CN115969994A - Core-shell structure nano ozone delivery controllable release system for targeting tumor, preparation method and application - Google Patents
Core-shell structure nano ozone delivery controllable release system for targeting tumor, preparation method and application Download PDFInfo
- Publication number
- CN115969994A CN115969994A CN202211223523.3A CN202211223523A CN115969994A CN 115969994 A CN115969994 A CN 115969994A CN 202211223523 A CN202211223523 A CN 202211223523A CN 115969994 A CN115969994 A CN 115969994A
- Authority
- CN
- China
- Prior art keywords
- nano
- ozone
- emulsion
- tumor
- shell structure
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 title claims abstract description 100
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 88
- 239000011258 core-shell material Substances 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 230000008685 targeting Effects 0.000 title claims abstract description 15
- 239000002105 nanoparticle Substances 0.000 claims abstract description 61
- 239000000839 emulsion Substances 0.000 claims abstract description 58
- TXEYQDLBPFQVAA-UHFFFAOYSA-N tetrafluoromethane Chemical compound FC(F)(F)F TXEYQDLBPFQVAA-UHFFFAOYSA-N 0.000 claims abstract description 25
- YHTTWXCDIRTOQX-FQJIPJFPSA-N (6S,9S,15S,18R,23R,26S,29S)-18-amino-6-(4-aminobutyl)-9,26-bis(carboxymethyl)-15-[3-(diaminomethylideneamino)propyl]-2,5,8,11,14,17,25,28-octaoxo-20,21-dithia-1,4,7,10,13,16,24,27-octazabicyclo[27.3.0]dotriacontane-23-carboxylic acid Chemical compound NCCCC[C@@H]1NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]2CCCN2C(=O)CNC1=O)C(O)=O YHTTWXCDIRTOQX-FQJIPJFPSA-N 0.000 claims abstract description 18
- 239000000243 solution Substances 0.000 claims abstract description 18
- 229960005540 iRGD Drugs 0.000 claims abstract description 17
- 229920001606 poly(lactic acid-co-glycolic acid) Polymers 0.000 claims abstract description 11
- 230000001804 emulsifying effect Effects 0.000 claims abstract description 7
- 241001465754 Metazoa Species 0.000 claims abstract description 4
- 125000004122 cyclic group Chemical group 0.000 claims abstract description 4
- 238000013270 controlled release Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 238000003756 stirring Methods 0.000 claims description 12
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000004945 emulsification Methods 0.000 claims description 10
- 238000009210 therapy by ultrasound Methods 0.000 claims description 8
- 230000009471 action Effects 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 5
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 238000002560 therapeutic procedure Methods 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 230000001225 therapeutic effect Effects 0.000 claims 1
- 210000004881 tumor cell Anatomy 0.000 abstract description 16
- 230000002147 killing effect Effects 0.000 abstract description 3
- 239000011259 mixed solution Substances 0.000 abstract 1
- 239000007789 gas Substances 0.000 description 18
- 230000000694 effects Effects 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 12
- 238000010586 diagram Methods 0.000 description 12
- 229920002873 Polyethylenimine Polymers 0.000 description 11
- 239000003642 reactive oxygen metabolite Substances 0.000 description 11
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 10
- 239000001301 oxygen Substances 0.000 description 10
- 229910052760 oxygen Inorganic materials 0.000 description 10
- 229920002125 Sokalan® Polymers 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 239000004584 polyacrylic acid Substances 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 230000006870 function Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 4
- 230000036542 oxidative stress Effects 0.000 description 4
- 238000001959 radiotherapy Methods 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- 208000022679 triple-negative breast carcinoma Diseases 0.000 description 4
- 230000022534 cell killing Effects 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 230000012202 endocytosis Effects 0.000 description 3
- 230000000149 penetrating effect Effects 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- 230000005778 DNA damage Effects 0.000 description 2
- 231100000277 DNA damage Toxicity 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 102000004207 Neuropilin-1 Human genes 0.000 description 2
- 108090000772 Neuropilin-1 Proteins 0.000 description 2
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002639 hyperbaric oxygen therapy Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 206010009244 Claustrophobia Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 206010048554 Endothelial dysfunction Diseases 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 241001180976 Nonea Species 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 229920002385 Sodium hyaluronate Polymers 0.000 description 1
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 125000003545 alkoxy group Chemical group 0.000 description 1
