm and 475nm can be emitted;
(3) GPLGVRGDG, CPENFFGRGDSG, VPLGVRGDK, PVGLIGRGDK, GGPLGVRGDK or GPQGIWGGCRGDK in RGD targeting peptide-bonded polyethylene glycol with acrylamide matrix metalloproteinase responsiveness can be responsively broken under the action of matrix metalloproteinase, so that the gel layer is destroyed and degraded, RGD tumor targeting peptide fragments are exposed, and the tumor position is efficiently enriched;
(4) the nitric oxide nano generator provided by the invention can be used for targeting a primary tumor or tumor metastasis of MMP (matrix-like tumor) with high expression through MMP responsiveness, performing an up-conversion luminescence process under near infrared light irradiation, exciting ANTP to generate nitric oxide, realizing accurate and controllable in-vivo delivery of nitric oxide, having the functions of targeting tumor microenvironment and effectively realizing timely, fixed-point and controllable release of nitric oxide with proper concentration under near infrared light irradiation, effectively treating tumors and simultaneously reducing systemic toxicity;
(5) the nitric oxide nano generator provided by the invention has small nano particle size, and is easy to leak to a tumor part through abnormal blood vessels at the tumor part, so that intra-tumor enrichment is realized.
(6) According to the preparation method of the nitric oxide nano generator, hydrophilic UCNP@PEG with PEG on the surface is taken as an inner core under specific reaction conditions, RGD targeting peptide bonded polyethylene glycol with functionalized monomer acrylamide matrix metalloproteinase responsiveness, an acrylic acid nitric oxide donor (ANTP), 2-methacryloyloxyethyl phosphorylcholine, a cross-linking agent N, N' -methyl bisacrylamide and an initiator ammonium persulfate undergo in-situ free radical polymerization, and an MMP responsive gel layer formed on the surface of the inner core is taken as an outer shell to jointly form the nitric oxide nano generator.
The invention is suitable for preparing the near infrared light response nitric oxide nano generator for targeting tumor, and the prepared near infrared light response nitric oxide nano generator for targeting tumor is suitable for treating tumor and antibacterial drugs.
Drawings
The invention will be described in more detail below with reference to the attached drawings and specific examples;
FIG. 1 is a schematic diagram of a tumor-targeted near infrared light responsive nitric oxide nano-generator and a preparation method thereof according to an embodiment of the present invention;
FIG. 2 is a schematic illustration of 4-nitro-3-trifluoromethyl-halogenobenzene derivative (NTP) of example 1 1 HNMR characterization map;
FIG. 3 shows an acrylamide-modified 4-nitro-3-trifluoromethyl-halogenobenzene derivative ANTP according to example 2 1 HNMR characterization map;
FIG. 4 is an ultraviolet characteristic absorption spectrum of NTP and ANTP in example 3;
FIG. 5 is a graph showing the variation of the ultraviolet absorbance of NTP under 365nm ultraviolet light in example 4;
FIG. 6 is a graph showing the variation of the ultraviolet absorbance and the nitric oxide release curve of the ANTP of example 4 after being irradiated with 365nm ultraviolet light;
FIG. 7 is an electron microscopy characterization of UCNP@PEG particles of example 7;
FIG. 8 is an up-conversion emission spectrum of UCNP@PEG in example 7;
FIG. 9 is an electron microscope characterization of the gel coated UCNP@N-pNO and UCNP@S-pNO of example 8 and example 9;
FIG. 10 is a size distribution of UCNP@PEG, UCNP@S-pNO, and UCNP@N-pNO in example 10;
FIG. 11 shows the surface potential of UCNP@PEG, UCNP@S-pNO, and UCNP@N-pNO in example 10;
FIG. 12 is a graph showing the nitric oxide release profile of UCNP@S-pNO in example 11 under near infrared light irradiation;
FIG. 13 is a graph showing the measurement of cytotoxicity of UCNP@S-pNO nanoparticles of example 1 under non-light using MTT colorimetric method in example 12;
FIG. 14 shows the detection of cytotoxicity of UCNP@S-pNO nanoparticles of example 1 under near infrared light irradiation using MTT colorimetric method in example 13.
