CN116059360A - Intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheet and preparation method and application thereof - Google Patents
Intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheet and preparation method and application thereof Download PDFInfo
- Publication number
- CN116059360A CN116059360A CN202310127719.0A CN202310127719A CN116059360A CN 116059360 A CN116059360 A CN 116059360A CN 202310127719 A CN202310127719 A CN 202310127719A CN 116059360 A CN116059360 A CN 116059360A
- Authority
- CN
- China
- Prior art keywords
- black phosphorus
- nano
- phosphorus nano
- bpnss
- sheet
- 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
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 title claims abstract description 198
- 239000002135 nanosheet Substances 0.000 title claims abstract description 131
- 239000000017 hydrogel Substances 0.000 title claims abstract description 79
- 239000002131 composite material Substances 0.000 title claims abstract description 60
- 230000004298 light response Effects 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 229920001223 polyethylene glycol Polymers 0.000 claims abstract description 167
- 239000006185 dispersion Substances 0.000 claims abstract description 74
- 239000002202 Polyethylene glycol Substances 0.000 claims abstract description 73
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 64
- 239000007788 liquid Substances 0.000 claims abstract description 63
- 239000003814 drug Substances 0.000 claims abstract description 53
- 150000004985 diamines Chemical class 0.000 claims abstract description 43
- 229940079593 drug Drugs 0.000 claims abstract description 42
- 238000002156 mixing Methods 0.000 claims abstract description 38
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 37
- 229940044683 chemotherapy drug Drugs 0.000 claims abstract description 29
- 239000000523 sample Substances 0.000 claims abstract description 28
- 230000008961 swelling Effects 0.000 claims abstract description 19
- 238000002604 ultrasonography Methods 0.000 claims abstract description 18
- 239000011159 matrix material Substances 0.000 claims abstract description 16
- 239000000843 powder Substances 0.000 claims abstract description 16
- 239000002064 nanoplatelet Substances 0.000 claims description 35
- 238000009210 therapy by ultrasound Methods 0.000 claims description 35
- 239000002244 precipitate Substances 0.000 claims description 31
- -1 diamine modified black phosphorus Chemical class 0.000 claims description 30
- 238000005119 centrifugation Methods 0.000 claims description 24
- 238000003760 magnetic stirring Methods 0.000 claims description 22
- 239000006228 supernatant Substances 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 19
- 201000008968 osteosarcoma Diseases 0.000 claims description 16
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 claims description 13
- 239000005457 ice water Substances 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 12
- 239000003960 organic solvent Substances 0.000 claims description 10
- 229920001983 poloxamer Polymers 0.000 claims description 9
- 229960000502 poloxamer Drugs 0.000 claims description 9
- 229940127089 cytotoxic agent Drugs 0.000 claims description 8
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 7
- 235000010413 sodium alginate Nutrition 0.000 claims description 7
- 229940005550 sodium alginate Drugs 0.000 claims description 7
- 239000000661 sodium alginate Substances 0.000 claims description 7
- 206010028980 Neoplasm Diseases 0.000 abstract description 16
- 238000011068 loading method Methods 0.000 abstract description 12
- 239000012221 photothermal agent Substances 0.000 abstract description 8
- 230000001976 improved effect Effects 0.000 abstract description 7
- 230000001788 irregular Effects 0.000 abstract description 7
- 239000002086 nanomaterial Substances 0.000 abstract description 4
- 239000003937 drug carrier Substances 0.000 abstract description 3
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 150
- 210000004027 cell Anatomy 0.000 description 46
- 229960004679 doxorubicin Drugs 0.000 description 27
- 230000003833 cell viability Effects 0.000 description 19
- 239000000499 gel Substances 0.000 description 18
- 239000008367 deionised water Substances 0.000 description 17
- 229910021641 deionized water Inorganic materials 0.000 description 17
- 238000011282 treatment Methods 0.000 description 17
- 238000005406 washing Methods 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 13
- 230000000694 effects Effects 0.000 description 13
- 210000004881 tumor cell Anatomy 0.000 description 13
- 239000008055 phosphate buffer solution Substances 0.000 description 11
- 238000002512 chemotherapy Methods 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 238000000338 in vitro Methods 0.000 description 9
- 239000013049 sediment Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 238000010609 cell counting kit-8 assay Methods 0.000 description 8
- 230000004048 modification Effects 0.000 description 7
- 238000012986 modification Methods 0.000 description 7
- 238000009792 diffusion process Methods 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 230000005855 radiation Effects 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 230000000259 anti-tumor effect Effects 0.000 description 5
- 201000011510 cancer Diseases 0.000 description 5
- 230000000973 chemotherapeutic effect Effects 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 230000001186 cumulative effect Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000002349 favourable effect Effects 0.000 description 5
- 239000012737 fresh medium Substances 0.000 description 5
- 238000011065 in-situ storage Methods 0.000 description 5
- 230000001960 triggered effect Effects 0.000 description 5
- 108010087230 Sincalide Proteins 0.000 description 4
- 238000003917 TEM image Methods 0.000 description 4
- HPZOOQSXPMEJBV-ODCFVKFUSA-N Tirilazad mesylate Chemical compound CS(O)(=O)=O.O=C([C@@H]1[C@@]2(C)CC=C3[C@@]4(C)C=CC(=O)C=C4CC[C@H]3[C@@H]2C[C@H]1C)CN(CC1)CCN1C(N=1)=CC(N2CCCC2)=NC=1N1CCCC1 HPZOOQSXPMEJBV-ODCFVKFUSA-N 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 230000003013 cytotoxicity Effects 0.000 description 4
- 231100000135 cytotoxicity Toxicity 0.000 description 4
- 238000001878 scanning electron micrograph Methods 0.000 description 4
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 230000002195 synergetic effect Effects 0.000 description 4
- 238000003556 assay Methods 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000036760 body temperature Effects 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000000857 drug effect Effects 0.000 description 3
- 230000012202 endocytosis Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000002147 killing effect Effects 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- 206010067484 Adverse reaction Diseases 0.000 description 2
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical group CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000006838 adverse reaction Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000013267 controlled drug release Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000004299 exfoliation Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 229910052698 phosphorus Inorganic materials 0.000 description 2
- 238000007626 photothermal therapy Methods 0.000 description 2
- 229920001993 poloxamer 188 Polymers 0.000 description 2
- 229940044519 poloxamer 188 Drugs 0.000 description 2
- 229920001992 poloxamer 407 Polymers 0.000 description 2
- 229940044476 poloxamer 407 Drugs 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000002271 resection Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 231100000057 systemic toxicity Toxicity 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 230000007675 toxicity by organ Effects 0.000 description 2
- 231100000155 toxicity by organ Toxicity 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 206010005949 Bone cancer Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 208000017787 Paraneoplastic neurologic syndrome Diseases 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000010478 bone regeneration Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000005918 in vitro anti-tumor Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000013532 laser treatment Methods 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 125000004437 phosphorous atom Chemical group 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910001392 phosphorus oxide Inorganic materials 0.000 description 1
- 238000002428 photodynamic therapy Methods 0.000 description 1
- 239000003504 photosensitizing agent Substances 0.000 description 1
- 238000011248 postoperative chemotherapy Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000002536 stromal cell Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- VSAISIQCTGDGPU-UHFFFAOYSA-N tetraphosphorus hexaoxide Chemical compound O1P(O2)OP3OP1OP2O3 VSAISIQCTGDGPU-UHFFFAOYSA-N 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- 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
Abstract
The invention belongs to the technical field of biomedical engineering nano materials, and provides an intelligent near infrared light response composite hydrogel based on black phosphorus nano sheets, and a preparation method and application thereof. Firstly, stripping black phosphorus powder into black phosphorus nano-sheets by water bath ultrasound and probe ultrasound to prepare dispersion liquid, wherein the black phosphorus nano-sheets are used as photo-thermal agents to convert light energy into heat energy and are used as drug carriers to efficiently load chemotherapeutic drugs; then polyethylene glycol diamine is adopted to modify the black phosphorus nano-sheet, so that the physiological stability and biocompatibility of the black phosphorus nano-sheet can be improved; and then loading the chemotherapeutic drugs, mixing with a matrix of the composite hydrogel and swelling, wherein the hydrogel has an irregular loose porous structure, and the black phosphorus nano-sheets are physically adsorbed on the surfaces of the cavities of the hydrogel, so that the black phosphorus nano-sheets can be prevented from being directly released into tumor tissues in a short time without control, and thus, the sustainable and slow release of the drugs is realized.
Description
Technical Field
The invention relates to the technical field of biomedical engineering nano materials, in particular to an intelligent near infrared light response composite hydrogel based on black phosphorus nano sheets, and a preparation method and application thereof.