- 210000003484 anatomy Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000000298 carbocyanine Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 108091092356 cellular DNA Proteins 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 210000005069 ears Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000008694 endothelial dysfunction Effects 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 201000000459 head and neck squamous cell carcinoma Diseases 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 108010021518 integrin beta5 Proteins 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 231100000783 metal toxicity Toxicity 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000002640 oxygen therapy Methods 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229950011087 perflunafene Drugs 0.000 description 1
- UWEYRJFJVCLAGH-IJWZVTFUSA-N perfluorodecalin Chemical compound FC1(F)C(F)(F)C(F)(F)C(F)(F)[C@@]2(F)C(F)(F)C(F)(F)C(F)(F)C(F)(F)[C@@]21F UWEYRJFJVCLAGH-IJWZVTFUSA-N 0.000 description 1
- 238000009520 phase I clinical trial Methods 0.000 description 1
- 208000019899 phobic disease Diseases 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 229920001601 polyetherimide Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000006950 reactive oxygen species formation Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 229940010747 sodium hyaluronate Drugs 0.000 description 1
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000007910 systemic administration Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 231100000057 systemic toxicity Toxicity 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 230000008728 vascular permeability Effects 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a core-shell structure nano ozone delivery controllable release system for targeting tumors, a preparation method and application, wherein the preparation method comprises the following steps: adding perfluorocarbon into a PVA solution, emulsifying, and adding a PLGA solution into an emulsion to obtain a layer of core-shell structure nano-particles B1; step 2: adding PEI into the B1 emulsion to obtain a nanoparticle B2 emulsion with a two-layer core-shell structure; and step 3: adding PAA into the B2 emulsion to obtain three-layer core-shell structure nanoparticle emulsion B3; and 4, step 4: adding B3 into a mixed solution of NHS, EDC and octapeptide cyclic iRGD to obtain a nano-gas delivery controllable release system emulsion with a four-layer core-shell structure; and 5: and introducing ozone to obtain the tumor-targeted nano ozone delivery controllable release system emulsion. The tumor-targeted nano ozone delivery system has a four-layer core-shell structure, can deliver ozone to tumor parts in human bodies or animal bodies in a targeted manner, can control the release of ozone by means of microwaves and the like, and achieves the purpose of killing tumor cells.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a core-shell structure nano ozone delivery controllable release system for targeting tumors, a preparation method and application.
Background
Increasing the yield of Reactive Oxygen Species (ROS) in tumor cells, breaking the redox balance in tumors and leading the tumors to generate oxidative stress, is one of the classic ideas for treating tumors. Currently there are two main approaches to delivering ROS to tumor cells:
1. oxygen is delivered. Oxygen is one of the main raw materials of ROS, and clinical high-pressure oxygen therapy is a treatment strategy for directly supplementing oxygen to tumors to improve the yield of ROS. High-pressure oxygen treatment and radiotherapy can further exert the synergistic effect, and in the ionizing radiation process, oxygen participates in the formation of ROS and alkoxy, and above components induce DNA chain breakage and DNA damage respectively and fix, can produce a large amount of ROS when alleviating tumour oxygen deficiency microenvironment, play the effect of radiotherapy sensitization. Clinical experiments with hyperbaric oxygen therapy in combination with radiotherapy have reported inhibition of head and neck squamous cell carcinoma (NCT 00474825) and metastatic brain tumor (NCT 01850563). However, systemic excess of ROS feedstock due to hyperbaric oxygen can lead to oxidative stress, irreversible cellular DNA damage, metabolic disorders, and endothelial dysfunction. Adverse effects of hyperbaric oxygen therapy have been reported to include damage to the ears, sinuses and lungs from hyperbaric pressure, as well as temporary myopic exacerbations, claustrophobia, oxygen poisoning, and the like. Perfluorocarbons and haemoglobin are capable of delivering oxygen specifically to tumors, however the oxygen carrying capacity of the above carriers is quite limited.
2. Delivery of hydrogen peroxide (H) 2 O 2 ). H has been confirmed in phase I clinical trials (NCT 02757651) 2 O 2 The safety and effectiveness of the co-injection of sodium hyaluronate gel into tumors in conjunction with radiotherapy, however, the administration mode of the intratumoral injection limits the application of the gel in the treatment of multifocal tumors or deep tumors. And H 2 O 2 The systemic toxicity of (A) is high, and the systemic administration of (A) is also limited.Related alternatives or e.g. using excess H in tumour cells 2 O 2 Metalloenzymes are delivered into tumor cells to induce a Fenton or Fenton-like reaction. But intracellular H 2 O 2 The substrate is limited, resulting in insufficient ROS production in tumor cells. Furthermore, the metal toxicity of the catalyst is also a non-negligible problem.