The specific embodiment is as follows:
the technical contents of the present invention are further described below with reference to examples: the following examples are illustrative, but not limiting, and are not intended to limit the scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention. The test methods used in the examples described below are generally carried out under conventional conditions or under conditions recommended by the manufacturer of the test materials and reagents used, unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The invention provides a near infrared light response nitric oxide nano generator for targeting tumor. The preparation flow and the response disintegration process of the nitric oxide nano generator are shown in figure 1:
as can be seen from fig. 1, the above-mentioned nitric oxide nano-generator uses the up-conversion luminescent rare earth material UCNP as the inner core; after the amphiphilization modification is carried out by using the propenyl PEG, a monomer ANTP which releases nitric oxide under the excitation of ultraviolet light and a Matrix Metalloproteinase (MMP) response targeting peptide are coated on the surface, and a gel layer shell is formed through polymerization.
The nitric oxide nano generator can specifically target a primary tumor or a tumor metastasis through MMP (matrix-like protein) response targeting peptide, and performs an up-conversion luminescence process under the irradiation of near infrared light to excite ANTP to generate nitric oxide, so that the aim of specifically generating nitric oxide is fulfilled.
In conclusion, the near infrared light response nitric oxide nano generator containing matrix metalloproteinase responsiveness is of a core-shell structure, and comprises an inner core of an allylated PEG modified up-conversion luminescent rare earth material UCNP, 2-methacryloyloxyethyl phosphorylcholine and RGD targeting peptide-bonded polyethylene glycol with MMP responsiveness, N' -methylacrylamide serving as a cross-linking agent and a gel layer formed by in-situ free radical polymerization taking ammonium persulfate as an initiator as an outer shell.
For further understanding of the present invention, the near infrared light responsive nitric oxide nano-generator, the preparation method and application thereof provided by the present invention are described below with reference to examples, and the scope of the present invention is not limited by the following examples.
Example 14 Synthesis of nitro-3-trifluoromethyl-halogenobenzene derivative (NTP) monomer
The embodiment is a synthetic method of 4-nitro-3-trifluoromethyl halogenated benzene derivative (NTP) monomer, which comprises the following specific steps:
2.43g (20 mmol) of 1, 6-hexamethylenediamine and 2.15g (20 mmol) of Na 2 CO 3 Adding into a flask containing 50mL of ethanol, refluxing at 80 ℃ for 15 minutes, taking out the flask, cooling, adding 60 mu L (4 mmol) of 5-chloro-2-nitrobenzotrifluoride, stirring, reacting for 3 days (monitoring the reaction progress by a thin layer chromatography silica gel plate), purifying by a column chromatography method, and removing the organic solvent by rotary evaporation to obtain yellow solid, wherein the characterization data are shown in figure 2: 1 HNMR(400MHz,MeOD)δ8.02(d,1H),6.98(d,1H),6.76(dd,J＝9.2,1H),3.21(t,2H),2.80-2.71(m,2H),1.74-1.62(m,2H),1.61-1.51(m,2H),1.50-1.37(m,4H)。
as can be seen from FIG. 2, the yellow solid product, namely 4-nitro-3-trifluoromethyl halogenated benzene derivative (NTP) monomer, was calculated to give a yield of 50%.
The specific reaction process is as follows:
EXAMPLE 2 preparation of acrylamide-modified 4-nitro-3-trifluoromethyl-halogenobenzene derivative (ANTP) monomer
The embodiment is a preparation method of an acrylamide modified 4-nitro-3-trifluoromethyl halogenated benzene derivative (ANTP) monomer, which comprises the following specific steps:
s1, dissolving 400mg of NTP in 5mL of dichloromethane, adding 152mg of triethylamine, and cooling to 0 ℃ to obtain a solution X;
s2, dissolving 118mg of acryloyl chloride in 5mL of dichloromethane to obtain a solution Y, dropwise adding the solution Y into the solution X, reacting for 10 hours (monitoring the reaction process by using a thin layer chromatography silica gel plate), and filtering to remove insoluble precipitate after the reaction is finished to obtain a solution Z;
s3, concentrating the solution Z, diluting with ethyl acetate, adding a saturated sodium chloride solution for washing for 3 times, purifying by using a column chromatography, and removing the organic solvent by rotary evaporation to obtain a yellow solid ANTP, wherein the characterization data of the yellow solid ANTP are shown in figure 3: 1 HNMR(400MHz,CDCl 3 )δ8.03(d,1H),6.91(d,1H),6.66(dd,1H),6.30(dd,1H),6.08(dd,1H),5.66(dd,2H),3.37(dd,2H),3.22(t,2H),1.62(ddd,2H),1.44(ddd,2H),1.25(s,4H)。
as can be seen from FIG. 3, the yellow solid product, namely the acrylamide modified 4-nitro-3-trifluoromethyl halogenated benzene derivative (ANTP) monomer, was calculated to give a yield of 65%.