Background
Malignant tumors seriously affect the life health of humans. Osteosarcoma is a common primary malignant bone tumor that originates in mesenchymal tissue and is characterized by malignant spindle-shaped stromal cells that produce bone-like tissue, frequently found in adolescents. At present, the clinical treatment mainly adopts the postoperative chemotherapy and radiotherapy of surgical resection (limb protection) in combination, and although patients are effectively improved after healing, the intractable and invasive properties of osteosarcoma tumors, the metastatic properties and the incompleteness of sarcoma surgical resection can promote tumor recurrence. The traditional chemotherapy mode has the problems of low bioavailability, drug resistance, systemic and organ toxicity, serious complications and the like which are difficult to solve, so that the multifunctional targeting nano-drug preparation based on the EPR effect becomes a research hot spot. However, heterogeneity of vasculature across species and tumor types and variability of other parameters of the tumor microenvironment lead to a controversial EPR effect. The insufficient accumulation of the nano-drug preparation at the tumor part and the poor pharmacokinetics are main factors affecting the drug effect. The tumor local administration technology has attracted wide attention because of the advantages of strong targeting, low adverse reaction and high curative effect.
Conventional photothermal agents have serious limitations such as low photothermal conversion efficiency (PTCE), low biosafety and non-biodegradability, which limit their clinical transformation and use. The advent of Black Phosphorus Nanoplatelets (BPNSs) now provides an opportunity to address this clinical challenge. Black Phosphorus (BP) is formed by stacking planar structures with unique P-atom corrugations, and layers are connected together by van der waals forces, so that a black phosphorus nanoplatelet can be prepared by a simple liquid phase exfoliation method. The unique photoelectric characteristic of black phosphorus, which has a tunable direct band gap dependent on the number of layers, tuning from 0.3eV for bulk to 2.0eV for single layer, allows for broad absorption bands from uv to nir and high photothermal conversion efficiency. The black phosphorus nano-sheet can be used for efficiently loading medicines due to an ultrathin two-dimensional structure and a lattice structure of a corrugated honeycomb plane, and an efficient nano-scale medicine delivery carrier is provided for photo-thermal-chemo-cooperative anti-tumor treatment. The black phosphorus can be rapidly degraded under the conditions of water and oxygen to generate nontoxic phosphorus oxide, P is an indispensable element in human body and bones, and the metabolic products generated by the degradation of the black phosphorus nano-sheets at the bone defect can complete the biological mineralization of phosphorus driving and calcium extraction so as to realize bone regeneration after osteosarcoma treatment, which indicates that the black phosphorus nano-sheets have good biocompatibility and biodegradability. The black phosphorus nano-sheet is used as a high-efficiency photosensitizer and can generate singlet oxygen for photodynamic therapy.
However, local injection of a single chemotherapeutic agent suffers from high loss rate, short duration of drug effect, insufficient local drug concentration, and the like, and frequent injection of invasive drugs is required, which brings endless pain to patients. Therefore, there is a need for an intelligent near-infrared light response composite hydrogel capable of realizing sustainable and slow release of drugs, convenient to use and even capable of promoting bone repair, which is used for photothermal-chemotherapy cooperative treatment of osteosarcoma.
Disclosure of Invention
The invention aims to provide an intelligent near-infrared light response composite hydrogel based on black phosphorus nano-sheets, and a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheets, which comprises the following steps:
(1) Mixing black phosphorus powder and an organic solvent, placing the mixture in an ice water bath, sequentially carrying out water bath ultrasonic treatment and probe ultrasonic treatment, centrifuging at 2000-4000 rpm, collecting supernatant, centrifuging the supernatant at a rotating speed of over 12000rpm, collecting precipitate, and finally mixing the precipitate with water to obtain black phosphorus nanosheet dispersion;
(2) Mixing the black phosphorus nano-sheet dispersion liquid obtained in the step (1) with polyethylene glycol diamine, sequentially performing first ultrasonic treatment, first magnetic stirring and first centrifugation, collecting precipitate, and adding water to obtain polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid;
(3) Mixing the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid obtained in the step (2) with a chemotherapeutic drug, sequentially carrying out second ultrasonic treatment, second magnetic stirring and second centrifugation, collecting precipitate, adding water, and obtaining the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid loaded with the chemotherapeutic drug;
(4) And (3) mixing the black phosphorus nano-sheet dispersion liquid modified by polyethylene glycol diamine and loaded with the chemotherapeutic drugs and the matrix of the composite hydrogel, and swelling to obtain the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheets.
Preferably, the volume ratio of the mass of the black phosphorus powder to the organic solvent in the step (1) is 1: (1-2) mg/mL.
Preferably, the power of the water bath ultrasonic in the step (1) is 200-500W, and the time of the water bath ultrasonic is 8-12 h; the power of the probe ultrasound is 180-720W, the time of the probe ultrasound is 8-12 h, and the on/off period of the probe ultrasound is 2-3 s/3s.
Preferably, the centrifugation time in the step (1) is 10-20 min at 2000-4000 rpm; the centrifugation time is 20-30 min at the rotating speed of 12000rpm or more.
Preferably, in the step (2), the mass ratio of the black phosphorus nanoplatelets to the polyethylene glycol diamine in the black phosphorus nanoplatelet dispersion is 1: (5-10).
Preferably, the mass ratio of the black phosphorus nanoplatelets to the chemotherapeutic agent in the polyethylene glycol diamine modified black phosphorus nanoplatelet dispersion liquid in the step (3) is 1: (1-8).
Preferably, the swelling temperature in the step (4) is 4 ℃, and the swelling time is more than or equal to 24 hours.
Preferably, the matrix of the composite hydrogel in the step (4) comprises, based on 100% of the mass of the composite hydrogel: poloxamer 40718-21%, poloxamer 1883-5% and sodium alginate 0.4-0.8%.
The invention also provides the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheet, which is prepared by the preparation method.
The invention also provides application of the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheet in preparing anti-osteosarcoma medicines.
The invention provides a preparation method of intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheets, which comprises the following steps: (1) Mixing black phosphorus powder and an organic solvent, placing the mixture in an ice water bath, sequentially carrying out water bath ultrasonic treatment and probe ultrasonic treatment, centrifuging at 2000-4000 rpm, collecting supernatant, centrifuging the supernatant at a rotating speed of over 12000rpm, collecting precipitate, and finally mixing the precipitate with water to obtain black phosphorus nanosheet dispersion; (2) Mixing the black phosphorus nano-sheet dispersion liquid obtained in the step (1) with polyethylene glycol diamine, sequentially performing first ultrasonic treatment, first magnetic stirring and first centrifugation, collecting precipitate, and adding water to obtain polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid; (3) Mixing the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid obtained in the step (2) with a chemotherapeutic drug, sequentially carrying out second ultrasonic treatment, second magnetic stirring and second centrifugation, collecting precipitate, adding water, and obtaining the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid loaded with the chemotherapeutic drug; (4) And (3) mixing the black phosphorus nano-sheet dispersion liquid modified by polyethylene glycol diamine and loaded with the chemotherapeutic drugs and the matrix of the composite hydrogel, and swelling to obtain the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheets. Firstly, stripping black phosphorus powder into black phosphorus nano-sheets by water bath ultrasound and probe ultrasound to prepare dispersion liquid, wherein the black phosphorus nano-sheets can be used as a photothermal agent to efficiently convert light energy into heat energy under 808nm near infrared light, and simultaneously are used as drug carriers to efficiently load chemotherapeutic drugs; then polyethylene glycol diamine is adopted to modify the black phosphorus nano-sheet, and the polyethylene glycol diamine is coated on the surface of the black phosphorus nano-sheet, so that the physiological stability and biocompatibility of the black phosphorus nano-sheet can be improved; and then loading the chemotherapeutic drugs, mixing with a matrix of the composite hydrogel and swelling, wherein the hydrogel has an irregular loose porous structure, and the black phosphorus nano-sheets are physically adsorbed on the surfaces of the cavities of the hydrogel, so that the black phosphorus nano-sheets can be prevented from being directly released into tumor tissues in a short time without control, and thus, the sustainable and slow release of the drugs is realized. The results of the examples show that after the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheet prepared by the preparation method provided by the invention is subjected to near infrared light triggering drug release for the last 96 hours, the accumulated drug release rates of PBS (phosphate buffer solution) at pH7.4 and pH5.0 respectively reach 72.8+/-1.9% and 82.0+/-1.6%.
The intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheet, which is prepared by the preparation method, can be injected near a osteosarcoma focus by an in-situ injection method, and can rapidly complete sol-gel phase transition at body temperature to form a semisolid medicine storage. Due to the three-dimensional blocking effect of physical adsorption and medicine diffusion of the hydrogel at body temperature, the black phosphorus nano-sheets loaded with the chemotherapeutic medicine, which are uniformly distributed in the cavity of the hydrogel, are released outwards very slowly, under the irradiation of 808nm near infrared light, the hydrogen bond in the composite hydrogel is destroyed by the rising of the environment temperature induced by the black phosphorus nano-sheets, the composite hydrogel structure is destroyed even because of being close to the other critical gel temperature, the molecular diffusion effect of the black phosphorus nano-sheets is enhanced by the rising of the temperature, the black phosphorus nano-sheets are released from the gel rapidly, when the near infrared laser is turned off, the heat of the gel part is dissipated, the temperature is reduced, the hydrogen bond in the composite hydrogel with thermal reversibility is recovered, the highly porous structure is reconstructed, the release speed of the black phosphorus nano-sheets is slowed down again, and the process can be repeatedly circulated by controlling the 808nm near infrared light switch according to the requirement of the administration time, so as to realize sustainable and intelligent near infrared light response release of the black phosphorus nano-sheets as required. Meanwhile, the black phosphorus nano-sheet entering the cells through endocytosis can release surface-loaded chemotherapeutic drugs under the induction of near infrared light and generate a large amount of heat at the same time, so that the synergistic killing of the osteosarcoma cells by photo-thermal-chemotherapy can be realized, and due to the drug storage function of the hydrogel, the combined treatment can be stably and repeatedly performed for a long time according to the condition requirement, so that the purpose of thoroughly killing tumor cells and effectively inhibiting the recurrence and metastasis of tumors can be achieved.
