CN111228215A - 一种自组装可成像丝素蛋白水凝胶的制备方法 - Google Patents
一种自组装可成像丝素蛋白水凝胶的制备方法 Download PDFInfo
- Publication number
- CN111228215A CN111228215A CN202010158842.5A CN202010158842A CN111228215A CN 111228215 A CN111228215 A CN 111228215A CN 202010158842 A CN202010158842 A CN 202010158842A CN 111228215 A CN111228215 A CN 111228215A
- Authority
- CN
- China
- Prior art keywords
- silk fibroin
- self
- hydrogel
- solution
- imageable
- 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
- 239000000017 hydrogel Substances 0.000 title claims abstract description 105
- 108010022355 Fibroins Proteins 0.000 title claims abstract description 66
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 26
- MIKKOBKEXMRYFQ-WZTVWXICSA-N meglumine amidotrizoate Chemical compound C[NH2+]C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.CC(=O)NC1=C(I)C(NC(C)=O)=C(I)C(C([O-])=O)=C1I MIKKOBKEXMRYFQ-WZTVWXICSA-N 0.000 claims abstract description 26
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims abstract description 24
- 229910052740 iodine Inorganic materials 0.000 claims abstract description 24
- 239000011630 iodine Substances 0.000 claims abstract description 24
- 239000002131 composite material Substances 0.000 claims abstract description 14
- 229920002565 Polyethylene Glycol 400 Polymers 0.000 claims abstract description 10
- JLFNLZLINWHATN-UHFFFAOYSA-N pentaethylene glycol Chemical compound OCCOCCOCCOCCOCCO JLFNLZLINWHATN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000126 substance Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 35
- CPKVUHPKYQGHMW-UHFFFAOYSA-N 1-ethenylpyrrolidin-2-one;molecular iodine Chemical compound II.C=CN1CCCC1=O CPKVUHPKYQGHMW-UHFFFAOYSA-N 0.000 claims description 20
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000001338 self-assembly Methods 0.000 claims description 6
- 229940079593 drug Drugs 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- YVPYQUNUQOZFHG-UHFFFAOYSA-N amidotrizoic acid Chemical compound CC(=O)NC1=C(I)C(NC(C)=O)=C(I)C(C(O)=O)=C1I YVPYQUNUQOZFHG-UHFFFAOYSA-N 0.000 claims description 4
- 230000000259 anti-tumor effect Effects 0.000 claims description 4
- 229960005423 diatrizoate Drugs 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 238000013268 sustained release Methods 0.000 claims description 3
- 239000012730 sustained-release form Substances 0.000 claims description 3
- 229960005133 diatrizoate meglumine Drugs 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims 3
- 238000007711 solidification Methods 0.000 claims 1
- 230000008023 solidification Effects 0.000 claims 1
- 238000002560 therapeutic procedure Methods 0.000 claims 1
- 230000006907 apoptotic process Effects 0.000 abstract description 5
- FAAHNQAYWKTLFD-UHFFFAOYSA-N 1-butan-2-ylpyrrolidin-2-one Chemical compound CCC(C)N1CCCC1=O FAAHNQAYWKTLFD-UHFFFAOYSA-N 0.000 abstract description 2
- 239000002246 antineoplastic agent Substances 0.000 abstract description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 2
- 229940041181 antineoplastic drug Drugs 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 13
- 239000000499 gel Substances 0.000 description 11
- 229920000153 Povidone-iodine Polymers 0.000 description 8
- 210000000056 organ Anatomy 0.000 description 8
- 201000008968 osteosarcoma Diseases 0.000 description 8
- 229960001621 povidone-iodine Drugs 0.000 description 8
- 102000003952 Caspase 3 Human genes 0.000 description 7
- 108090000397 Caspase 3 Proteins 0.000 description 7
- 101710179684 Poly [ADP-ribose] polymerase Proteins 0.000 description 7
- 102100023712 Poly [ADP-ribose] polymerase 1 Human genes 0.000 description 7
- 239000002202 Polyethylene glycol Substances 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 229920001223 polyethylene glycol Polymers 0.000 description 7
- 229920000776 Poly(Adenosine diphosphate-ribose) polymerase Polymers 0.000 description 6
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 5
- 238000011532 immunohistochemical staining Methods 0.000 description 5
- 238000002441 X-ray diffraction Methods 0.000 description 4
- 239000012620 biological material Substances 0.000 description 4
- 230000029918 bioluminescence Effects 0.000 description 4
- 238000005415 bioluminescence Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000011580 nude mouse model Methods 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 231100000057 systemic toxicity Toxicity 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003181 biological factor Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 230000008876 conformational transition Effects 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 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
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
Images
Classifications
-
- 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
- A61K31/74—Synthetic polymeric materials
- A61K31/785—Polymers containing nitrogen
- A61K31/787—Polymers containing nitrogen containing heterocyclic rings having nitrogen as a ring hetero atom
- A61K31/79—Polymers of vinyl pyrrolidone
-
- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- 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
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
- A61K9/0009—Galenical forms characterised by the drug release technique; Application systems commanded by energy involving or responsive to electricity, magnetism or acoustic waves; Galenical aspects of sonophoresis, iontophoresis, electroporation or electroosmosis
-
- 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
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
- C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
- C08J3/075—Macromolecular gels
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2439/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by a single or double bond to nitrogen or by a heterocyclic ring containing nitrogen; Derivatives of such polymers
- C08J2439/04—Homopolymers or copolymers of monomers containing heterocyclic rings having nitrogen as ring member
- C08J2439/06—Homopolymers or copolymers of N-vinyl-pyrrolidones
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dispersion Chemistry (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Polymers & Plastics (AREA)
- Dermatology (AREA)
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明涉及一种可用于治疗肿瘤的自组装可成像丝素蛋白水凝胶的制备方法。本发明将丝素蛋白溶液、PEG400溶液、聚维酮碘(PVP‑I)溶液、泛影葡胺(MD)溶液于室温下混合，静置，所述共混液经过约38min后自组装，形成复合水凝胶。本发明制备的自组装可成像丝素蛋白水凝胶可以在X线引导下通过注射器直接注入肿瘤内部，用于向肿瘤组织内部传递单质碘，促使肿瘤细胞发生凋亡，实现治疗肿瘤的目的。本发明制备方法简单易行，可长期保存，可用于抗肿瘤药物领域。
Description
技术领域
本发明涉及一种简便的医用自组装可成像丝素蛋白水凝胶的制备方法及其应用，具体涉及一种以天然来源的丝素蛋白和人工合成的聚维酮碘为基础制备的自组装可成像丝素蛋白水凝胶的方法及其在组织再生修复、肿瘤治疗、预防感染、药物缓释等医疗领域的应用，属于生物材料和生物医学领域。
背景技术
水凝胶具有良好三维立体网络结构，适合于包载转运药物、细胞因子等，由于水凝胶中含有大量的水分，分子链可随意伸展，能够保持较好的流体性质，所以水凝胶可以更容易植入到肿瘤组织内部并且可以在肿瘤内部弥散均匀地分布，被广泛地应用骨肉瘤等肿瘤治疗领域。近年来，生物医学及组织工程领域研究方向将不同的生物材料复合丝素蛋白，制备出新型的生物材料-丝素蛋白水凝胶。丝素蛋白复合水凝胶因其优异的生物相容性、稳定的解缓速度、优良的力学强度等优点引起研究人员的重视，具有广阔的临床应用前景。
丝素蛋白溶液经物理和化学处理后可形成水凝胶，聚乙二醇(PEG)是一种有机化合物。将聚乙二醇与丝素蛋白混合可形成丝素蛋白凝胶。它可作为生物因子和药物的缓释体系，此外，丝素蛋白水凝胶可以通过注射器直接注射到特定部位，这一特性允许丝素水凝胶以一种微创的方式通过注射进入体内，然后将药物或生物因子输送到局部病灶。
聚维酮碘(PVP-I)能有效杀灭细菌、病毒、真菌和原生动物。作为广谱抗菌剂，它通常用于皮肤消毒和清洁伤口。PVP-I可释放游离碘单质，对微生物有较强的杀灭作用。同样，碘单质也是一种有效的杀瘤剂，作为一种抗肿瘤治疗手段，在临床上也越来越受欢迎。
发明内容
本发明所要解决的技术问题是应用简单的操作方法制备出一种可用于治疗肿瘤的自组装可成像丝素蛋白水凝胶。
本发明制备上述目标水凝胶的技术方法如下：将丝素蛋白冻干粉溶解于去离子水中，充分震荡后，制得一定浓度的丝素蛋白水溶液。将PVP-I溶液与丝素蛋白水溶液吸入一侧注射器，PEG400溶液与泛影葡胺溶液吸入另一侧注射器，将两个注射器使用双头鲁尔接头对接，通过多次互推溶液形成共混液，所述的共混液经过约37min后凝固形成一种可成像丝素蛋白复合水凝胶。
本发明的发明点在于是：
(1)安全无毒，无明显过敏反应：本复合生物材料中，应用的丝素蛋白是一种生物相容性良好的材料，而医用聚维酮碘是一种临床常用的消毒药剂，广泛的临床应用证实其安全无毒。
(2)促进丝素蛋白形成水凝胶。单纯丝素蛋白溶液自然状态下不易形成水凝胶，加入PEG400溶液，可促进丝素蛋白形成水凝胶。
(3)良好的抗肿瘤效果。本发明由于采用聚维酮碘，将聚维酮碘控制在有效浓度比例时，能够使复合水凝胶具有明显的抗肿瘤效果。将复合水凝胶在X光引导下注射入裸鼠皮下骨肉瘤模型内，一段时间后，可以看到肿瘤体积的增长明显减缓，经小动物成像仪检测生物发光的荧光强度也较对照组和单纯凝胶组明显降低。说明自组装可成像丝素蛋白水凝胶实现了抑制肿瘤增长的作用。为了进一步验证自组装水凝胶对骨肉瘤的治疗效果，我们通过免疫组化染色实验观察肿瘤组织中caspase-3和PARP蛋白表达水平来评估水凝胶传递的碘在体内是否诱导细胞凋亡。我们发现复合水凝胶处理组肿瘤组织中PARP和caspase-3的蛋白表达明显高于对照组和单纯凝胶治疗组。这些结果表明碘可以在体内诱导骨肉瘤组织发生凋亡。
(4)良好的生物安全性。本发明将生物相容性良好的丝素蛋白和临床常用的聚维酮碘复合制备水凝胶。虽然高浓度的聚维酮碘对机体细胞具有一定的损伤效果，但制备复合水凝胶时，控制聚维酮碘浓度，使复合水凝胶释放的有效碘浓度不会对机体造成损伤。在生物安全实验中，为了验证自组装水凝胶的生物安全性及碘对是否对全身系统产生毒性，对心脏、肝脏、脾脏、肺脏、肾脏、胰腺、甲状腺等主要器官进行了HE染色，分析各个器官经自组装水凝胶治疗后是否发生病理损伤。HE染色结果显示，各个主要器官均未出现显著病理改变。说明包载碘的自组装水凝胶不仅能抑制体内肿瘤生长，起到局部治疗骨肉瘤的作用，而且不会引起全身系统毒副作用，具有良好的生物安全性。
(5)持续释放有效碘，缓慢降解。本发明将聚维酮碘与丝素蛋白混合，形成复合水凝胶，使得其能够向周围环境缓慢释放复合的有效碘，且降解时间延长。
本发明的应用前景：由于该水凝胶原料简单，制备方法方便，可大量生产，并且性能优异，故拥有良好的临床应用的潜力。在X光引导下局部注入肿瘤组织内，可起到明显治疗作用。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例共同用于解释本发明，并不构成对本发明的限制。在附图中：
图1为本发明中的注射器及连接器的结构示意图。
图2a至2c为所制备自组装可成像丝素蛋白水凝胶。图2a为各组分溶液混合后随着时间的增加，溶液变得更加粘稠和不透明；图2b为自组装水凝胶形成时间；图2c为自组装水凝胶形成时间表。
图3为包载碘的丝素蛋白水凝胶碘释放曲线
图4a至4b为自组装丝素蛋白水凝胶FTIR和XRD分析。图4a为自组装水凝胶FTIR数据；图4b为自组装水凝胶XRD数据。
图5a至5c为自组装丝素蛋白水凝胶的可注射性及机械性能分析，图5a为将水凝胶推出注射器所需要施加的力；图5b为自组装可成像丝素蛋白水凝胶可以通过注射器轻松推出；图5c为水凝胶机械性能分析。
图6a至6c为水凝胶的微观结构。图6a为PEG-Silk水凝胶；图6b为PEG-Silk/MD水凝胶；图6c为PEG-Iodine-Silk/MD水凝胶。图中比例尺为50μm。
图7a至7d为碘可以抑制体内骨肉瘤的生长。图7a为裸鼠体内肿瘤生物荧光表达水平；图7b为注射水凝胶21天后肿瘤外观；图7c为在X线引导下向肿瘤组织内分别注射生理盐水、PEG-Silk/MD水凝天胶和PEG-Iodine-Silk/MD水凝胶；图7d为注射水凝胶21天后肿瘤体积大小。
图8a至8c为免疫组织化学染色和全身毒性评估。图8a为肿瘤及主要器官HE染色(*为丝水凝胶；
显示肿瘤组织坏死),(放大倍数为200x)；图8b为三组肿瘤组织Caspase3和PARP免疫组织化学染色(放大倍数为200x)；图8c为三组肿瘤组织中Caspase3和PARP表达水平。
附图标记：1.注射器；2.连接器。
具体实施方式
以下结合附图对本发明实施方式做进一步阐述。
实施例1
以上所述，仅为本发明的具体实施案例，本发明的保护范围并不局限于此，任何熟悉本技术的技术人员在本发明所述的技术规范内，对本发明的修改或替换，都应在本发明的保护范围之内。
将丝素蛋白冻干粉溶解于去离子水中，充分震荡5-10分钟后，制得质量体积比60％的丝素蛋白水溶液，室温保存。
抽取相应溶液：丝素蛋白水溶液与PVP-I溶液吸入一侧注射器，PEG400溶液与泛影葡胺(MD)溶液吸入另一侧注射器，其中各成分比例为：PEG 400为40％(W/V)，丝素蛋白为13.5％(W/V)，MD为18％(V/V)，PVP-I为19.5％(V/V)，其余用超纯水补足。将两个注射器使用双头鲁尔接头对接，通过快速多次互推溶液约一分钟左右，形成1ml的共混液，采用OD值测成胶时间，结果参考图2a至2c，可见共混液经过约38min后凝固形成水凝胶。将所述水凝胶置于一侧的注射器内，缓慢将水凝胶从注射器中取出。在无菌条件下，将水凝胶置于PBS中进行有效碘的缓释量的测定。
水凝胶体外有效碘的释放采用紫外可见分光光度计测量OD值来确定，以反映水凝胶中碘的释放情况。首先使用碘单质、无水乙醇和超纯水制备不同浓度的碘溶液，浓度范围为从0到100μM。在37℃下测量OD值，计算标准曲线。将各组水凝胶样品(1cm3)(n＝3)浸泡在40ml的超纯水中。分别在0、3、6、9、12、24、48、72、96、120和144h，取各组浸泡水凝胶的溶液加入到96孔板中，每孔200μl。测量结束后将各孔中的液体全部移回相应的各组液体中。计算各组样品OD值的平均值，再根据标准曲线计算碘浓度,并绘制碘释放曲线。结果显示，在前12小时内有一次快速释放，在接下来的6天，碘的释放变得缓慢而稳定，在第7天释放达到最高水平29.3μM。释放7天后的累积释放量约为初始碘含量的31.3％。具体实验结果如图所示3。
为了分析残留的PEG溶剂、泛影葡胺和PVP-I是否影响凝胶构象转变,采用傅立叶变换红外光谱(FTIR)对复合凝胶进行检测。可见PEG残留对丝素蛋白结构的影响可以忽略不计，PVP-I和MD的存在也不影响丝素蛋白的结构。具体结果参照图4a至4b。
采用X射线衍射(XRD)对水凝胶的结晶度进行测定，可发现XRD分析结果进一步证实了FTIR结果，PVP-I和泛影葡胺存在对丝素蛋白结晶度的影响可以忽略不记。具体结果参照图4a至4b。
水凝胶的注射性能是通过测量1ml注射器通过25G针头喷出200μl体积水凝胶时所需要的力来测定，结果显示：自组装可注射水凝胶可以很容易地用注射器推出。PEG400、60％丝素蛋白溶液、PVP-I溶液和泛影葡胺溶液(MD)混合30-40分钟后，检测到PEG-Silk水凝胶、PEG-Silk/MD水凝胶和PEG-Iodine-Silk/MD水凝胶三种水凝胶所需注射力开始明显增大。注射力在测量结束时(60-70min)达到50N，但仍然可以缓慢增加。采用水凝胶无限制承受压缩应力直至实验样品发生形变来评估其机械性能。结果表明：PEG-Silk水凝胶、PEG-Silk/MD水凝胶和PEG-Iodine-Silk/MD水凝胶三种水凝胶的抗压强度、弹性模量和断裂应变率相似，因为三组水凝胶中丝素和PEG的浓度相同。凝胶的这些特性使它更容易被注射，因此也更容易在体外向病灶局部注射。具体实验结果如图5a至5c。
用扫描电镜(SEM)观察含13.5％丝素蛋白的水凝胶冻干后的多孔形貌。可见凝胶整体形态为微孔状，微孔形态细长，具有较高的孔隙率和较好的联通性。微孔孔壁菲薄而且孔径从50μm到100μm不等。PEG的存在可能改变了冻干过程中丝凝胶的冷冻速率，从而形成了凝胶的孔结构。由于各组水凝胶中丝素蛋白和PEG400的浓度相同，所以三组水凝胶的孔径相似。具体实验结果参考图6a至6c。
为了确定碘对体内骨肉瘤的影响，将转染了荧光素酶的MG-63细胞注射入裸鼠腋窝皮下。当肿瘤形成时，在X线引导下，实验组注射两种水凝胶(PEG-Silk/MD水凝胶和PEG-Iodine-Silk/MD水凝胶)，对照组注射生理盐水。水凝胶含有泛影葡胺(MD)，因此，通过X线可以判断复合丝素蛋白水凝胶的注射量、分布和分散情况。结果显示，注射PEG-Iodine-Silk/MD水凝胶组小鼠的肿瘤生长速度明显慢于其他两组小鼠。体内生物发光成像实验中，注射PEG-Iodine-Silk/MD水凝胶组小鼠的生物荧光水平明显低于PEG-Iodine-Silk/MD水凝胶组和生理盐水组。结果表明，碘对小鼠肿瘤生长有明显的抑制作用。具体实验结果参考图7a至7d。
PARP和caspase-3表达水平与肿瘤细胞凋亡有关。通过免疫组织化学染色进一步检测PARP和caspase-3在不同处理组肿瘤组织中的表达水平。结果表明，在裸小鼠中，碘处理增加了骨肉瘤中PARP和caspase 3的表达，促进了凋亡的发生。各组注射后第21天处死小鼠，取各组重要器官，包括心脏、肝脏、脾脏、肺脏、肾脏、胰腺和甲状腺。各器官组织进行HE染色观察各器官的病理变化，以检测自组装水凝胶全身毒性。在HE染色结果中各器官未见明显形态学改变，因此，PEG-Iodine-Silk/MD水凝胶无全身毒性，在体内具有良好的生物相容性和生物安全性。此外，PEG-Iodine-Silk/MD水凝胶周围出现大量坏死的肿瘤组织。这些结果与免疫组化染色一致。具体实验结果参考图8a至8c。
Claims (10)
1.一种可用于治疗肿瘤的自组装可成像丝素蛋白水凝胶的制备方法，其特征在于，包括以下步骤：
将冻干的丝素蛋白粉装入容器中，加去离子水并震荡5-10分钟，制成一定浓度的丝素蛋白水溶液；准备PVP-I溶液、PEG400溶液、泛影葡胺溶液，置于室温保存；
将所述的丝素蛋白水溶液与PVP-I溶液吸入一侧注射器，PEG400溶液与泛影葡胺溶液吸入另一侧注射器，注射器内溶液的体积不超过注射器体积的一半，再将两个注射器以双头鲁尔接头相连，之后交替互推至充分混合得到共混液，所述共混液静置，经过约37min凝固形成所述自组装可成像丝素蛋白水凝胶。
2.根据权利要求1所述的自组装可成像丝素蛋白水凝胶的制备方法,其特征在于：丝素蛋白溶液质量体积比为60％。
3.根据权利要求1所述的自组装可成像丝素蛋白水凝胶的制备方法，其特征在于：所述冻干的丝素蛋白粉室温条件下完全溶解的时间为10-30分钟。
4.根据权利要求1所述的自组装可成像丝素蛋白水凝胶的制备方法，其特征在于：所述PVP-I溶液碘单质含量为5g/L。
5.根据权利要求1所述的自组装可成像丝素蛋白水凝胶的制备方法，其特征在于：以混合液总体积为1ml为例，混合液中各成分比例为：PEG 400为40％(W/V)，丝素蛋白为13.5％(W/V)，MD为18％(V/V)，PVP-I为19.5％(V/V)，其余用超纯水补足。
6.根据权利要求1至5任一项所述的自组装可成像丝素蛋白水凝胶的制备方法，其特征在于：各种溶液按一定比例分别吸入两个注射器中，快速互推注射器10-20次或40秒以上。
7.根据权利要求1至6所述的自组装可成像丝素蛋白水凝胶的制备方法，其特征在于：将混合液于室温下静置，约38min后自组装，形成复合水凝胶。
8.根据权利要求1至7所述的自组装可成像丝素蛋白水凝胶的制备方法，其特征在于：所述共混液凝固形成丝素蛋白水凝胶的过程中，可将共混液置入一个容器中，还可直接置于一侧的注射器中，去掉另一侧的空注射器和连接器，在X线引导下体内注射或者涂抹填充。
9.一种自组装可成像丝素蛋白水凝胶，其特征在于：其使用权利要求1至8之一的自组装可成像丝素蛋白水凝胶制备方法制得。
10.一种可应用于抗肿瘤治疗、药物缓释医疗领域的自组装可成像丝素蛋白水凝胶，是一种根据权利要求1至9任一项所述的制备方法制得的自组装可成像丝素蛋白水凝胶。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010158842.5A CN111228215A (zh) | 2020-03-09 | 2020-03-09 | 一种自组装可成像丝素蛋白水凝胶的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010158842.5A CN111228215A (zh) | 2020-03-09 | 2020-03-09 | 一种自组装可成像丝素蛋白水凝胶的制备方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111228215A true CN111228215A (zh) | 2020-06-05 |
Family
ID=70867661
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010158842.5A Pending CN111228215A (zh) | 2020-03-09 | 2020-03-09 | 一种自组装可成像丝素蛋白水凝胶的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111228215A (zh) |
Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5514379A (en) * | 1992-08-07 | 1996-05-07 | The General Hospital Corporation | Hydrogel compositions and methods of use |
| CN102220017A (zh) * | 2011-05-05 | 2011-10-19 | 苏州大学 | 一种可注射成型的丝素蛋白水凝胶及其制备方法 |
| CN103289107A (zh) * | 2013-05-28 | 2013-09-11 | 苏州丝美特生物技术有限公司 | 一种可注射丝素蛋白原位凝胶的制备方法及其应用 |
| CN104644545A (zh) * | 2015-02-27 | 2015-05-27 | 苏州丝美特生物技术有限公司 | 一种治疗内耳疾病的控释缓释丝素蛋白凝胶制剂 |
| CN104815357A (zh) * | 2015-04-27 | 2015-08-05 | 复旦大学 | 一种无机有机杂化的三维互穿网络水凝胶材料及其制备方法和应用 |
| CN105407927A (zh) * | 2013-05-24 | 2016-03-16 | 丹麦技术大学 | 用于引导放射治疗的凝胶配方 |
| US20160136241A1 (en) * | 2014-02-28 | 2016-05-19 | Union Hospital, Tongji Medical College, Huazhong University Of Science And Technology | Methods of preparing and using sericin hydrogel |
| CN105597104A (zh) * | 2014-12-16 | 2016-05-25 | 苏州丝美特生物技术有限公司 | 一种用于药物控释的丝素-载药纳微米颗粒的制备方法 |
| WO2016079331A1 (en) * | 2014-11-21 | 2016-05-26 | Technical University Of Denmark | Gel formulations for enhancing the effect of radiotherapy |
| WO2016079332A1 (en) * | 2014-11-21 | 2016-05-26 | Technical University Of Denmark | Gel formulations for improving immunotherapy |
| CN106492270A (zh) * | 2016-11-28 | 2017-03-15 | 哈尔滨工业大学 | 一种用于镁合金与丝素蛋白之间连接的方法 |
| US20170107264A1 (en) * | 2014-03-27 | 2017-04-20 | Simatech Incorporation | Freeze-dried powder of high molecular weight silk fibroin, preparation method therefor and use thereof |
| US20170360912A1 (en) * | 2014-12-17 | 2017-12-21 | Socovar, L.P. | Chitosan-based hydrogel and applications thereof. |
| CN108785752A (zh) * | 2018-06-29 | 2018-11-13 | 苏州丝美特生物技术有限公司 | 丝素蛋白基多孔材料及其制备方法 |
| CN109589447A (zh) * | 2018-11-13 | 2019-04-09 | 王岩松 | 一种医用可注射抗菌复合原位水凝胶的制备方法及其应用 |
-
2020
- 2020-03-09 CN CN202010158842.5A patent/CN111228215A/zh active Pending
Patent Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5514379A (en) * | 1992-08-07 | 1996-05-07 | The General Hospital Corporation | Hydrogel compositions and methods of use |
| CN102220017A (zh) * | 2011-05-05 | 2011-10-19 | 苏州大学 | 一种可注射成型的丝素蛋白水凝胶及其制备方法 |
| CN105407927A (zh) * | 2013-05-24 | 2016-03-16 | 丹麦技术大学 | 用于引导放射治疗的凝胶配方 |
| CN103289107A (zh) * | 2013-05-28 | 2013-09-11 | 苏州丝美特生物技术有限公司 | 一种可注射丝素蛋白原位凝胶的制备方法及其应用 |
| US20160136241A1 (en) * | 2014-02-28 | 2016-05-19 | Union Hospital, Tongji Medical College, Huazhong University Of Science And Technology | Methods of preparing and using sericin hydrogel |
| US20170107264A1 (en) * | 2014-03-27 | 2017-04-20 | Simatech Incorporation | Freeze-dried powder of high molecular weight silk fibroin, preparation method therefor and use thereof |
| WO2016079332A1 (en) * | 2014-11-21 | 2016-05-26 | Technical University Of Denmark | Gel formulations for improving immunotherapy |
| WO2016079331A1 (en) * | 2014-11-21 | 2016-05-26 | Technical University Of Denmark | Gel formulations for enhancing the effect of radiotherapy |
| CN105597104A (zh) * | 2014-12-16 | 2016-05-25 | 苏州丝美特生物技术有限公司 | 一种用于药物控释的丝素-载药纳微米颗粒的制备方法 |
| US20170360912A1 (en) * | 2014-12-17 | 2017-12-21 | Socovar, L.P. | Chitosan-based hydrogel and applications thereof. |
| CN104644545A (zh) * | 2015-02-27 | 2015-05-27 | 苏州丝美特生物技术有限公司 | 一种治疗内耳疾病的控释缓释丝素蛋白凝胶制剂 |
| CN104815357A (zh) * | 2015-04-27 | 2015-08-05 | 复旦大学 | 一种无机有机杂化的三维互穿网络水凝胶材料及其制备方法和应用 |
| CN106492270A (zh) * | 2016-11-28 | 2017-03-15 | 哈尔滨工业大学 | 一种用于镁合金与丝素蛋白之间连接的方法 |
| CN108785752A (zh) * | 2018-06-29 | 2018-11-13 | 苏州丝美特生物技术有限公司 | 丝素蛋白基多孔材料及其制备方法 |
| CN109589447A (zh) * | 2018-11-13 | 2019-04-09 | 王岩松 | 一种医用可注射抗菌复合原位水凝胶的制备方法及其应用 |
Non-Patent Citations (1)
| Title |
|---|
| 高振利等: "60%泛影葡胺皮下注射治疗家兔淋巴瘘病理形态计量研究", 《中华泌尿外科杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Oxidation triggered formation of polydopamine-modified carboxymethyl cellulose hydrogel for anti-recurrence of tumor | |
| Yin et al. | Swellable silk fibroin microneedles for transdermal drug delivery | |
| Zhu et al. | L‐Arg‐rich amphiphilic dendritic peptide as a versatile NO donor for NO/photodynamic synergistic treatment of bacterial infections and promoting wound healing | |
| Deng et al. | Facile and eco-friendly fabrication of polysaccharides-based nanocomposite hydrogel for photothermal treatment of wound infection | |
| Shi et al. | Schiff based injectable hydrogel for in situ pH-triggered delivery of doxorubicin for breast tumor treatment | |
| Zhang et al. | A multifunctional antibacterial coating on bone implants for osteosarcoma therapy and enhanced osteointegration | |
| Zhang et al. | Dual stimuli-responsive smart fibrous membranes for efficient photothermal/photodynamic/chemo-therapy of drug-resistant bacterial infection | |
| Dziubla et al. | Evaluation of porous networks of poly (2-hydroxyethyl methacrylate) as interfacial drug delivery devices | |
| CN105473622B (zh) | 用于制造成形的交联的透明质酸产物的方法 | |
| Ran et al. | Erythrocyte membrane-camouflaged nanoworms with on-demand antibiotic release for eradicating biofilms using near-infrared irradiation | |
| CN110215438B (zh) | 双载蒽环类药物及光敏剂介孔硅纳米粒的制备方法与应用 | |
| CN113018507B (zh) | 一种具有光热性能的抗菌水凝胶及其制备方法和应用 | |
| CN107141345A (zh) | 一种角蛋白生物大分子一氧化氮供体及其合成与应用 | |
| Miao et al. | Thermochromic polyvinyl alcohol‐iodine hydrogels with safe threshold temperature for infectious wound healing | |
| CN105169476B (zh) | 一种医用原位凝胶的制备方法及其应用 | |
| Li et al. | A gene-coated microneedle patch based on industrialized ultrasonic spraying technology with a polycation vector to improve antitumor efficacy | |
| Qian et al. | Dual-modal imaging and synergistic spinal tumor therapy enabled by hierarchical-structured nanofibers with cascade release and postoperative anti-adhesion | |
| Priddy‐Arrington et al. | Characterization and Optimization of Injectable In Situ Crosslinked Chitosan‐Genipin Hydrogels | |
| Han et al. | An antibacterial membrane based on Janus bacterial cellulose with nano-sized copper oxide through polydopamine conjugation for infectious wound healing | |
| Shakiba et al. | A bi‐functional nanofibrous composite membrane for wound healing applications | |
| Lei et al. | Gelatinase-responsive biodegradable targeted microneedle patch for abscess wound treatment of S. aureus infection | |
| CN113304325B (zh) | 一种集可注射与抗菌于一体的双功能水凝胶及其制备方法和用途 | |
| Lee et al. | Enhancing the anti-bacterial activity of nanofibrous polyurethane membranes by incorporating glycyrrhizic acid-conjugated β-Cyclodextrin | |
| Zhang et al. | Doxorubicin-loaded boron-rich polymer nanoparticles for orthotopically implanted liver tumor treatment | |
| CN111228215A (zh) | 一种自组装可成像丝素蛋白水凝胶的制备方法 |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200605 |
|
| RJ01 | Rejection of invention patent application after publication |