The specific reaction process is as follows:
as can be seen from the combination of examples 1 and 2, the specific scheme for synthesizing ANTP is as follows:
EXAMPLE 3 characterization of the ultraviolet absorption curve of NTP and ANTP
The embodiment is an ultraviolet absorption measurement experiment of NTP and ANTP, and comprises the following specific steps:
after the compounds were prepared in the methods of example 1 and example 2, ethanol solutions of NTP and ANTP were prepared at concentrations of 0.5mg/mL, and the ultraviolet absorption of the two compounds, NTP and ANTP, was measured by an ultraviolet spectrophotometer, respectively, and the results are shown in FIG. 4.
As can be seen from fig. 4, the highest absorption peak of NTP appears at 404nm, while the highest absorption peak of ANTP appears as a red shift of 408nm; from this, it was found that the modification of the NTP by acrylation successfully achieved ANTP.
EXAMPLE 4 ultraviolet light triggered NTP and ANTP release of nitric oxide
(1) Ultraviolet absorption change detection of NTP after ultraviolet illumination:
the ultraviolet absorption peak of the 4-nitro-3-trifluoromethyl halogenated benzene derivative (NTP) monomer prepared in example 1 was detected using an ultraviolet spectrophotometer. After the NTP is irradiated by ultraviolet light, light rearrangement from nitro to nitrite occurs, then O-N bond is broken to generate phenoxy free radical and nitric oxide, ultraviolet absorption can be obviously changed, and based on the principle, the nitric oxide release amount is calculated through the change of ultraviolet absorption peaks of NTP monomers before and after ultraviolet light irradiation. The specific experimental steps are as follows:
s1, measuring a baseline by using ethanol;
s2, dispersing an NTP monomer in ethanol with the concentration of 0.5mg/mL, and detecting an ultraviolet absorption peak when the NTP monomer is not irradiated by ultraviolet light;
s3, using a 365nm laser with illumination power of 1.5W/cm 2 Illumination is carried out on the NTP ethanol solution in the S2 under the condition, each illumination is stopped for 1min, illumination time is calculated in an accumulated mode, and ultraviolet absorption patterns of NTP in 0min,2min,4min,6min,11min,26min,36min,46min,56min,71min and 90min are measured in sequence, wherein the result is shown in figure 5;
as can be seen from fig. 5, the ultraviolet absorption value at 404nm gradually decreases with the illumination time, which indicates that the structure of NTP changes, and thus it can be seen that NTP can release nitric oxide after ultraviolet illumination.
(2) Detection of ultraviolet absorption change of ANTP after ultraviolet light irradiation:
the ultraviolet absorption peak of the acrylamide-modified 4-nitro-3-trifluoromethyl halogenated benzene derivative (ANTP) monomer prepared in example 2 was detected using an ultraviolet spectrophotometer. After the ANTP is irradiated by ultraviolet light, the light rearrangement from nitro to nitrite occurs, and then O-N bond is broken to generate phenoxy free radical and nitric oxide, and the ultraviolet absorption can be obviously changed. Based on this principle, the amount of nitric oxide released by the ant p monomer is calculated by the change of its ultraviolet absorption peaks before and after the irradiation with ultraviolet light. The specific experimental steps are as follows:
s1, measuring a baseline by using ethanol;
s2, dispersing an ANTP monomer in ethanol with the concentration of 0.5mg/mL, and detecting an ultraviolet absorption peak of the ANTP when the ANTP monomer is not irradiated by ultraviolet light;
s3, using a 365nm laser with illumination power of 1.5W/cm 2 Illumination is carried out on the ANTP ethanol solution in the S2 under the condition, each illumination is stopped for 1min, illumination time is calculated in an accumulated mode, and ultraviolet absorption patterns of the ANTP are measured in sequence at 0min,2min,4min,6min,11min,26min,36min,46min,56min,71min and 90min, and the result is shown in figure 6;
as can be seen from fig. 6, the ultraviolet absorption value at 404nm gradually decreases with the increase of the illumination time, and the ultraviolet absorption value at 538nm gradually increases, so that the ant p can release nitric oxide after the illumination with ultraviolet light.
