CN115252798A - 一种pH敏感水凝胶及其制备方法和应用 - Google Patents
一种pH敏感水凝胶及其制备方法和应用 Download PDFInfo
- Publication number
- CN115252798A CN115252798A CN202210487551.XA CN202210487551A CN115252798A CN 115252798 A CN115252798 A CN 115252798A CN 202210487551 A CN202210487551 A CN 202210487551A CN 115252798 A CN115252798 A CN 115252798A
- Authority
- CN
- China
- Prior art keywords
- hydrogel
- sensitive hydrogel
- sensitive
- dopamine
- cells
- 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 101
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims abstract description 42
- 239000003814 drug Substances 0.000 claims abstract description 40
- 229940079593 drug Drugs 0.000 claims abstract description 35
- 229960003638 dopamine Drugs 0.000 claims abstract description 22
- 229920001661 Chitosan Polymers 0.000 claims abstract description 19
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims abstract description 19
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims abstract description 19
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims abstract description 15
- 239000002246 antineoplastic agent Substances 0.000 claims abstract description 6
- 229940041181 antineoplastic drug Drugs 0.000 claims abstract description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 12
- 238000004132 cross linking Methods 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 2
- 230000000259 anti-tumor effect Effects 0.000 claims 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 16
- 206010028980 Neoplasm Diseases 0.000 abstract description 15
- 210000004881 tumor cell Anatomy 0.000 abstract description 7
- 238000002512 chemotherapy Methods 0.000 abstract description 5
- 238000011068 loading method Methods 0.000 abstract description 3
- 230000001839 systemic circulation Effects 0.000 abstract description 3
- 239000000853 adhesive Substances 0.000 abstract 1
- 230000001070 adhesive effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 35
- ONDYALNGTUAJDX-UHFFFAOYSA-N tasquinimod Chemical compound OC=1C=2C(OC)=CC=CC=2N(C)C(=O)C=1C(=O)N(C)C1=CC=C(C(F)(F)F)C=C1 ONDYALNGTUAJDX-UHFFFAOYSA-N 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 229950001899 tasquinimod Drugs 0.000 description 9