Drawings
FIG. 1 is a TEM image of BPNSs prepared in example 1 of the present invention;
FIG. 2 is a TEM image of BPNSs-PEG prepared in example 1 of the present invention;
FIG. 3 is an SEM image of BPNSs-PEG/DOX@Hydrogel prepared in example 1 of the present invention;
FIG. 4 is an SEM image of BPNSs-PEG/DOX in the internal microcavity of a gel of BPNSs-PEG/DOX@Hydrogel prepared in example 1 of the present invention;
FIG. 5 is an EDS diagram of BPNSs-PEG/DOX in the microcavity inside the gel of BPNSs-PEG/DOX@Hydrogel prepared in example 1 of the present invention;
FIG. 6 is a graph showing the temperature change with irradiation time under 808nm laser irradiation at different power densities for BPNSs-PEG/DOX@Hydrogel prepared in example 1 of the present invention;
FIG. 7 shows deionized water, blank hydrogels and various concentrations of BPNSs-PEG/DOX@Hydrogel prepared in example 1 of the present invention at a power density of 1W/cm 2 A plot of temperature as a function of irradiation time for 808nm laser radiation;
FIG. 8 is a graph of photo-thermal stability of BPNSs-PEG/DOX@Hydrogel prepared in example 1 of the present invention;
FIG. 9 shows the power densities of 1W at 0 day and 7 days, respectively, for BPNSs, BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel prepared in example 1 of the present invention/cm 2 Temperature dependence under 808nm laser radiation irradiation time variation of irradiation time variation of;
FIG. 10 shows the power density of BPNSs-PEG/DOX@Hydrogel prepared in example 1 of the present invention at 1W/cm 2 A temperature change curve graph of natural cooling after photo-thermal temperature rise and laser closing under 808nm laser radiation;
FIG. 11 is a plot of t-ln (θ) curve-fitted linearly to the cooling curve of FIG. 10;
FIG. 12 is a graph showing cumulative release kinetics of DOX under near infrared light triggering at various pH values for BPNSs-PEG/DOX prepared in example 1 of the present invention;
FIG. 13 is a graph showing the cumulative release profile of DOX at different pH values and near infrared light triggering for BPNSs-PEG/DOX@Hydrogel prepared in example 1 of the present invention;
FIG. 14 is a bar graph of cell viability after 48h incubation of Hydrogel and BPNSs, BPNSs-PEG, BPNSs-PEG@Hydrogel prepared in example 1 of the present invention with different cancer cells;
FIG. 15 is a bar graph of cell viability after 48h incubation of BPNSs, BPNSs-PEG and BPNSs-PEG@Hydrogel prepared in example 1 of the present invention with K7M2-WT cells at varying concentrations of BP;
FIG. 16 shows BPNSs, BPNSs-PEG and BPNSs-PEG@Hydrogel prepared in example 1 of the present invention incubated with K7M2-WT cells for 4h at various concentrations of BP, then at a power density of 1.0W/cm 2 For 10min, and then continuing to incubate the cell viability histogram after 20 h;
FIG. 17 is a bar graph of cell viability after 24h incubation of K7M2-WT cells with free DOX, BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel prepared in example 1 of the present invention at various concentrations of DOX;
FIG. 18 shows the incubation of free DOX, BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel with K7M2-WT cells prepared in example 1 of the present invention for 4h, followed by laser-free irradiation and a power density of 1.0W/cm, respectively 2 Is irradiated for 10min with a laser at 808nm and then incubated for a further 20 h.
Detailed Description
The invention provides a preparation method of intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheets, which comprises the following steps:
(1) Mixing black phosphorus powder and an organic solvent, placing the mixture in an ice water bath, sequentially carrying out water bath ultrasonic treatment and probe ultrasonic treatment, centrifuging at 2000-4000 rpm, collecting supernatant, centrifuging the supernatant at a rotating speed of over 12000rpm, collecting precipitate, and finally mixing the precipitate with water to obtain black phosphorus nanosheet dispersion;
(2) Mixing the black phosphorus nano-sheet dispersion liquid obtained in the step (1) with polyethylene glycol diamine, sequentially performing first ultrasonic treatment, first magnetic stirring and first centrifugation, collecting precipitate, and adding water to obtain polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid;
(3) Mixing the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid obtained in the step (2) with a chemotherapeutic drug, sequentially carrying out second ultrasonic treatment, second magnetic stirring and second centrifugation, collecting precipitate, adding water, and obtaining the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid loaded with the chemotherapeutic drug;
(4) And (3) mixing the black phosphorus nano-sheet dispersion liquid modified by polyethylene glycol diamine and loaded with the chemotherapeutic drugs and the matrix of the composite hydrogel, and swelling to obtain the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheets.
In the present invention, all raw materials are commercially available products unless otherwise specified.
The invention mixes black phosphorus powder and organic solvent, then puts them into ice water bath, then sequentially carries on water bath ultrasonic and probe ultrasonic, then centrifugates and collects supernatant fluid at 2000-4000 rpm, centrifugates and collects sediment at 12000rpm, finally mixes sediment with water to obtain black phosphorus nanometer sheet dispersion liquid.
The invention is not particularly limited in the operation of mixing the black phosphorus powder and the organic solvent, and the technical scheme of solid-liquid mixing, which is well known to those skilled in the art, can be adopted.
In the present invention, the volume ratio of the mass of the black phosphorus powder to the organic solvent is preferably 1: (1-2) mg/mL, more preferably 1:2mg/mL.
In the present invention, the organic solvent is preferably N-methylpyrrolidone.
After the mixing is completed, the mixture obtained by mixing is placed in an ice-water bath, and then water bath ultrasonic and probe ultrasonic are sequentially carried out. The invention peels off the black phosphorus by combining water bath ultrasonic and probe ultrasonic, is favorable for obtaining the black phosphorus nano-sheet with small average hydrodynamic size, has the advantages of good dispersity, large specific surface area and large drug loading capacity, and has higher extinction coefficient and photo-thermal conversion efficiency, on the other hand, the invention is favorable for penetrating into tumor cells deeply, entering the tumor cells through endocytosis, cooperatively killing the tumor cells by photo-thermal effect and release of photo-thermal triggering chemotherapeutic drugs on the surface of the black phosphorus nano-sheet in the tumor cells, and in addition, the physiological stability and biocompatibility of the tumor cells can be improved by coating polyethylene glycol diamine on the surface of the black phosphorus nano-sheet.
The temperature of the ice water bath is not particularly limited, and the ice water bath commonly used in the field is adopted.
The operation of the water bath ultrasonic wave and the probe ultrasonic wave is not particularly limited, and the technical schemes of the water bath ultrasonic wave and the probe ultrasonic wave which are well known to the person skilled in the art can be adopted.
In the invention, the power of the water bath ultrasonic wave is preferably 200-500W, more preferably 250-400W; the time of the water bath ultrasonic treatment is preferably 8-12 hours, more preferably 8-10 hours.
In the invention, the power of the ultrasonic wave of the probe is preferably 180-720W, more preferably 180-540W; the ultrasonic time of the probe is preferably 8-12 hours, more preferably 8-10 hours; the on/off period of the probe ultrasound is preferably 2 to 3s/3s, more preferably 3s/3s.
After the probe ultrasound is completed, the invention centrifugates the probe ultrasound product at 2000-4000 rpm and collects the supernatant, and then centrifugates the supernatant at a rotation speed of over 12000rpm and collects the sediment. The invention is favorable for obtaining the high-quality black phosphorus nanosheets with nanoscale dimensions and atomic thickness by centrifuging at a lower rotating speed and then centrifuging at an increased rotating speed.
In the present invention, the time for the centrifugation at 2000 to 4000rpm is preferably 10 to 20 minutes, more preferably 10 to 15 minutes.
In the present invention, the centrifugation time at 12000rpm or more is preferably 20 to 30 minutes, more preferably 20 to 25 minutes.
After the precipitate is obtained, the precipitate is preferably washed, and then the precipitate and water are mixed to obtain the black phosphorus nano-sheet dispersion liquid. The washing operation is not particularly limited in the present invention, and a washing method known to those skilled in the art may be adopted. In the present invention, the agent for washing is preferably deionized water; the number of times of washing is preferably 2 to 3.