In summary, it is known that ant p can realize light-controlled release of nitric oxide.
Example 5 acrylamido MMP responsive RGD targeting peptide-bonded polyethylene glycol
This example is a method for preparing an acrylamido MMP responsive RGD targeting peptide (exemplified by GPLGVRGDG sequence) conjugated polyethylene glycol with a polyethylene glycol of molecular weight 2000 comprising the following steps, performed in sequence:
s1, 0.3g (0.06 mmol) of azidated polyethylene glycol (PEG) 2000 -N 3 ) 62mg (0.072 mmol) of alkynylated MMP peptide (sequence: alkynyl-GPLGVRGDDG), 31mg (0.18 mmol) of N, N, N' -Pentamethyldiethylenetriamine (PMDETA) are dissolved in 3mL of anhydrous N, N-dimethylformamide to give a solution α;
s2, freezing the solution alpha into solid in liquid nitrogen, vacuumizing, filling nitrogen, repeating for 3 times, and adding 26mg (0.18 mmol) of catalyst CuBr under the protection of nitrogen atmosphere to obtain a reaction system beta;
s3, the reaction system beta is subjected to freeze-pump-thawing cycle twice and then degassing, is sealed under vacuum condition, and reacts for 24 hours at 40 ℃ to obtain a reaction system gamma;
s4, precipitating the reaction system gamma into cold diethyl ether, centrifuging for 20min under the condition of 12000r/min, collecting precipitate, and drying overnight under vacuum to obtain solid delta;
s5, dissolving the solid delta in water, filling into a dialysis bag (MWCO: 3500 Da), dialyzing in ultrapure water for 3 days, and freeze-drying to obtain white powder, namely polypeptide PEG-GPLGVRGDG-NH with MMP responsiveness 2 Calculated, the yield was 55.8%;
s6, taking 55.4mg of polypeptide PEG-GPLGVRGDG-NH with MMP responsiveness 2 Dissolving in 1mL sodium borate buffer solution (20 mM, pH 8.0-8.5), slowly adding 7.2mg of acrylic acid-N-succinimidyl ester into the reaction solution under intense stirring, and reacting at 4 ℃ for 8 hours to obtain solid epsilon;
s7, dialyzing the solid epsilon in PBS phosphate buffer solution (pH=7.4) with the concentration of 20mM for 24 hours, and then freeze-drying to obtain white powder, namely RGD targeting peptide-bonded polyethylene glycol (PEG-GPLGVRGDG-Alkene) with the acrylamide MMP responsiveness, wherein the yield is 52.6% through calculation.
Example 6 preparation and characterization of upconversion fluorescent nanoparticle UCNP
The embodiment is a preparation method of up-conversion fluorescent nanoparticle UCNP, comprising the following steps in sequence:
s1, taking 2mL of acetic acid hydrate Y (CH) containing yttrium (III) 3 CO 2 ) 3 Ytterbium (III) acetate hydrate Yb (CH) 3 CO 2 ) 3 And thulium acetate hydrate Tm (CH) 3 COOH) 3 Is [ n (Y) 3+ ):n(Yb 3+ ):n(Tm 3+ )＝79:20:1]Mixing with 90% Oleic Acid (OA), and reacting at 150deg.C for 30 min;
y (CH) in the present embodiment 3 CO 2 ) 3 The concentration of Yb (CH) is 0.395mol/L 3 CO 2 ) 3 The concentration of (C) is 0.1mol/L, tm (CH) 3 COOH) 3 The concentration of (C) is 0.005mol/L;
s2, adding 90% of 1-Octadecene (ODE) into the solution for continuous reaction for 30 minutes, cooling to 50 ℃, adding sodium hydroxide-methanol with the concentration of 0.17M and ammonium fluoride (NH) with the concentration of 0.27M 4 F) After the temperature of the methanol mixed solution is raised to 100 ℃, the mixture is kept for 30 minutes under vacuum to obtain a reaction system zeta;
s3, heating the reaction system zeta to 290 ℃, reacting for 2 hours under a nitrogen flow, cooling to room temperature, and washing with ethanol to obtain the up-conversion fluorescent nanoparticle UCNP.