Ozone is an ideal ROS raw material, and the low-dose microwave can achieve the purpose of releasing ozone adsorbed on perfluorocarbon and promote the ozone to generate hydroxyl free radicals more quickly. The activity of generating ROS by ozone is greater than that of oxygen and H 2 O 2 Delivery of the same amount of ozone to the tumor can achieve satisfactory ROS production compared to the latter two. However, ozone is unstable and strongly oxidizing, and direct input of ozone into the body poses a high risk of toxicity. Finding a proper ozone carrier and realizing the in vivo delivery and the controlled release of the ozone is beneficial to applying the ozone in clinical work.
Disclosure of Invention
The invention provides a high-efficiency stable tumor-targeted core-shell structure nano ozone delivery controllable release system, a preparation method and application aiming at the problems in the prior art.
The technical scheme adopted by the invention is as follows:
a preparation method of a core-shell structure nano ozone delivery controllable release system for targeting tumors comprises the following steps:
step 1: adding perfluorocarbon into a PVA solution, and emulsifying to obtain emulsion A1; adding a PLGA solution into the emulsion A1, emulsifying for the second time to obtain an emulsion A2, and volatilizing the solvent to obtain a nano particle B1 with a layer of core-shell structure;
and 2, step: adding polyethyleneimine PEI into the nanoparticle B1 emulsion obtained in the step 1, and fully stirring for reaction to obtain a nanoparticle B2 emulsion with a two-layer core-shell structure;
and 3, step 3: adding polyacrylic acid (PAA) into the nano-particle emulsion B2 obtained in the step 2, and fully stirring and reacting to obtain nano-particle emulsion B3 with a three-layer core-shell structure;
and 4, step 4: and (3) adding the nano-particle emulsion B3 obtained in the step (3) into a buffer solution of a mixture of N-hydroxysuccinimide NHS, 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and octapeptide cyclic iRGD, and fully stirring for reaction to obtain the nano-gas delivery controllable release system emulsion with the target tumor function and the four-layer core-shell structure.
Further, the emulsification process in the step 1 is as follows:
carrying out ultrasonic treatment on the solution with the ultrasonic power of 50W for multiple times; first, ultrasound t 1 Time, then pause t 2 Time, cycle N1 times altogether.
Further, the secondary emulsification process in the step 1 is as follows:
carrying out ultrasonic treatment on the solution with the ultrasonic power of 50W for multiple times; first of all, ultrasonic t 1 Time then pause t 2 Time, cycle N2 times in total.
Further, the mass ratio of the perfluorocarbon to the PVA in the step 1 is 5;
further, the mass ratio of the PEI to the perfluorocarbon is 1.
Further, the mass ratio of NHS, EDC and iRGD in step 4 is 1.
A controllable release system for delivering nano ozone with a core-shell structure for targeting tumors is provided, wherein the delivery system is nanoparticles with a four-layer core-shell structure, an innermost shell is PLGA, a second shell is PEI, a third shell is PAA, and an outermost layer is iRGD; the outermost layer iRGD has a tumor targeting function; the emulsion in the innermost shell is perfluorocarbon; the average particle size of the nano particles is 150-250 nm; the releasing means includes microwaves and the like.
The application of a core-shell structure nano ozone delivery controllable release system for targeting tumors is an auxiliary preparation for delivering ozone to a target site of a human body or an animal body in microwave therapy.
An application method comprises the steps of introducing ozone gas into nano ozone delivery system emulsion targeted to tumors at the speed of 0.5NL/min and 2min/mL to obtain ozone-rich targeted nano delivery system emulsion, incubating the ozone-rich targeted nano delivery system emulsion and tumor cells together, and controllably releasing ozone under the action of microwaves by the ozone-rich targeted nano delivery system to kill the tumor cells.
The invention has the beneficial effects that:
(1) The tumor-targeted nano-gas delivery controllable release system obtained by the invention loads ozone, specifically delivers the ozone to the tumor and promotes endocytosis of the tumor, and under the action of microwaves, the ozone can be rapidly released in a large quantity and can be also decomposed into hydroxyl free radicals at the same time, so that the redox balance in the tumor cells is broken, the oxidative stress of the tumor cells is realized, and the effect of obviously killing the tumor cells is achieved;
(2) According to the invention, the perfluorocarbon emulsion is wrapped in a four-layer core-shell structure, and perfluorocarbon is combined with ozone by the principle of similarity and intermiscibility, so that the ozone load capacity and particle stability of the nanoparticles are increased.