- 230000005764 inhibitory process Effects 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 230000002378 acidificating effect Effects 0.000 description 7
- 102100030708 GTPase KRas Human genes 0.000 description 6
- 101000584612 Homo sapiens GTPase KRas Proteins 0.000 description 6
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 6
- 201000005202 lung cancer Diseases 0.000 description 6
- 208000020816 lung neoplasm Diseases 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000022131 cell cycle Effects 0.000 description 5
- 210000002889 endothelial cell Anatomy 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 5
- 210000003606 umbilical vein Anatomy 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 239000012148 binding buffer Substances 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 230000008961 swelling Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000010370 cell cloning Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000009701 normal cell proliferation Effects 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 101150105104 Kras gene Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical group OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000003783 cell cycle assay Methods 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 201000005249 lung adenocarcinoma Diseases 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 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 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000002522 swelling effect Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Images
Classifications
-
- 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
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4704—2-Quinolinones, e.g. carbostyril
-
- 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
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
-
- 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
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Inorganic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Neurosurgery (AREA)
- Dermatology (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明涉及一种pH敏感水凝胶及其制备方法和应用,将壳聚糖和聚乙烯吡咯烷酮制备得到水凝胶,再交联多巴胺得到pH敏感水凝胶,具有较强的粘附力和内聚力,用于负载抗肿瘤药物时能够高效的与肿瘤细胞进行结合,通过其pH敏感的特性,特异性的识别肿瘤细胞,维持肿瘤部位局部高浓度的药物,减少药物流入全身循环,减少化疗的副作用。