EXAMPLE 7 preparation of an acrylamido polyethylene glycol modified UCNP (UCNP@PEG)
The embodiment is a preparation method of an acrylamide polyethylene glycol modified UCNP (UCNP@PEG), comprising the following steps in sequence:
s1, UCNP precipitation is obtained by mixing ethanol and centrifuging UCNP dispersed by cyclohexane;
s2. Adding a mixture of ethanol and HCl (0.2M) after collecting UCNP precipitate, and centrifuging at 16000rpm for 25min twice to obtain solid eta;
s3, washing the solid eta with ethanol and dispersing the solid eta in ultrapure water, diluting UCNP to a sodium borate solution with the pH value of 8, adding 0.2mg of carboxyl-PEG-acrylamide, reacting overnight, washing the solid eta with deionized water for 5 times, and finally dispersing the solid eta in 1mL of deionized water to obtain UCNP modified by acrylamide polyethylene glycol (UCNP@PEG);
s4, dispersing UCNP@PEG in 4mL of water for electron microscope characterization, wherein as shown in figure 7, the morphology of the UCNP@PEG is uniformly elliptical, and the particle size is about 20nm;
the up-conversion emission spectrum of UCNP@PEG is shown in FIG. 8, and it can be seen that: the excitation wavelength of UCNP@PEG is 980nm, and the emission peaks are 365nm and 475nm, which shows that under the irradiation of excitation light 980nm, UCNP@PEG can emit ultraviolet light 365nm and 475 nm.
EXAMPLE 8 preparation of UCNP@N-pNO System
The embodiment is a preparation method of a UCNP@N-pNO system, which comprises the following steps in sequence:
s1, ANTP (10 mg) and PEG 2000 Alkene (3.34 mg), N-methylenebisacrylamide (0.86 mg) and 2-methacryloyloxyethyl phosphorylcholine (4.29 mg) were dissolved in 0.5mL of ultrapure water to obtain a solution I;
s2, diluting 500 mu L of UCNP@PEG with the concentration of 2mg/mL into 5mL of ethanol, and adding 15 mu L of ammonium persulfate with the concentration of 133.3mg/mL and 4 mu L of N, N, N ', N' -tetramethyl ethylenediamine to obtain a solution II;
s3, dropwise adding the solution II into the solution I, culturing for 2 hours under a nitrogen atmosphere, centrifuging for 15 minutes at 12000rpm, and cleaning with ultrapure water for 3 times to obtain the UCNP@N-pNO system.
Wherein, ANTP, 2-methacryloyloxyethyl phosphorylcholine and PEG in S1 2000 -molar ratio of Alkene and N, N-methylenebisacrylamide = 5:5:0.2:1;
the electron microscope data of UCNP@N-pNO system are shown in FIG. 9: the morphology of UCNP@N-pNO with non-MMP responsiveness shows that a layer of uniform gel layer exists on the surface of UCNP, and the particle size is about 40 nm.
EXAMPLE 9 preparation of UCNP@S-pNO System
The embodiment is a preparation method of a UCNP@S-pNO system, which comprises the following steps in sequence:
s1, ANTP (10 mg) and PEG 2000 -GPLGVRGDG-Alkene (3.34 mg), N-methylenebisacrylamide (0.86 mg) and 2-methacryloyloxyethyl phosphorylcholine (4.29 mg) were dissolved in 0.5mL of ultrapure water to obtain a solution I';
s2, diluting 500 mu L of UCNP@PEG with the concentration of 2mg/mL into 5mL of ethanol, and adding 15 mu L of ammonium persulfate with the concentration of 133.3mg/mL and 4 mu L of N, N, N ', N ' -tetramethyl ethylenediamine to obtain a solution II ';
s3, dropwise adding the solution II 'into the solution I', culturing for 2 hours under a nitrogen atmosphere, centrifuging at 12000rpm for 15min, and washing with ultrapure water for 3 times to obtain the UCNP@S-pNO system.
Wherein, ANTP, 2-methacryloyloxyethyl phosphorylcholine, N-methylenebisacrylamide and PEG in S1 2000 -GPLGVRGDG-Alkene molar ratio = 5:5:1:0.2.