In the present invention, the amount of water to be used is preferably determined according to the desired concentration of black phosphorus nanoplatelets in the black phosphorus nanoplatelet dispersion.
After the black phosphorus nano-sheet dispersion liquid is obtained, the black phosphorus nano-sheet dispersion liquid and polyethylene glycol diamine are mixed and then sequentially subjected to first ultrasonic treatment, first magnetic stirring and first centrifugation, and then the precipitate is collected and added with water to obtain the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid. According to the invention, the polyethylene glycol diamine is adopted to modify the black phosphorus nano-sheet, and the polyethylene glycol diamine is coated on the surface of the black phosphorus nano-sheet, so that the physiological stability and biocompatibility of the black phosphorus nano-sheet can be improved.
The invention is not particularly limited in the operation of mixing the black phosphorus nanoplatelet dispersion liquid and polyethylene glycol diamine, and a mixing mode well known to those skilled in the art can be adopted.
In the present invention, the molecular weight of the polyethylene glycol diamine is preferably 2000 to 5000Da. The invention adopts polyethylene glycol diamine with molecular weight in the above range, which is beneficial to the functional modification of the black phosphorus nano-sheet surface.
In the invention, the mass ratio of the black phosphorus nanoplatelets to the polyethylene glycol diamine in the black phosphorus nanoplatelet dispersion liquid is preferably 1: (5 to 10), more preferably 1:5. the invention preferably controls the mass ratio of the black phosphorus nanosheets and the polyethylene glycol diamine in the black phosphorus nanosheet dispersion liquid within the range, and is favorable for obtaining the polyethylene glycol diamine modified black phosphorus nanosheets with good dispersibility, controllable size and excellent quality.
After the mixing is completed, the mixture obtained by mixing is sequentially subjected to first ultrasonic treatment, first magnetic stirring and first centrifugation, and then sediment is collected and added with water to obtain the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid.
The operations of the first ultrasonic treatment, the first magnetic stirring and the first centrifugation are not particularly limited, and the technical schemes of ultrasonic treatment, magnetic stirring and centrifugation, which are well known to those skilled in the art, are adopted. In the invention, the power of the first ultrasonic treatment is preferably 200-500 Hz; the time of the first ultrasonic treatment is preferably 20 to 40 minutes. In the invention, the rotating speed of the first magnetic stirring is preferably 500-1200 rpm; the temperature of the first magnetic stirring is preferably normal temperature; the time of the first magnetic stirring is preferably 4-6 hours. In the present invention, the rotational speed of the first centrifuge is preferably 13000 to 14000rpm; the time of the first centrifugation is preferably 20 to 30 minutes.
After the sediment is collected, the sediment is preferably washed firstly, and then water is added to obtain the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid. The washing operation is not particularly limited in the present invention, and a washing method known to those skilled in the art may be adopted. In the present invention, the agent for washing is preferably deionized water; the number of times of washing is preferably 2 to 3.
After the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid is obtained, the invention mixes the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid with the chemotherapeutic medicine, sequentially carries out second ultrasonic treatment, second magnetic stirring and second centrifugation, then collects sediment and adds water to obtain the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid loaded with the chemotherapeutic medicine.
The invention is not particularly limited in the operation of mixing the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid and the chemotherapeutic drug, and the mixing mode well known to those skilled in the art can be adopted.
In the invention, the mass ratio of the black phosphorus nano-sheet to the chemotherapeutic agent in the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid is preferably 1: (1 to 8), more preferably 1: (1-4). The invention preferably controls the mass ratio of the black phosphorus nano-sheet and the chemotherapeutic agent in the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid within the range, is favorable for loading the chemotherapeutic agent on the surface of the black phosphorus nano-sheet, and ensures higher drug loading rate and encapsulation rate.
In the present invention, the chemotherapeutic agent is preferably doxorubicin.
After the mixing is completed, the mixture obtained by mixing is sequentially subjected to second ultrasonic treatment, second magnetic stirring and second centrifugation, and then the precipitate is collected and added with water to obtain the black phosphorus nano-sheet dispersion liquid modified by polyethylene glycol diamine and loaded with the chemotherapeutic drugs.
The operations of the second ultrasonic treatment, the second magnetic stirring and the second centrifugation are not particularly limited, and the technical schemes of ultrasonic treatment, magnetic stirring and centrifugation, which are well known to those skilled in the art, are adopted. In the invention, the power of the second ultrasonic treatment is preferably 200-500 Hz; the time of the second ultrasonic treatment is preferably 20 to 40 minutes. In the invention, the rotating speed of the second magnetic stirring is preferably 500-1200 rpm; the temperature of the second magnetic stirring is preferably normal temperature; the time of the second magnetic stirring is preferably 24-26 hours. In the present invention, the rotation speed of the second centrifuge is preferably 13000 to 14000rpm; the time of the second centrifugation is preferably 20 to 30 minutes.
After the sediment is collected, the sediment is preferably washed firstly, and then water is added to obtain the black phosphorus nano-sheet dispersion liquid modified by polyethylene glycol diamine and loaded with the chemotherapeutic drugs. The washing operation is not particularly limited in the present invention, and a washing method known to those skilled in the art may be adopted. In the present invention, the agent for washing is preferably deionized water; the number of times of washing is preferably 2 to 3.
After the black phosphorus nano-sheet dispersion liquid of polyethylene glycol diamine modification and loading of the chemotherapeutic drugs is obtained, the black phosphorus nano-sheet dispersion liquid of polyethylene glycol diamine modification and loading of the chemotherapeutic drugs and a matrix of the composite hydrogel are mixed and then swelled, so that the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheets is obtained. According to the invention, the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid loaded with the chemotherapeutic drugs is mixed with the matrix of the composite hydrogel and swelled, the hydrogel has an irregular loose porous structure, and the black phosphorus nano-sheets can be prevented from being directly released into tumor tissues in a short time by physical adsorption on the surfaces of the cavities of the hydrogel, so that the sustainable and slow release of the drugs is realized.
The invention is not particularly limited in the operation of mixing the polyethylene glycol diamine modified and chemotherapeutic drug loaded black phosphorus nanosheet dispersion liquid and the matrix of the composite hydrogel, and adopts a mixing technical scheme well known to those skilled in the art.
In the present invention, the mixing of the black phosphorus nanoplatelet dispersion modified with polyethylene glycol diamine and loaded with a chemotherapeutic agent and the matrix of the composite hydrogel is preferably performed under stirring. In the present invention, the stirring environment is preferably an ice water bath; the stirring time is preferably 4 to 6 hours.
In the present invention, the matrix of the composite hydrogel preferably comprises, based on 100% by mass of the composite hydrogel: poloxamer 40718-21%, poloxamer 1883-5% and sodium alginate 0.4-0.8%. The molecular weight of poloxamer 407 and poloxamer 188 in the invention is smaller than 20000, so that the poloxamer can be directly discharged from the body through the kidney, and the sodium alginate can be directly biodegraded. In the invention, after the matrix of the composite hydrogel is mixed with the black phosphorus nano-sheet dispersion liquid modified by polyethylene glycol diamine and loaded with the chemotherapeutic drugs, the composite hydrogel is formed by utilizing water in the dispersion liquid.
The composite hydrogel can quickly complete sol-gel phase transition at body temperature to form a semisolid medicine storage library; the black phosphorus nano-sheet in the gel cavity rapidly generates heat by absorbing 808nm near infrared light, the switch of the near infrared light is intelligently triggered as required, the temperature rise and fall are realized to promote the hydrogel to complete the reversible expansion-contraction process, the sustainable and intelligent light-controlled drug release purpose is achieved, the drug release mode enables the drug concentration of the focus part to be maintained above a threshold value for a long time, and the heat generated by the black phosphorus nano-sheet is cooperated to kill tumor cells more thoroughly.
The swelling operation is not particularly limited in the present invention, and a technical scheme of swelling of hydrogel, which is well known to those skilled in the art, may be adopted.
In the present invention, the swelling temperature is preferably 4 ℃; the swelling time is preferably not less than 24 hours. The invention preferably controls the swelling temperature and time within the above ranges, ensuring the adequate swelling of the composite hydrogel. The invention prepares the inverse reversible three-dimensional physical crosslinking temperature-sensitive hydrogel by swelling at low temperature, and can form a drug reservoir in situ on a focus.
Firstly, stripping black phosphorus powder into black phosphorus nano-sheets by water bath ultrasound and probe ultrasound to prepare dispersion liquid, wherein the black phosphorus nano-sheets can be used as a photothermal agent to efficiently convert light energy into heat energy under 808nm near infrared light, and simultaneously are used as drug carriers to efficiently load chemotherapeutic drugs; then polyethylene glycol diamine is adopted to modify the black phosphorus nano-sheet, and the polyethylene glycol diamine is coated on the surface of the black phosphorus nano-sheet, so that the physiological stability and biocompatibility of the black phosphorus nano-sheet can be improved; and then loading the chemotherapeutic drugs, mixing with a matrix of the composite hydrogel and swelling, wherein the hydrogel has an irregular loose porous structure, and the black phosphorus nano-sheets are physically adsorbed on the surfaces of the cavities of the hydrogel, so that the black phosphorus nano-sheets can be prevented from being directly released into tumor tissues in a short time without control, and thus, the sustainable and slow release of the drugs is realized.
The invention also provides the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheet, which is prepared by the preparation method.
In the invention, the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheet preferably comprises composite hydrogel and the black phosphorus nano-sheet which is embedded in the composite hydrogel, is modified by polyethylene glycol diamine and is loaded with a chemotherapeutic medicine.
The intelligent near infrared light response composite hydrogel based on the black phosphorus nano sheet has the characteristics of high photo-thermal conversion efficiency, good photo-thermal stability, excellent biocompatibility and biodegradability, can realize sustainable and intelligent light-operated release of medicines through near infrared light induction, can realize the in-situ photo-thermal-chemotherapy cooperative treatment of osteosarcoma, and has huge clinical conversion application value. In addition, the intelligent near infrared light response composite hydrogel based on the black phosphorus nano sheet provided by the invention has the advantages of capability of realizing in-situ sustainable and intelligent near infrared light response drug release of osteosarcoma, realization of photo-thermal-chemical cooperative treatment of osteosarcoma, strong targeting, low systemic and organ toxicity, long duration of drug effect, small invasiveness, convenient application and high cure rate.
The invention also provides application of the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheet in preparing anti-osteosarcoma medicines.
The application of the intelligent near infrared light response composite hydrogel based on the black phosphorus nanosheets in preparing the osteosarcoma resistant medicament is not particularly limited, and the application of biomedical engineering nanomaterials well known to the skilled in the art in preparing the osteosarcoma resistant medicament can be adopted.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
(1) Dispersing 45mg of black phosphorus powder in 90 mLN-methylpyrrolidone, transferring the mixture into an ice water bath, performing ultrasonic treatment in a water bath under 300W for 10 hours, performing ultrasonic treatment in a probe under 360W for 10 hours, performing ultrasonic treatment on the probe for 3s/3s, performing centrifugal treatment at 4000rpm for 10 minutes, collecting supernatant, performing centrifugal treatment at 13000rpm for 20 minutes, collecting precipitate, washing with deionized water for 2 times, and finally adding deionized water to the washed precipitate to obtain black phosphorus nano-sheet dispersion with BP concentration of 200 mu g/mL, and marking as BPNSs; wherein the mass volume ratio of the black phosphorus powder to the N-methyl pyrrolidone is 1:2mg/mL;
(2) Taking 10mL of black phosphorus nanosheet dispersion liquid with BP concentration of 200 mug/mL prepared in the step (1), adding 10mg of polyethylene glycol diamine, sequentially carrying out ultrasonic treatment for 30min at 300Hz, magnetically stirring for 4h at normal temperature according to a rotating speed of 1000rpm, centrifuging for 20min at 13000rpm to obtain a precipitate, washing with deionized water for 2 times, and finally adding deionized water to the washed precipitate to obtain polyethylene glycol diamine modified black phosphorus nanosheet dispersion liquid with BP concentration of 200 mug/mL, which is denoted as BPNSs-PEG; wherein, the mass ratio of the black phosphorus nano-sheet to the polyethylene glycol diamine in the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid is 1:5, a step of;
(3) Taking 10mL of polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid with BP concentration of 200 mug/mL prepared in the step (2), adding 8mg of doxorubicin, sequentially carrying out ultrasonic treatment for 30min at 300Hz, magnetically stirring at normal temperature for 24h at a rotating speed of 1000rpm, centrifuging at 13000rpm for 20min to obtain a precipitate, washing with deionized water for 2 times, and finally adding deionized water into the washed precipitate to obtain polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid with BP concentration of 200 mug/mL and carrying doxorubicin, and marking as BPNSs-PEG/DOX; wherein, the mass ratio of the black phosphorus nano-sheet to the doxorubicin in the polyethylene glycol diamine modified and doxorubicin loaded black phosphorus nano-sheet dispersion liquid is 1:4, a step of;
(4) Taking 7.74mL of polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid with the BP concentration of 200 mug/mL and loaded with doxorubicin, adding 1.8g of poloxamer 407, 0.4g of poloxamer 188 and 0.06g of sodium alginate (based on 100% of the mass of the composite hydrogel, the matrix of the composite hydrogel is poloxamer 40718%, poloxamer 1884% and sodium alginate 0.6%), stirring for 4 hours in an ice water bath, and then fully swelling for 24 hours at 4 ℃ to obtain intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheet with the BP concentration of 200 mug/mL, which is marked as BPNSs-PEG/DOX@hydrogel.
FIG. 1 is a TEM image of BPNSs prepared in example 1. As can be seen from FIG. 1, the BPNSs are in an irregular ultrathin two-dimensional sheet shape, edges are provided with edges, the surface color is lighter and uniform, the transverse dimension is 100-400 nm, and the nanoscale sheet structure of the BPNSs is beneficial to surface modification and drug loading.
FIG. 2 is a TEM image of BPNSs-PEG prepared in example 1. As can be seen from fig. 2, BPNSs-PEG has no obvious corners and a darker surface color due to the polyethylene glycol diamine forming a carbon film on the surface of BPNSs.
FIG. 3 is an SEM image of BPNSs-PEG/DOX@Hydrogel prepared in example 1. As can be seen from FIG. 3, the BPNSs-PEG/DOX@Hydrogel has an irregular loose porous structure inside, has an irregular microcavity and a porous structure on the microcavity, and has a pore diameter of 10-20 μm, and the whole body of the BPNSs-PEG/DOX@Hydrogel has a three-dimensional physical crosslinked network structure. The structure is highly porous and the pores are communicated with each other, which is beneficial to the diffusion and release of the medicine.
FIG. 4 is an SEM image of BPNSs-PEG/DOX in the microcavities inside the gel of BPNSs-PEG/DOX@Hydrogel prepared in example 1. As can be seen from FIG. 4, BPNSs-PEG/DOX is adsorbed in the gel microcavity.
The elemental analysis of the surface of BPNSs-PEG/DOX in the internal microcavities of the gel of BPNSs-PEG/DOX@Hydrogel prepared in example 1 was performed using an X-ray energy chromatograph (EDS), and the results are shown in FIG. 5. As can be seen from FIG. 5, the sheet structure contains P, C, O, na elements, P element is attributed to BPNSs, C element and O element are attributed to PEG and DOX on the surface of BPNSs, and Na element is attributed to Na in sodium alginate + ，Na + The appearance on the surface of BPNSs may be due to Na + Electrostatically binds to the remaining negative charge sites on the surface of BPNSs.
Comparative example 1
45mg of black phosphorus powder was dispersed in 90 mLN-methylpyrrolidone, then the mixture was transferred to an ice water bath, then sonicated in a 300W water bath for 10 hours, then centrifuged at 4000rpm for 10 minutes and the supernatant was collected, then centrifuged at 13000rpm for 20 minutes and the precipitate was collected, then washed with deionized water for 2 times, and finally deionized water was added to the washed precipitate to obtain a black phosphorus nanoplatelet dispersion having a BP concentration of 200. Mu.g/mL.
Comparative example 2
45mg of black phosphorus powder was dispersed in 90 mLN-methylpyrrolidone, then the mixture was transferred to an ice water bath, then probe-sonicated at 360W for 10 hours with an on/off period of 3s/3s, then centrifuged at 4000rpm for 10min and the supernatant was collected, then centrifuged at 13000rpm for 20min and the precipitate was collected, then washed with deionized water for 2 times, and finally deionized water was added to the washed precipitate to obtain a black phosphorus nanoplatelet dispersion having a BP concentration of 200 μg/mL.
Table 1 example 1 and pair preparation of the mixture according to the ratio of 1 to 2 average hydrodynamic size of BPNSs of (c)
| Liquid phase stripping mode | Average hydrodynamic size/nm of BPNSs | |
| Comparative example 1 | Water bath ultrasonic treatment for 10h | 275.7±3.4 |
| Comparative example 2 | Ultrasonic probe for 10h | 262.3±2.2 |
| Example 1 | Water bath ultrasonic 10 h+probe ultrasonic 10h | 251.7±1.8 |
As can be taken from table 1, the average hydrodynamic size of BPNSs was obtained using different liquid phase exfoliation modes: 10h of water bath ultrasound + 10h of probe ultrasound < 10h of water bath ultrasound.
Example 2
The difference from example 1 is that 2mg of doxorubicin is added in step (3), and the mass ratio of the black phosphorus nanoplatelets to the doxorubicin in the doxorubicin-loaded black phosphorus nanoplatelet dispersion liquid modified by polyethylene glycol diamine is 1:1 and the rest of the procedure is the same as in example 1.
Example 3
The difference from example 1 is that 4mg of doxorubicin is added in step (3), and the mass ratio of the black phosphorus nanoplatelets to the doxorubicin in the doxorubicin-loaded black phosphorus nanoplatelet dispersion liquid modified by polyethylene glycol diamine is 1:2, the rest of the procedure is the same as in example 1.
Example 4
The difference from example 1 is that 6mg of doxorubicin is added in step (3), and the mass ratio of the black phosphorus nanoplatelets to the doxorubicin in the doxorubicin-loaded black phosphorus nanoplatelet dispersion liquid modified by polyethylene glycol diamine is 1:3, the rest of the procedure is the same as in example 1.
Example 5
The difference from example 1 is that 10mg of doxorubicin is added in step (3), and the mass ratio of the black phosphorus nanoplatelets to the doxorubicin in the doxorubicin-loaded black phosphorus nanoplatelet dispersion liquid modified by polyethylene glycol diamine is 1:5, the rest of the procedure is the same as in example 1.
Example 6
The difference from example 1 is that 12mg of doxorubicin is added in step (3), and the mass ratio of the black phosphorus nanoplatelets to the doxorubicin in the doxorubicin-loaded black phosphorus nanoplatelet dispersion liquid modified by polyethylene glycol diamine is 1:6, the rest of the procedure is the same as in example 1.
Example 7
The difference from example 1 is that 14mg of doxorubicin is added in step (3), and the mass ratio of the black phosphorus nanoplatelets to the doxorubicin in the doxorubicin-loaded black phosphorus nanoplatelet dispersion liquid modified by polyethylene glycol diamine is 1:7, the rest of the procedure is the same as in example 1.
Example 8
The difference from example 1 is that 16mg of doxorubicin is added in step (3), and the mass ratio of the black phosphorus nanoplatelets to the doxorubicin in the doxorubicin-loaded black phosphorus nanoplatelet dispersion liquid modified by polyethylene glycol diamine is 1:8, the rest of the procedure is the same as in example 1.
TABLE 2 drug loading rates of BPNSs-PEG in examples 1-8
As can be seen from Table 2, the capacity of BPNSs-PEG to load DOX increased significantly as the dose of DOX increased. When the dose of DOX was 8mg, the capacity of the black phosphorus nanoplatelets to load DOX was almost saturated.
Performance testing
1. Photothermal performance test
To test the photothermal performance of BPNSs-PEG/dox@hydro gel, different materials were added to a mini-transparent vial, exposed to 808nm near infrared laser, ensuring that the spot was irradiated at the center of the solution, incubated at a incubator of 23 ℃ before irradiation, the temperature at the center of the solution was monitored using a thermocouple thermometer, the temperature was recorded every 30s and plotted as a function of irradiation time.
With power densities of 0.5W/cm respectively 2 、1.0W/cm 2 、1.5W/cm 2 And 2.0W/cm 2 As a result of the irradiation of BPNSs-PEG/dox@hydro gel for 10min (BP concentration of 100 μg/mL) with near infrared light, the temperature was continuously increased with an increase in irradiation time, and the tendency of temperature increase was greater with an increase in irradiation intensity, as shown in fig. 6, the photo-thermal effect had time dependence and power dependence. The laser power density was 1.0W/cm 2 At this point, the temperature of the BPNSs-PEG/DOX@Hydrogel was raised to 52.3℃to a temperature sufficient to thermally ablate tumor cells.
BPNSs-PEG/DOX@Hydrogel containing varying BP (10. Mu.g/mL, 25. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL and 200. Mu.g/mL) concentrations were exposed to a power density of 1.0W/cm 2 Is irradiated for 10min under 808nm near infrared laser, and deionized water is usedAs compared with the blank hydrogel, the result is that the temperature is continuously increased with the increase of the irradiation time, and as the BP concentration is increased, the trend of the temperature increase is larger, and the photothermal effect also has the photothermal agent dose dependence, as shown in fig. 7. Under the same laser treatment, the temperature of BPNSs-PEG/DOX@Hydrogel with BP concentration of 200 μg/mL was raised to 61.8 ℃, demonstrating that trace amounts (200 μg/mL) of BP can rapidly and effectively convert light energy into heat energy, in sharp contrast to deionized water and blank gel which are raised only to 30.5 ℃ and 31.6 ℃, indicating that photothermal therapy without BP present is difficult to effectively kill tumor cells.
The power density is 1.0W/cm 2 BPNSs-PEG/DOX@Hydrogel (BP concentration 100. Mu.g/mL) was irradiated with near infrared laser light at 808nm for 10min, and then the laser was turned off and cooled to room temperature for 15min. The rapid heating and cooling of the nano preparation are realized by switching laser, the same operation lasts for 5 cycles, and the result is shown in figure 8, the highest temperature of BPBPNSs-PEG/DOX@Hydrogel can reach about 52.7 ℃ each time, and the photo-thermal stability of the nano preparation under the cyclic irradiation is fully reflected.
To further investigate the photo-thermal stability of BPNSs-PEG/DOX@Hydrogel over time, BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel (BP concentrations were 100. Mu.g/mL) were exposed to air, using a power density of 1.0W/cm at two time points after preparation (0 day) and 7 days, respectively 2 The temperature was recorded as a function of irradiation time by irradiation with a near infrared laser at 808 nm. As shown in fig. 9, after the BPNSs were left for 7 days, the laser irradiation temperature reached only 35.7 ℃, which was far less than the photothermal treatment temperature requirement, and the photothermal effect of the BPNSs was significantly reduced relative to a decrease of 14.0 ℃ in day 0 due to degradation thereof upon prolonged exposure to deionized water and air. The photo-thermal effects of the BPNSs-PEG/DOX and the BPNSs-PEG/DOX@Hydrogel are not affected, and the PEG modification on the surface of the BPNSs can greatly enhance the stability of the BPNSs, so that the photo-thermal stability of the BPNSs in a long time is improved.
The photo-thermal conversion efficiency is an important index for evaluating the photo-thermal conversion capability of the material, and the photo-thermal conversion efficiency of BPNSs-PEG/DOX@Hydrogel with the BP concentration of 100 mug/mL is measured. Determination of BPNSs-PEG/DOX@Hydrogel (mass 1 g) absorbance at 808nm, followed by exposure to a power density of 1.0W/cm 2 The laser is turned off for cooling for 15min under the near infrared laser of 808nm, the temperature is recorded every 30s, and a function chart of the temperature change along with time is drawn, as shown in fig. 10. A non-quantitative driving force temperature theta was introduced, and a t-ln (theta) graph was drawn using the relationship between temperature and time during cooling, as shown in fig. 11. The photo-thermal conversion efficiency of BPNSs-PEG/DOX@Hydrogel calculated from the relevant parameters obtained in FIGS. 10 and 11 was 40.2% slightly higher than that of the literature (Qia M, wang D, liang W, et al Novel concept ofthe smart NIR-light-controlled drug release of black phosphorus nanostructure for cancer therapy [ J)]Proceedings of the National Academy of Sciences,2018,115 (3): 501-506.) the photothermal conversion efficiency of BPNSs reported in (38.8%).
The experiment shows that the BPNSs-PEG/DOX@Hydrogel has excellent photo-thermal conversion performance, photo-thermal stability and high photo-thermal conversion efficiency.
PH/Intelligent near infrared light responsive drug Release test
In vitro release of DOX from BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel was evaluated using a dialysis bag diffusion method, 2mL PNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel (BP concentration 200. Mu.g/mL) were packaged in dialysis bags (MWCO=3500 Da), immersed in 18mL centrifuge tubes containing PBS buffers of different pH (pH 7.4 and 5.0, respectively), and placed in a thermostatted shaker at 37 ℃. 2mL of release medium was removed at 2, 4, 6, 12, 24, 36, 48, 72, 96h, respectively, and 2mL of fresh PBS at 37℃was supplemented to maintain a constant volume. For near infrared light triggered DOX release experiments, after fresh PBS was replenished at fixed time points in the non-irradiated group, the rapid exposure was at a power density of 1.0W/cm 2 After 10min of irradiation with 808nm near infrared laser, 2mL of release medium was collected, an equal volume of 37 ℃ fresh PBS was added, the other conditions were completely identical, and the experiment was repeated 3 times. The amount of DOX released was determined by an ultraviolet-visible spectrophotometer.
The cumulative release dynamic results of BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel under the triggering of near infrared light at different pH values are shown in FIGS. 12 and 13, when the pH value of PBS is 7.4, the cumulative release rates of BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel reach 36.5+/-1.5% and 23.0+/-1.2% respectively after 96 hours, and when the PBS is exposed to a lower pH value (pH value is 5.0), the cumulative release rates of DOX are respectively increased to 52.0+/-1.7% and 31.0+/-1.6% in the same time. DOX releases faster under slightly acidic conditions. In the near infrared light triggered drug release test, after the BPNSs-PEG/DOX is subjected to near infrared light triggered drug release again for 24 hours, the accumulated drug release rates of PBS at pH7.4 and pH5.0 reach 72.2+/-2.2% and 82.4+/-1.9%, respectively, and after the last 72 hours, the drug release rates are increased by 15.6% and 10.6%, the final accumulated release rates reach 87.8+/-2.1% and 92.8+/-1.6%, and the drug release rate of the BPNSs-PEG/DOX is too high in the first 24 hours of near infrared light triggered drug release. After the last near infrared light triggering drug release of BPNSs-PEG/DOX@Hydrogel is performed for 96 hours, the accumulated drug release rates of PBS at pH7.4 and pH5.0 respectively reach 72.8+/-1.9% and 82.0+/-1.6%, compared with the BPNSs-PEG/DOX, the gel structure slows down the release speed of DOX under the condition of near infrared light irradiation or not, and the excessive release of DOX under the condition of light response is avoided, so that the release time of the drug is prolonged, and the release amount of the drug is better and continuously controlled. BPNSs-PEG/DOX@Hydrogel is a drug storage library, an in-vitro near infrared laser irradiation switch is an optical switch for releasing drugs, and by adjusting in-vitro near infrared light irradiation, slow release and controlled release of the drugs can be well realized, accumulation of DOX at tumor focus positions is increased, and adverse reactions are weakened.
3. Cell compatibility test
The toxicity of the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheets on some common human cancer cells (namely Hela (human cervical cancer cells), A549 (human lung cancer cells), MCF-7 (human breast cancer cells) and HepG2 (human liver cancer cells)) is studied, various cancer cells are sown in 96-well plates at the density of 8000 pieces/well, and the supernatant is discarded after 24 hours of culture. mu.L of fresh medium and cells containing BPNSs (BP concentration: 200. Mu.g/mL), BPNSs-PEG (BP concentration: 200. Mu.g/mL), hydrogel (Vgel/V medium=1/9), BPNSs-PEG@Hydrogel (BP concentration: 200. Mu.g/mL and Vgel/V medium=1/9) were incubated for 48 hours, the supernatant was discarded, PBS was washed 2 times, and 100. Mu.L of 10% CCK-8 reagent (10. Mu.L of a mixture of CCK-8 reagent and 90. Mu.L of LDMEM medium) was added thereto, and further incubated for 1 to 2 hours. Absorbance was measured at 450nm using a microplate reader. The cell viability of untreated cells was 100%. Cell viability was calculated according to the following formula: cell viability (%) = (experimental well absorbance-blank well absorbance)/(control well absorbance-blank well absorbance) ×100%. All samples from CCK-8 experiments were provided with 6 multiplex wells. As a result, as shown in FIG. 14, even though the BP concentration was as high as 200. Mu.g/mL, the cell viability was higher than 90%, and it was confirmed that the intelligent near infrared light responsive composite hydrogel based on black phosphorus nanoplatelets had low toxicity to human cancer cells.
It is also desirable to evaluate its toxicity to mouse osteosarcoma osteoblasts (K7M 2-WT). K7M2-WT cells were seeded at 8000 cells/well in 96-well plates, the supernatant was discarded after 24h incubation, 100. Mu.L of fresh medium containing various concentrations of BP (1, 5, 10, 25, 50, 100, 200. Mu.g/mL, respectively), BPNSs-PEG, BPNSs-PEG@Hydrogel (V gel/V medium=1/9) was incubated with K7M2-WT cells for 48h, and then CCK-8 assay was performed (assay calculation method same). The cell viability of untreated cells was 100%. As shown in FIG. 15, the cell viability was over 90%, and the cell viability was 92.6%, 93.2% and 93.9% respectively, without significant cytotoxicity, when the BP concentration was as high as 200. Mu.g/mL. The intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheet has excellent in-vitro biocompatibility.
4. In vitro antitumor therapy test
In vitro photothermal therapy test
Based on the excellent photothermal conversion performance of BP, the in vitro photothermal treatment effect of BPNSs-PEG@Hydrogel as a single photothermal agent on K7M2-WT cells was evaluated by CCK-8 experiment. K7M2-WT cells were seeded at 10000 cells/well in 96-well plates, the supernatant was discarded after 24h of culture, 100. Mu.L of fresh medium containing various concentrations of BP (0, 1, 5, 10, 25, 50, 100, 200. Mu.g/mL, respectively), BPNSs-PEG, BPNSs-PEG@Hydrogel (V gel/V medium=1/9) was incubated with K7M2-WT cells for 4h, and then the power density was 1.0W/cm 2 808nm near redAnd (5) irradiating the external laser for 10min. The area of each well was completely covered by the laser spot and incubated for a further 20h. The relative viability of the cells was then determined using the CCK-8 assay (assay calculation methods are as above). The cell viability of untreated cells was 100%. As shown in fig. 16, the BP-based nanoformulation exhibited a dose-dependent photothermal anti-tumor effect. When BP concentration was 100. Mu.g/mL, the cell viability of BPNSs-PEG@Hydrogel treatment was only 13.0% by near infrared light irradiation, and there was no significant difference between the BPNSs (cell viability: 12.4%) and the BPNSs-PEG (cell viability: 13.9%) treatments. The BP concentration was increased to 200. Mu.g/mL, and almost all cells were killed. Near infrared light radiation (BP concentration of 0. Mu.g/mL) did not affect the growth of K7M2-WT cells. These results clearly demonstrate that the strong photothermal antitumor effect of BPNSs is not affected by the surface modification and embedding adsorption of PEG in the cavities of hydrogels.
In vitro chemotherapy test
Based on the excellent drug loading capacity of BP, the in vitro chemotherapeutic effect of BP nano-preparation as a drug delivery platform of chemotherapeutic drug DOX on K7M2-WT cells was evaluated. K7M2-WT cells were seeded at 10000 cells/well in 96-well plates, the supernatant was discarded after 24h of culture, 100. Mu.L of fresh medium containing various concentrations of DOX ( concentrations 1, 5, 10, 25, 50, 100, 200. Mu.g/mL, respectively) free DOX, BPNSs-PEG/DOX@Hydrogel (V gel/V medium=1/9) was incubated with K7M2-WT cells for 24h, and then the relative viability of the cells was determined by CCK-8 assay (assay calculation method as above). The cell viability of untreated cells was 100%. As a result, as shown in FIG. 17, the cell viability was decreased as the DOX concentration was increased, and the concentration-dependent cytotoxicity was exhibited. DOX shows a higher tumor cell killing compared to BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel, which may be attributed to the rapid diffusion of DOX into tumor cells, whereas BPNSs-PEG/DOX requires slow entry into cells by endocytosis and release of DOX in cells, on which BPNSs-PEG/DOX@Hydrogel also requires slow diffusion from the lumen of the gel into the cell medium.
In vitro photothermal therapy-chemotherapy synergistic anti-tumor therapy test
Next, the DOX-loaded BP preparation was continuously evaluated for its synergistic anti-tumor effect on K7M2-WT cells under irradiation of near infrared light. K7M2-WT cells were seeded at 10000 cells/well in 96-well plates, the supernatant was discarded after 24h incubation, 100. Mu.L of fresh medium containing different concentrations of DOX (1, 5, 10, 25. Mu.g/mL, respectively) free DOX, BPNSs-PEG/DOX@Hydrogel (V gel/V medium=1/9) was incubated with K7M2-WT cells for 4h, and then a power density of 1.0W/cm was used 2 Is irradiated with near infrared light of 808nm for 10min. The area of each well was completely covered by the laser spot and incubated for a further 20h. The relative viability of the cells was then determined using the CCK-8 assay. The non-near infrared light irradiation group is the same as the near infrared light irradiation group except for near infrared light treatment. The cell viability of untreated cells was 100%. The results are shown in FIG. 18, where there is no significant difference in cytotoxicity in the presence or absence of the photothermal agent BPNSs, DOX, indicating that near infrared light irradiation in the absence of the photothermal agent does not promote tumor cell death. And BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel exert stronger cytotoxicity under near infrared light irradiation than those of the non-irradiated group. The DOX content of BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel increases as the concentration of BPNSs on its load platform increases. The effect of the combined treatment of the BPNSs-PEG/DOX and the BPNSs-PEG/DOX@Hydrogel under the irradiation of near infrared light is more remarkable along with the increase of BP concentration compared with that of the single chemotherapy without irradiation, which is attributed to the fact that the higher concentration BP can make the temperature rise effect of a cell area stronger under the triggering of the near infrared light, the generated high temperature can quickly thermally ablate tumor cells, and the release of DOX is quickened to promote the enhancement of the chemotherapy effect. Cell viability after treatment of cells with BPNSs-PEG/DOX and BPNSs-PEG/DOX@Hydrogel at a DOX concentration of 25. Mu.g/mL was 6.7% and 5.8%, respectively, under near infrared radiation. Has the advantages of small dosage and short action time compared with the single chemotherapy.
From the above examples, it can be seen that the intelligent near infrared light response composite hydrogel based on black phosphorus nanoplatelets prepared by the preparation method provided by the invention realizes sustainable and slow release of the drug, and realizes in-situ release of the drug with pH/intelligent near infrared light response by slightly acidic environment of tumor tissue and 808nm near infrared light (NIR) radiation, thereby achieving the aim of light-heat-chemotherapy synergistic treatment of osteosarcoma.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A preparation method of intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheets comprises the following steps:
(1) Mixing black phosphorus powder and an organic solvent, placing the mixture in an ice water bath, sequentially carrying out water bath ultrasonic treatment and probe ultrasonic treatment, centrifuging at 2000-4000 rpm, collecting supernatant, centrifuging the supernatant at a rotating speed of over 12000rpm, collecting precipitate, and finally mixing the precipitate with water to obtain black phosphorus nanosheet dispersion;
(2) Mixing the black phosphorus nano-sheet dispersion liquid obtained in the step (1) with polyethylene glycol diamine, sequentially performing first ultrasonic treatment, first magnetic stirring and first centrifugation, collecting precipitate, and adding water to obtain polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid;
(3) Mixing the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid obtained in the step (2) with a chemotherapeutic drug, sequentially carrying out second ultrasonic treatment, second magnetic stirring and second centrifugation, collecting precipitate, adding water, and obtaining the polyethylene glycol diamine modified black phosphorus nano-sheet dispersion liquid loaded with the chemotherapeutic drug;
(4) And (3) mixing the black phosphorus nano-sheet dispersion liquid modified by polyethylene glycol diamine and loaded with the chemotherapeutic drugs and the matrix of the composite hydrogel, and swelling to obtain the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheets.
2. The method according to claim 1, wherein the volume ratio of the mass of the black phosphorus powder to the organic solvent in the step (1) is 1: (1-2) mg/mL.
3. The preparation method according to claim 1, wherein the power of the water bath ultrasonic wave in the step (1) is 200-500W, and the time of the water bath ultrasonic wave is 8-12 h; the power of the probe ultrasound is 180-720W, the time of the probe ultrasound is 8-12 h, and the on/off period of the probe ultrasound is 2-3 s/3s.
4. The method according to claim 1, wherein the centrifugation time at 2000 to 4000rpm in the step (1) is 10 to 20 minutes; the centrifugation time is 20-30 min at the rotating speed of 12000rpm or more.
5. The preparation method according to claim 1, wherein the mass ratio of the black phosphorus nanoplatelets to the polyethylene glycol diamine in the black phosphorus nanoplatelet dispersion in the step (2) is 1: (5-10).
6. The method according to claim 1, wherein the mass ratio of the black phosphorus nanoplatelets to the chemotherapeutic agent in the polyethylene glycol diamine modified black phosphorus nanoplatelet dispersion in the step (3) is 1: (1-8).
7. The method according to claim 1, wherein the swelling temperature in the step (4) is 4 ℃ and the swelling time is not less than 24 hours.
8. The method according to claim 1, wherein the matrix of the composite hydrogel in the step (4) comprises, based on 100% of the mass of the composite hydrogel: poloxamer 40718-21%, poloxamer 1883-5% and sodium alginate 0.4-0.8%.
9. The intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheets prepared by the preparation method of any one of claims 1 to 8.
10. The application of the intelligent near infrared light response composite hydrogel based on the black phosphorus nano-sheet in preparing anti-osteosarcoma medicines.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310127719.0A CN116059360A (en) | 2023-02-16 | 2023-02-16 | Intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheet and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310127719.0A CN116059360A (en) | 2023-02-16 | 2023-02-16 | Intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheet and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116059360A true CN116059360A (en) | 2023-05-05 |
Family
ID=86169721
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310127719.0A Pending CN116059360A (en) | 2023-02-16 | 2023-02-16 | Intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheet and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116059360A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116919886A (en) * | 2023-07-21 | 2023-10-24 | 中国海洋大学 | Injectable photo-thermal hydrogel based on black pigment, and preparation method and application thereof |
| CN117065036A (en) * | 2023-09-28 | 2023-11-17 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Nanometer preparation of vanadium carbide coated doxorubicin and application thereof |
| CN117065036B (en) * | 2023-09-28 | 2024-04-26 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Nanometer preparation of vanadium carbide coated doxorubicin and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104758931A (en) * | 2015-03-17 | 2015-07-08 | 郑州大学 | Preparation method of temperature-sensitive hydrogel based on functionalized graphene oxide and application thereof |
-
2023
- 2023-02-16 CN CN202310127719.0A patent/CN116059360A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104758931A (en) * | 2015-03-17 | 2015-07-08 | 郑州大学 | Preparation method of temperature-sensitive hydrogel based on functionalized graphene oxide and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 朱铁梁;胡霞;张莉;陈孝储;郭一沙;陈莉;张岭;: "黄芩苷温敏凝胶的处方筛选及体外释放研究", 中草药, no. 11, 20 September 2012 (2012-09-20), pages 2166 * |
| 王玮;叶放;周建平;: "泊洛沙姆407原位凝胶作为药物载体的研究进展", 中国新药杂志, no. 08, 30 April 2009 (2009-04-30), pages 701 * |
| 阿依妮尕尔·艾力等: "功能化载药黑磷纳米片的制备表征及抗骨肉瘤活性评价", 中国医院药学杂志, vol. 41, no. 23, 31 December 2021 (2021-12-31), pages 2388 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116919886A (en) * | 2023-07-21 | 2023-10-24 | 中国海洋大学 | Injectable photo-thermal hydrogel based on black pigment, and preparation method and application thereof |
| CN116919886B (en) * | 2023-07-21 | 2024-01-30 | 中国海洋大学 | Injectable photo-thermal hydrogel based on black pigment, and preparation method and application thereof |
| CN117065036A (en) * | 2023-09-28 | 2023-11-17 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Nanometer preparation of vanadium carbide coated doxorubicin and application thereof |
| CN117065036B (en) * | 2023-09-28 | 2024-04-26 | 广州医科大学附属第三医院（广州重症孕产妇救治中心、广州柔济医院） | Nanometer preparation of vanadium carbide coated doxorubicin and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2019114066A1 (en) | Black phosphorus-based hydrogel near-infrared light-controllable drug release system and preparation method therefor | |
| Niu et al. | A multifunctional bioactive glass-ceramic nanodrug for post-surgical infection/cancer therapy-tissue regeneration | |
| Du et al. | Polymeric photothermal agents for cancer therapy: recent progress and clinical potential | |
| Chen et al. | Temperature-regulating phase change fiber scaffold toward mild photothermal–chemotherapy | |
| CN111671914B (en) | Near infrared light response nanoparticle and controlled release system | |
| CN108653754B (en) | Hyaluronic acid targeted polydopamine coated phase-change type liquid fluorocarbon nano ultrasonic contrast agent | |
| Ruan et al. | Transdermal delivery of multifunctional CaO 2@ Mn-PDA nanoformulations by microneedles for NIR-induced synergistic therapy against skin melanoma | |
| Xu et al. | Balancing the toxicity, photothermal effect, and promotion of osteogenesis: Photothermal scaffolds for malignant bone tumor therapy | |
| CN110101860B (en) | Bismuth-doped metal sulfide nanoflower and preparation method thereof | |
| CN116059360A (en) | Intelligent near infrared light response composite hydrogel based on black phosphorus nano-sheet and preparation method and application thereof | |
| CN109091674B (en) | Multifunctional drug carrier and preparation method and application thereof | |
| CN108030922B (en) | Temperature-sensitive gold nanocage, preparation method and application thereof, drug-loading temperature-sensitive gold nanocage and preparation method thereof | |
| Qi et al. | Injectable and self-healing nanocomposite hydrogel loading needle-like nano-hydroxyapatite and graphene oxide for synergistic tumour proliferation inhibition and photothermal therapy | |
| WO2021083370A1 (en) | Preparation and use of nanomaterial specifically activating immune system | |
| Chen et al. | Glucose oxidase-loaded colloidal stable WS2 nanobowls for combined starvation/photothermal therapy of colorectal tumors | |
| Pan et al. | Mesoporous porphyrinic metal-organic framework nanoparticles/3D nanofibrous scaffold as a versatile platform for bone tumor therapy | |
| Liu et al. | Multifunctional 3D-printed bioceramic scaffolds: Recent strategies for osteosarcoma treatment | |
| CN113018435A (en) | Stable targeted photo-thermal black phosphorus nanosheet preparation and preparation method and application thereof | |
| CN110496097B (en) | Biodegradable hydrogel capable of promoting tissue repair and releasing nano hydroxyapatite by temperature control | |
| KR102513560B1 (en) | Pharmaceutical composition for the treatment of cancer comprising phycocyanin-anticancer agent conjugate, composition for photodynamic therapy comprising the same and manufacturing method thereof | |
| CN112972366A (en) | Injectable photothermal chemotherapy sensitization drug-loaded hydrogel and preparation method thereof | |
| CN116271062A (en) | Temperature-sensitive hydrogel based on folic acid-mediated black phosphorus nanosheets, and preparation method and application thereof | |
| Zhao et al. | Combination of chemotherapy and photothermal methods for in vitro ablation of MCF-7 cancer cells using crinkly core–shell structure MoS2/C@ SiO2 nanospheres | |
| CN110279650A (en) | A kind of controllable medicine-releasing system of hydrogel near infrared light based on tellurium alkene | |
| López et al. | Obtaining of SiO2 nanostructured materials for local drug delivery of methotrexate |
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 |