Description
技术领域
本发明属于生物医药领域,尤其是涉及一种pH敏感水凝胶,制备方法及应用。
背景技术
最近的一项研究表明,肺腺癌最突出亚型中有三分之一的患者携带KRAS基因突变。目前临床上对KRAS突变型肺癌的治疗多以化疗为主,但化疗不能保证肿瘤局灶组织内的较高药物浓度,需要频繁注射以维持有效的药物浓度。为了解决这些问题,提高治疗效率,我们提出了一种由多巴胺修饰的pH敏感水凝胶系统,负载化疗药物Tasquinimod持续高效抑制KRAS突变型肺癌细胞的增殖。
发明内容
为解决上述技术问题,本发明提供一种pH敏感水凝胶,制备方法及应用。
本发明采用的技术方案是:一种pH敏感水凝胶制备方法,壳聚糖和聚乙烯吡咯烷酮制备得到水凝胶,再交联多巴胺得到pH敏感水凝胶。
优选地,壳聚糖、聚乙烯吡咯烷酮和多巴胺的质量比为12-17:10-15:3-8,优选为15:13:5。
优选地,具体制备方法如下:
向壳聚糖溶液中加入聚乙烯吡咯烷酮搅拌混匀,加入甲醛反应得到水凝胶;
将多巴胺溶解于Tris-HCl溶液中,加入到水凝胶中浸泡,过滤得到多巴胺修饰的pH敏感水凝胶。
优选地,先通过酸调节壳聚糖溶液pH值到5-7,再加入聚乙烯吡咯烷酮。
pH敏感水凝胶制备方法制备得到的pH敏感水凝胶。
pH敏感水凝胶在抗肿瘤药物中的应用。
优选地,将药物负载在pH敏感水凝胶中。
优选地,在pH为5-6.5条件下释放药物,优选在pH为6的条件下释放药物。
本发明具有的优点和积极效果是:多巴胺与水凝胶的结合使得水凝胶中出现了氨基或巯基,增强了粘附力和内聚力,能够高效的与肿瘤细胞进行结合,通过其pH敏感的特性,特异性的识别肿瘤细胞,维持肿瘤部位局部高浓度的药物,减少药物流入全身循环,减少化疗的副作用。
附图说明
图1是制备得到的水凝胶形态;
图2是水凝胶交联速率;
图3是不同pH条件下药物释放速率;
图4是水凝胶溶胀性能;
图5是人脐静脉内皮细胞存活率;
图6是细胞染色结果;
图7是肿瘤活性抑制率;
图8是肿瘤活性抑制率;
图9各实验组的细胞克隆形成结果图;
图10克隆形成结果的量化图;
图11是流式细胞仪检测各组细胞的细胞周期;
图12细胞周期结果的量化图。
具体实施方式
下面结合附图对本发明的实施例做出说明。
本发明涉及一种pH敏感水凝胶及其制备方法,壳聚糖和聚乙烯吡咯烷酮制备得到水凝胶,再交联多巴胺得到pH敏感水凝胶。水凝胶是亲水聚合物,在体内外均具有良好的生物相容性,目前基于这些特性的应用范围从细胞培养到生物支架和注射给药。由壳聚糖和聚乙烯吡咯烷酮制备得到水凝胶,加入多巴胺后,多巴胺的邻苯二酚结构与水凝胶材料结合后,容易氧化为醌或半醌结构,然后发生Michael或者Schiff反应,水凝胶中出现氨基或巯基,从而增强了粘附力和内聚力。
其制备方法具体如下:
步骤一:先通过乙酸调整壳聚糖溶液pH值至5-7,优选调整pH值为6,加入聚乙烯吡咯烷酮搅拌混匀,加入甲醛反应得到水凝胶甲醛;乙酸和甲醛能够促进交联,同时甲醛还能够中和调节pH的乙酸;
步骤二:将多巴胺溶解于Tris-HCl溶液中,加入到水凝胶中浸泡,过滤得到多巴胺修饰的pH敏感水凝胶;
其中壳聚糖、聚乙烯吡咯烷酮和多巴胺的摩尔质量比为12-17:10-15:3-8,当比例为15:13:5时,效果最佳。
这种pH敏感水凝胶能够用于负载药物,例如负载抗肿瘤药物,将药物负载在这种pH敏感水凝胶中后,能够通过其对肿瘤细胞的粘附性提高药物作用效果;并且其药物释放效果容易受到环境pH影响,中性pH环境中药物释放速度低于酸性pH环境中药物释放速度,尤其pH值为5-6.5时,释放效果最佳。基于这一特性,负载药物的pH敏感水凝胶可以在倾向于低pH的肿瘤微环境中快速、大量地释放药物,解决了现有技术中存在治疗种肿瘤局灶组织内的药物浓度不高,需要频繁注射以维持有效的药物浓度等问题,能够维持肿瘤部位局部高浓度的药物,减少药物流入全身循环,减少化疗的副作用。
下面结合附图对本发明方案做出说明,其中,未具体说明操作步骤的实验方法,均按照相应商品说明书进行,实施例中所用到的仪器、试剂、耗材如无特殊说明,均可从商业公司购买得到。
实施例1:pH敏感水凝胶的制备
1.1水凝胶的合成
将0.15g壳聚糖、15mL双蒸水和2mL H+(3mol/L)加入5ml圆底烧瓶中搅拌均匀。加入0.13g聚乙烯吡咯烷酮,搅拌并抽真空。加入0.2ml甲醛(37%),室温下反应24小时得到水凝胶,程透明状。
1.2交联多巴胺
将0.050g多巴胺加入10ml Tris-HCl溶液中,搅拌溶解;加入之前合成的水凝胶1.0g,室温浸泡48h,过滤干燥得到pH敏感水凝胶,如图1所示,呈棕色。
另外制备壳聚糖水凝胶、PVP水凝胶、壳聚糖+PVP混合水凝胶作为对照样,与上述方法制备得到的pH敏感水凝胶比对。在合成过程中量化水凝胶完全凝固所需的时间,发现加入多巴胺后水凝胶的形成时间缩短一半,如图2所示。这表明多巴胺导致的交联速度更快,负载药物后,材料可以快速组肿瘤生长部位,防止它转移到非处理地点。
实施例2:水凝胶药物释放能力评估
模拟肿瘤周围环境,分别将负载了Tasquinmomod药物的pH敏感水凝胶至于中性pH环境中和酸性pH环境中,通过检测Tasquinmomod的OD值,评估药物释放水平,得到该物质的释药曲线。
如图3所示,发现在中性pH水平下,药物释放速度相对较慢,第16天释放量约为总负荷的45%,pH值为7.35-7.45时尤其明显。相反,在酸性pH水平下,药物释放速率加快,总释放量更大,在第16天达就能够到约70%。这些数据表明,该材料具有一定的pH敏感性,并突出说明它可以在倾向于低pH的肿瘤微环境中快速、大量地释放药物。
进一步的,测试水凝胶的溶胀性能,等体积的冻干水凝胶在37℃的PBS(pH 为7.45/pH为6)中浸泡。每10min取出膨胀水凝胶称重(n=3),膨胀比计算公式如下:膨胀比(%)=(Wt-W0)/W0×100%,其中Wt为膨胀水凝胶的重量, W0为冻干水凝胶的初始重量。如图4所示,pH为6条件下,水凝胶吸水膨胀,随后出现溶解,但是pH为7.45环境下,吸水后并未出现明显的溶解,发现水凝胶在酸性环境下溶解能力比中性环境下有着显著提升,故水凝胶对酸性条件更为敏感,在酸性条件下更易溶解。人体环境一般为中性环境,肿瘤处一般呈偏酸性,本方案制得的水溶胶在肿瘤处更容易溶解释药。
实施例3:水凝胶对正常细胞增殖影响
分别取壳聚糖水凝胶、PVP水凝胶、壳聚糖+PVP混合水凝胶和pH敏感水凝胶,将4种水凝胶与人脐静脉内皮细胞混合培养5天,评估材料的细胞毒性。小鼠成纤维细胞(L929)和人脐静脉内皮细胞(HUVEC)在DMEM和RPMI 1640培养基中培养。所有培养基均含有10%的胎牛血清,同时细胞保持在37℃,在5% CO2和95%湿度的环境中。将细胞接种于96孔板中,培养24小时。将4种质量相同的水凝胶加入相应的培养基中,过滤灭菌,用得到的浓度为1mg/mL无菌母液进行实验操作。将无菌母液稀释至200、100、50、25或0μg/mL(不加样品)。每孔加100μL无菌液,与细胞共培养1、3和5天。最后用CCK-8法测定细胞活力,用分光光度计测定光密度(OD)。
如图5所示,所有组的人脐静脉内皮细胞存活率全天都在90%以上,证明材料具有良好的生物相容性。如图6所示,进行活死细胞染色同时证明所有的实验材料在人脐静脉内皮细胞中均表现出良好的生物相容性。说明水凝胶对正常细胞增殖无影响,材料的生物安全性并支持了材料的临床应用价值。
实施例4:低浓度Tasquinimod联合水凝胶对A549细胞增殖的影响
与传统的一次性注射方法相比,水凝胶的优势在于缓释:在局部治疗环境中保持较高的药物浓度,可获得优越的治疗效果。因此我们比较水凝胶缓释与一次性给药的疗效。载药释放实验:将负载Tasquinimod的pH敏感和不敏感水凝胶分别置于10ml PBS(5.5/7.35)的两个pH梯度中,并释放16天。每隔2天,用移液管抽取各组100μl的溶液,加入96孔板中,提取后立即加入100μl PBS补充实验液。用酶标仪定量分析结果。体外抑瘤效果试验:实验成分分为四组:第一组(多巴胺修饰pH敏感水凝胶+2.5μmol Tasquinimod)、第二组(多巴胺修饰 pH敏感水凝胶+10μmolTasquinimod)、第三组(5μmol Tasquinimod)和第四组 (10μmolTasquinimod)。为了检测肿瘤细胞的活性,将4组载抗肿瘤药物 Tasquinimod的水凝胶组与肿瘤细胞体外共培养。
将4种不同浓度的Tasquinimod(0,2.5μmol,5μmol和10μmol)进行梯度检测,并在处理A549 KRAS突变肺癌细胞5天后评估其细胞毒性和生存能力。如图7所示,发现2.5μmol的Tasquinimod对肿瘤活性的抑制超过30%,浓度越高,抑制效果越明显。
为了评价凝胶的释放性能,我们采用2.5μmol和10μmol梯度进行了实验。通过使用不同浓度的水凝胶和直接给药体外治疗KRAS突变型肺癌细胞,我们发现水凝胶比一次性给药具有更好的抑制效果,并显示出更高的抑制峰。如图8 所示,在2.5μmol时表现出显著的抑制作用,在10μmol时表现出更明显的抑制作用。
采用克隆形成实验,将水凝胶给药组与直接一次性给药组进行比较。如图 9-10所示,发现该药物显著抑制细胞克隆,且在水凝胶载药组抑制效果更明显。
实施例5:低浓度Tasquinimod复合水凝胶有效抑制A549细胞的细胞周期
细胞周期试验:将细胞以每孔1.5×106个细胞的速度铺板于6孔板中,并使其生长到对数生长期。贴壁50~80%后,将细胞转移到含药培养基中。孵育相应时间后,进行流式细胞术,每个样品分析的细胞数约为106个。
细胞处理:胰蛋白酶处理细胞,含血清培养基中和,1000rpm离心3分钟,重悬,预冷PBS洗涤两次。
细胞固定:将细胞离心后去除上清,同时缓慢加入3ml预冷的70%无水乙醇并轻轻吹散细胞,-20℃固定过夜,然后进行流式细胞术检测。用预冷PBS洗涤固定细胞2次,2000rpm离心5min,重悬于200μL PBS中,终体积约为400 μL。为了减少细胞损失,使用1.5ml离心容量。轻轻拍打离心机底部,使细胞适当分散,避免细胞结块。在重悬后的细胞中加入20μg/L RNase,37℃水浴培养30分钟。加入20μL的碘化丙啶(PI)至终浓度50ug/mL,在室温下避光下染色30分钟。流式细胞术,染色后24小时内进行,细胞充分混合。在488nm 激发波长下检测到红色荧光并评估光散射。同时采用合适的分析软件进行细胞 DNA含量分析和光散射分析。
细胞凋亡试验:用预冷PBS缓冲液洗涤细胞2次,在1×Binding buffer中重悬,1×106细胞/ml悬液。将细胞悬液100μL加入Falcon试管中,轻轻混合,室温(20-25℃)避光放置15min。用1×Binding Buffer冲洗细胞1次,取上清液。 0.5μg的SAv-FITC试剂溶于100μl的1×Binding Buffer中,加入试管中轻轻混合。加入5μL PI,室温(20-25℃)避光染色15min。每个试管加入400μL 1×Binding Buffer,1小时内用流式细胞仪定量细胞凋亡。
如图11-12所示,采用流式细胞仪检测各组细胞的细胞周期,结果证明Tasquinimod组细胞分裂能力弱于对照组且细胞周期延长,细胞G1期和S期发生改变,这与药物的作用方式有关。
以聚乙烯吡咯烷酮和壳聚糖为基础研制pH敏感水凝胶,在水凝胶中加入多巴胺使其具有优异的凝胶形成和支持特性。同时多巴胺的加入,交联水凝胶可以比基础水凝胶更快地凝固,并且在不影响其pH敏感性的情况下负载更大数量的药物。本发明发现Tasquinimod可以抑制KRAS突变型肺癌细胞的增殖,与聚乙烯吡咯烷酮-壳聚糖-多巴胺pH敏感水凝胶系统联合使用时增强抑制癌细胞增殖作用。这种利用生物材料抑制KRAS突变型肺癌的新方法具有效率高、副作用少等优点,具有一定的临床价值意义。
以上对本发明的实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等,均应仍归属于本发明的专利涵盖范围之内。
Claims (9)
1.一种pH敏感水凝胶制备方法,其特征在于:壳聚糖和聚乙烯吡咯烷酮制备得到水凝胶,再交联多巴胺得到pH敏感水凝胶。
2.根据权利要求1所述的pH敏感水凝胶制备方法,其特征在于:壳聚糖、聚乙烯吡咯烷酮和多巴胺的质量比为12-17:10-15:3-8。
3.根据权利要求2所述的pH敏感水凝胶制备方法,其特征在于:壳聚糖、聚乙烯吡咯烷酮和多巴胺的质量比为15:13:5。
4.根据权利要求1-3中任一所述的pH敏感水凝胶制备方法,其特征在于:具体制备方法如下:
向壳聚糖溶液中加入聚乙烯吡咯烷酮搅拌混匀,加入甲醛反应得到水凝胶;
将多巴胺溶解于Tris-HCl溶液中,加入到水凝胶中浸泡,过滤得到多巴胺修饰的pH敏感水凝胶。
5.根据权利要求4所述的pH敏感水凝胶制备方法,其特征在于:先通过酸调节壳聚糖溶液pH值再加入聚乙烯吡咯烷酮。
6.权利要求1-5中任一所述的pH敏感水凝胶制备方法制备得到的pH敏感水凝胶。
7.权利要求6所述的pH敏感水凝胶在制备抗肿瘤药物中的应用。
8.根据权利要求7所述的pH敏感水凝胶在制备抗肿瘤药物中的应用,其特征在于:将药物负载在pH敏感水凝胶中。
9.根据权利要求8所述的pH敏感水凝胶在制备抗肿瘤药物中的应用,其特征在于:在pH值为5-6.5条件下释放药物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210487551.XA CN115252798A (zh) | 2022-05-06 | 2022-05-06 | 一种pH敏感水凝胶及其制备方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210487551.XA CN115252798A (zh) | 2022-05-06 | 2022-05-06 | 一种pH敏感水凝胶及其制备方法和应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN115252798A true CN115252798A (zh) | 2022-11-01 |
Family
ID=83759192
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210487551.XA Pending CN115252798A (zh) | 2022-05-06 | 2022-05-06 | 一种pH敏感水凝胶及其制备方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115252798A (zh) |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1417248A (zh) * | 2002-12-06 | 2003-05-14 | 暨南大学 | 壳聚糖接枝聚乙烯吡咯烷酮及其制备方法和用于制备生物降解材料的方法 |
KR20030060458A (ko) * | 2002-01-09 | 2003-07-16 | 한국원자력연구소 | 상처 치료용 수화겔의 제조방법 |
CN102504291A (zh) * | 2011-10-28 | 2012-06-20 | 北京联合大学生物化学工程学院 | pH敏感型壳聚糖交联聚维酮凝胶及其制备方法和应用 |
KR101829136B1 (ko) * | 2016-08-11 | 2018-02-13 | 가톨릭대학교 산학협력단 | 가시광선 광경화 수용성 키토산 유도체, 키토산 하이드로겔 및 이의 제조방법 |
CN108434514A (zh) * | 2018-04-18 | 2018-08-24 | 华中农业大学 | 一种兼具抑菌与生物诱导作用的胶原水凝胶制备方法 |
CN109180970A (zh) * | 2018-08-30 | 2019-01-11 | 武汉理工大学 | 一种柠檬酸交联壳聚糖和多巴胺的水凝胶及其制备方法 |
WO2021158013A1 (en) * | 2020-02-04 | 2021-08-12 | Korea Atomic Energy Research Institute | Hydrogel and method for preparing the same |
CN113350483A (zh) * | 2021-06-04 | 2021-09-07 | 南京鼓楼医院 | 一种用于疤痕修复的载白喉毒素的微针及其制备方法 |
-
2022
- 2022-05-06 CN CN202210487551.XA patent/CN115252798A/zh active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20030060458A (ko) * | 2002-01-09 | 2003-07-16 | 한국원자력연구소 | 상처 치료용 수화겔의 제조방법 |
CN1417248A (zh) * | 2002-12-06 | 2003-05-14 | 暨南大学 | 壳聚糖接枝聚乙烯吡咯烷酮及其制备方法和用于制备生物降解材料的方法 |
CN102504291A (zh) * | 2011-10-28 | 2012-06-20 | 北京联合大学生物化学工程学院 | pH敏感型壳聚糖交联聚维酮凝胶及其制备方法和应用 |
KR101829136B1 (ko) * | 2016-08-11 | 2018-02-13 | 가톨릭대학교 산학협력단 | 가시광선 광경화 수용성 키토산 유도체, 키토산 하이드로겔 및 이의 제조방법 |
CN108434514A (zh) * | 2018-04-18 | 2018-08-24 | 华中农业大学 | 一种兼具抑菌与生物诱导作用的胶原水凝胶制备方法 |
CN109180970A (zh) * | 2018-08-30 | 2019-01-11 | 武汉理工大学 | 一种柠檬酸交联壳聚糖和多巴胺的水凝胶及其制备方法 |
WO2021158013A1 (en) * | 2020-02-04 | 2021-08-12 | Korea Atomic Energy Research Institute | Hydrogel and method for preparing the same |
CN113350483A (zh) * | 2021-06-04 | 2021-09-07 | 南京鼓楼医院 | 一种用于疤痕修复的载白喉毒素的微针及其制备方法 |
Non-Patent Citations (2)
Title |
---|
JUN XU: "A Tasquinomod-loaded dopamine modified pH sensitive hydrogel is effective at inhibiting the proliferation of KRAS mutant lung cancer cells", 《JOURNAL OF APPLIED BIOMATERIALS & FUNCTIONAL MATERIALS》, vol. 20, 28 January 2022 (2022-01-28), pages 1 - 8 * |
陈欣: "聚乙烯吡咯烷酮/壳聚糖水凝胶的制备与表征", 《湖北理工学院学报》, vol. 32, no. 4, 31 August 2016 (2016-08-31), pages 26 - 34 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Koutsopoulos et al. | Long-term three-dimensional neural tissue cultures in functionalized self-assembling peptide hydrogels, matrigel and collagen I | |
Yang et al. | Three-dimensional-engineered matrix to study cancer stem cells and tumorsphere formation: effect of matrix modulus | |
Jeon et al. | The effect of oxidation on the degradation of photocrosslinkable alginate hydrogels | |
Tsai et al. | Enhancement of human adipose-derived stem cell spheroid differentiation in an in situ enzyme-crosslinked gelatin hydrogel | |
Han et al. | Chitosan-hyaluronan based 3D co-culture platform for studying the crosstalk of lung cancer cells and mesenchymal stem cells | |
De Luca et al. | Positively charged polymers modulate the fate of human mesenchymal stromal cells via ephrinB2/EphB4 signaling | |
Nakod et al. | Three‐dimensional biomimetic hyaluronic acid hydrogels to investigate glioblastoma stem cell behaviors | |
Martínez‐Ramos et al. | Three‐dimensional constructs using hyaluronan cell carrier as a tool for the study of cancer stem cells | |
Scanga et al. | Biomaterials for neural-tissue engineering—Chitosan supports the survival, migration, and differentiation of adult-derived neural stem and progenitor cells | |
KR20100044173A (ko) | 줄기 세포에 대해 펩타이드 연결된 세포 기질 및 이의 사용 방법 | |
CN113774027A (zh) | 一种蜂巢状GelMA微球在构建肿瘤模型中的应用 | |
Rezaeeyazdi et al. | Engineering hyaluronic acid-based cryogels for CD44-mediated breast tumor reconstruction | |
Jiang et al. | Brain microenvironment responsive and pro‐angiogenic extracellular vesicle‐hydrogel for promoting neurobehavioral recovery in type 2 diabetic mice after stroke | |
Ozturk et al. | Development and characterization of cancer stem cell‐based tumoroids as an osteosarcoma model | |
Shrimali et al. | Efficient in situ gene delivery via PEG diacrylate matrices | |
US20240026286A1 (en) | Method of producing cancer stem cells | |
US20230201114A1 (en) | A synthetic hydrogel and its use for immunotherapy and 3d-printing | |
Villa et al. | P (NIPAAM–co‐HEMA) thermoresponsive hydrogels: an alternative approach for muscle cell sheet engineering | |
KR101293618B1 (ko) | 생분해성 합성고분자와 공중합을 통한 세포주사형 하이드로겔 및 이의 제조방법 | |
EP3017301A2 (en) | Microtissues | |
Kleinberger et al. | Synthetic polycations with controlled charge density and molecular weight as building blocks for biomaterials | |
US9029148B2 (en) | Methods for the preparation of fibroblasts | |
CN115252798A (zh) | 一种pH敏感水凝胶及其制备方法和应用 | |
KR101233072B1 (ko) | 연골조직 재건 또는 재생을 위한 나노 복합체 및 이를 이용한 연골세포로의 분화방법 | |
Zhu et al. | ECM-inspired peptide dendrimer microgels with human MSCs encapsulation for systemic lupus erythematosus treatment |
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 |