The morphology of the MMP-responsive near infrared light responsive nitric oxide nano generator gel in this example was observed using a transmission electron microscope, and the specific steps were:
sucking 10 mu L of the UCNP@S-pNO system prepared in the S3, placing the UCNP@S-pNO system on a transmission electron microscope sample preparation copper wire, standing the copper wire in an ultra-clean workbench at room temperature for airing, and observing the appearance of a sample under the condition of 200kV by using a normal temperature transmission electron microscope, wherein the appearance is shown in FIG. 9;
as can be seen from fig. 9: the UCNP@S-pNO gel particles have the advantages that the surface of the UCNP inner core with high lining degree in the UCNP@S-pNO gel particles is provided with a layer of organic matter coating layer with lower lining degree, so the result shows that the UCNP@S-pNO gel particles with the UCNP inner core and the surface coated with the organic gel layer are successfully prepared.
Example 10 characterization of particle size potential Properties of UCNP@S-pNO System
The particle size and potential properties of the MMP-responsive near infrared light responsive nitric oxide nano generator gels in examples 8 and 9 were measured using a malvern zetasizer dynamic light scattering nano-sizer, the samples prepared according to examples 8 and 9 were dispersed in ultrapure water, respectively, and 300 μl was sucked up and added to a nano-particle size measuring sample cell and a potential measuring sample cell, respectively, and the particle size and potential properties of the nano-gels were measured under conditions of a measuring temperature of 25 ℃, an equilibrium time of 120s, and a measuring medium of water.
As shown in fig. 10 and 11, the MMP-responsive near infrared light responsive nitric oxide nano-generator ucnp@s-pNO gel particles of examples 8 and 9, respectively, were a nano-particle size distribution image and a surface potential image. From the image, UCNP@S-pNO gel particles have uniform particle size, about 50nm, surface potential of-25 mV and strong electronegativity.
Example 11 near infrared light triggered kinetics of nitric oxide release by UCNP@S-pNO
The ultraviolet absorption peak of the matrix metalloproteinase-responsive near-infrared light-responsive nitric oxide nano-generator gel UCNP@S-pNO prepared in example 9 was detected by using an ultraviolet spectrophotometer, and the amount of released nitric oxide was calculated by the change of the ultraviolet absorption peak of the UCNP@S-pNO nano-gel before and after irradiation with near-infrared light. The specific experimental steps are as follows:
s1, measuring a base line by using ultrapure water;
s2, dispersing the nanogel UCNP@S-pNO in ultrapure water, and detecting an ultraviolet absorption peak which is not irradiated by near infrared light;
s3, using 980nm laser with the illumination power of 1.5W/cm in ice bath 2 Illumination is carried out under the condition, each illumination time is 1min, the illumination is stopped for 1min, the illumination time is calculated in an accumulated way, and each accumulated illumination time is 5min, the ultraviolet absorption spectrum of UCNP@S-pNO nanogel is detected;
s4, counting according to the reduction degree of the ultraviolet absorption peak at 385nm to obtain a UCNP@S-pNO released nitric oxide kinetic profile.
Results of the UCNP@S-pNO release nitric oxide kinetic profile are shown in FIG. 12. From the images, the ucnp@s-pNO gel particles released nitric oxide rapidly 20min before illumination, followed by a slow release rate, reaching plateau at about 50min, indicating that ucnp@s-pNO gel particles prepared according to example 9 were indeed capable of controllably and responsively releasing nitric oxide under near infrared light (980 nm laser).
Example 12UCNP@S-pNO cytotoxicity assay
The embodiment is a UCNP@S-pNO cytotoxicity detection experiment, which specifically comprises the following steps in sequence:
s1, culturing murine breast cancer 4T1 cells in a DMEM complete medium containing 10% fetal calf serum and 1% double antibodies, wherein each well is 5 multiplied by 10 3 Cells were seeded at a density of 100. Mu.L per 100. Mu.L in 96-well plates at 37℃with 5% CO 2 Culturing for 24 hours under the condition that the cell density reaches about 70%;
s2, the UCNP@S-pNO gel particles prepared in the example 9 and UCNP@N-pNO gel particles used for control are dispersed in a DMEM complete medium containing 10% of fetal calf serum and 1% of diab at a concentration of 0.5mg/mL to prepare a stock solution, and the stock solution is diluted by 1/2 of the medium to obtain 9 concentrations;
s3, sucking out the original culture medium in the S1, adding diluted diluents with various concentrations for continuous culture for 48 hours, sucking out the diluents containing gel particles, adding MTT diluent with the concentration of 1mg/mL (diluted by DMEM complete culture medium containing 10% fetal calf serum and 1% double antibody), and incubating for 4 hours at 37 ℃ to form blue-black formazan;
s4, carefully sucking out the supernatant, adding 100 mu LDMSO into each hole, incubating for 15min at 37 ℃, measuring absorbance at 570nm by using a multifunctional enzyme-labeled instrument ID5, and calculating the cell survival rate by using the absorbance;
the calculation formula is as follows: cell viability (%) = (OD sample -OD blank /OD PBS -OD blank )×100％
Wherein OD sample Absorbance for the experimental group; OD (optical density) blank Absorbance for the blank group without cells added; OD (optical density) PBS Absorbance for control group with cells but without gel particles.
As shown in FIG. 13, the concentration of UCNP@S-pNO gel particles prepared in example 9 reaches 250 mug/mL, and the cell activity is still higher than 90%, which indicates that UCNP@S-pNO has good biocompatibility.
Example 13 detection of cytotoxicity after near-infrared light Combined UCNP@S-pNO treatment
The embodiment is a cytotoxicity detection experiment after the treatment of the near infrared light and UCNP@S-pNO, and specifically comprises the following steps of:
s1, culturing murine breast cancer 4T1 cells in a DMEM complete medium containing 10% fetal calf serum and 1% double antibodies, wherein each well is 5 multiplied by 10 3 Cells were seeded at a density of 100. Mu.L per 100. Mu.L in 96-well plates at 37℃with 5% CO 2 Culturing for 24 hours under the condition that the cell density reaches about 70%;
s2, UCNP@S-pNO gel particles prepared in example 9 and UCNP@N-pNO gel particles used for control were dispersed in a DMEM complete medium containing 10% fetal bovine serum and 1% diabody at a concentration of 0.5mg/mL to prepare a stock solution. Diluting the stock solution with the culture medium by 1/2, and diluting to 9 concentrations;
s3, sucking out the original culture medium in the S1, adding diluted diluents with various concentrations for continuous culture for 48 hours, and using a 980nm laser with illumination power of 1.5W/cm 2 Illuminating under the condition that each time of illumination is 1min, stopping for 1min, and accumulating illumination time for 10min;
s4, sucking out the diluent containing gel particles after illumination, adding MTT diluent with the concentration of 1mg/mL (diluted by DMEM complete medium containing 10% of fetal calf serum and 1% of double antibody), and incubating for 4 hours at 37 ℃ to form blue-black formazan;
s5, carefully sucking out the supernatant, adding 100 mu LDMSO into each hole, incubating for 15min at 37 ℃, measuring absorbance at 570nm by using a multifunctional enzyme-labeled instrument ID5, and calculating the cell survival rate by using the absorbance;
the calculation formula is as follows: cell viability (%) = (OD sample -OD blank /OD PBS -OD blank )×100％
Wherein OD sample Absorbance for the experimental group; OD (optical density) blank Absorbance for the blank group without cells added; OD (optical density) PBS Absorbance for control group with cells but without gel particles.
The UCNP@S-pNO gel particles of example 9 were indeed capable of exhibiting significant cytotoxicity upon irradiation with near infrared light (980 nm laser) as shown in FIG. 14, and it was found from FIG. 14 that the NO produced by the UCNP@S-pNO gel particles was capable of causing cytotoxicity.

Claims (7)

1. A near infrared light responsive nitric oxide nano generator for targeting tumor, characterized in that the nitric oxide nano generator is of a core-shell structure;
the core-shell structure is characterized in that the shell is a gel layer formed by in-situ free radical polymerization of a functional monomer, a cross-linking agent and an initiator, and the inner core is an up-conversion luminescent rare earth material modified by acrylated polyethylene glycol;
the size of the inner core is 20-60 nm, and the size of the outer shell is 10-20 nm;
the functional monomers comprise 2-methacryloxyethyl phosphorylcholine, an acrylated nitric oxide donor and an RGD targeting peptide-bonded polyethylene glycol which is acrylamide matrix metalloproteinase responsive;
the cross-linking agent is N, N-methylene bisacrylamide;
the initiator is ammonium persulfate;
the up-conversion luminescent rare earth material modified by the propenyl polyethylene glycol is UCNP@PEG;
among the RGD targeting peptide-bonded polyethylene glycol to which the acrylamide matrix metalloproteinase is responsive, the amino acid sequence of the polypeptide is GPLGVRGDG, CPENFFGRGDSG, VPLGVRGDK, PVGLIGRGDK, GGPLGVRGDK or GPQGIWGGCRGDK;
the acrylated nitric oxide donor is an acrylamide modified 4-nitro-3-trifluoromethyl halogenated benzene derivative ANTP, and the structural formula is as follows:
。
2. the tumor-targeted near infrared light responsive nitric oxide nano-generator of claim 1, wherein said 2-methacryloxyethyl phosphorylcholine, acrylated nitric oxide donor, acrylamido matrix metalloproteinase responsive RGD targeting peptide-bound polyethylene glycol and N, N-methylenebisacrylamide are in a molar ratio of 5:5:0.2:1.
3. the tumor-targeted near infrared light responsive nitric oxide nano-generator according to claim 1 or 2, wherein said polyethylene glycol has a molecular weight of 500-5000.
4. A method for preparing a tumor-targeted near infrared light responsive nitric oxide nano-generator according to any of claims 1-3, comprising the following steps, performed in sequence:
s1, synthesizing UCNP modified by hydrophobic oleic acid through a solvothermal method;
s2, exchanging oleic acid ligand on the surface of the oleic acid modified UCNP by using a ligand exchange method and using carboxyl-PEG-acrylamide to obtain hydrophilic UCNP@PEG with the surface modified by PEG;
s3, resuspension the hydrophilic UCNP@PEG, adding a functional monomer, a cross-linking agent and an initiator to perform in-situ free radical polymerization, wherein an MMP responsive gel layer formed on the surface of the inner core is taken as an outer shell, and the tumor-targeted near infrared light response nitric oxide nano generator is obtained.
5. The method of claim 4, wherein the method of preparing the acrylated nitric oxide donor comprises the steps of:
s1, taking 5-chloro-2-nitro benzotrifluoride and 1, 6-hexamethylenediamine to carry out halogenation reaction to obtain a 4-nitro-3-trifluoromethyl halogenated benzene derivative;
and reacting the amino group of the S2.4-nitro-3-trifluoromethyl halogenated benzene derivative with acryloyl chloride to obtain the acrylamide modified 4-nitro-3-trifluoromethyl halogenated benzene derivative, namely the acrylated nitric oxide donor.
6. The method for preparing a tumor-targeted near infrared light-responsive nitric oxide nano-generator according to claim 4 or 5, wherein the preparation method of the RGD targeting peptide-bonded polyethylene glycol with the matrix metalloproteinase responsiveness is as follows:
s1, taking azido polyethylene glycol and alkynyl matrix metalloproteinase response peptide to bond under the catalysis of CuBr, and obtaining RGD targeting peptide-bonded polyethylene glycol PEG-GPLGVRGDG-NH with amino matrix metalloproteinase responsiveness 2 、PEG-CPENFFGRGDSG-NH 2 、PEG-VPLGVRTGDK-NH 2 、PEG-PVGLIGRGDK-NH 2 、PEG-GGPLGVRGDK-NH 2 Or PEG-GPQGIWGGCRGDK-NH 2 ；
S2, reacting the RGD targeting peptide-bonded polyethylene glycol of the amination matrix metalloproteinase with acrylic acid-N-succinimidyl ester in sodium borate buffer solution to obtain the RGD targeting peptide-bonded polyethylene glycol PEG-GPLGVRGDG-Alkene, PEG-CPENFFGRGDSG-Alkene, PEG-VPLGVRTGDK-Alkene, PEG-PVGLIGRGDK-Alkene, PEG-GGPLGVRGDK-Alkene or PEG-GPQGIWGGCRGDK-Alkene of the acrylamide matrix metalloproteinase.
7. Use of a tumor-targeted near infrared light responsive nitric oxide nano-generator according to any of claims 1-3 for the preparation of a therapeutic tumor and an antibacterial drug.
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