(3) The tumor penetrating peptide iRGD in the nano-gas delivery controllable release system is a bifunctional ligand containing a tumor targeting motif (RGD) and a tissue penetrating motif CendR, can target tumors, and promotes local penetration and endocytosis of nanoparticles. The iRGD-coupled nanoparticles can obviously promote the enrichment of tumor parts of the nanoparticles by targeting highly expressed alpha v beta 3/alpha v beta 5 integrin receptors and neuropilin-1 (NRP-1) in tumor tissues, so that the effective load of the drugs on the tumor parts is improved; meanwhile, the tissue penetrating motif CendR can stimulate vascular permeability in a VEGF-like manner, effectively promote the nanoparticles to pass through an endothelial cell barrier from tumor vessels to enter tumor parenchyma, and can target and deliver the nanoparticles to a tumor site in a human body or an animal body.
(4) The ozone release means in the nano-gas delivery controllable release system of the invention includes but is not limited to microwaves, and the ozone can be controllably released by controlling the irradiation time and power of the microwaves.
Drawings
FIG. 1 is a schematic diagram of the synthesis process and the test of the synthesis result of the nano ozone delivery controlled release system of the present invention. In the figure, PFD is perfluorodecalin; PEI is polyethyleneimine; PAA polyacrylic acid; iRGD is an octameric peptide loop, and Ozone is Ozone.
Fig. 2 is the in vivo biological distribution diagram of the tumor-targeted nano ozone delivery controllable release system obtained by the invention. And DiR is lipophilic dialkyl carbocyanine dye.
Fig. 3 is a diagram of the cell killing effect of the tumor-targeted nano ozone delivery controlled release system obtained by the present invention. 4T1 is a mouse triple negative breast cancer cell line; MDA-MB-468 is a triple negative breast cancer cell line; blank is Blank control group; iPP is a group of nanoparticles without ozone; MW is microwave group; iPP @ MW is the set of microwave-controlled ozone-free nanoparticle-releasing gases, iPPO is the set of ozone-containing nanoparticles, and iPPO @ MW is the set of microwave-controlled ozone-containing nanoparticle-releasing gases (set of nanoozone delivery controlled-release systems).
FIG. 4 is a schematic diagram showing the effect of the tumor-targeted nano ozone delivery controllable release system on a mouse subcutaneous tumor model. 4T1 is a mouse triple negative breast cancer cell line; MDA-MB-468 is a human triple negative breast cancer cell line; blank is Blank control group; iPP is a group of nanoparticles without ozone; MW is a microwave group, iPO is an ozone-containing nanoparticle group, and anti-PD-1 is anti-PD-1 group; iPPO @ MW is the microwave controlled ozone-containing nanoparticle release ozone group (nano ozone delivery controlled release system group); the anti-PD-1+ iPO reverse MW is the anti-PD-1 combined nano ozone delivery controllable release system group.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
A preparation method of a core-shell structure nano ozone delivery controllable release system for targeting tumors comprises the following steps:
step 1: adding perfluorocarbon into a PVA solution, and emulsifying to obtain emulsion A1; adding a PLGA solution into the emulsion A1, carrying out secondary emulsification to obtain emulsion A2, and volatilizing the solvent to obtain nano-particles B1 with a layer of core-shell structure; the mass ratio of perfluorocarbon to PVA is 3:1-5, the mass ratio of PLGA to perfluorocarbon is 1:7-1;
the emulsification process is as follows:
carrying out ultrasonic treatment on the solution with the ultrasonic power of 50W for multiple times; first of all, ultrasonic t 1 Time then pause t 2 Time, cycle N1 times altogether.
The secondary emulsification process is as follows:
carrying out ultrasonic treatment on the solution with the ultrasonic power of 50W for multiple times; first of all, ultrasonic t 1 Time, then pause t 2 Time, cycle N2 times in total.
Step 2: adding PEI into the nanoparticle B1 emulsion obtained in the step 1, and fully stirring for reaction to obtain a nanoparticle B2 emulsion with a two-layer core-shell structure; the mass ratio of the PEI to the perfluorocarbon is 5:9-5.
And step 3: adding PAA into the nano-particle B2 emulsion obtained in the step 2, and fully stirring for reaction to obtain nano-particle emulsion B3 with a three-layer core-shell structure; the mass ratio of PAA to perfluorocarbon is 1:9-1.
And 4, step 4: and (3) adding the nano-particle emulsion B3 obtained in the step (3) into a buffer solution of a mixture of N-hydroxysuccinimide NHS, 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and octatomic peptide cyclic iRGD, and fully stirring for reaction to obtain the nano-gas delivery controllable release system emulsion with the four-layer core-shell structure and the target tumor function. The mass ratio of NHS, EDC and iRGD is 1.
a tumor-targeted core-shell structure nano ozone delivery controllable release system obtained by the preparation method is characterized in that the delivery system is nanoparticles with a four-layer core-shell structure, an innermost shell is PLGA, a second shell is PEI, a third shell is PAA, and an outermost layer is iRGD; the iRGD has a tumor targeting function; the emulsion in the innermost shell is perfluorocarbon; the average particle size of the nano particles is 150-250 nm; the releasing means includes microwaves and the like.
Fig. 1 is a schematic diagram of the test results of the tumor-targeted nano ozone delivery system obtained in this example. Fig. 1 a is a schematic diagram of the change of the product during the preparation process, and it can be seen from the diagram that the finally obtained nano ozone delivery system has a granular structure composed of four core-shell structures, and PLGA, PEI, PAA and iRGD are respectively coated outside perfluorocarbon. When in use, ozone gas is introduced into the nano-particles and adsorbed in the perfluorocarbon. FIG. 1B is a scanning electron microscope and transmission electron microscope image of the observed nano-gas delivery system, from which it can be seen that the obtained nano-ozone delivery system is in a spherical structure with a diameter of 150-250 nm. Figure 1C is a graphical representation of the particle diameter size change of the nanoozone delivery system at different pH conditions. Fig. 1D is a schematic diagram of the potential of the nanoparticles under different pH conditions, and it can be seen that the resulting nanoozone delivery system has better stability. Fig. 1E is a schematic diagram of the release of ozone from a nano ozone delivery system under the action of microwaves of different powers, and it can be seen that perfluorocarbons can controllably release ozone from the power under the action of microwaves. FIG. 1F is a diagram showing the ability of perfluorocarbons to release hydroxyl radicals under microwave irradiation as determined by the terephthalic acid assay, showing that a large number of hydroxyl radicals can be released under microwave irradiation.
A preparation method of a core-shell structure nano ozone delivery system for targeting tumors comprises the following steps:
step 1: adding 200ul of perfluorocarbon into 6ml of PVA solution, and emulsifying to obtain emulsion A1; dissolving 30mg of PLGA in 2ml of dichloromethane, adding the emulsion A1, emulsifying for the second time to obtain emulsion A2, and volatilizing the solvent to obtain the nano-particles B1 with a layer of core-shell structure; emulsion A2 was rotary evaporated under reduced pressure and the dichloromethane evaporated.
The emulsification process is as follows:
carrying out ultrasonic treatment on the solution with the ultrasonic power of 50W for multiple times; sonication was first carried out for 5s, then paused for 2s, for a total of 20 cycles.
The secondary emulsification process is as follows:
carrying out ultrasonic treatment on the solution at the ultrasonic power of 50W for multiple times; sonication was first carried out for 5s, then paused for 2s, for a total of 20 cycles.
Step 2: adding 100mg of PEI into the nanoparticle B1 emulsion obtained in the step 1, and fully stirring for 20 minutes at 37 ℃ to obtain a nanoparticle B2 emulsion with a two-layer core-shell structure;
and step 3: and (3) adding 20mg of PAA into the nano-particle B2 emulsion obtained in the step (2), and fully stirring and reacting at 37 ℃ to obtain nano-particle emulsion B3 with a three-layer core-shell structure.
And 4, step 4: and (3) adding the nanoparticle emulsion B3 obtained in the step (3) into PBS buffer solution of a mixture of 90mg of N-hydroxysuccinimide NHS, 90mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and 90mg of octapeptide cyclo iRGD, and fully stirring for reaction to obtain the nano gas delivery system emulsion with the four-layer core-shell structure and the target tumor function.
And (5) introducing ozone into the nano gas delivery system emulsion of the targeted tumor obtained in the step (4) to obtain the targeted nano particles rich in ozone.
The in vivo distribution of a tumor-targeting core-shell structure nano-gas delivery controllable release system comprises the following steps:
step 1: the female Balb/C mice were injected subcutaneously into the left back of the body at 5X 10 5 The 4T1 cells establish a mouse subcutaneous tumor model.
Step 2: when tumor is mentioned, the tumor size reaches 500mm 3 In the process, after the nano gas delivery particles loaded with the fluorescent dye DiR are injected into tail veins, the distribution condition of the nano particles is observed at different time points by using the excitation light of 748nm and the emission light of 780 nm.
Fig. 2 is the in vivo biodistribution diagram of the tumor-targeted nano ozone delivery controlled release system obtained according to the steps. It can be seen that the nanoparticles of the present invention can be effectively accumulated at the tumor site, and can be accumulated at the tumor site as early as 1 hour after intravenous infusion, reaching a peak at 24 hours.
An in vitro cell killing effect detection of a tumor-targeted core-shell structure nano ozone delivery controllable release system comprises the following steps:
step 1: the 4T1 and MDA-MB-468 cell lines were plated in 96-well plates at 1000/well and the cell supernatants at 100. Mu.L/well.
