CN111110861B - 一种基于人血清白蛋白的载药颗粒及其制备方法 - Google Patents
一种基于人血清白蛋白的载药颗粒及其制备方法 Download PDFInfo
- Publication number
- CN111110861B CN111110861B CN202010110186.1A CN202010110186A CN111110861B CN 111110861 B CN111110861 B CN 111110861B CN 202010110186 A CN202010110186 A CN 202010110186A CN 111110861 B CN111110861 B CN 111110861B
- Authority
- CN
- China
- Prior art keywords
- serum albumin
- human serum
- drug
- based drug
- ddc
- 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.)
- Active
Links
- 102000008100 Human Serum Albumin Human genes 0.000 title claims abstract description 57
- 108091006905 Human Serum Albumin Proteins 0.000 title claims abstract description 57
- 239000003814 drug Substances 0.000 title claims abstract description 51
- 229940079593 drug Drugs 0.000 title claims abstract description 46
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 239000008187 granular material Substances 0.000 title description 3
- 239000002245 particle Substances 0.000 claims abstract description 35
- 150000001875 compounds Chemical class 0.000 claims abstract description 15
- 239000008017 pharmaceutical colorant Substances 0.000 claims abstract description 9
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 53
- 239000000243 solution Substances 0.000 claims description 29
- 229960003180 glutathione Drugs 0.000 claims description 26
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 20
- 239000008055 phosphate buffer solution Substances 0.000 claims description 20
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 13
- 108010024636 Glutathione Proteins 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 12
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 claims description 11
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 10
- 229910052786 argon Inorganic materials 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000000502 dialysis Methods 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 5
- 238000000520 microinjection Methods 0.000 claims description 5
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 4
- 238000000108 ultra-filtration Methods 0.000 claims description 4
- 230000001133 acceleration Effects 0.000 claims description 2
- 239000007789 gas Substances 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 abstract description 31
- 230000000259 anti-tumor effect Effects 0.000 abstract description 10
- 230000008685 targeting Effects 0.000 abstract description 7
- 206010006187 Breast cancer Diseases 0.000 abstract description 6
- 208000026310 Breast neoplasm Diseases 0.000 abstract description 6
- 231100000419 toxicity Toxicity 0.000 abstract description 5
- 230000001988 toxicity Effects 0.000 abstract description 5
- 238000004020 luminiscence type Methods 0.000 abstract description 3
- 238000012544 monitoring process Methods 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 10
- 238000001727 in vivo Methods 0.000 description 10
- 230000009467 reduction Effects 0.000 description 10
- 239000002246 antineoplastic agent Substances 0.000 description 8
- 229940041181 antineoplastic drug Drugs 0.000 description 5
- 229940002612 prodrug Drugs 0.000 description 5
- 239000000651 prodrug Substances 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 230000005918 in vitro anti-tumor Effects 0.000 description 4
- 230000005917 in vivo anti-tumor Effects 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 231100000002 MTT assay Toxicity 0.000 description 2
- 238000000134 MTT assay Methods 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000005415 bioluminescence Methods 0.000 description 2
- 230000029918 bioluminescence Effects 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 125000002228 disulfide group Chemical group 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- 231100000374 pneumotoxicity Toxicity 0.000 description 2
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 125000003396 thiol group Chemical group [H]S* 0.000 description 2
- 206010015548 Euthanasia Diseases 0.000 description 1
- 206010061924 Pulmonary toxicity Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 125000005587 carbonate group Chemical group 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 231100000259 cardiotoxicity Toxicity 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000009513 drug distribution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- ZXQYGBMAQZUVMI-GCMPRSNUSA-N gamma-cyhalothrin Chemical compound CC1(C)[C@@H](\C=C(/Cl)C(F)(F)F)[C@H]1C(=O)O[C@H](C#N)C1=CC=CC(OC=2C=CC=CC=2)=C1 ZXQYGBMAQZUVMI-GCMPRSNUSA-N 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 231100000417 nephrotoxicity Toxicity 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000007047 pulmonary toxicity Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 231100000974 spleenotoxicity Toxicity 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 231100000057 systemic toxicity Toxicity 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
-
- 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
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- 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
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nanotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicinal Preparation (AREA)
Abstract
本发明公开了一种基于人血清白蛋白的载药颗粒(DDC‑HSA)，包括作为载体分子的人血清白蛋白(HSA)和负载在所述人血清白蛋白上的药物染料化合物(DDC)，所述基于人血清白蛋白的载药颗粒(DDC‑HSA)具有如式(I)所示结构。本发明还公开了上述载药颗粒的制备方法。本申请制得的DDC‑HSA实现了乳腺癌的肿瘤靶向，具有良好的抗肿瘤功效和低的生理毒性，可以很好地用作抗肿瘤试剂。本申请制得的DDC‑HSA一旦被富含肿瘤微环境的GSH诱导，药物的释放伴随着1:1的染料发光，实现了无创监测药物在瘤体内的释放，还可以用于肿瘤的精准个性化治疗。
Description
技术领域
本发明涉及抗肿瘤药物制剂领域，尤其涉及一种基于人血清白蛋白的载药颗粒及其制备方法。
背景技术
前药代表一种衍生自母体药物的化合物，具有改善的可用药性。前药本身是无活性的，但可以在体内微环境中被激活。前药可以调整体内分布，调节药代动力学并最大程度降低母体药物的副作用，因此目前被认为是改良常规临床药物以提高治疗效率的有用策略。例如，富含肿瘤微环境的谷胱甘肽(GSH)可被用作引发抗肿瘤前药再转化的触发因素，最终将导致针对性和局部性的药物释放。以无创方式实时监测前药系统中药物的活化或转化已引起越来越多的关注，因为精确的时空药物释放信息可以帮助指导实现个性化的精准治疗。
在目前的治疗学系统中，用于肿瘤(乳腺癌)的药物染料化合物(DDC)是一种使用光学方法(近红外(NIR)光)报告体内即时药物释放的范例。DDC的结构如下：
由上述结构可知，DDC抗肿瘤药由SN-38、“OFF”状态的NIR染料和中间连接结构组成，可以被异常因子分解，释放药物的同时以1：1模式打开NIR染料以立即表明药物的命运。然而，母体抗肿瘤药物SN-38和选定的NIR染料通常溶解度低且肿瘤靶向能力差，抗肿瘤药物在肿瘤组织的积累少、浓度低，治疗效果有限。
因此，本领域的技术人员致力于开发一种溶解度高、肿瘤靶向性好、生理毒性低的DDC载药系统。
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是现有的DDC存在的溶解度低、肿瘤靶向能力差，使得抗肿瘤药物SN-38在肿瘤组织的积累少、浓度低，治疗效果有限等。
人血清白蛋白(HSA)在生理活动和代谢中起着至关重要的作用，并具有待运输物质的疏水结合袋。人血清白蛋白(HSA)作为抗肿瘤药物的载体具有显著的前景：延长半衰期，可调节生物分布，较少的副作用和靶向肿瘤的作用。在本发明中，发明人通过可逆的二硫键交换反应成功地将DDC分子共价连接到HSA中，形成了特殊的“特洛伊木马”，被分解时药物释放和染料还原严格按照1：1模式进行。
为实现上述目的，本发明提供了一种基于人血清白蛋白的载药颗粒(DDC-HSA)，包括作为载体分子的人血清白蛋白(HSA)和负载在所述人血清白蛋白上的药物染料化合物(DDC)，所述基于人血清白蛋白的载药颗粒(DDC-HSA)具有如式(I)所示结构，所述药物染料化合物(DDC)具有如式(II)所示结构：
在本发明的较佳实施方式中，所述基于人血清白蛋白的载药颗粒(DDC-HSA)的粒径为220±87nm。
本发明还提供了一种上述基于人血清白蛋白的载药颗粒(DDC-HSA)的制备方法，包括如下步骤：
a、将人血清白蛋白溶于含有谷胱甘肽的磷酸盐缓冲溶液中，在37℃搅拌均匀，所得溶液在氩气保护下于37℃用磷酸盐缓冲溶液透析，以去除过量的谷胱甘肽，其中，所述磷酸盐缓冲溶液的pH为7.4、浓度为10mM；
b、将步骤a所得的溶液在氩气下转移至反应瓶，并在37℃下在剧烈搅拌下使用微量注射泵逐滴添加药物染料化合物的二甲基亚砜溶液，所得溶液用磷酸盐缓冲溶液透析，以除去离去基团吡啶和未反应的药物染料化合物，所述磷酸盐缓冲溶液的pH为7.4、浓度为10mM；
c、在室温搅拌下将步骤b所得的溶液转移至双氧水中并保持一段时间、超滤以除去过量的H2O2和无机盐、冷冻干燥，得到所述基于人血清白蛋白的载药颗粒(DDC-HSA)。
在本发明的较佳实施方式中，所述步骤a具体为：将25mg人血清白蛋白溶于含有谷胱甘肽的磷酸盐缓冲溶液中，在37℃搅拌1h，所得溶液在氩气保护下于37℃用磷酸盐缓冲溶液透析12h，以去除过量的谷胱甘肽，其中，所述磷酸盐缓冲溶液的pH为7.4、浓度为10mM，所述谷胱甘肽在所述磷酸盐缓冲溶液中的浓度为5mM。
在本发明的较佳实施方式中，所述步骤b具体为：将所述步骤a所得的溶液在氩气下转移至反应瓶，并在37℃下在剧烈搅拌下使用微量注射泵逐滴添加0.5ml浓度为2mg/mL的药物染料化合物的二甲基亚砜溶液，所得溶液用磷酸盐缓冲溶液透析12h，以除去离去基团吡啶和未反应的药物染料化合物，所述磷酸盐缓冲溶液的pH为7.4、浓度为10mM。
在本发明的较佳实施方式中，述步骤c具体为：在室温搅拌下将所述步骤b所得的溶液转移至20mL3％双氧水中并保持0.5h、4℃超滤以除去过量的H2O2和无机盐、冷冻干燥，得到所述基于人血清白蛋白的载药颗粒(DDC-HSA)。
在本发明的较佳实施方式中，在执行所述步骤a和b中的透析步骤时所采用的透析袋的截留分子量为8K。
在本发明的较佳实施方式中，在执行所述步骤c中的超滤步骤时，所采用的超滤膜的分子量为10k、离心力是重力加速度的3倍。
本发明提供的基于人血清白蛋白的载药颗粒(DDC-HSA)及其制备方法具有以下技术效果：
1、本申请制得的DDC-HSA实现了乳腺癌的肿瘤靶向，具有良好的抗肿瘤功效和低的生理毒性，可以很好地用作抗肿瘤试剂，这是由于HSA的靶向性实现了所负载的DDC在肿瘤组织的释入，通过改善母体药物的体内分布从而改善肿瘤的局部药物浓度，增强了抗肿瘤作用。
2、本申请制得的DDC-HSA一旦被富含肿瘤微环境的GSH诱导，药物的释放伴随着1:1的染料发光，实现了无创监测药物在瘤体内的释放，还可以用于肿瘤的精准个性化治疗。
3、本发明提供了一种具有乳腺癌治疗作用的颗粒及合成方法，为乳腺癌治疗提供了新的途径，该途径将治疗和诊断结合在一起，具有潜在的临床意义。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是本发明实施例1制得的基于人血清白蛋白的载药颗粒(DDC-HSA)的核磁共振氢谱图。
图2是DDC-HSA透射电镜图像。
图3是DDC-HSA纳米粒粒径分布图。
图4是DDC-HSA中药物释放和染料还原机理图。
图5是DDC-HSA中SN-38随时间的释放曲线图。
图6是DDC-HSA中染料随时间的还原曲线图。
图7是DDC-HSA中SN-38释放与染料还原的线性关系图(在GSH浓度为10mM下测定)。
图8是体外抗肿瘤效果图。
图9是体内抗肿瘤效果图，以肿瘤体积表示。
图10是体内抗肿瘤效果图，以肿瘤重量表示。
图11是体外细胞毒性实验图。
图12是心脏、肝脏、脾、肺和肾脏毒性实验图。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。需说明的是，在不冲突的情况下，以下实施例及实施例中的特征可以相互组合。
基于人血清白蛋白的载药颗粒(DDC-HSA)的合成反应式如下：
其中作为起始原料的DDC中的NIR染料(DDC右边基团)处于沉默状态(在λex/em＝679/730nm处关闭)，归因于吸电子碳酸酯基团。HSA拥有35个半胱氨酸残基，其中34个形成分子内二硫键。合成路线中，先使用GSH裂解二硫键以留下反应性硫醇基，多余的GSH使用透析法去除，然后通过二硫键交换反应将DDC与具有扩大的口袋的被还原的HSA连接，其中吡啶部分起离去基团的作用，最后将残留的硫醇基团氧化成二硫键以封闭HSA的口袋，从而获得高稳定性的基于人血清白蛋白的载药颗粒(DDC-HSA)。
实施例1
基于人血清白蛋白的载药颗粒(DDC-HSA)的合成
步骤a：将25mg人血清白蛋白冻干粉溶于含有谷胱甘肽(5mM)的磷酸盐缓冲溶液7.4(PBS7.4，10mL，10mM)中，在37℃搅拌1h，所得溶液在氩气保护下于37℃用PBS7.4(1L*4)透析(MW 8K)12h，以去除过量的谷胱甘肽；
步骤b：将步骤a所得的溶液在氩气下转移至反应瓶，并在37℃下在剧烈搅拌下使用微量注射泵逐滴添加0.5ml浓度为2mg/mL的DDC的二甲基亚砜溶液，所得溶液用PBS7.4(1L*4)透析(MW 8K)12h，以除去离去基团吡啶和未反应的DDC。
步骤c：在室温搅拌下将步骤b所得的溶液转移至双氧水(3％水溶液，20mL)中并保持0.5h，然后溶液在4℃超滤(10K，3倍重力离心)以除去过量的H2O2和无机盐，冷冻干燥，得到目标产物基于人血清白蛋白的载药颗粒(DDC-HSA)。
DDC在水中的溶解度很差，而DDC-HSA可以很好地分散在水中，这是由于DDC是包裹在HSA的口袋里的，从而具有较好的水溶性。
图1示出了上述制得的基于人血清白蛋白的载药颗粒(DDC-HSA)的核磁共振氢谱图；图2示出了上述制得的DDC-HSA透射电镜图像，由图2可以看出，DDC-HSA呈球形；图3示出了上述制得的DDC-HSA粒径分布图，粒径为220±87nm。
实施例2
基于人血清白蛋白的载药颗粒(DDC-HSA)中的药物转化和染料响应效能
DDC-HSA在GSH作用下，双向崩解药物释放及染料还原机理如图4所示。
用HPLC测定了经不同浓度(2μΜ、1mM、10mM)GSH的PBS7.4溶液处理的DDC-HSA中释放的SN-38的累积百分比与时间的关系，图5示出了SN-38释放曲线；用荧光光谱仪测定了经不同浓度(2μΜ、1mM、10mM)GSH的PBS7.4溶液处理的DDC-HSA中还原的染料的荧光强度，图6示出了染料还原曲线。
由图5和6可以看出，在10mM GSH存在下15小时后完成约75％的药物释放和染料还原，该GSH浓度用于模拟肿瘤细胞内微环境条件。浓度较低的GSH存在时，药物释放率和染料还原率要低得多(1mM GSH，50％/15h，模拟细胞外条件；2μM GSH，30％/15h，模拟血浆条件；10％，PBS 7.4)。分别选择SN-38释放曲线和染料还原曲线，发现所有GSH浓度组均具有精细的同步性，图7示出了，以10mM GSH为例，SN-38释放和染料还原的线性关系。
实施例3
体外和体内抗肿瘤效果检测
体外抗肿瘤效果检测
本实施例通过MTT测定法评估体外抗肿瘤功效。将MDA-MB-231细胞以每孔103个细胞的密度接种在96孔板中，并孵育直至达到80-90％的细胞密度。用梯度浓度的不同制剂处理细胞24小时。然后，除去培养基，并用Hank′s冲洗细胞，然后加入100mL MTT溶液(0.5mg/mL)。将该悬浮液在37℃下孵育4小时，然后除去MTT溶液。添加DMSO(100μL)以在37℃振荡器上溶解甲臢晶体15分钟。使用微孔板分光光度计在570nm处记甲臢的吸光度，以提供IC50值的数据。未经处理的细胞用作对照。抗肿瘤效果图如图8所示。
体内抗肿瘤效果检测
通过将5×107MDA-MB-231/luci细胞注入第二个乳垫，原位移植乳腺癌裸鼠模型。7天后，根据可反映初始肿瘤大小的体内实时生物发光IVIS系统扫描的生物发光信号将小鼠随机分为4组(n＝5)(IVIS发光强度约为105)。然后在第0、3、6和9天以5mg/kg SN-38的等价剂量以尾静脉注射给小鼠给药。每三天记录一次体重和肿瘤体积。使用游标卡尺测量肿瘤大小，并计算为体积＝(肿瘤长度)×(肿瘤宽度)2/2。当肿瘤大小超过2000mm3时，将严格执行安乐死，仔细切除肿瘤以进一步称重。抗肿瘤效果如图9和10所示，其中在20天的治疗过程中每两天记录一次数据，n＝5，*P<0.1，**P<0.01和***P<0.001。在肿瘤体积和重量方面，DDC-HSA在所有组中均具有最好的抗肿瘤功效，肿瘤大小得到了很明显的控制。
从图8-10所示的体外和体内的结果表明，DDC-HSA可以很好地用作抗肿瘤试剂，这是由于HSA的靶向性实现所负载的DDC在肿瘤组织的释入，通过改善母体药物的体内分布从而改善肿瘤的局部药物浓度以增强抗肿瘤作用。
实施例4
生理毒性检测
生物安全性是临床应用的前提。通过MTT测定法评估了所有制剂对HEK 293细胞的体外细胞毒性，结果如图11所示，其中载体HSA如所预测的对HEK 293细胞几乎没有细胞毒性，DDC-HSA对HEK 293细胞的毒性相对于SN-38略有降低。由于不确定的体内分布，全身注射化疗药物可能导致不可忽视的副作用。通过对心脏、肝脏、脾、肺和肾脏切片的组织化学分析，探索了体内的生理毒性，结果如图12所示，可以看出SN-38和DDC治疗组的肺部组织病理学病变和异常，表明潜在的肺部毒性，对于DDC-HSA，未观察到对实体器官的明显损害，表明低的全身毒性，这是由于HSA改善药物的体内分布所致。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
Claims (7)
1.一种基于人血清白蛋白的载药颗粒，其特征在于，包括作为载体分子的人血清白蛋白HSA和负载在所述人血清白蛋白上的药物染料化合物DDC，所述基于人血清白蛋白的载药颗粒具有如式（）所示结构，所述药物染料化合物具有如式（/>）所示结构：
（）
（）
所述基于人血清白蛋白的载药颗粒通过以下方法制备：
a、将人血清白蛋白溶于含有谷胱甘肽的磷酸盐缓冲溶液中，在37℃搅拌均匀，所得溶液在氩气保护下于37℃用磷酸盐缓冲溶液透析，以去除过量的谷胱甘肽，其中，所述磷酸盐缓冲溶液的pH为7.4、浓度为10mM；
b、将步骤a所得的溶液在氩气下转移至反应瓶 ，并在37℃下在剧烈搅拌下使用微量注射泵逐滴添加药物染料化合物的二甲基亚砜溶液，所得溶液用磷酸盐缓冲溶液透析，以除去离去基团吡啶和未反应的药物染料化合物，所述磷酸盐缓冲溶液的pH为7.4、浓度为10mM；
c、在室温搅拌下将步骤b所得的溶液转移至双氧水中并保持一段时间、超滤以除去过量的H2O2和无机盐、冷冻干燥，得到所述基于人血清白蛋白的载药颗粒。
2.如权利要求1所述的基于人血清白蛋白的载药颗粒，其特征在于，所述基于人血清白蛋白的载药颗粒的粒径为220±87nm。
3.如权利要求1所述的基于人血清白蛋白的载药颗粒，其特征在于，所述步骤a具体为：将25mg人血清白蛋白溶于含有谷胱甘肽的磷酸盐缓冲溶液中，在37℃搅拌1h，所得溶液在氩气保护下于37℃用磷酸盐缓冲溶液透析12h，以去除过量的谷胱甘肽，其中，所述磷酸盐缓冲溶液的pH为7.4、浓度为10mM，所述谷胱甘肽在所述磷酸盐缓冲溶液中的浓度为5mM。
4.如权利要求1所述的基于人血清白蛋白的载药颗粒，其特征在于，所述步骤b具体为：将所述步骤a所得的溶液在氩气下转移至反应瓶，并在37℃下在剧烈搅拌下使用微量注射泵逐滴添加0.5ml浓度为2mg/mL的药物染料化合物的二甲基亚砜溶液，所得溶液用磷酸盐缓冲溶液透析12h，以除去离去基团吡啶和未反应的药物染料化合物，所述磷酸盐缓冲溶液的pH为7.4、浓度为10mM。
5.如权利要求1所述的基于人血清白蛋白的载药颗粒，其特征在于，所述步骤c具体为：在室温搅拌下将所述步骤b所得的溶液转移至20mL3％双氧水中并保持0.5h、4℃超滤以除去过量的H2O2和无机盐、冷冻干燥，得到所述基于人血清白蛋白的载药颗粒。
6.如权利要求1所述的基于人血清白蛋白的载药颗粒，其特征在于，在执行所述步骤a和b中的透析步骤时所采用的透析袋的截留分子量为8K。
7.如权利要求1所述的基于人血清白蛋白的载药颗粒，其特征在于，在执行所述步骤c中的超滤步骤时，所采用的超滤膜的分子量为10k、离心力是重力加速度的3倍。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010110186.1A CN111110861B (zh) | 2020-02-23 | 2020-02-23 | 一种基于人血清白蛋白的载药颗粒及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010110186.1A CN111110861B (zh) | 2020-02-23 | 2020-02-23 | 一种基于人血清白蛋白的载药颗粒及其制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111110861A CN111110861A (zh) | 2020-05-08 |
| CN111110861B true CN111110861B (zh) | 2023-08-25 |
Family
ID=70492140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010110186.1A Active CN111110861B (zh) | 2020-02-23 | 2020-02-23 | 一种基于人血清白蛋白的载药颗粒及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111110861B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114917238B (zh) * | 2022-05-30 | 2024-02-02 | 郑州大学 | 胰腺癌化疗-免疫治疗递药系统的制备方法及其应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013513609A (ja) * | 2009-12-11 | 2013-04-22 | バイオリテック ファーマ マーケティング リミテッド | 光力学的療法のためのヒト血清アルブミンをベースにしたナノ粒子キャリアシステム |
| CN105997943A (zh) * | 2016-06-24 | 2016-10-12 | 浙江大学 | 一种人血清白蛋白负载喜树碱类药物的纳米颗粒及其制备方法和应用 |
| CN108478805A (zh) * | 2018-02-28 | 2018-09-04 | 湖南大学 | 一种新型白蛋白-siRNA复合纳米颗粒及其制备方法和应用 |
-
2020
- 2020-02-23 CN CN202010110186.1A patent/CN111110861B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013513609A (ja) * | 2009-12-11 | 2013-04-22 | バイオリテック ファーマ マーケティング リミテッド | 光力学的療法のためのヒト血清アルブミンをベースにしたナノ粒子キャリアシステム |
| CN105997943A (zh) * | 2016-06-24 | 2016-10-12 | 浙江大学 | 一种人血清白蛋白负载喜树碱类药物的纳米颗粒及其制备方法和应用 |
| CN108478805A (zh) * | 2018-02-28 | 2018-09-04 | 湖南大学 | 一种新型白蛋白-siRNA复合纳米颗粒及其制备方法和应用 |
Non-Patent Citations (1)
| Title |
|---|
| Tao Sun et al..A targeting theranostics nanomedicine as an alternative approach for hyperthermia perfusion.Biomaterials.2018,第183卷摘要，Fig 3. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111110861A (zh) | 2020-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Schmidt et al. | Engineering microrobots for targeted cancer therapies from a medical perspective | |
| Li et al. | Ultrasmall MoS2 nanodots-doped biodegradable SiO2 nanoparticles for clearable FL/CT/MSOT imaging-guided PTT/PDT combination tumor therapy | |
| Li et al. | Nanosized Janus AuNR-Pt motor for enhancing NIR-II photoacoustic imaging of deep tumor and Pt2+ ion-based chemotherapy | |
| Cui et al. | Multi-stimuli responsive smart chitosan-based microcapsules for targeted drug delivery and triggered drug release | |
| US8241393B2 (en) | Methods and articles for gold nanoparticle production | |
| Peng et al. | “One-for-all”-type, biodegradable prussian blue/manganese dioxide hybrid nanocrystal for trimodal imaging-guided photothermal therapy and oxygen regulation of breast cancer | |
| US20100233219A1 (en) | Inorganic nanoparticle compromising an active substance immobilized on the surface and a polymer | |
| Oroojalian et al. | Current trends in stimuli-responsive nanotheranostics based on metal–organic frameworks for cancer therapy | |
| Chee et al. | Superparamagnetic iron oxide nanoparticles for drug delivery | |
| CN109157662B (zh) | 一种人血清白蛋白-阿霉素交联物纳米颗粒及其应用 | |
| Wang et al. | Chemically engineering cells for precision medicine | |
| Oh et al. | Macrophage-mediated exocytosis of elongated nanoparticles improves hepatic excretion and cancer phototherapy | |
| Ma et al. | In vivo synergistic tumor therapies based on copper sulfide photothermal therapeutic nanoplatforms | |
| Mohammed et al. | Gold Nanoparticle: Synthesis, Functionalization, Enhancement, Drug Delivery and Therapy: A Review. | |
| Hu et al. | Dynamic nanoassembly-based drug delivery system (DNDDS): learning from nature | |
| US9023370B2 (en) | Conjugates comprising nanoparticles coated with platinum containing compounds | |
| CN111110861B (zh) | 一种基于人血清白蛋白的载药颗粒及其制备方法 | |
| Sadeghi et al. | Bioorthogonal catalysis for treatment of solid tumors using thermostable, self-assembling, single enzyme nanoparticles and natural product conversion with indole-3-acetic acid | |
| Liu et al. | Nano-bio interactions: A major principle in the dynamic biological processes of nano-assemblies | |
| Zhou et al. | Mitochondria-localized self-reporting small-molecule-decorated theranostic agents for cancer-organelle transporting and imaging | |
| Yu et al. | Surface membrane coating as a versatile platform for modifying antitumor nanoparticles | |
| Wang et al. | Stimuli-responsive nanocarrier delivery systems for Pt-based antitumor complexes: A review | |
| Singh et al. | Bioinspired microrobots: Opportunities and challenges in targeted cancer therapy | |
| Baik et al. | 3-Diethylaminopropyl-bearing glycol chitosan as a protein drug carrier | |
| Ji et al. | Stimuli-responsive delivery strategies for controllable gene editing in tumor therapeutics |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |