TWI510256B - Used in the treatment of thrombotic diseases of magnetic nano drugs - Google Patents
Used in the treatment of thrombotic diseases of magnetic nano drugs Download PDFInfo
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- TWI510256B TWI510256B TW101110199A TW101110199A TWI510256B TW I510256 B TWI510256 B TW I510256B TW 101110199 A TW101110199 A TW 101110199A TW 101110199 A TW101110199 A TW 101110199A TW I510256 B TWI510256 B TW I510256B
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- drug
- mnps
- nanomedicine
- spanh
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Classifications
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- 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
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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- 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
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
- A61K31/37—Coumarins, e.g. psoralen
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- 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
- A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4365—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
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- A61K31/60—Salicylic acid; Derivatives thereof
- A61K31/612—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid
- A61K31/616—Salicylic acid; Derivatives thereof having the hydroxy group in position 2 esterified, e.g. salicylsulfuric acid by carboxylic acids, e.g. acetylsalicylic acid
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
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- 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
- A61K47/6923—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 an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
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- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Description
本發明係關於一種應用於治療血栓性疾病的磁性奈米藥物,特別係指一種可以快速作用於血栓部位並發揮集中治療效果的磁性奈米藥物。
現代人由於飲食習慣和過於忙碌缺乏運動等因素越來越容易發生血管窄化和阻塞的症狀,血管阻塞是引發心血管疾病、身體局部中風或是腦中風的重要因素之一,因此,如何清除在血管中造成血管阻塞的血塊便顯得益發重要。
目前較常使用於治療血栓性疾病的藥物例如重組組織型胞漿素原活化劑(recombinant-tissue type plasminogen activator,以下簡稱rt-PA),其半衰期通常僅有20-30分鐘,因此人體在使用藥物後,藥物在體內的有效藥量及時效皆十分有限,而無法快速即時且有效的將血管中的血栓溶解掉,所以替代性的做法便是用高濃度的藥物來治療病患,但此作法卻有引發大量出血的風險,有鑑於此,一些藥物載體已被用來攜帶此類藥物,以延長藥物在體內的循環時間,例如脂體(liposome)[Thromb Haemost,vol.90,p.64-70(2003)]與高分子奈米粒子[Biomaterials,vol.29,p.228-237(2008)]等,但卻無法被快速導引及集中於血塊處。
隨後所開發出具有磁性之奈米粒子現在也已逐漸被廣泛應用於此類藥物之載體,藉由外加磁場將藥物導引並集中於血栓處,增加局部藥物濃度,提升其療效[Journal of
Magnetism and Magnetic Materials,vol.311,p.376-378(2007);Biomaterials,vol.30,5125-5130(2009);Biomaterials,vol.30,p.3343-3351(2009);Thrombosis Research,vol.121,p.799-811(2008)],雖然藥物可被磁場導引並集中於血栓處,但此法之應用卻極受藥物釋放的效率及速率所限制。
為了克服前述所使用的藥物載體於藥物釋放過程所產生的多種影響,以及目前治療血栓方法上的各種漏失,本發明人極盡思量,終於開發出本發明應用於治療血栓性疾病的磁性奈米藥物。
本發明提供了一種可以不需要進行外科手術,不使用介面活性劑、分散劑和交聯劑等有毒化學品,同時又具備有磁性的奈米藥物,其係將可以溶解血栓的藥物固定於磁性奈米複合物的表面,所形成的磁性奈米藥物除具有超順磁性的特性外,由於單位磁性奈米複合物的表面積很大,提升每一單位磁性奈米複合物可鍵結的藥物量,因此可藉由外加磁場的導引下將藥物於短時間內引導至到達患部,增加局部藥物濃度,此一磁性奈米藥物可以藉由磁場引導至血栓分布的部位,而達到強化局部治療效果。
為達上述目的,本發明應用於治療血栓性疾病的磁性奈米藥物包括有由磁性粒子組成的核心層、包覆於核心層外且由羧基化聚苯胺高分子組成的外殼層,利用1-乙基-3-(3-二甲基氨基丙基)碳醯二亞胺鹽酸
(1-ethyl-3-(3-dimethylaminepropyl)carbodiimidehydrochlo-ride,以下簡稱EDC)與N-羥基琥珀醯亞胺磺酸鈉鹽(N-hydroxysulfosuccinimide sodium salt,以下簡稱sulfo-NHS)可將具有治療血栓性疾病的藥物藉由共價鍵固定在外殼層上,本發明可選用之治療血栓性疾病的藥物,在一實施例中,此藥物包括有重組組織型胞漿素原活化劑(recombinant-tissue type plasminogen activator,以下簡稱rt-PA)、組織型胞漿素原活化劑(tissue-type plasminogen activator,t-PA)、阿斯匹靈(aspirin)、氯吡多(Clopidogrel)、雙嘧達莫(Dipyridamole)、低份子肝素(Fraxiparine)、華法林(Warfarin)或肝素(heparin)。
本發明的磁性奈米藥物,不但具有熱穩定性,更可以均勻的分散於水中,加上此磁性奈米藥物具有超順磁特性,可藉由外加磁場將藥物導引集中於特定部位,增加局部治療濃度。在藥物安定性方面,本發明之磁性奈米藥物在25℃下儲存35天後,依然保有約73%的藥物活性。再由體外毒性測試可知,本發明中應用於攜帶血栓溶解藥物的SPAnH/MNPs對血管內皮細胞並不具生物毒性。由體外人血血栓溶解測試可知,本發明之磁性奈米藥物在磁場的導引下可以比目前使用血栓溶解藥物更能夠快速有效的將血塊溶解。是故,本發明應用於於治療血栓性疾病的磁性奈米藥物是一製備簡單、無毒、可被磁導、可集中治療且兼俱發展潛力之血栓溶解新藥。
為使審查委員得以更加了解本發明,特以下列實施例進行說明。
請參考第1圖所示,本發明應用於治療血栓性疾病的磁性奈米藥物,其包括有一核心層1、一外殼層2和一藥物層3,核心層1係一具有磁性之粒子,其粒徑可小於10奈米,外殼層2係包覆於核心層外1,其材質可選用羧基化聚苯胺高分子,藥物層3係共價鍵結於外殼層2外之治療血栓性疾病藥物。
實施例一:係說明製備本發明應用於治療血栓性疾病的磁性奈米藥物中磁性奈米粒子(Magnetic nano particles,以下簡稱MNPs)和SPAnH/MNPs磁性奈米複合物的方法
MNPs製備:利用共沉澱法製備磁性奈米粒子Fe3O4(MNPs),在室溫下將0.7公克(g)(濃度為4.32×10-3mole)的三氯化鐵(以下簡稱為FeCl3)、1.07g(濃度為6.48×10-3mole)的四水氯化亞鐵(以下簡稱為FeCl2.4H2O)和400毫升(mL)二次水加入三頸瓶中,在填充氮氣的環境下以磁石攪拌5分鐘,使FeCl3和FeCl2.4H2O完全溶解後,將20mL濃度為0.864N之氫氧化鈉(NaOH)水溶液加入三頸瓶中,加熱至溫度達到80℃,即可生成含有MNPs的溶液。
MNPs分離:使含有MNPs的溶液冷卻,並於超音波震盪器中震盪,使MNPs均勻分散於水溶液中。將此混合水溶液倒入分液漏斗中,在漏斗外以強力磁鐵吸附磁粒子,讓漏斗中的水溶液由漏斗下方流出,得以和磁粒子分離。最後,加入二次水反覆萃洗磁粒子數次,直至水溶液呈中性、
無色為止,其粒徑大小約為5-10奈米(nm)。
SPAnH/MNPs磁性奈米複合物的製備:取10mL的含有MNPs的水溶液(濃度為每毫升6.4毫克MNPs,mg/mL)與4mL濃度為4.9mg/mL的聚[苯胺-共聚-鈉N-(1-丁酸)苯胺](poly[aniline-co-sodiumN-(1-one-butyric acid)aniline],以下簡稱SPAnNa)水溶液均勻混合以形成一混合溶液,將此混合溶液置於超音波震盪器進行均質,同時滴入濃度為0.5M的鹽酸(HCl),當SPAnNa遇到酸會行聚集反應,同時會將MNPs包覆起來,形成以MNPs為核心,SPAnH為外殼的SPAnH/MNPs磁性奈米複合物,將SPAnH/MNPs磁性奈米複合物從溶液中分離出來,並分散於二次水中,製備成水溶液狀態的SPAnH/MNPs磁性奈米複合物備用,此SPAnH/MNPs磁性奈米複合物的粒徑約為15nm。
請參考第2圖所示,其中標線4表示MNPs的紅外線光譜曲線,標線5表示SPAnH/MNPs磁性奈米複合物的紅外線光譜曲線,由圖中可看出在波數為586(1/公分,cm-1)時,有出現MNPs中的鐵氧鍵結(F-O)伸縮震動現象;而SPAnH/MNPs磁性奈米複合物之紅外線光譜圖在波數為582cm-1處依然有鐵氧鍵結(Fe-O,νFe-O)發生伸縮振動,在波數2844和2927cm-1處有飽和對稱及非對稱碳氫鍵結(C-H,νC-H)出現伸縮振動,在波數為1707cm-1處有羧基團(-COOH)的碳氧雙鍵(C=O,νC=O)出現伸縮振動,在波數為3410cm-1處則有羧基團(-COOH)的氫氧鍵結(O-H,νO-H)發生伸縮振動,這表示MNPs外的確包覆有一層高分子
(SPAnH),且SPAnH/MNPs磁性奈米複合物的外層同時具有-NH基和羧基(-COOH)的官能基,十分適於應用於生物及藥物的固定。
再請參考第3圖所示,係說明利用超導量子干涉儀(Superconducting Quantum Interference Device,SQUID)量測MNPs的磁化結果,其中標線6為MNPs磁性奈米子的磁滯曲線,標線7為SPAnH/MNPs磁性奈米複合物的磁滯曲線,由圖出可看出MNPs磁性奈米子的飽和磁化強度為66.2emu/g,其飽和磁化強度皆會隨著磁場強度增強而增加且會通過原點,具有超順磁的特性,而SPAnH/MNPs磁性奈米複合物的磁化狀況,由圖中可看出SPAnH/MNPs磁性奈米複合物的飽和磁化強度為37.6emu/g,具有超順磁的特性。
實施例二:係說明本發明應用於治療血栓性疾病的磁性奈米藥物中之SPAnH/MNPs磁性奈米複合物的生物毒性和對細胞生長的影響
細胞株的準備:將人類臍帶靜脈內皮細胞(human umbilical vein endothelial cell,以下簡稱HUVEC)置於含有5%二氧化碳的37℃恆溫培養箱中以M199(含有2.2mg/mL碳酸氫鈉、10%胎牛血清(fetal bovine serum,FBS)、50μg/mL建它黴素(gentamycin)、50μg/mL盤尼西林(penicillin)、50μg/mL鏈黴素(streptomycin)、25U/mL肝素(heparin)與30μg/mL內皮細胞生長補充劑(ECGS)培養液進行細胞培養繼代,培養繼代前須先在細胞培養盤上塗佈一層1%明膠(gelatin),之後再加入2mL含有0.2mg/mL胰蛋白酶(trypsin)與0.08mg/mL EDTA的溶液於
細胞繼代培養之培養皿中,靜置二分鐘,使貼附之細胞自盤壁脫落,並將溶液移置離心管中,在8℃下以1500rpm之轉速離心8分鐘,離心後將上層液移除,並將HUVEC均勻分散於M199培養液中。
生物毒性試驗:
(1)於塗佈有1% gelatin的96孔槽多孔培養盤之每個孔槽中放入150μL,具有10,000顆細胞混合液,將其置入37℃、5%二氧化碳潮濕培養箱中,讓細胞進行貼附生長,隔天於每個孔槽中加入50μL分散於M199培養液的奈米磁複合物溶液,並將其置入37℃、5%二氧化碳潮濕培養箱中,置入奈米磁複合物後的隔天開始進行繼數觀察細胞生長,進行細胞計數前係先移除M199培養液,加入120μL XTT反應液,保存於培養箱反應3小時,之後每孔槽取出100μL已反應的XTT反應液置於96孔槽計數多孔盤中,並用ELISA reader(BIO-TEK,model EL 808)於490nm測其OD值,評估此材料是否具有生物毒性。
(2)於直徑3.5cm的培養盤塗佈1% gelatin並加入2mL含有20,000顆的細胞混合液,置於37℃的5%二氧化碳潮濕培養箱中讓細胞進行貼附生長,隔天於培養盤中加入100μL分散於M199培養液的奈米磁複合物溶液,並於置入奈米磁複合物後的隔天開始將培養盤中的液體,之後以1mL HBSS清洗,清洗後再加入1mL Live/Dead染劑對細胞進行染色30分鐘,之後再以漢氏平衡鹽溶液(簡稱HBSS)清洗乾淨,以共軛焦顯微鏡(德國萊卡,型號TCS SP2)觀察細胞生長趨勢。
請參見第4圖所示,HUVEC在含有濃度為25-200μg/mL的SPAnH/MNPs複合物之環境下培養1-7天所得到之細胞粒線體活性相對應培養時間的表現,並以不含有SPAnH/MNPs複合物之細胞培養環境做為控制組。
在經過一天培養後,在含有SPAnH/MNPs複合物環境下培養之HUVEC細胞數量皆比控制組少,且細胞數隨著加入的SPAnH/MNPs複合物濃度的增加而更明顯減少,可能是因為SPAnH/MNPs複合物的加入改變培養液的本質,造成HUVEC細胞不適應而逐漸脫離貼附基材而出現細胞數目的降低。
當HUVEC於含有25-150μg/mL SPAnH/MNPs複合物環境下進行培養,雖然在第一天約有15%細胞死亡率(150μg/mL SPAnH/MNPs複合物),但剩餘細胞到了第二天細胞開始適應環境而貼附生長,且在第二天到第五天開始進行對數生長增殖,此時期細胞快速分裂增殖使其細胞數快速增加,第五天過後因培養基裡的營養物質已不夠讓大量細胞群體繼續快速分裂增殖,因此進入緩慢生長期直至第七天,由第二天至第五天之細胞對數增殖期計算出細胞生長速率與控制組並無差異,因此SPAnH/MNPs複合物在此濃度範圍對HUVEC並未造成明顯的生物毒性。反之,當SPAnH/MNPs複合物濃度提高至200μg/mL,細胞在第一天的死亡率即達30%,且在第二天至第五天亦表現出緩慢的生長速率,這可能是因為每個孔槽中的細胞數已無法承受及代謝如此高濃度的SPAnH/MNPs複合物,而導致細胞無法正常代謝增殖。
若是利用倒立式共軛焦顯微鏡觀察細胞存活及生長增殖的狀態,培養第一天後,HUVEC在含有SPAnH/MNPs複合物環境下培養之細胞數量比對照組少,但細胞數目隨著培養天數增加而增加,並與對照組一樣的生長趨勢,這說明SPAnH/MNPs複合物並不具生物毒性,與細胞生長曲線圖的結果相符合。
實施例三:係說明本發明應用於治療血栓性疾病的磁性奈米藥物的製備方法
取12mg 1-乙基-3-(3-二甲基氨基丙基)碳醯二亞胺鹽酸(1-ethyl-3-(3-dimethylaminepropyl)carbodiimide-hydrochloride,以下簡稱EDC)與24mg N-羥基琥珀醯亞胺磺酸鈉鹽(N-hydroxysulfosuccinimide sodium salt,以下簡稱sulfo-NHS)溶在濃度為0.5M、酸鹼值為6.3的2-嗎啉乙磺酸緩衝液(2-Morpholinoethanesulfonic acid,pH=6.3,以下簡稱MES buffer)中以形成一混合反應溶液,取0.2mL混合反應溶液和0.2mL SPAnH/MNPs磁性奈米複合物溶液在25℃下震盪反應一小時後以一強磁性磁鐵進行分離,並以MES buffer清洗,加入重組組織型胞漿素原活化劑(recombinant-tissue type plasminogen activator,以下簡稱rt-PA)藥物在25℃下反應兩小時以進行藥物固定化反應,再以一強磁性磁鐵進行分離並取出澄清液,並以PBS buffer清洗此澄清液後進行分離,即可獲得含有本發明應用於治療血栓性疾病的磁性奈米藥物(以下簡稱rt-PA/SPAnH/MNPs)之澄清溶液。
實施例四:係說明本發明應用於治療血栓性疾病的磁
性奈米藥物上不同濃度的藥物固定化功效及藥物相對活性變化
係將0.15、0.30、0.40、0.50、0.70毫克之rt-PA利用實施例三的方法使其與SPAnH/MNPs磁性奈米複合物作用,以確認添加不同重量rt-PA時,SPAnH/MNPs磁性奈米複合物的最大化固定功效和所形成的磁性奈米藥物之相對活性。
檢測時係將添加有不同重量rt-PA後所形成含有磁性奈米藥物的澄清液和蛋白質分析染劑均勻混和反應,再使用分光光度計在波長為595奈米(nm)的條件下檢測吸光值,以取得游離rt-PA的含量,並換算得到被固定在SPAnH/MNPs磁性奈米複合物的rt-PA藥物總量,其結果如表1和第4圖所示,結果顯示當添加的rt-PA藥物重量達到0.70毫克時,可以被固定在SPAnH/MNPs磁性奈米複合物的rt-PA藥物重量沒有再出現等比增加,而是出現漸趨穩定的情形,表示此時SPAnH/MNPs磁性奈米複合物上幾乎所有羧基都已經鍵結有rt-PA藥物,但是因為鍵結太多rt-PA藥物卻也會產生立體障礙,而使得rt-PA藥物的活性部分會因為立體結構的緣故而無法參與反應,因此,較佳的狀況下是在每毫克SPAnH/MNPs磁性奈米複合物鍵結276.2微克的rt-PA藥物。
實施例五:係說明本發明應用於治療血栓性疾病的磁性奈米藥物於不同溫度下之安定性試驗結果
將rt-PA藥物與本發明固定在磁性奈米複合物上的磁性奈米藥物儲存在4℃及25℃下1至35天後,利用蛋白質分析方法檢測藥物剩餘活性,以評估本發明磁性奈米藥物的穩定性。
請參考第5圖所示,rt-PA藥物以25℃保存至第21天已失去其原本的活性,若是儲存在4℃的環境下,於保存第35天時還可保有約32%的活性;但本發明的磁性奈米藥物以25℃保存於第35天仍具有約73%的活性,若是以4℃的溫度保存下,即使至保存第35天時,鍵結在磁性奈米複合物上的rt-PA藥物還可保有約78%的活性,雖然將rt-PA藥物固定於磁性奈米複合物上有可能會使得原本的藥物活性降低,但卻能防止rt-PA藥物產生游離的現象,也可以使得藥物分子的形態較不易出現改變,藉此便能增加藥物穩定性,增加藥物保存的有效時間。
實施例六:係說明本發明應用於治療血栓性疾病的磁性奈米藥物之體外靜態溶血試驗結果
血塊之至被:取長度為35釐米(mm)、直徑為5mm的聚乙烯軟管(PE tube),其軟管之一端先封閉後,注入0.5毫升(mL)人類血液並立即封住另一端開口,將裝有血液的聚乙烯軟管放置於37℃下進行凝血反應24小時後,取出凝固的血條切成每塊為1mm×2mm的血塊。
血漿的製備:取4.5mL的人類血液與0.5mL檸檬酸鈉放置於離心管中,以轉速6000rpm進行離心20分鐘,並取出上清液,此上清液即為血漿(platelet poor plasma,以下簡稱PPP)。
標準曲線製備:於5mL的樣品瓶中分別加入100微升(μL)濃度分別為0.2、0.4、0.6、0.8、1.0mg/mL的rt-PA藥物以、一塊血塊、800μL生理食鹽水及100μL PPP,於37℃下震盪(150rpm)反應30分鐘後,取出上層液體,利用分光光度計以405nm之波長檢測血色素的吸收值並建立標準曲線。
溶血試驗:於5mL的樣品瓶中分別加入30μg的rt-PA藥物或61.9μg本發明的磁性奈米藥物、一塊血塊、800μL生理食鹽水及100μL PPP,於37℃下,於樣品瓶底部施加外部磁場或不施加磁場的情況下震盪(150rpm)反應30分鐘後,取出上層液體,利用分光光度計以405nm之波長檢測血色素的吸收值,並比對標準曲線計算溶血效果。
請參考第8A-8B圖所示,其中第8A圖表示進行反應前、第8B圖表示進行反應後的溶血情形,標號(a)為控制組(未加藥物)、(b)為rt-PA藥物的溶血情形、(c)為本發明的磁性奈米藥物在未施加外部磁場下的溶血情形、(d)為本發明中磁性奈米複合物在未施加外部磁場下的溶血情形、(e)為磁性奈米藥物在施加外部磁場下的溶血情形和(f)為本發明中磁性奈米複合物在施加外部磁場下的溶血情形。
本發明的磁性奈米藥物在未施加外部磁場下進行反應後的吸收值為0.558,經由標準曲線換算可知完成作用的藥
量約為41.03μg,藥物作用率為66.28%,且由圖中可以明顯看出,其所產生的血色素顏色較rt-PA藥物作用後血色素為深,因此可知本發明的磁性奈米藥物參與溶血反應的劑量較高。
若於樣品瓶底部施加磁場時,則本發明的磁性奈米藥物會因為磁場作用被導引至瓶底,使血塊周圍會佈滿本發明的磁性奈米藥物,一旦血塊因為震盪而搖晃滾動時,更多本發明的磁性奈米藥物便可因此與血塊接觸,使血塊表面的胞漿素原(plasminogen)更快速被活化為胞漿素(plasmin)而發揮溶血效果,因此藥物作用率可以提高至86.12%(吸收值為0.725,作用藥量約為53.31μg),且其參與溶血反應所產生的血色素顏色較rt-PA物與無施加磁場之磁性奈米藥物深,顯見本發明之磁性奈米藥物在具有外部施加磁場的導引下,更能有效發揮血塊溶解的功能。
此外,無論在有無施加外部磁場的環境下,SPAnH/MNPs磁性奈米複合物並不會對血塊產生溶血的作用。
實施例七:係說明本發明應用於治療血栓性疾病的磁性奈米藥物之體外動態溶血試驗結果
請參考第6圖所示,本實施例中所使用的血塊和血漿其製備方式與實施例六相同,其主要係利用使用一蠕動式幫浦(圖中未示)樞接一玻璃滴管A的方式進行體外模擬以評估本發明之磁性奈米藥物於體內血管中流動並溶解血塊的功效,本實施例中蠕動式幫浦的流速係設定為0.18mL/min,其先將PPP/生理食鹽水溶液(體積比=1/4)輸送至
玻璃滴管A的管筒A1處,玻璃低管A的左端為壓力緩衝出口,右端模擬血管拴塞;隨後將2mm×5mm血塊B栓塞於玻璃滴管內徑縮小A3處;啟動蠕動式幫浦待系統達穩定流態(steady state)後,分別取100μL rt-PA藥物(250μg)、本發明之磁性奈米藥物(250μg)和SPAnH/MNPs磁性奈米複合物分別於血塊後方10cm箭頭處緩慢注入,並搭配使用強力磁鐵將該些藥物或磁性複合物引導至血塊(血栓)周圍,並計算溶解血塊使其可以由玻璃滴管右方尖端處A2完全流出的時間。
請參考第7圖所示,其中標號14為對照組、標號15為本發明中磁性奈米複合物、標號16為習用之重組組織型胞漿素原活化劑藥物和標號17為本發明之磁性奈米藥物的體外溶血試驗結果,由圖中可看出,習用之重組組織型胞漿素原活化劑藥物並無法在短時間內流到血塊處將血塊表面的胞漿素原活化為胞漿素,因此約需要39分鐘血塊才會被溶解而從玻璃滴管右方尖端處A2的出口流出,而本發明之磁性奈米藥物在外加磁場將其導引至血塊處的情況下,可在較短時間內達到溶解血塊效果,溶解血塊使其流出的時間僅需約9分鐘,大幅減少血塊溶解所需的時間。
綜上所述,本發明之應用於治療血栓性疾病的磁性奈米藥物在製造上的步驟及方式皆非常簡單,每毫克磁性奈米粒子可固定0~430μg rt-PA,且固定於磁性奈米粒子上之rt-PA的活性可維持在52.8~95.6%,若是儲存在溫度為4℃或25℃的條件下35天後,藥物活性分別較純rt-PA提升約46.0%或72.3%,在靜態溶血栓測試下,施加磁場後可
以使得血栓溶解率增加約19.8%,在動態溶血栓測試下,施加磁場後則可使血栓溶解時間提早約30分鐘。且此磁性奈米藥物對於血管內皮細胞並不會產生毒性、具有良好的藥物穩定性、可均勻的分散於水中,並具有超順磁性,因此可藉由外加磁場將藥物導引集中於特定部位,增加局部治療濃度,以提升血栓治療效果。
1‧‧‧核心層
2‧‧‧外殼層
3‧‧‧藥物
4‧‧‧磁性奈米粒子的紅外線光譜曲線
5‧‧‧磁性奈米複合物的紅外線光曲線
6‧‧‧磁性奈米粒子之磁滯曲線
7‧‧‧磁性奈米複合物之磁滯曲線
8‧‧‧不同重量重組組織型胞漿素原活化劑於磁性奈米複合物的固定化效率曲線
9‧‧‧不同重量重組組織型胞漿素原活化劑固定於磁性奈米複合物後的相對活性曲線
10‧‧‧本發明之磁性奈米藥物保存在4℃之相對活性變化曲線
11‧‧‧本發明之磁性奈米藥物保存在25℃之相對活性變化曲線
12‧‧‧習用之重組組織型胞漿素原活化劑藥物保存在4℃之相對活性變化曲線
13‧‧‧習用之重組組織型胞漿素原活化劑藥物保存在25℃之相對活性變化曲線
14‧‧‧對照組
15‧‧‧磁性奈米複合物
16‧‧‧重組組織型胞漿素原活化劑藥物
17‧‧‧磁性奈米藥物
附件一為體外靜態血栓溶解圖。
第1圖係本發明應用於治療血栓性疾病的磁性奈米藥物之結構示意圖。
第2圖係本發明應用於治療血栓性疾病的磁性奈米藥物中磁性奈米粒子和磁性奈米複合物之紅外線圖譜。
第3圖係本發明應用於治療血栓性疾病的磁性奈米藥物中磁性奈米粒子和磁性奈米複合物之磁滯曲線圖。
第4圖係將不同重量重組組織型胞漿素原活化劑與磁性奈米複合物反應後的固定效率和固定後的藥物相對活性圖。
第5圖係本發明之磁性奈米藥物和習用之重組組織型胞漿素原活化劑藥物在不同溫度下進行保存之活性變化曲線圖。
第6圖為本發明所使用之體外模擬試驗裝置結構示意圖。
第7圖為係本發明之磁性奈米藥物、磁性奈米複合物和習用之重組組織型胞漿素原活化劑藥物於體外模擬試驗中溶血試驗結果長條圖。
第8A-8B圖分別為本發明之磁性奈米藥物、磁性奈米複合物和習用之重組組織型胞漿素原活化劑藥物於體外靜態溶血試驗反應前、後之體外靜態血栓溶解圖。
1‧‧‧核心層
2‧‧‧外殼層
3‧‧‧藥物
Claims (5)
- 一種應用於治療血栓性疾病的磁性奈米藥物,其包括有:一核心層,係由粒徑小於10奈米的磁性粒子組成;及一外殼層,係包覆於該核心層外部,並由羧基化聚苯胺高分子組成;一血栓治療藥物,係以共價鍵結方式結合於該外殼層;其中,該羧基化聚苯胺高分子具有下列化學式:
- 如申請專利範圍第1項所述的磁性奈米藥物,其中組成該核心層之該磁性粒子係選自包含有下列成分之群組:四氧化三鐵、三氧化二鐵和鎳。
- 如申請專利範圍第1項所述的磁性奈米藥物,其中該磁性奈米藥物之粒徑介於25至50奈米之間。
- 如申請專利範圍第3項所述的磁性奈米藥物,其中 該血栓治療藥物係在20至25℃之溫度條件下鍵結於該外殼層。
- 如申請專利範圍第4項所述的磁性奈米藥物,其中該血栓治療藥物係重組組織型胞漿素原活化劑(recombinant tissue-type plasminogen activator,rt-PA)、組織型胞漿素原活化劑(tissue-type plasminogen activator,t-PA)、阿斯匹靈(aspirin)、氯吡多(Clopidogrel)、雙嘧達莫(Dipyridamole)、低份子肝素(Fraxiparine)、華法林(Warfarin)或肝素(heparin)。
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