Step 2: after 24 hours, cells were divided into blank control groups, noneA set of ozone-containing nanoparticles, a set of microwave-controlled ozone-free nanoparticle-releasing gas, a set of ozone-containing nanoparticles, a set of microwaves, and a set of microwave-controlled ozone-containing nanoparticles-releasing ozone (set of nanoozone-delivery-controllable release systems), each set having 6 multiple wells. The administration concentration of the nano particles is 200 mu g/mL, and the microwave power is 0.5W/cm 2 And 2 minutes.
And step 3: after 4 hours of group treatment, thiazole blue (MTT, 5mg/mL, 10. Mu.L) was added to each well of the 96-well plate and incubated for 4 hours in the dark.
And 4, step 4: cell supernatants were aspirated, 100. Mu.L of Tris-SDS solution was added to each well, incubated for 4 hours in the dark, absorbance read at 570nm, cell activity calculated, and comparisons between groups were made.
FIG. 3 is a graph showing the cell killing effect of the nano-ozone delivery system obtained as described above under the action of low-dose microwaves. From FIG. 4, it can be seen that 4T1 and MDA-MB-468 cells treated by the nano ozone delivery controlled release system group were effectively inhibited from proliferation.
An in vivo tumor inhibition effect of a tumor-targeting core-shell structure nano-gas delivery controllable release system comprises the following steps:
step 1: 5X 10 subcutaneous injection on both dorsal sides of female Balb/C mice 5 4T1 cells establish a mouse subcutaneous tumor model.
Step 2: measuring the tumor volume every 2 days until the tumor volume reaches 100mm 3 When the mice were divided into a blank control group, a nanoparticle group without ozone, a nanoparticle group with ozone, a microwave group, an anti-PD-1 group, a group in which ozone-containing nanoparticles are controlled by microwave to release ozone (a nano-ozone delivery controlled release system group), and an anti-PD-1 combined nano-ozone delivery controlled release system group, each group consisting of 6 mice. The administration concentration of the nano particles is 200 mug/mL, and the administration is carried out in tail vein; the microwave power is 0.5W/cm 2 2min, only left tumor was irradiated; the administration concentration of the anti-PD-1 is 2.5mg/kg, and the intraperitoneal administration is carried out.
And 3, step 3: tumor volumes were measured every 2 days and recorded, and the mean tumor volume reached 1500mm in any group of mice 3 Mice were sacrificed at time. Dissecting mouse tumor, taking picture and weighingAnd (4) heavy. Tumor growth curves, tumor weights, tumor volumes were compared for each group of mice.
FIG. 4 is a schematic diagram showing the effect of the tumor-targeted nano ozone delivery controllable release system obtained according to the above steps on a mouse subcutaneous tumor model. Fig. 4A is a treatment flow chart. From the mouse tumor anatomy figures, tumor growth curves, tumor volumes and tumor quality figures of fig. 4B-4H, it can be seen that the nano ozone delivery controlled release system group can effectively inhibit the growth of tumor tissues on the treatment side, and the combined immunosuppressant anti-PD-1 can achieve the growth inhibition effect on bilateral tumors.
When the tumor-targeted nano ozone delivery controllable system is prepared, perfluorocarbon is prepared into emulsion at first, and then the emulsion is wrapped in a four-layer core-shell structure; the perfluorocarbon is combined with ozone by the similarity and intermiscibility principle, so that the ozone load capacity and the particle stability of the nano particles are increased; the nano gas delivery system loads ozone, the ozone is delivered to the tumor in a targeted mode through the specificity of the iRGD functional group, and the endocytosis of the tumor is promoted, under the action of microwaves, the ozone can be released in a large amount, the speed of decomposing into hydroxyl free radicals can be accelerated, the redox balance in the tumor cells is broken, the oxidative stress of the tumor cells is realized, and the effect of obviously killing the tumor cells is achieved; the content of hydroxyl free radicals in the tumor cells is increased through the controllable release of ozone, and the generation of immunogenic death of the tumor cells is promoted.
Claims (9)
1. A preparation method of a core-shell structure nano ozone delivery controllable release system for targeting tumors is characterized by comprising the following steps:
step 1: adding perfluorocarbon into a PVA solution, and emulsifying to obtain emulsion A1; adding a PLGA solution into the emulsion A1, carrying out secondary emulsification to obtain emulsion A2, and volatilizing the solvent to obtain nano-particles B1 with a layer of core-shell structure;
step 2: adding PEI into the nanoparticle B1 emulsion obtained in the step 1, and fully stirring for reaction to obtain a nanoparticle B2 emulsion with a two-layer core-shell structure;
and step 3: adding PAA into the nano-particle B2 emulsion obtained in the step 2, and fully stirring for reaction to obtain nano-particle emulsion B3 with a three-layer core-shell structure;
and 4, step 4: adding the nano-particle emulsion B3 obtained in the step 3 into a buffer solution of a mixture of N-hydroxysuccinimide NHS, 1-ethyl- (3-dimethylaminopropyl) carbodiimide EDC and octatomic peptide cyclic iRGD, fully stirring for reaction, and introducing ozone to obtain a nano-gas delivery controllable release system emulsion B4 with a tumor-targeted four-layer core-shell structure;
and 5: and (3) introducing ozone into the nano gas delivery system emulsion of the targeted tumor to obtain the nano ozone delivery controllable release system emulsion of the targeted tumor.
2. The preparation method of the tumor-targeting core-shell structure nano ozone delivery controllable release system according to claim 1, wherein the emulsification process in the step 1 is as follows:
carrying out ultrasonic treatment on the solution with the ultrasonic power of 50W for multiple times; first of all, ultrasonic t 1 Time, then pause t 2 Time, cycle N1 times in total.
3. The preparation method of the tumor-targeting core-shell structure nano ozone delivery controllable release system according to claim 1, wherein the secondary emulsification process in the step 1 is as follows:
carrying out ultrasonic treatment on the solution with the ultrasonic power of 50W for multiple times; first of all, ultrasonic t 1 Time, then pause t 2 Time, cycle N2 times in total.
4. The preparation method of the tumor-targeting nucleocapsid structure nano ozone delivery controllable release system according to claim 1, characterized in that the mass ratio of the perfluorocarbon to PVA in the step 1 is 3:1-5, the mass ratio of PLGA to perfluorocarbon is 1:7-1;
5. the preparation method of the tumor-targeting core-shell structure nano ozone delivery controlled release system according to claim 1, wherein the mass ratio of PEI and perfluorocarbon is 5:9-5, and the mass ratio of PAA and perfluorocarbon is 1:9-1.
6. The method for preparing the core-shell structure nano ozone delivery controlled release system according to claim 1, wherein the mass ratio of NHS, EDC and iRGD in the step 4 is 1.
7. The tumor-targeting core-shell structure nano ozone delivery controllable release system obtained by any one of the preparation methods of claims 1 to 6, wherein the delivery system is a nanoparticle with a four-layer core-shell structure, the inner core is a perfluorocarbon droplet, the innermost shell is PLGA, the second shell is PEI, the third shell is PAA, and the outermost layer is iRGD; the average particle size of the nano particles is 150-250 nm; the releasing means includes microwaves and the like.
8. Use of the tumor targeting nucleocapsid structure nanoozone delivery controlled release system according to claim 7 for the delivery of ozone to an adjunctive therapeutic formulation of a target site in a human or animal body in microwave therapy.
9. The use method of the application as claimed in claim 8, wherein ozone gas is introduced into the nano ozone delivery system emulsion targeted to tumor at a speed of 0.5NL/min and 2min/mL to obtain the ozone-rich targeted nano delivery controlled release system emulsion, and the ozone-rich targeted nano delivery system can release ozone under the action of microwave.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211223523.3A CN115969994A (en) | 2022-10-08 | 2022-10-08 | Core-shell structure nano ozone delivery controllable release system for targeting tumor, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211223523.3A CN115969994A (en) | 2022-10-08 | 2022-10-08 | Core-shell structure nano ozone delivery controllable release system for targeting tumor, preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115969994A true CN115969994A (en) | 2023-04-18 |
Family
ID=85972688
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211223523.3A Pending CN115969994A (en) | 2022-10-08 | 2022-10-08 | Core-shell structure nano ozone delivery controllable release system for targeting tumor, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115969994A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1431909A (en) * | 2000-01-19 | 2003-07-23 | B·于 | Combinations for treating neoplasms |
| CN101549153A (en) * | 2008-04-02 | 2009-10-07 | 于保法 | Composite and method for self-treating tumor |
| RU2010102087A (en) * | 2010-01-25 | 2011-07-27 | Елена Александровна Аркадьева (RU) | METHOD FOR INTEGRATED TREATMENT OF TUMOR DISEASES BASED ON OZONE THERAPY |
| CN113101269A (en) * | 2021-04-15 | 2021-07-13 | 四川大学华西医院 | Delivery system based on nano-liposome, preparation method and application |
| WO2022186802A1 (en) * | 2021-03-01 | 2022-09-09 | Sabanci Ahmet Uemit | Liposomal ozone nanosolutions |
-
2022
- 2022-10-08 CN CN202211223523.3A patent/CN115969994A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1431909A (en) * | 2000-01-19 | 2003-07-23 | B·于 | Combinations for treating neoplasms |
| CN101549153A (en) * | 2008-04-02 | 2009-10-07 | 于保法 | Composite and method for self-treating tumor |
| RU2010102087A (en) * | 2010-01-25 | 2011-07-27 | Елена Александровна Аркадьева (RU) | METHOD FOR INTEGRATED TREATMENT OF TUMOR DISEASES BASED ON OZONE THERAPY |
| WO2022186802A1 (en) * | 2021-03-01 | 2022-09-09 | Sabanci Ahmet Uemit | Liposomal ozone nanosolutions |
| CN113101269A (en) * | 2021-04-15 | 2021-07-13 | 四川大学华西医院 | Delivery system based on nano-liposome, preparation method and application |
Non-Patent Citations (4)
| Title |
|---|
| LINLIN SONG等: "Improvement of TNBC immune checkpoint blockade with a microwave-controlled ozone release nanosystem", JOURNAL OF CONTROLLED RELEASE, vol. 351, 12 October 2022 (2022-10-12), pages 954 - 969 * |
| LINLIN SONG等: "Improvement of TNBC immune checkpoint blockade with a microwave-controlled ozone release nanosystem", JOURNAL OF CONTROLLED RELEASE, vol. 351, 30 September 2022 (2022-09-30), pages 954 - 969 * |
| YANCHU LI等: "Ozone therapy for breast cancer: an integrative literature review", INTEGRATIVE CANCER THERAPIES, vol. 23, 23 January 2024 (2024-01-23), pages 1 - 9 * |
| 邱世香等: "臭氧治疗肿瘤的研究进展", 西部医学, vol. 31, no. 12, 31 December 2019 (2019-12-31), pages 1966 - 1969 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sahu et al. | Improving cancer therapy through the nanomaterials-assisted alleviation of hypoxia | |
| Gao et al. | Recent advancement of imidazolate framework (ZIF-8) based nanoformulations for synergistic tumor therapy | |
| Wei et al. | Oxygen self-sufficient photodynamic therapy | |
| Zhu et al. | Free radical as a double-edged sword in disease: Deriving strategic opportunities for nanotherapeutics | |
| Yang et al. | Recent advances in nanosized metal organic frameworks for drug delivery and tumor therapy | |
| CN109846856B (en) | Bio-enzyme catalysis gas production anti-tumor bionic nanoparticle and preparation method thereof | |
| Glass et al. | Redox potential and ROS-mediated nanomedicines for improving cancer therapy | |
| Ruan et al. | Nanomaterials for tumor hypoxia relief to improve the efficacy of ROS-generated cancer therapy | |
| CN113599520B (en) | Porphyrin lipid-perfluorocarbon nano preparation and preparation method and application thereof | |
| CN113101269B (en) | Delivery system based on nano-liposome, preparation method and application | |
| CN105126113B (en) | A kind of preparation method and applications of hollow mesoporous copper sulfide/Artesunate nanoparticle of Surface-modified by Transferrin | |
| Du et al. | Ultrasound-targeted delivery technology: a novel strategy for tumor-targeted therapy | |
| Han et al. | The development of live microorganism-based oxygen shuttles for enhanced hypoxic tumor therapy | |
| Yi et al. | Manipulate tumor hypoxia for improved photodynamic therapy using nanomaterials | |
| Chai et al. | Carbon monoxide therapy: a promising strategy for cancer | |
| Zhang et al. | Reactive oxygen species-based nanotherapeutics for head and neck squamous cell carcinoma | |
| CN109125723A (en) | Compound sound sensitiser, preparation method, application, application method, purposes and pharmaceutical composition | |
| Ma et al. | Multifunctional nano MOF drug delivery platform in combination therapy | |
| Yoon et al. | Macrophage-reprogramming upconverting nanoparticles for enhanced TAM-mediated antitumor therapy of hypoxic breast cancer | |
| Li et al. | Cell Membrane-based Biomimetic Technology for Cancer Phototherapy: Mechanisms, Recent Advances and Perspectives | |
| CN112641942B (en) | Near-infrared light control nano gas diagnosis and treatment agent and preparation method and application thereof | |
| CN109620801B (en) | Composite nano micelle for multi-mode treatment of nasopharyngeal carcinoma and preparation method and application thereof | |
| CN110115763B (en) | Near-infrared light activated multifunctional liposome and preparation method and application thereof | |
| CN106606783B (en) | A kind of targeting is passed altogether to be released the drug of photosensitizer and chemotherapeutics and passs release system | |
| CN115969994A (en) | Core-shell structure nano ozone delivery controllable release system for targeting tumor, preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |