TWI764564B - Nanocarriers and their applications - Google Patents

Abstract

本發明揭示一種奈米載體以及一種包含有該奈米載體的藥物遞送系統。本發明亦揭示該藥物遞送系統可被用來治療和/或預防眼睛疾病。The present invention discloses a nanocarrier and a drug delivery system comprising the nanocarrier. The present invention also discloses that the drug delivery system can be used to treat and/or prevent eye diseases.

Description

奈米載體及其應用Nanocarriers and their applications

本發明是有關於一種奈米載體(nanocarrier)以及一種包含有該奈米載體的藥物遞送系統(drug delivery system)。本發明亦有關於使用該藥物遞送系統來治療和/或預防眼睛疾病(ocular disorder)。The present invention relates to a nanocarrier and a drug delivery system comprising the nanocarrier. The present invention also relates to the use of the drug delivery system for the treatment and/or prevention of ocular disorders.

眼睛是視覺的器官，它的視覺功能會因老化(aging)或長期使用電子產品而逐漸惡化，進而導致各種不同的眼睛疾病(ocular disorder)，造成視力與生活品質的下降。常見的眼睛疾病包括：青光眼(glaucoma)、白內障(cataract)、葡萄膜炎(uveitis)、黃斑退化(macular degeneration)、眼內炎(endophthalmitis)、糖尿病視網膜病變(diabetic retinopathy)以及乾眼症(xerophthalmia)等。The eye is the organ of vision, and its visual function will gradually deteriorate due to aging or long-term use of electronic products, which in turn leads to various ocular disorders, resulting in a decline in vision and quality of life. Common eye diseases include: glaucoma, cataract, uveitis, macular degeneration, endophthalmitis, diabetic retinopathy, and xerophthalmia )Wait.

由於複雜且特殊的眼睛構造會限制眼睛藥物(ophthalmic drug)的進入，因此如何將眼睛藥物順利地遞送到眼睛[包括眼睛的前段(anterior segment)和後段(posterior segment)]一直是眼睛藥物開發過程中最具挑戰性的課題。造成此限制的主要因素包括： (1)   靜態屏障(static barriers)，包含分層的角膜(cornea)與鞏膜(sclera)，以及血液-房水屏障(blood-aqueous barriers)； (2)   動態屏障(dynamic barriers)，包含結膜血流(conjunctival blood flow)、淋巴清除(lymphatic clearance)、淚液排出(tear drainage)，以及藥物外排泵(drug efflux pumps)；以及 (3)   代謝屏障(metabolic barriers)，包含酵素，例如，細胞色素P450 (cytochrome P450)以及溶酶體酶(lysosomal enzymes)。 How to smoothly deliver ophthalmic drugs to the eye (including the anterior segment and posterior segment of the eye) has been an ophthalmic drug development process because the complex and special ocular structure restricts the entry of ophthalmic drugs. the most challenging subject. The main factors contributing to this limitation include: (1) Static barriers, including layered cornea and sclera, and blood-aqueous barriers; (2) dynamic barriers, including conjunctival blood flow, lymphatic clearance, tear drainage, and drug efflux pumps; and (3) Metabolic barriers, including enzymes such as cytochrome P450 and lysosomal enzymes.

目前最廣為使用的非侵入性治療方法--局部滴注(topical instillation)也因面臨上述所列舉的諸多因素而使得滴入眼睛的藥物通常最多只有5%的滴注劑量能夠到達眼睛內部，導致生物可利用性(bioavailability)受到了大幅的限制。因此，通常只能藉由施予大量藥物及增加給藥次數的方式來達到預定的療效，但是大量給藥常伴隨著眼睛紅腫與刺痛，甚至可能影響呼吸或心臟系統而造成全身性副作用。At present, the most widely used non-invasive treatment method, topical instillation, also faces many factors listed above, so that only 5% of the instilled dose of the drug instilled into the eye can reach the inside of the eye at most. As a result, bioavailability has been greatly restricted. Therefore, the intended therapeutic effect can usually only be achieved by administering a large amount of drugs and increasing the number of administrations. However, large doses are often accompanied by redness, swelling and stinging of the eyes, and may even affect the respiratory or cardiac system and cause systemic side effects.

此外，利用注射方式[諸如玻璃體內注射(intravitreal injection)與前房內注射(intracameral injection)]來將藥物直接注射入眼睛內部雖然可以大幅提升藥物的生物可利用性並且避免過度用藥所衍生出的全身性副作用，但仍存在有下面的缺點：當藥效持續時間較短時，會因必須頻繁地多次注射而降低了病患的藥物順從性(drug compliance)，且經常注射亦容易引起不良反應，例如，眼內炎、眼內出血(hemophthalmus)或視網膜剝離(retinal detachment)等眼部傷害。In addition, the use of injection methods (such as intravitreal injection and intracameral injection) to inject drugs directly into the eye can greatly improve the bioavailability of the drug and avoid overmedication. Systemic side effects, but there are still the following disadvantages: when the duration of the drug effect is short, it will reduce the patient's drug compliance (drug compliance) due to frequent multiple injections, and frequent injections are also likely to cause adverse effects Reactions, eg, ocular injuries such as endophthalmitis, hemophthalmus, or retinal detachment.

近年來，本技術領域的研究人員已有嘗試使用具高黏著性而對淚液沖洗有強抵抗力之優點的奈米粒子(nanoparticle)作為藥物載體(drug carrier)，以延長藥物遞送進入眼內時間，但目前的效果仍十分有限。In recent years, researchers in the field of technology have tried to use nanoparticles with the advantages of high adhesion and strong resistance to tear flushing as drug carriers to prolong the time of drug delivery into the eye , but the current effect is still very limited.

因此，本技藝中仍然存在有一需要去發展出一種能夠準確地將眼睛藥物遞送到眼內的目標位址並且具有持續釋放效用的遞送系統。Therefore, there remains a need in the art to develop a delivery system capable of accurately delivering an ocular drug to a target site in the eye and having a sustained release effect.

發明概要Summary of Invention

於是，在第一個方面，本發明提供一種奈米載體，其包含有： 一金屬氧化物中空奈米球體，其中該金屬氧化物是選自於由下列所構成的群組：CeO ₂、CuO、ZnO、NiO、AuO、Fe ₃O ₄，以及它們的組合；以及 接枝於該金屬氧化物中空奈米球體的複數個腺苷A2A受體拮抗劑(adenosine A2A receptor antagonist)以及複數個緊密型連結調節子(tight junction modulator)。 Thus, in a first aspect, the present invention provides a nanocarrier comprising: a metal oxide hollow nanosphere, wherein the metal oxide is selected from the group consisting of: CeO ₂ , CuO , ZnO, NiO, AuO, Fe ₃ O ₄ , and combinations thereof; and a plurality of adenosine A2A receptor antagonists and a plurality of compact types grafted on the metal oxide hollow nanospheres Tight junction modulator.

在第二個方面，本發明提供一種藥物遞送系統，其包含有一如上所述的奈米載體以及一眼睛藥物。In a second aspect, the present invention provides a drug delivery system comprising a nanocarrier as described above and an ophthalmic drug.

在第三個方面，本發明提供一種如上所述的藥物遞送系統供應用於製備一用來治療和/或預防眼睛疾病之醫藥品的用途。In a third aspect, the present invention provides the use of a drug delivery system as described above for the preparation of a medicament for the treatment and/or prevention of eye diseases.

在第四個方面，本發明提供一種用於治療和/或預防一具有或被懷疑具有眼睛疾病之個體的方法，其包括對該個體投予一如上所述的藥物遞送系統。In a fourth aspect, the present invention provides a method for treating and/or preventing an individual having or suspected of having an eye disease comprising administering to the individual a drug delivery system as described above.

發明的詳細說明Detailed description of the invention

要被瞭解的是：若有任何一件前案刊物在此被引述，該前案刊物不構成一個下述承認：在台灣或任何其他國家之中，該前案刊物形成本技藝中的常見一般知識之一部分。It is to be understood that if any antecedent publication is cited herein, that antecedent publication does not constitute an admission that, in Taiwan or any other country, the antecedent publication forms a common general practice in the art part of knowledge.

為了這本說明書之目的，將被清楚地瞭解的是：文字“包含有(comprising)”意指“包含但不限於”，以及文字“包括(comprises)”具有一對應的意義。For the purposes of this specification, it will be clearly understood that the word "comprising" means "including but not limited to" and that the word "comprises" has a corresponding meaning.

除非另外有所定義，在本文中所使用的所有技術性與科學術語具有熟悉本發明所屬技藝的人士所共同瞭解的意義。一熟悉本技藝者會認知到許多與那些被描述於本文中者相似或等效的方法和材料，它們可被用於實施本發明。當然，本發明決不受到所描述的方法和材料之限制。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Of course, the present invention is in no way limited by the methods and materials described.

在開發可用於治療和/或預防眼睛疾病(ocular disorder)的藥物遞送系統(drug delivery system)上，申請人經戮力研究結果發現，以腺苷A2A受體拮抗劑(adenosine A2A receptor antagonist)(例如ZM241385)與緊密型連結調節子(tight junction modulator)[例如幾丁聚糖(chitosan)]來進行官能基化(functionalization)所製得的氧化鈰中空奈米球體(ceria hollow nanosphere)能夠準確地將所欲遞送的藥物遞送到眼內的目標位址並且能夠達到持續釋放該藥物的效用。In the development of a drug delivery system that can be used for the treatment and/or prevention of ocular disorders, the applicant has made great efforts to study and found that adenosine A2A receptor antagonist (adenosine A2A receptor antagonist) ( Ceria hollow nanospheres prepared by functionalization with tight junction modulators (such as chitosan) such as ZM241385 can accurately The drug to be delivered is delivered to the target site in the eye and the effect of sustained release of the drug can be achieved.

因此，本發明提供一種奈米載體(nanocarrier)，其包含有： 一金屬氧化物中空奈米球體，其中該金屬氧化物是選自於由下列所構成的群組：CeO ₂、CuO、ZnO、NiO、AuO、Fe ₃O ₄，以及它們的組合；以及 接枝於該金屬氧化物中空奈米球體的複數個腺苷A2A受體拮抗劑以及複數個緊密型連結調節子。 Therefore, the present invention provides a nanocarrier, comprising: a metal oxide hollow nanosphere, wherein the metal oxide is selected from the group consisting of: CeO ₂ , CuO, ZnO, NiO, AuO, Fe ₃ O ₄ , and combinations thereof; and a plurality of adenosine A2A receptor antagonists and a plurality of tight junction regulators grafted on the metal oxide hollow nanospheres.

較佳地，該金屬氧化物是CeO ₂。 Preferably, the metal oxide is CeO ₂ .

依據本發明，該金屬氧化物中空奈米球體可具有一範圍落在20 nm至150 nm內的粒徑。According to the present invention, the metal oxide hollow nanospheres may have a particle size ranging from 20 nm to 150 nm.

較佳地，該金屬氧化物中空奈米球體是粒徑約為60 nm的氧化鈰中空奈米球體。Preferably, the metal oxide hollow nanospheres are cerium oxide hollow nanospheres with a particle size of about 60 nm.

依據本發明，該氧化鈰中空奈米球體可以採用熟習此項技藝者所詳知且慣用的技術而被製得。在此方面，可以參考，例如，Strandwitz N.C. and Stucky G.D. (2009), Chem. Mater., 19:4577-4582。 According to the present invention, the cerium oxide hollow nanospheres can be prepared using techniques well known and commonly used by those skilled in the art. In this regard, reference may be made to, for example, Strandwitz NC and Stucky GD (2009), Chem. Mater ., 19:4577-4582.

依據本發明，適用於製備本發明的氧化鈰中空奈米球體的模板(template)包括，但不限於：四乙基正矽酸鹽(tetraethyl orthosilicate, TEOS)、葡萄糖(glucose)，以及蔗糖(sucrose)。在本發明的一個較佳具體例中，該模板是TEOS。According to the present invention, templates suitable for preparing the cerium oxide hollow nanospheres of the present invention include, but are not limited to: tetraethyl orthosilicate (TEOS), glucose, and sucrose ). In a preferred embodiment of the present invention, the template is TEOS.

如本文中所使用的，術語“腺苷A2A受體拮抗劑(adenosine A2A receptor antagonist)”意欲涵蓋任何已知可結合至腺苷A2A受體(adenosine A2A receptor)(較佳地為位於眼內者)的配位子(ligand)。依據本發明，該等腺苷A2A受體拮抗劑可選自於由下列所構成的群組：ZM241385、SCH58261、KW6002，以及它們的組合。在本發明的一個較佳具體例中，該等腺苷A2A受體拮抗劑是ZM241385。As used herein, the term "adenosine A2A receptor antagonist" is intended to encompass any known binding to the adenosine A2A receptor, preferably in the eye ) ligand. According to the present invention, the adenosine A2A receptor antagonists may be selected from the group consisting of ZM241385, SCH58261, KW6002, and combinations thereof. In a preferred embodiment of the present invention, the adenosine A2A receptor antagonists are ZM241385.

如本文中所使用的，術語“緊密型連結調節子(tight junction modulator)”意欲涵蓋任何已知可打開緊密型連結(tight junction)(較佳地為位於眼內者)的分子。依據本發明，該等緊密型連結調節子可選自於由下列所構成的群組：幾丁聚糖[例如，三甲基幾丁聚糖(trimethyl chitosan)、二甲基乙基幾丁聚糖(dimethylethyl chitosan)、二乙基甲基幾丁聚糖(diethylmethyl chitosan)、三乙基幾丁聚糖(triethyl chitosan)以及N-三甲基幾丁聚糖氯化物(N-trimethyl chitosan chloride, TMC)]、聚-L-離胺酸(Poly-L-lysine)、聚-L-精胺酸(Poly-L-arginine)、魚精蛋白(protamine)、聚乙烯亞胺(polyethylenimine)、胺端基聚醯胺樹枝狀聚合物(amine terminated polyamidoamine dendrimer, PAMAM-NH ₂)、聚(2-胺乙基丙烯醯胺)[poly(2-aminoethyl acrylamide)]、四級化的聚(甲基丙烯酸二甲胺乙酯)[quaternized poly(dimethylaminoethyl methacrylate)]，以及它們的組合。在本發明的一個較佳具體例中，該等緊密型連結調節子是幾丁聚糖。 As used herein, the term "tight junction modulator" is intended to encompass any molecule known to open tight junctions, preferably those located in the eye. According to the present invention, the tight junction regulators may be selected from the group consisting of: chitosan [eg, trimethyl chitosan, dimethyl ethyl chitosan Sugar (dimethylethyl chitosan), diethylmethyl chitosan (diethylmethyl chitosan), triethyl chitosan (triethyl chitosan) and N-trimethyl chitosan chloride (N-trimethyl chitosan chloride, TMC)], poly-L-lysine (Poly-L-lysine), poly-L-arginine (Poly-L-arginine), protamine (protamine), polyethyleneimine (polyethylenimine), amine amine terminated polyamidoamine dendrimer (PAMAM-NH ₂ ), poly(2-aminoethyl acrylamide) [poly(2-aminoethyl acrylamide)], quaternized poly(methyl) dimethylaminoethyl acrylate) [quaternized poly(dimethylaminoethyl methacrylate)], and combinations thereof. In a preferred embodiment of the present invention, the tight junction regulators are chitosan.

依據本發明，該等腺苷A2A受體拮抗劑與緊密型連結調節子的用量比可介於1：1 (w/w)至1：300 (w/w)之間。較佳地，該等腺苷A2A受體拮抗劑與緊密型連結調節子的用量比可介於1：1 (w/w)至1：10 (w/w)之間。在本發明的一個較佳具體例中，該等腺苷A2A受體拮抗劑與緊密型連結調節子的用量比是1：2 (w/w)。According to the present invention, the dosage ratio of these adenosine A2A receptor antagonists to tight junction modulators may be between 1:1 (w/w) to 1:300 (w/w). Preferably, the dosage ratio of these adenosine A2A receptor antagonists to tight junction modulators may be between 1:1 (w/w) to 1:10 (w/w). In a preferred embodiment of the present invention, the dosage ratio of the adenosine A2A receptor antagonists to the tight junction modulator is 1:2 (w/w).

依據本發明，該等腺苷A2A受體拮抗劑以及緊密型連結調節子的接枝可以採用熟習此項技藝者所詳知且慣用的技術來進行。在此方面，可以參考，例如，Ma D.H. et al. (2010), Biomaterials, 25:6647-6658。 In accordance with the present invention, the grafting of the adenosine A2A receptor antagonists and tight junction regulators can be carried out using techniques well known and commonly used by those skilled in the art. In this regard, reference may be made to, for example, Ma DH et al . (2010), Biomaterials , 25:6647-6658.

較佳地，該金屬氧化物中空奈米球體可被聚乙二醇化(PEGylation)以供該等腺苷A2A受體拮抗劑與緊密型連結調節子的接枝。Preferably, the metal oxide hollow nanospheres can be PEGylation for grafting of the adenosine A2A receptor antagonists to tight junction regulators.

可瞭解到的是，有關該接枝的操作條件會進一步隨著所使用的金屬氧化物中空奈米球體、腺苷A2A受體拮抗劑與緊密型連結調節子的種類與用量等因素而被變動，以便達致最佳的反應效果。而這些操作條件的選擇是熟習此項技藝者能例行性地自行決定的。It can be understood that the operating conditions for the grafting will be further changed according to factors such as the types and amounts of metal oxide hollow nanospheres, adenosine A2A receptor antagonists and tight junction regulators used. , in order to achieve the best response effect. The selection of these operating conditions is routinely determined by those skilled in the art.

依據本發明，該接枝是在一範圍落在16℃至26℃的溫度下被進行歷時18至24小時。在本發明的一個較佳具體例中，該接枝是在室溫(約25℃)下被進行歷時24小時。According to the invention, the grafting is carried out for 18 to 24 hours at a temperature in the range of 16°C to 26°C. In a preferred embodiment of the present invention, the grafting is carried out at room temperature (about 25°C) for 24 hours.

本發明亦提供一種藥物遞送系統，其包含有一如上所述的奈米載體以及一眼睛藥物。The present invention also provides a drug delivery system comprising a nanocarrier as described above and an ophthalmic drug.

依據本發明，該奈米載體與該眼睛藥物的用量比可介於1：0.2 (w/w)至1：0.5 (w/w)之間。在本發明的一個較佳具體例中，該奈米載體與該眼睛藥物的用量比是1：0.3 (w/w)。According to the present invention, the dosage ratio of the nanocarrier to the eye drug can be between 1:0.2 (w/w) to 1:0.5 (w/w). In a preferred embodiment of the present invention, the dosage ratio of the nanocarrier to the eye drug is 1:0.3 (w/w).

依據本發明，該眼睛藥物可以是一選自於由下列所構成之群組中的藥劑：降眼壓藥劑(ocular hypotensive agent)、抗青光眼藥劑(anti-glaucoma agent)、Rho-關聯性蛋白激酶(ROCK)抑制劑[Rho-associated protein kinase (ROCK) inhibitor]、多巴胺拮抗劑(dopaminergic antagonist)、抗傳染劑(anti-infective agent)、抗發炎劑(anti-inflammatory agent)、抗病毒劑(antiviral agent)、生長因子(growth factor)、黏液促泌素(mucus secretogogue)、血管生成抑制劑(angiogenesis inhibitor)、肥大細胞穩定劑(mast cell stabilizer)、免疫調節劑(immunomodulatory agent)，以及它們的組合。According to the present invention, the ocular drug may be an agent selected from the group consisting of: ocular hypotensive agent, anti-glaucoma agent, Rho-associated protein kinase (ROCK) inhibitor [Rho-associated protein kinase (ROCK) inhibitor], dopaminergic antagonist, anti-infective agent, anti-inflammatory agent, antiviral agent agent), growth factor, mucus secretogogue, angiogenesis inhibitor, mast cell stabilizer, immunomodulatory agent, and combinations thereof .

另擇地，該眼睛藥物可選自於由下列所構成的群組：硝酸毛果芸香鹼(pilocarpine nitrate)、Y-27632、蒂目羅(Timolol)、卡特洛(Carteolol)、溴莫尼定(Brimonidine)、多佐胺(Dorzolamide)、布林佐胺(Brinzolamide)、乙醯偶氮胺(Acetazolamide)、拉坦前列素(Latanoprost)、曲伏前列素(Travoprost)、比馬前列素(Bimatoprost)、甘露醇(Mannitol)、異山梨醇(Isosorbide)、環孢素(cyclosporine)(例如，Restasis ^®、Cyclokat ^®以及Cequa ^®)、維替泊芬(verteporfin)(例如Visudyne ^®)、維生素A棕櫚酸鹽與維生素E (vitamin A palmitate and vitamin E)(例如Lacrisek ^®)、維生素B12 (vitamin B12)(例如Artelac Rebalance)、蘭尼單抗(ranibizumab)(例如Lucentis ^®)、柔癌捕(aflibercept)(例如Eylea ^®)、溴珠單抗(brolucizumab)(例如Beovu ^®)、貝伐珠單抗(bevacizumab)(例如Avastin ^®)，以及它們的組合。 Alternatively, the eye drug may be selected from the group consisting of: pilocarpine nitrate, Y-27632, Timolol, Carteolol, Brimonidine , Dorzolamide, Brinzolamide, Acetazolamide, Latanoprost, Travoprost, Bimatoprost, Manna Mannitol, Isosorbide, Cyclosporine (eg Restasis ^® , Cyclokat ^® and Cequa ^® ), Verteporfin (eg Visudyne ^® ), Vitamin A Palmitate and Vitamin E (vitamin A palmitate and vitamin E) (eg Lacrisek ^® ), vitamin B12 (eg Artelac Rebalance), ranibizumab (eg Lucentis ^® ), aflibercept (eg Eylea) ^® ), brolucizumab (eg, Beovu ^® ), bevacizumab (eg, Avastin ^® ), and combinations thereof.

在本發明的一個較佳具體例中，該眼睛藥物是硝酸毛果芸香鹼。在本發明的另一個較佳具體例中，該眼睛藥物是Y-27632。In a preferred embodiment of the present invention, the eye drug is pilocarpine nitrate. In another preferred embodiment of the present invention, the eye medicine is Y-27632.

依據本發明的藥物遞送系統經由動物實驗而被證實能夠有效地改善帶有青光眼(glaucoma)的白兔的眼睛的眼內壓(intraocular pressure, IOP)，因而被預期可供應用於製備一用來治療和/或預防眼睛疾病之醫藥品的用途。此外，本發明亦提供一種用於治療和/或預防一具有或被懷疑具有眼睛疾病之個體的方法，其包括對該個體投予一如上所述的藥物遞送系統。The drug delivery system according to the present invention has been confirmed to be effective in improving the intraocular pressure (IOP) of the eyes of white rabbits with glaucoma through animal experiments, and thus is expected to be applicable to the preparation of a Use of medicinal products for the treatment and/or prevention of eye diseases. In addition, the present invention also provides a method for treating and/or preventing an individual having or suspected of having an eye disease comprising administering to the individual a drug delivery system as described above.

依據本發明，該眼睛疾病可選自於由下列所構成的群組：眼前段疾病(anterior segment eye disease)、眼後段疾病(posterior segment eye disease)，以及它們的組合。According to the present invention, the eye disease may be selected from the group consisting of: anterior segment eye disease, posterior segment eye disease, and combinations thereof.

另擇地，該眼睛疾病可選自於由下列所構成的群組：青光眼、白內障(cataract)、葡萄膜炎(uveitis)、黃斑退化(macular degeneration)、眼內炎(endophthalmitis)、糖尿病視網膜病變(diabetic retinopathy)、乾眼症(xerophthalmia)，以及它們的組合。在本發明的一個較佳具體例中，該眼睛疾病是青光眼。Alternatively, the eye disease may be selected from the group consisting of: glaucoma, cataract, uveitis, macular degeneration, endophthalmitis, diabetic retinopathy (diabetic retinopathy), xerophthalmia, and combinations thereof. In a preferred embodiment of the present invention, the eye disease is glaucoma.

如本文中所使用的，術語“治療(treating)”或“治療(treatment)”眼睛疾病意指該眼睛疾病的嚴重性(severity)或該眼睛疾病的症狀(symptom)被減少(reduced)，或是該眼睛疾病被部分地(partially)或完全地(entirely)消除(eliminated)。As used herein, the term "treating" or "treatment" of an eye disease means that the severity of the eye disease or the symptoms of the eye disease is reduced, or Is that the eye disease is partially or completely eliminated.

如本文中所使用的，術語“預防(preventing)”或“預防(prevention)”眼睛疾病意指一個體在還沒有被診斷具有該眼睛疾病時，消除(eliminate)或減少(reduce)該眼睛疾病的發生率(incidence)，以及減緩(slow)、延遲(delay)、控制(control)或減少(decrease)該眼睛疾病的可能性(likelihood)或機率(probability)。As used herein, the term "preventing" or "prevention" of eye disease means to eliminate or reduce the eye disease when an individual has not been diagnosed with the eye disease The incidence (incidence), and slowing (slow), delay (delay), control (control) or reduce (decrease) the likelihood (likelihood) or probability (probability) of the eye disease.

如本文中所使用的，術語“個體(subject)”意指任何感興趣的哺乳類動物，諸如人(humans)、猴子(monkeys)、牛(cows)、綿羊(sheeps)、馬(horses)、豬(pigs)、山羊(goats)、狗(dogs)、貓(cats)、小鼠(mice)以及大鼠(rats)。As used herein, the term "subject" means any mammal of interest, such as humans, monkeys, cows, sheeps, horses, pigs (pigs), goats (goats), dogs (dogs), cats (cats), mice (mice) and rats (rats).

依據本發明，該醫藥品可利用熟習此技藝者所詳知的技術而被製造成一適合於眼內(intraocular)投藥的劑型。In accordance with the present invention, the medicinal product can be manufactured in a dosage form suitable for intraocular administration using techniques well known to those skilled in the art.

依據本發明，該醫藥品可進一步包含有一被廣泛地使用於藥物製造技術之藥學上可接受的載劑(pharmaceutically acceptable carrier)。例如，該藥學上可接受的載劑可包含一或多種選自於下列的試劑：溶劑(solvent)(諸如無菌水)、緩衝液(buffer)[諸如眼科均衡鹽溶液(ophthalmic balanced salt solution)、磷酸鹽緩衝生理鹽水(Phosphate Buffered Saline, PBS)、林格氏液(Ringer’s solution)以及漢克氏溶液(Hank’s solution)]、乳化劑(emulsifier)、懸浮劑(suspending agent)、分解劑(decomposer)、pH調整劑(pH adjusting agent)、安定劑(stabilizing agent)、螯合劑(chelating agent)、防腐劑(preservative)、稀釋劑(diluent)、吸收延遲劑(absorption delaying agent)、脂質體(liposome)以及類似之物。有關這些試劑的選用與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。According to the present invention, the pharmaceutical product may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of a solvent (such as sterile water), a buffer (such as an ophthalmic balanced salt solution, Phosphate Buffered Saline (Phosphate Buffered Saline, PBS), Ringer's solution and Hank's solution], emulsifier (emulsifier), suspending agent (suspending agent), decomposer (decomposer) , pH adjusting agent, stabilizing agent, chelating agent, preservative, diluent, absorption delaying agent, liposome and the like. The selection and quantity of these reagents are within the scope of the expertise and routine skills of those skilled in the art.

較佳地，該醫藥品被製造成適合於眼內滴注(intraocular instillation)的劑型，這包括，但不限於：滴劑(drop)、乳劑(emulsion)、凝膠(gel)、軟膏(ointment)、乳霜(cream)、噴霧(spray)，以及懸浮液(suspension)。Preferably, the medicinal product is manufactured in a dosage form suitable for intraocular instillation, including, but not limited to: drops, emulsions, gels, ointments ), creams, sprays, and suspensions.

另擇地，該醫藥品可被製造成適合於眼內注射(intraocular injection)的劑型，這包括，但不限於：注射品(injection)[例如，無菌的水性溶液(sterile aqueous solution)或分散液(dispersion)]。Alternatively, the medicinal product may be manufactured in a dosage form suitable for intraocular injection including, but not limited to: injection [eg, sterile aqueous solution or dispersion] (dispersion)].

依據本發明，適用於本發明的眼內注射包括，但不限於：玻璃體內注射(intravitreal injection)、前房內注射(intracameral injection)、視網膜內注射(intra-retinal injection)、視網膜下注射(subretinal injection)，以及脈絡膜上腔注射(suprachoroidal injection)。According to the present invention, intraocular injections suitable for use in the present invention include, but are not limited to: intravitreal injection, intracameral injection, intra-retinal injection, subretinal injection injection), and suprachoroidal injection.

依據本發明的藥物遞送系統的投藥劑量與投藥次數會視下列因素而變化：要被治療的疾病之嚴重性、投藥途徑，以及要被治療的個體之年齡、身體狀況與反應。一般而言，依據本發明的醫藥品可呈單一劑量或是分成數個劑量的形式而被投藥。 較佳實施例之詳細說明 The dosage and frequency of administration of the drug delivery system according to the present invention will vary depending on the severity of the disease to be treated, the route of administration, and the age, physical condition and response of the individual to be treated. In general, a pharmaceutical product according to the present invention may be administered in a single dose or divided into several doses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

本發明將就下面的實施例來做進一步說明，但應瞭解的是，該等實施例僅是供例示說明用，而不應被解釋為本發明的實施上的限制。 實施例 一般實驗材料： 1.  下列實驗材料是購自於Sigma-Aldrich：硝酸鈰(cerium nitrate)、四乙基正矽酸鹽(tetraethyl orthosilicate, TEOS)、乙醇、乙二醇(ethylene glycol)、氫氧化銨(ammonium hydroxide)(28% NH ₃配於H ₂O中)、氫氧化鈉(sodium hydroxide)、1-乙基-3-(3-二甲基胺基丙基)碳二亞胺(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, EDC)、過氧化氫(hydrogen peroxide, HP)、(4-(-2-[7-胺基-2-{2-呋喃基}{1,2,4}***并{2,3-a}{1,3,5}三嗪-5-基-胺基]乙基)酚(4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol)(ZM241385)、硝酸毛果芸香鹼(pilocarpine nitrate)，Y-27632以及α-胰凝乳蛋白酶(α-chymotrypsin)。 2.  下列實驗材料是購自於Specific Polymers (Castries, France)：磷酸酯-聚乙二醇(PEG)-COOH [Phosphonate-polyethylene glycol (PEG)-COOH, PO-PEG-COOH](MW 1200 g mol ^-1; ref. SP-1P-10-001)。 3.  下列實驗材料是購自於Fluka (Milwaukee, WI, USA)：幾丁聚糖(chitosan)(源自於蟹殼)(Cat. No. 28191)。 4.  實驗動物： The present invention will be further described with respect to the following embodiments, but it should be understood that these embodiments are only for illustration purposes and should not be construed as limitations on the implementation of the present invention. EXAMPLES General experimental materials: 1. The following experimental materials were purchased from Sigma-Aldrich: cerium nitrate, tetraethyl orthosilicate (TEOS), ethanol, ethylene glycol, Ammonium hydroxide (28% _NH3 in _H2O ), sodium hydroxide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, EDC), hydrogen peroxide (HP), (4-(-2-[7-amino-2-{2-furanyl}{ 1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol (4-(-2-[7-amino-2 -{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol)(ZM241385), pilocarpine nitrate , Y-27632 and α-chymotrypsin (α-chymotrypsin). 2. The following experimental materials were purchased from Specific Polymers (Castries, France): Phosphate-polyethylene glycol (PEG)-COOH [Phosphonate-polyethylene (PEG)-COOH, PO-PEG-COOH] (MW 1200 g mol ^-1 ; ref. SP-1P-10-001). 3. The following experimental materials were purchased from Fluka (Milwaukee, WI, USA): Chitosan (derived from crab shell) (Cat. No. 28191). 4. Experimental animals:

在下面實施例中所使用的紐西蘭白兔(New Zealand white rabbits)(16至20週大，體重約為3至3.5 kg)是購自於國家實驗動物繁殖及研究中心(National Laboratory Animal Breeding and Research Center)。所有的實驗動物被飼養於一個光照與黑暗各為12小時、溫度維持在20至24℃以及濕度維持在55至65%的獨立空調的動物房內，而且水分與飼料被充分地供給。有關實驗動物的飼養環境、處理以及一切實驗程序均符合國家衛生研究院(National Institutes of Health, NIH)的實驗動物飼養管理及使用規範(Guide for the Care and Use of Laboratory Animals)以及視覺與眼科學研究協會(Association for Research in Vision and Ophthalmology)的準則。 一般實驗方法： 1.  統計學分析(statistical analysis)： New Zealand white rabbits (16 to 20 weeks old, weighing approximately 3 to 3.5 kg) used in the following examples were purchased from National Laboratory Animal Breeding and Research Center). All experimental animals were housed in an individually air-conditioned animal room with 12 hours of light and 12 hours of darkness, temperature maintained at 20 to 24°C, and humidity maintained at 55 to 65%, and water and feed were adequately supplied. The breeding environment, handling and all experimental procedures of laboratory animals comply with the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals and the Vision and Ophthalmology Guidelines of the Association for Research in Vision and Ophthalmology. General experimental methods: 1. Statistical analysis:

在下面的實施例中，實驗數據是以“平均值(mean)±標準差(standard deviation, SD)”來表示。所有的數據是藉由單因子變異數分析(one-way analysis of variance, ANOVA)，繼而以Newman-Keuls事後檢定(Newman-Keuls post hoc test)來作分析，俾以評估各組之間的差異性。若所得到的統計分析結果是 p＜0.05，這表示有統計學顯著性(statistical significance)。 實施例 1. 製備本發明的氧化鈰中空奈米球體 (ceria hollow nanosphere) 實驗方法： A. 氧化鈰中空奈米球體的合成： In the following examples, experimental data are expressed as "mean ± standard deviation (SD)". All data were analyzed by one-way analysis of variance (ANOVA) followed by Newman-Keuls post hoc test to assess differences between groups sex. Statistical significance was indicated if the result of the statistical analysis obtained was p < 0.05. Example 1. Experimental method for preparing ceria hollow nanospheres of the present invention: A. Synthesis of ceria hollow nanospheres:

首先，將8 mL的TEOS與280 mL的乙醇混合並攪拌均勻，繼而對所形成的混合物添加56 mL的去離子水以及8.4 mL的氫氧化銨，並於室溫下進行攪拌歷時24小時。之後，以10,000 rpm來進行離心歷時10分鐘，接著移除上澄液並將所得到的沉澱物(pellets)重新散浮於99%乙醇中，然後置於一溫度被設定為65℃的烘箱中進行乾燥歷時6小時，藉此而得到氧化矽奈米粒子(silica NPs)。First, 8 mL of TEOS was mixed with 280 mL of ethanol and stirred well, then 56 mL of deionized water and 8.4 mL of ammonium hydroxide were added to the resulting mixture and stirred at room temperature for 24 hours. After that, centrifugation was performed at 10,000 rpm for 10 minutes, then the supernatant was removed and the resulting pellets were resuspended in 99% ethanol, and then placed in an oven set at 65°C Drying was performed for 6 hours, thereby obtaining silica nanoparticles (silica NPs).

之後，將300 mg的氧化矽奈米粒子添加至45 mL的乙二醇中，並進行超音波處理(ultrasonication)歷時30分鐘以避免奈米粒子的聚集，藉此而得到氧化矽分散液(silica dispersion)。接著，將2.25 mL的1 M硝酸鈰添加至該氧化矽分散液中並予以攪拌歷時10分鐘，所得到的混合物被置於一溫度被設定為130℃的高壓釜(autoclave)中進行加熱歷時7小時以形成氧化矽核-氧化鈰殼(silica core-ceria shell)的結構。待冷卻至室溫之後，以15,000 rpm來進行離心歷時10分鐘，接著移除上澄液並以99%乙醇予以清洗所得到的沉澱物，藉此而分離出具有氧化矽核-氧化鈰殼之結構的奈米粒子(以下簡稱為SiO ₂-CeO ₂-NPs)。 After that, 300 mg of silica nanoparticles were added to 45 mL of ethylene glycol, and ultrasonication (ultrasonication) was performed for 30 minutes to avoid aggregation of nanoparticles, thereby obtaining a silica dispersion (silica). dispersion). Next, 2.25 mL of 1 M cerium nitrate was added to the silica dispersion and stirred for 10 minutes, and the resulting mixture was placed in an autoclave whose temperature was set to 130° C. for 7 hours to form a silica core-ceria shell structure. After cooling to room temperature, centrifugation was performed at 15,000 rpm for 10 minutes, then the supernatant was removed and the resulting precipitate was washed with 99% ethanol, thereby separating the silica core-cerium oxide shell. Structured nanoparticles (hereinafter referred to as SiO ₂ -CeO ₂ -NPs).

接著，以5 M的氫氧化鈉溶液作為蝕刻劑(etchant)來對該SiO ₂-CeO ₂-NPs進行蝕刻(etching)歷時48小時，並在進行蝕刻的第24小時之時有更換新鮮的蝕刻劑，俾以移除氧化矽核(silica core)。之後，以10,000 rpm來進行離心歷時10分鐘，接著移除上澄液並將所得到的沉澱物重新散浮於99%乙醇中，然後置於一溫度被設定為70℃的烘箱中進行烘乾歷時4-5小時，藉此而得到不帶有官能基的氧化鈰中空奈米球體(以下簡稱為CeO ₂-HNS)。 B. CeO ₂-HNS 的雙官能基化 (dual functionalization) ： Next, the SiO ₂ -CeO ₂ -NPs was etched with 5 M sodium hydroxide solution as an etchant for 48 hours, and was replaced with fresh etching at the 24th hour of the etching. agent to remove the silica core. After that, centrifugation was performed at 10,000 rpm for 10 minutes, then the supernatant was removed and the resulting precipitate was resuspended in 99% ethanol, and then placed in an oven set at 70°C for drying It took 4-5 hours to obtain cerium oxide hollow nanospheres without functional groups (hereinafter referred to as CeO ₂ -HNS). B. Dual functionalization of CeO ₂ -HNS :

將CeO ₂-HNS與PO-PEG-COOH以一為1：10的重量比進行混合，繼而散浮於等量的去離子水中，並以1 N的鹽酸將所形成的混合液的pH值調整至3，然後使用超濾盤(Ultrafiltration Disc)(Milipore，30 kDa NMW，Cat. No. PLTK07610)來進行超過濾(ultrafiltration)以移除液體，而得到經PEG修飾的CeO ₂-HNS。 CeO ₂ -HNS and PO-PEG-COOH were mixed in a weight ratio of 1:10, then dispersed in an equal amount of deionized water, and the pH of the resulting mixture was adjusted with 1 N hydrochloric acid. To 3, Ultrafiltration Disc (Milipore, 30 kDa NMW, Cat. No. PLTK07610) was then used to perform ultrafiltration to remove liquid to obtain PEG-modified CeO2 _- HNS.

之後，將1 g之該經PEG修飾的CeO ₂-HNS散浮於去離子水中，接而添加至含有0.26 g之EDC的MES緩衝液(5 mL)中並予以攪拌歷時6小時，繼而分別混合以具有下面表1所示之含有不同重量比(亦即1:10、1:2以及1:1)之ZM241385與幾丁聚糖的MES緩衝液(5 mL)。於室溫下進行接枝反應(grafting reaction)歷時24小時之後，所得到的3種混合物分別被置於50℃下進行沉澱，以移除未鍵結的ZM241385與幾丁聚糖，繼而收集沉澱物並以去離子水予以清洗3次，然後散浮於去離子水中並以15,000 rpm來進行離心歷時10分鐘，移除上澄液，而所得到的沉澱物即為ZM241385/幾丁聚糖之雙官能基化的氧化鈰中空奈米球體(以下簡稱為DF-CeO ₂-HNS)。最後，於-50℃下進行冷凍乾燥歷時24小時，而得到呈凍乾粉末(lyophilized powder)之3種本發明的DF-CeO ₂-HNS，亦即DF-CeO ₂-HNS1至3。 表1.   用於製備DF-CeO ₂-HNS1至3的MES緩衝液的配方 DF-CeO ₂-HNS 各個組分的用量(g) ZM241385 幾丁聚糖 1 0.15 1.5 2 0.75 1.5 3 1.5 1.5 實施例 2. 本發明之氧化鈰中空奈米球體的物化性質分析 After that, 1 g of the PEG-modified CeO ₂ -HNS was dispersed in deionized water, then added to MES buffer (5 mL) containing 0.26 g of EDC and stirred for 6 hours, and then mixed separately MES buffer (5 mL) containing ZM241385 and chitosan in different weight ratios (ie 1:10, 1:2 and 1:1) as shown in Table 1 below. After the grafting reaction was carried out at room temperature for 24 hours, the obtained three mixtures were respectively placed at 50°C for precipitation to remove unbound ZM241385 and chitosan, and then the precipitates were collected. The material was washed 3 times with deionized water, then dispersed in deionized water and centrifuged at 15,000 rpm for 10 minutes, the supernatant was removed, and the obtained precipitate was ZM241385/chitosan. Difunctionalized cerium oxide hollow nanospheres (hereinafter referred to as DF-CeO ₂ -HNS). Finally, freeze-drying was performed at -50°C for 24 hours to obtain three DF-CeO ₂ -HNSs of the present invention, namely DF-CeO ₂ -HNS1 to 3, as lyophilized powders. Table 1. Recipes for MES Buffers for Preparation of DF-CeO2 _- HNS1 to 3 DF-CeO ₂ -HNS Amount of each component (g) ZM241385 Chitosan 1 0.15 1.5 2 0.75 1.5 3 1.5 1.5 Example 2. Analysis of physicochemical properties of cerium oxide hollow nanospheres of the present invention

在上面實施例1當中所得到的氧化鈰中空奈米球體CeO ₂-HNS以及DF-CeO ₂-HNS1至3被拿來進行下列物化性質的分析 。 A. 傅立葉轉換紅外線光譜 (Fourier Transform Infrared Spectroscopy, FTIR) 分析： The cerium oxide hollow nanospheres CeO ₂ -HNS and DF-CeO ₂ -HNS 1 to 3 obtained in the above Example 1 were used to analyze the following physicochemical properties . A. Fourier Transform Infrared Spectroscopy (FTIR) analysis:

使用一FT-730 ATR/FTIR光譜儀(FT-730 ATR/FTIR spectrophotometer)(Horiba, Japan)來對CeO ₂-HNS以及DF-CeO ₂-HNS1至3進行FTIR分析，並記錄在解析度(resolution)為8 cm ^-1時所測得之波數範圍落在800-3700 cm ^-1內的FTIR光譜(FTIR spectra)。而所得到的結果被顯示於圖1中。 FTIR analysis of CeO ₂ -HNS and DF-CeO ₂ -HNS 1 to 3 was performed using a FT-730 ATR/FTIR spectrophotometer (Horiba, Japan) and recorded at resolution FTIR spectra with wavenumbers in the range of 800-3700 cm ^-1 measured at 8 cm ^-1 . The results obtained are shown in FIG. 1 .

從圖1可見，DF-CeO ₂-HNS1至3在波數為3436 cm ^-1處皆有出現特徵峰(characteristic peak)，亦即有偵測到OH基團，而CeO ₂-HNS則沒有出現此特徵峰，這表示DF-CeO ₂-HNS1至3確實具有ZM241385與幾丁聚糖這兩種官能基。 B. 電子顯微鏡觀察： It can be seen from Figure 1 that DF-CeO ₂ -HNS1 to 3 all have characteristic peaks at the wavenumber of 3436 cm ^-1 , that is, OH groups are detected, while CeO ₂ -HNS does not appear This characteristic peak indicates that DF-CeO ₂ -HNS1 to 3 indeed have two functional groups, ZM241385 and chitosan. B. Electron microscope observation:

使用一穿透式電子顯微鏡(transmission electron microscopy, TEM)(購自於JEOL, Ltd，型號為JSM-1200EX II)來對CeO ₂-HNS以及DF-CeO ₂-HNS1至3進行觀察與拍照，結果如圖2所示，DF-CeO ₂-HNS1至3所具有之多晶形(polycrystalline)與中空的結構是相同於CeO ₂-HNS所具者。而DF-CeO ₂-HNS1至3的粒徑(皆約為62 nm)則是略大於CeO ₂-HNS所具者(約為54 nm)。由此可見，CeO ₂-HNS的粒徑會因雙官能基化而變大。 C. X- 射線繞射 (XRD) 分析 [X-ray diffraction (XRD) analysis]： A transmission electron microscope (TEM) (purchased from JEOL, Ltd, model JSM-1200EX II) was used to observe and photograph CeO ₂ -HNS and DF-CeO ₂ -HNS1 to 3, and the results As shown in FIG. 2 , the polycrystalline and hollow structures of DF-CeO ₂ -HNS1 to 3 are the same as those of CeO ₂ -HNS. The particle sizes of DF-CeO ₂ -HNS1 to 3 (all about 62 nm) are slightly larger than those of CeO ₂ -HNS (about 54 nm). It can be seen that the particle size of CeO ₂ -HNS increases due to bifunctionalization. C. X -ray diffraction (XRD) analysis [X-ray diffraction (XRD) analysis] :

使用一Bruker AXS D8 Advance X-射線繞射儀(Bruker AXS D8 Advance X-ray diffractometer)(Karksruhe, Germany)並依據下面表2中所示的操作條件來對CeO ₂-HNS以及DF-CeO ₂-HNS1至3進行XRD分析。而所得到的結果被顯示於圖3中。 表2.   X-射線繞射儀的操作條件 操作參數 條件 X-射線光源 銅靶(Cu Kα，波長=0.154056 nm) 光源能量 40 kV-30 mA 掃描範圍 2θ：10°~80° 掃描速度 1°/分鐘 掃描步長(step size) 0.3° CeO 2 -HNS and DF-CeO ₂ -HNS and DF-CeO ₂ - HNS1 to 3 were subjected to XRD analysis. The results obtained are shown in FIG. 3 . Table 2. Operating Conditions of X-ray Diffraction Apparatus Operating parameters condition X-ray light source Copper target (Cu Kα, wavelength=0.154056 nm) light source energy 40kV-30mA Scan range 2θ: 10°~80° Scan speed 1°/min Scan step size (step size) 0.3°

從圖3可見，CeO ₂-HNS的XRD圖譜出現有4個特徵衍射峰(characteristic diffraction peaks)，分別對應於CeO ₂的(111)、(200)、(220)以及(311)晶面，這表示：CeO ₂-HNS存在有結構完整的氧化鈰。至於DF-CeO ₂-HNS1至3，則在2 θ為15-35°的範圍內觀察到有寬化波峰(broadened peaks)，這表示：DF-CeO ₂-HNS1至3已具有晶格結構的改變。 D. ζ 電位 (zeta potential) 的測定： It can be seen from Figure 3 that the XRD pattern of CeO ₂ -HNS has 4 characteristic diffraction peaks, which correspond to the (111), (200), (220) and (311) crystal planes of CeO ₂ respectively. It means that CeO ₂ -HNS has structurally complete cerium oxide. As for DF-CeO ₂ -HNS1 to 3, broadened peaks were observed in the range of 2θ of 15-35°, which means that DF-CeO ₂ -HNS1 to 3 have lattice structures. Change. D. Determination of zeta potential :

將適量之CeO ₂-HNS以及DF-CeO ₂-HNS1至3分別溶於10 mM的磷酸緩衝液(phosphate buffer, PB)(pH 7.4)中以作為待測樣品，繼而使用一Zetasizer Nano ZS分析儀(Zetasizer Nano ZS analyzer)(Malvern Instruments, Worcestershire, UK)來對該等待測樣品進行ζ電位的測定。而所得到的結果被顯示於圖4中。 Appropriate amounts of CeO ₂ -HNS and DF-CeO ₂ -HNS1 to 3 were respectively dissolved in 10 mM phosphate buffer (PB) (pH 7.4) as the samples to be tested, and then a Zetasizer Nano ZS analyzer was used. (Zetasizer Nano ZS analyzer) (Malvern Instruments, Worcestershire, UK) to measure the zeta potential of the sample to be tested. The results obtained are shown in FIG. 4 .

從圖4可見，CeO ₂-HNS以及DF-CeO ₂-HNS1至3皆有測得ζ電位，並且CeO ₂-HNS的ζ電位是明顯高於DF-CeO ₂-HNS1至3所具者。由此可見，CeO ₂-HNS表面所帶有的正電荷會因雙官能基化而降低。 實施例 3. 本發明之氧化鈰中空奈米球體的活體內生物相容性 ( in vivobiocompatibilty) 的測試 實驗材料： 1.  製備含有不同氧化鈰中空奈米球體的試驗溶液： It can be seen from FIG. 4 that the zeta potentials of CeO ₂ -HNS and DF-CeO ₂ -HNS1 to 3 are all measured, and the zeta potential of CeO ₂ -HNS is significantly higher than that of DF-CeO ₂ -HNS1 to 3. It can be seen that the positive charge on the CeO ₂ -HNS surface will be reduced due to the bifunctionalization. Example 3. Test materials for in vivo biocompatibility of the cerium oxide hollow nanospheres of the present invention : 1. Prepare test solutions containing different cerium oxide hollow nanospheres:

將適量之依據上面實施例1當中所得到的CeO ₂-HNS以及DF-CeO ₂-HNS1至3分別溶於磷酸鹽緩衝生理食鹽水(phosphate buffered saline, PBS)中，而得到濃度為1 mg/mL的試驗溶液。 實驗方法： Appropriate amounts of CeO ₂ -HNS and DF-CeO ₂ -HNS1 to 3 obtained in Example 1 above were respectively dissolved in phosphate buffered saline (PBS) to obtain a concentration of 1 mg/ mL of test solution. experimental method:

首先，將紐西蘭白兔隨機地分成5組(每組n=6)，其中包括1個正常對照組、1個比較實驗組以及3個實驗組(亦即實驗組1至3)，繼而以舒泰(Zoletil, Virbac, Carros, France)(劑量為2.5 mg/kg體重)與鹽酸賽拉嗪(xylazine hydrochloride)(劑量為1 mg/kg體重)的肌肉內注射(intramuscular injection)而被麻醉，然後將50 μL之含有CeO ₂-HNS的試驗溶液滴注(instillation)至比較實驗組的白兔的眼表(ocular surface)中，以及將50 μL之含有DF-CeO ₂-HNS1至3的試驗溶液分別滴注至實驗組1至3的白兔的眼表中。至於正常對照組的白兔則不作任何處理。 First, New Zealand white rabbits were randomly divided into 5 groups (n=6 in each group), including 1 normal control group, 1 comparative experimental group and 3 experimental groups (ie experimental groups 1 to 3), and then Anesthetized with an intramuscular injection of Zoletil (Virbac, Carros, France) (2.5 mg/kg body weight) and xylazine hydrochloride (1 mg/kg body weight) , and then 50 μL of the test solution containing CeO ₂ -HNS was instilled into the ocular surface of the white rabbits of the comparative experimental group, and 50 μL of the test solution containing DF-CeO ₂ -HNS1 to 3 was instilled. The test solutions were instilled into the ocular surfaces of the white rabbits of experimental groups 1 to 3, respectively. As for the white rabbits in the normal control group, no treatment was given.

在進行滴注之後的第7天，藉由CO ₂來犧牲白兔並使用手術刀片而從各組白兔的眼睛中取出角膜組織(corneal tissue)，接著，對所得到之各組白兔的角膜組織各取大約0.2 g的組織樣品，並在室溫下以4%三聚甲醛(paraformaldehyde)(配於PBS中)來進行固定(fixation)歷時30分鐘，繼而將經固定的組織樣品以石蠟(paraffin)予以包埋(embedding)，然後進行切片處理，藉此而得到具有一厚度為5 μm的組織切片(tissue sections)。 On the 7th day after the instillation, the rabbits were sacrificed by CO ₂ and the corneal tissue was removed from the eyes of the rabbits in each group using a scalpel blade. About 0.2 g of each corneal tissue sample was taken and fixed with 4% paraformaldehyde (in PBS) for 30 minutes at room temperature, and the fixed tissue samples were then paraffinized. Paraffin was embedded, and then sliced, thereby obtaining tissue sections with a thickness of 5 μm.

之後，所得到的組織切片藉由使用蘇木精-伊紅(hematoxylin-eosin)並且依據熟習此項技藝者所詳知且慣用的技術來進行染色，繼而使用一光學顯微鏡(Carl Zeiss, Oberkochen, Germany)並在一為10倍的放大倍率下，隨機地從該組織切片中選出1個區域來進行拍照並觀察角膜組織的變化，以及進行中心角膜厚度(central corneal thickness)的量測。 結果： The resulting tissue sections were then stained by using hematoxylin-eosin and according to techniques well known and customary to those skilled in the art, followed by an optical microscope (Carl Zeiss, Oberkochen, Germany) and at a magnification of 10 times, a region was randomly selected from the tissue section to take pictures and observe changes in corneal tissue, as well as measure the central corneal thickness. result:

圖5顯示各組白兔的眼表在被滴注以不同氧化鈰中空奈米球體之後的第7天，藉由蘇木精與伊紅染色所觀察到之角膜組織的結果。從圖5可見，各組的角膜組織皆呈現出整齊且明確分層的形態，並且沒有觀察到有發炎細胞的浸潤(infiltration of inflammatory cells)、組織完整性的缺失(loss of tissue integrity)以及水腫(edema)的情形。Figure 5 shows the results of corneal tissue observed by hematoxylin and eosin staining on the ocular surface of each group of white rabbits on the 7th day after instillation with different cerium oxide hollow nanospheres. As can be seen from Figure 5, the corneal tissue in each group showed a neat and well-defined layered morphology, and no infiltration of inflammatory cells, loss of tissue integrity and edema were observed. (edema).

圖6顯示各組白兔的眼表在被滴注以不同氧化鈰中空奈米球體之後的第7天所測得的中心角膜厚度(μm)。從圖6可見，各組白兔的中心角膜厚度不具有差異性，皆可測得約為400 μm的中心角膜厚度。Figure 6 shows the central corneal thickness (μm) measured on the 7th day after the ocular surface of each group of white rabbits was instilled with different cerium oxide hollow nanospheres. It can be seen from Figure 6 that there is no difference in the central corneal thickness of each group of white rabbits, and the central corneal thickness of about 400 μm can be measured.

這些實驗結果顯示：CeO ₂-HNS在進行雙官能基化後仍不會影響角膜組織的形態，而具有高度的生物相容性。 實施例 4. 本發明之氧化鈰中空奈米球體的細胞標靶能力 (cellular targeting capability) 之評估 實驗材料： 1.  兔子睫狀體細胞(rabbit ciliary body cells)的分離與培養： These experimental results show that CeO ₂ -HNS does not affect the morphology of corneal tissue after bifunctionalization, and has a high degree of biocompatibility. Example 4. Evaluation of the cellular targeting capability of the cerium oxide hollow nanospheres of the present invention Experimental materials: 1. Isolation and culture of rabbit ciliary body cells:

在本實施例中所使用的兔子睫狀體細胞大體上是參考Cilluffo M.C. et al. (1993), Exp. Eye Res., 57:513-526當中所述的方法而從紐西蘭白兔的睫狀體中被分離出，並以添加有5%胎牛血清(Fetal Bovine Serum, FBS)、3 mM L-麩醯胺酸(L-glutamine)、50 μg/mL建它黴素(gentamicin)、100 μg/mL康那黴素(kanamycin)、0.49 μM腺嘌呤HCl (adenine HCl)、520 nM膽固醇(cholesterol)、5 μM O-磷酸乙醇胺(O-phosphoethanolamine)、1 μM FeSO ₄、3.3 μM核糖(ribose)、150 μM丙酮酸鈉(sodium pyruvate)、1.9 μM ATP、5 μM乙醇胺(ethanolamine)以及0.79 mg/mL牛血清白蛋白(bovine serum albumin, BSA)的NCTC-135培養基(Gibco)來進行培養(37℃、5% CO ₂)。 實驗方法： Rabbit ciliary cells used in this example were substantially obtained from New Zealand white rabbits with reference to the method described in Cilluffo MC et al . (1993), Exp. Eye Res ., 57:513-526 The ciliary body was isolated and supplemented with 5% Fetal Bovine Serum (FBS), 3 mM L-glutamine, 50 μg/mL gentamicin , 100 μg/mL kanamycin, 0.49 μM adenine HCl, 520 nM cholesterol (cholesterol), 5 μM O-phosphoethanolamine, 1 μM FeSO ₄ , 3.3 μM ribose (ribose), 150 μM sodium pyruvate (sodium pyruvate), 1.9 μM ATP, 5 μM ethanolamine (ethanolamine), and 0.79 mg/mL bovine serum albumin (BSA) in NCTC-135 medium (Gibco) Incubate (37°C, 5% CO ₂ ). experimental method:

首先，將兔子睫狀體細胞分成5組，其中包括1個正常對照組、1個比較實驗組以及3個實驗組(亦即實驗組1至3)。將各組細胞分別以一為5×10 ⁴細胞/井的數量培養於含有300 µL的NCTC-135培養基(添加有5% FBS、3 mM L-麩醯胺酸、50 μg/mL建它黴素、100 μg/mL康那黴素、0.49 μM腺嘌呤HCl、520 nM膽固醇、5 μM O-磷酸乙醇胺、1 μM FeSO ₄、3.3 μM核糖、150 μM丙酮酸鈉、1.9 μM ATP、5 μM乙醇胺以及0.79 mg/mL BSA)的24-井培養盤(24-well plate)中，接著在培養箱(37℃、5% CO ₂)中進行培養歷時24小時。 First, the rabbit ciliary body cells were divided into 5 groups, including 1 normal control group, 1 comparative experimental group, and 3 experimental groups (ie, experimental groups 1 to 3). Cells in each group were cultured in 300 µL of NCTC-135 medium (supplemented with ⁵ % FBS, 3 mM L-glutamic acid, 50 µg/mL Kenta spp. pyruvate, 100 μg/mL kanamycin, 0.49 μM adenine HCl, 520 nM cholesterol, 5 μM O-phosphoethanolamine, 1 μM FeSO ₄ , 3.3 μM ribose, 150 μM sodium pyruvate, 1.9 μM ATP, 5 μM ethanolamine and 0.79 mg/mL BSA) in a 24-well plate, followed by incubation in an incubator (37°C, 5% CO ₂ ) for 24 hours.

之後，將各組的細胞培養物分別更換以新鮮的培養基，然後將300 μL之依據上面實施例3的“實驗材料”的第1項當中所得到之含有CeO ₂-HNS的試驗溶液添加至比較實驗組中，以及將300 μL之依據上面實施例3的“實驗材料”的第1項當中所得到之含有DF-CeO ₂-HNS1至3的試驗溶液分別添加至實驗組1至3中。至於正常對照組的細胞培養物則不作任何處理。 After that, the cell cultures of each group were respectively replaced with fresh medium, and then 300 μL of the CeO ₂ -HNS-containing test solution obtained in item 1 of "Experimental Materials" in Example 3 above was added to the comparison In the experimental group, and 300 μL of the test solution containing DF-CeO ₂ -HNS1 to 3 obtained in item 1 of "Experimental Materials" in Example 3 above were added to the experimental groups 1 to 3, respectively. The cell cultures of the normal control group were left untreated.

各組細胞在培養箱(37℃、5% CO ₂)中進行培養歷時24小時之後，移除培養基並以PBS來清洗細胞2次，然後使用抗ZO-1抗體(購自於Zymed Laboratories Inc.)作為一次抗體(primary antibody)並於4℃下作用歷時1小時，繼而使用綴合有螢光素異硫氰酸鹽(fluorescein isothiocyanate, FITC)的抗體(購自於Chemicon International, Inc.)作為二次抗體(secondary antibody)並於室溫下進行避光作用歷時2小時。之後，使用一螢光顯微鏡(型號為Axiovert 200M，廠牌為Carl Zeiss)並在一為10倍的放大倍率下來進行觀察以及拍照。而有關經FITC染色的細胞培養物之平均螢光強度(mean fluorescence intensity)是使用ImageJ軟體來作測定。 結果： After each group of cells was cultured in an incubator (37°C, 5% CO ₂ ) for 24 hours, the medium was removed and the cells were washed twice with PBS before using an anti-ZO-1 antibody (purchased from Zymed Laboratories Inc. ) as a primary antibody and reacted at 4°C for 1 hour, followed by the use of a fluorescein isothiocyanate (FITC)-conjugated antibody (purchased from Chemicon International, Inc.) as secondary antibody and protected from light for 2 hours at room temperature. Afterwards, observation and photographing were performed using a fluorescence microscope (model Axiovert 200M, brand Carl Zeiss) at a magnification of 10 times. The mean fluorescence intensity of FITC-stained cell cultures was determined using ImageJ software. result:

圖7顯示各組細胞培養物所測得的平均螢光強度(a.u.)。從圖7可見，與正常對照組相較之下，比較實驗組的平均螢光強度只有些微的增加，而實驗組1至3的平均螢光強度則皆有明顯的增加，其中又以實驗組2的增加幅度最為明顯。這個實驗結果顯示：本發明之氧化鈰中空奈米球體確實能夠標靶至睫狀體，特別是以1：2 (w/w)的ZM241385與幾丁聚糖來進行官能基化所製得之氧化鈰中空奈米球體展現最優異的睫狀體-標靶能力(ciliary body-targeting capability)。 實施例 5. 本發明之氧化鈰中空奈米球體的藥物承載能力之測量 Figure 7 shows the average fluorescence intensity (au) measured for each group of cell cultures. As can be seen from Figure 7, compared with the normal control group, the average fluorescence intensity of the experimental group only slightly increased, while the average fluorescence intensity of the experimental groups 1 to 3 increased significantly. 2 has the most obvious increase. The experimental results show that the cerium oxide hollow nanospheres of the present invention can indeed target the ciliary body, especially those prepared by functionalizing 1:2 (w/w) ZM241385 and chitosan. Cerium oxide hollow nanospheres exhibit the most excellent ciliary body-targeting capability. Example 5. Measurement of the drug-carrying capacity of the cerium oxide hollow nanospheres of the present invention

在本實施例中，申請人選用硝酸毛果芸香鹼作為藥物範例來測量本發明之氧化鈰中空奈米球體的藥物承載能力。 實驗方法： In this example, the applicant selected pilocarpine nitrate as a drug example to measure the drug-carrying capacity of the cerium oxide hollow nanospheres of the present invention. experimental method:

將適量之依據上面實施例3的實驗材料的第1項當中所得到之含有CeO ₂-HNS以及DF-CeO ₂-HNS1與2的試驗溶液分別混合以20 mg/mL的硝酸毛果芸香鹼溶液[氧化鈰中空奈米球體與硝酸毛果芸香鹼的用量比約為1：0.3 (w/w)]，繼而分別加入至含有1.5 mL均衡鹽溶液(balanced salt solution, BSS)的小瓶(vials)中，並適時地添加以HCl溶液來將pH值控制在5.5。所得到的混合物被進行一音波處理(sonication)歷時2小時，然後在室溫下進行攪拌歷時24小時，繼而以NaOH溶液來將pH值調整至7.4，並且持續進行攪拌歷時12小時。接著，以15,000 rpm來進行離心歷時10分鐘，取出上澄液，而所得到的沉澱物以BSS予以清洗3次，藉此而得到承載有毛果芸香鹼的CeO ₂-HNS以及DF-CeO ₂-HNS1至2。 An appropriate amount of the test solutions containing CeO ₂ -HNS and DF-CeO ₂ -HNS 1 and 2 obtained in item 1 of the experimental materials in Example 3 above were mixed with a 20 mg/mL pilocarpine nitrate solution [cerium oxide]. The dosage ratio of hollow nanospheres to pilocarpine nitrate is about 1:0.3 (w/w)], and then respectively added to vials containing 1.5 mL of balanced salt solution (BSS), and added in time. The pH was controlled at 5.5 with HCl solution. The resulting mixture was sonicated for 2 hours, then stirred at room temperature for 24 hours, then adjusted to pH 7.4 with NaOH solution, and stirring continued for 12 hours. Next, centrifugation was performed at 15,000 rpm for 10 minutes, the supernatant was taken out, and the obtained precipitate was washed 3 times with BSS, thereby obtaining CeO ₂ -HNS and DF-CeO ₂ -HNS1 to carry pilocarpine. 2.

之後，藉由高效能液相層析(high performance liquid chromatography, HPLC)分析來測定各個上澄液中未被包埋的(entrapped)毛果芸香鹼的含量，藉此而換算出CeO ₂-HNS以及DF-CeO ₂-HNS1與2所承載的毛果芸香鹼的含量。所使用的HPLC分析儀器如下：Hitachi高效能液相層析系統(Hitachi high performance liquid chromatography system, Japan)、UV偵測器(L2400, Hitachi)；分析管柱為Mighty RP-18 (Kanto Chemical)，長度：250 mm × 4.6 mm。而HPLC操作條件被顯示於下面的表3中。 表3.    HPLC的操作條件 操作參數 條件 偵測波長 UV-216 nm 移動相 5%磷酸二氫鉀(potassium dihydrogen phosphate)/甲醇(methanol)，85：15 (v/v) 流速(mL/分鐘) 0.7 Afterwards, the content of unembedded (entrapped) pilocarpine in each supernatant was determined by high performance liquid chromatography (HPLC) analysis, thereby converting CeO ₂ -HNS and DF- Content of pilocarpine carried by CeO ₂ -HNS1 and 2. The HPLC analytical instruments used are as follows: Hitachi high performance liquid chromatography system (Hitachi high performance liquid chromatography system, Japan), UV detector (L2400, Hitachi); analytical column is Mighty RP-18 (Kanto Chemical), Length: 250 mm × 4.6 mm. While the HPLC operating conditions are shown in Table 3 below. Table 3. Operating Conditions for HPLC Operating parameters condition Detection wavelength UV-216 nm mobile phase 5% potassium dihydrogen phosphate (potassium dihydrogen phosphate)/methanol (methanol), 85:15 (v/v) Flow rate (mL/min) 0.7

此外，為供比對，使用不同濃度的毛果芸香鹼(0.1-500 μg/mL)(購自於Sigma-Aldrich)來作為對照標準品(control standard)並進行相同的HPLC分析。In addition, for comparison, different concentrations of pilocarpine (0.1-500 μg/mL) (purchased from Sigma-Aldrich) were used as control standards and the same HPLC analysis was performed.

各個氧化鈰中空奈米球體的藥物承載量百分比(%)是藉由將所使用的各個氧化鈰中空奈米球體的重量(g)及其所承載的毛果芸香鹼含量(g)代入下列公式(1)而被計算出： 公式 (1) ： A ＝ (B/C) × 100其中：A＝藥物承載量百分比(%) B＝各個氧化鈰中空奈米球體所承載的毛果芸香鹼含量 C＝各個氧化鈰中空奈米球體的重量 The drug loading percentage (%) of each cerium oxide hollow nanosphere is calculated by substituting the weight (g) of each cerium oxide hollow nanosphere used and the pilocarpine content (g) carried by it into the following formula (1) And it is calculated: formula (1) : A = (B/C) × 100 where: A = drug loading percentage (%) B = pilocarpine content carried by each cerium oxide hollow nanosphere C = each cerium oxide hollow Weight of Nanospheres

之後，所得到的實驗數據是依據上面“一般實驗方法”的第1項「統計學分析」當中所述方法來進行分析。 結果： After that, the obtained experimental data were analyzed according to the method described in the item 1 "Statistical Analysis" of "General Experimental Methods" above. result:

圖8顯示各個氧化鈰中空奈米球體的藥物承載量百分比(%)。從圖8可見，DF-CeO ₂-HNS1與2的藥物承載量百分比皆是顯著地高於CeO ₂-HNS所具者，其中又以DF-CeO ₂-HNS2具有最高的藥物承載量。這個實驗結果顯示：本發明之氧化鈰中空奈米球體能夠有效地承載眼睛藥物，特別是以1：2 (w/w)的ZM241385與幾丁聚糖來進行官能基化所製得的氧化鈰中空奈米球體。因此，申請人進一步選用DF-CeO ₂-HNS2來進行下面的試驗。 實施例 6. 本發明承載有眼睛藥物之氧化鈰中空奈米球體對於青光眼 ( glaucoma) 的治療效用之評估 A. 青光眼的誘發 (Induction of glaucoma) ： Figure 8 shows the drug loading percentage (%) of each cerium oxide hollow nanosphere. It can be seen from Figure 8 that the drug loading percentages of DF-CeO ₂ -HNS1 and 2 are significantly higher than those of CeO ₂ -HNS, and DF-CeO ₂ -HNS2 has the highest drug loading. The experimental results show that the cerium oxide hollow nanospheres of the present invention can effectively carry eye medicines, especially cerium oxide prepared by functionalization of ZM241385 and chitosan at 1:2 (w/w) Hollow Nanospheres. Therefore, the applicant further selected DF-CeO ₂ -HNS2 for the following experiments. Example 6. Evaluation of the therapeutic efficacy of cerium oxide hollow nanospheres carrying ophthalmic drugs of the present invention for glaucoma A. Induction of glaucoma :

有關青光眼的誘發是參考Nguyen D.D. et al.(2019), Adv. Healthc. Mater., doi: 10.1002/adhm.201900702.當中所述的方法來進行。簡言之，使用30G針頭將0.1 mg/mL的α-胰凝乳蛋白酶注射至紐西蘭白兔的雙眼當中之一者的眼後房(posterior chamber)中，繼而予以觀察歷時4週。當經α-胰凝乳蛋白酶注射的眼睛的絕對眼內壓(absolute intraocular pressure, absolute IOP)高於20 mmHg時，表示青光眼被誘發。 B. 硝酸毛果芸香鹼的眼內給藥 (ophthalmic administration) ： The induction of glaucoma was carried out with reference to the method described in Nguyen DD et al. (2019), Adv. Healthc. Mater. , doi: 10.1002/adhm.201900702. Briefly, 0.1 mg/mL alpha-chymotrypsin was injected into the posterior chamber of one of the eyes of New Zealand white rabbits using a 30G needle and then observed for 4 weeks. Glaucoma was indicated when the absolute intraocular pressure (absolute IOP) of the alpha-chymotrypsin injected eye was higher than 20 mmHg. B. Intraocular Administration of Pilocarpine Nitrate :

首先，對依據上面實施例5當中所得到之承載有毛果芸香鹼的CeO ₂-HNS與DF-CeO ₂-HNS2分別加入20 μL之1 mM的FITC並予以充分混合均勻，繼而於4℃下靜置隔夜，而分別得到綴合有FITC之承載有毛果芸香鹼的CeO ₂-HNS與DF-CeO ₂-HNS2。 First, 20 μL of 1 mM FITC was added to the CeO ₂ -HNS and DF-CeO ₂ -HNS2 carrying pilocarpine obtained according to Example 5 above, and the mixture was well mixed, and then left at 4°C overnight. , and FITC-conjugated CeO ₂ -HNS and DF-CeO ₂ -HNS2 loaded with pilocarpine were obtained, respectively.

接著，將24隻之依據上面第A項當中所述的方法來進行青光眼的誘發而帶有青光眼的紐西蘭白兔隨機地分成4組(每組n=6)，其中包括1個病理對照組、1個比較實驗組以及2個實驗組(亦即實驗組1與2)，繼而以舒泰(劑量為2.5 mg/kg體重)與鹽酸賽拉嗪(劑量為1 mg/kg體重)的肌肉內注射而被麻醉，然後將20 mg/mL的硝酸毛果芸香鹼溶液(劑量為50 μL/隻)滴注至比較實驗組的白兔的眼表中，以及將綴合有FITC之承載有毛果芸香鹼的CeO ₂-HNS與DF-CeO ₂-HNS2 (劑量為50 μL/隻)分別滴注至實驗組1與2的白兔的眼表中。至於病理對照組的白兔則不作任何處理。 Next, 24 New Zealand white rabbits with glaucoma induced by the method described in item A above were randomly divided into 4 groups (n=6 in each group), including 1 pathological control group, 1 comparative experimental group, and 2 experimental groups (ie, experimental groups 1 and 2), followed by sutai (2.5 mg/kg body weight) and xylazine hydrochloride (1 mg/kg body weight) The mice were anesthetized by intramuscular injection, and then 20 mg/mL pilocarpine nitrate solution (dose of 50 μL/rabbit) was instilled into the ocular surface of the white rabbits in the comparative experimental group, and FITC-conjugated pilocarpine-loaded CeO ₂ -HNS and DF-CeO ₂ -HNS2 (dose of 50 μL/rabbit) were instilled into the ocular surfaces of white rabbits in experimental groups 1 and 2, respectively. As for the white rabbits in the pathological control group, no treatment was given.

另外，以沒有進行青光眼的誘發以及硝酸毛果芸香鹼的眼內給藥之正常的紐西蘭白兔作為正常對照組。 C. 眼內壓 (intraocular pressure, IOP) 的相對改變 (relative change) 的 測定： In addition, normal New Zealand white rabbits without glaucoma induction and intraocular administration of pilocarpine nitrate were used as a normal control group. C. Determination of relative change in intraocular pressure (IOP) :

在眼內給藥之前(亦即第0小時)以及眼內給藥之後的第4與12小時以及第1、3、5與7天，藉由使用一希厄茨眼壓計(Schiotz tonometer)(購自於AMANN Ophthalmic Instruments，Germany，型號為2993-00)來測量病理對照組、比較實驗組以及實驗組1與2的白兔的雙眼(亦即一帶有青光眼的眼睛以及一未經任何處理的正常眼睛)的眼內壓。接著，眼內壓的相對改變是藉由將所測得的眼內壓(mmHg)代入下列公式(2)而被計算出： 公式 (2) ： D ＝ E － F其中：D=眼內壓的相對改變 E=在帶有青光眼的眼睛中所測得的眼內壓 F=在正常的眼睛中所測得的眼內壓 Before intraocular administration (ie, hour 0) and at 4 and 12 hours and 1, 3, 5, and 7 days after intraocular administration, by using a Schiotz tonometer (purchased from AMANN Ophthalmic Instruments, Germany, model 2993-00) to measure both eyes of rabbits in the pathological control group, the comparative experimental group, and experimental groups 1 and 2 (ie, one eye with glaucoma and one without any intraocular pressure of normal eyes treated). Next, the relative change in intraocular pressure is calculated by substituting the measured intraocular pressure (mmHg) into the following equation (2): Equation (2) : D = E - F where: D = intraocular pressure Relative change of E = intraocular pressure measured in eye with glaucoma F = intraocular pressure measured in normal eye

所得到的實驗結果被顯示於圖9中。從圖9可見，在眼內給藥之後的第4小時至第7天，比較實驗組與實驗組1的白兔的眼內壓的相對改變會隨著時間而被顯著地增加，並且逐漸趨近於病理對照組所具者。相對地，實驗組2的白兔的眼內壓的相對改變皆維持在0 mmHg左右。這個實驗結果顯示：使用本發明之氧化鈰中空奈米球體來遞送硝酸毛果芸香鹼，能夠有效地在眼前房內持續釋放硝酸毛果芸香鹼，以使其長期發揮治療青光眼的效用。The obtained experimental results are shown in FIG. 9 . As can be seen from Figure 9, from the 4th hour to the 7th day after the intraocular administration, the relative changes in the intraocular pressure of the rabbits comparing the experimental group and the experimental group 1 were significantly increased with time, and gradually tended to close to those of the pathological control group. In contrast, the relative changes of the intraocular pressure of the white rabbits in the experimental group 2 were all maintained at about 0 mmHg. The experimental results show that using the cerium oxide hollow nanospheres of the present invention to deliver pilocarpine nitrate can effectively and continuously release pilocarpine nitrate in the anterior chamber, so that it can play a long-term effect on treating glaucoma.

另外，申請人亦有針對各組白兔的眼睛進行平均角膜曲率數值(mean keratometric value)的測定，而結果顯示，使用本發明之氧化鈰中空奈米球體來遞送硝酸毛果芸香鹼，能夠使平均角膜曲率數值回復至接近正常數值(數據未顯示)。In addition, the applicant has also measured the mean keratometric value of the eyes of each group of white rabbits, and the results show that the use of the cerium oxide hollow nanospheres of the present invention to deliver pilocarpine nitrate can make the mean keratometric value Values returned to near normal values (data not shown).

這些實驗結果顯示：使用本發明之氧化鈰中空奈米球體來遞送硝酸毛果芸香鹼能夠有效地降低IOP並修復角膜畸變(corneal aberration)，而發揮青光眼的治療效用。 D. 免疫螢光染色 (Immunofluorescence staining) ： These experimental results show that using the cerium oxide hollow nanospheres of the present invention to deliver pilocarpine nitrate can effectively reduce IOP and repair corneal aberration, thereby exerting therapeutic effects on glaucoma. D. Immunofluorescence staining :

在眼內給藥之後的第7天，藉由CO ₂來犧牲白兔並使用手術刀片而從正常對照組、比較實驗組以及實驗組1與2的白兔的眼睛中取出大約0.2 g的睫狀體組織(ciliary body tissue)，並在室溫下以4%三聚甲醛(配於PBS中)來進行固定歷時30分鐘，繼而將經固定的組織樣品以石蠟予以包埋，然後進行切片處理，藉此而得到具有一厚度為12 μm的組織切片。 On the 7th day after the intraocular administration, the rabbits were sacrificed by _CO2 and approximately 0.2 g of ciliary ciliary was removed from the eyes of the normal control group, the comparison experimental group, and the experimental groups 1 and 2 using a scalpel blade. Ciliary body tissue was fixed with 4% paraformaldehyde (in PBS) for 30 minutes at room temperature, and the fixed tissue samples were then embedded in paraffin and sectioned , thereby obtaining a tissue section with a thickness of 12 μm.

所得到的組織切片在以PBS予以清洗3次之後，被浸泡在一封阻緩衝液(blocking buffer)[含有配於PBS中的0.3 Triton-X與3% FBS)中反應歷時1小時，以PBS予以清洗3次，繼而加入綴合有FITC之山羊抗-兔子IgG抗體[Goat Anti-Rabbit IgG Antibody conjugated with FITC](廠牌為Chemicon International，Cat. No. AP307F)並在室溫下進行培育歷時2小時，以PBS予以清洗3次，然後以4’,6-二脒基-2-苯基吲哚(4’,6-diamidino-2-phenylindole, DAPI)來進行細胞核染色。之後，使用一螢光顯微鏡(型號為Axiovert 200M，廠牌為Carl Zeiss)並在一為10倍的放大倍率下來進行觀察以及拍照。而有關經FITC染色的組織切片之平均螢光強度是使用ImageJ軟體來作測定。The obtained tissue sections were washed 3 times with PBS, then immersed in blocking buffer (containing 0.3 Triton-X and 3% FBS in PBS) for 1 hour and reacted with PBS. It was washed 3 times, then FITC-conjugated goat anti-rabbit IgG antibody [Goat Anti-Rabbit IgG Antibody conjugated with FITC] (brand: Chemicon International, Cat. No. AP307F) was added and incubated at room temperature for a period of time After 2 hours, the cells were washed three times with PBS, and then nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Afterwards, observation and photographing were performed using a fluorescence microscope (model Axiovert 200M, brand Carl Zeiss) at a magnification of 10 times. The mean fluorescence intensity of FITC-stained tissue sections was determined using ImageJ software.

所得到的實驗結果被顯示於圖10中。從圖10可見，與正常對照組相較之下，比較實驗組與實驗組1的平均螢光強度只有些微的增加，而實驗組2的平均螢光強度則有顯著的增加。這個實驗結果顯示：本發明之氧化鈰中空奈米球體能夠有效地將硝酸毛果芸香鹼遞送至睫狀體中 。 E. 承載有 Y-27632 的氧化鈰中空奈米球體在降低 眼內壓上的效用 評估： The obtained experimental results are shown in FIG. 10 . As can be seen from Figure 10, compared with the normal control group, the average fluorescence intensity of the experimental group and the experimental group 1 only slightly increased, while the average fluorescence intensity of the experimental group 2 increased significantly. The experimental results show that the cerium oxide hollow nanospheres of the present invention can effectively deliver pilocarpine nitrate to the ciliary body . E. Evaluation of the efficacy of Y-27632- loaded cerium oxide hollow nanospheres in reducing intraocular pressure :

首先，大體上參照上面實施例5當中所述的方法來製備承載有Y-27632的DF-CeO ₂-HNS2，不同之處在於：以Y-27632來替代硝酸毛果芸香鹼。接著，參照上面第A至C項當中所述的方法來對白兔進行青光眼的誘發、承載有Y-27632的DF-CeO ₂-HNS2的眼內給藥以及眼內壓的量測與計算。而所得到的結果顯示：使用本發明之氧化鈰中空奈米球體來遞送Y-27632，能夠使白兔的眼內壓的相對改變維持在0 mmHg左右長達10天(數據未顯示)。 First, DF-CeO2 _- HNS2 loaded with Y-27632 was prepared substantially as described in Example 5 above, except that pilocarpine nitrate was replaced with Y-27632. Next, induction of glaucoma, intraocular administration of Y-27632-loaded DF-CeO ₂ -HNS2, and measurement and calculation of intraocular pressure were performed in white rabbits with reference to the methods described in items A to C above. The obtained results show that using the cerium oxide hollow nanospheres of the present invention to deliver Y-27632 can maintain the relative change of intraocular pressure at around 0 mmHg for up to 10 days in white rabbits (data not shown).

綜合以上的實驗結果，申請人認為：本發明之氧化鈰中空奈米球體可以有效地將所欲遞送的藥物遞送至眼內組織(例如睫狀體)中，並且能夠使該藥物被有效地釋放，以使其長期發揮治療眼睛疾病的效用。Based on the above experimental results, the applicant believes that the cerium oxide hollow nanospheres of the present invention can effectively deliver the drug to be delivered to the intraocular tissue (such as the ciliary body), and can effectively release the drug , so that it can play a long-term effect on the treatment of eye diseases.

於本說明書中被引述之所有專利和文獻以其整體被併入本案作為參考資料。若有所衝突時，本案詳細說明(包含界定在內)將佔上風。All patents and documents cited in this specification are incorporated by reference in their entirety. In the event of conflict, the detailed description of the case (including definitions) will prevail.

雖然本發明已參考上述特定的具體例被描述，明顯地在不背離本發明之範圍和精神之下可作出很多的修改和變化。因此意欲的是，本發明僅受如隨文檢附之申請專利範圍所示者之限制。Although the present invention has been described with reference to the specific embodiments above, it will be apparent that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the present invention be limited only as indicated by the scope of the appended claims.

本發明的上述以及其它目的、特徵與優點，在參照以下的詳細說明與較佳實施例和隨文檢附的圖式後，將變得明顯，其中： 圖1顯示各個氧化鈰中空奈米球體藉由傅立葉轉換紅外線光譜分析所得到的光譜圖，其中CeO ₂-HNS表示不帶有官能基的氧化鈰中空奈米球體；以及DF-CeO ₂-HNS1至3表示以不同重量比(亦即1:10、1:2以及1:1)的ZM241385與幾丁聚糖來進行官能基化所製得之氧化鈰中空奈米球體； 圖2顯示各個氧化鈰中空奈米球體藉由穿透式電子顯微鏡所觀察到的結果，其中CeO ₂-HNS表示不帶有官能基的氧化鈰中空奈米球體；以及DF-CeO ₂-HNS1至3表示以不同重量比(亦即1:10、1:2以及1:1)的ZM241385與幾丁聚糖來進行官能基化所製得之氧化鈰中空奈米球體； 圖3顯示各個氧化鈰中空奈米球體藉由X-射線繞射(XRD)分析所得到的XRD圖譜，其中CeO ₂-HNS表示不帶有官能基的氧化鈰中空奈米球體；以及DF-CeO ₂-HNS1至3表示以不同重量比(亦即1:10、1:2以及1:1)的ZM241385與幾丁聚糖來進行官能基化所製得之氧化鈰中空奈米球體； 圖4顯示各個氧化鈰中空奈米球體所測得的ζ電位(mV)，其中CeO ₂-HNS表示不帶有官能基的氧化鈰中空奈米球體；以及DF-CeO ₂-HNS1至3表示以不同重量比(亦即1:10、1:2以及1:1)的ZM241385與幾丁聚糖來進行官能基化所製得之氧化鈰中空奈米球體； 圖5顯示各組白兔的眼表在被滴注以不同氧化鈰中空奈米球體之後的第7天，藉由蘇木精與伊紅染色所觀察到之角膜組織的結果，其中正常對照組表示不作任何處理的白兔；比較實驗組表示被處理以含有CeO ₂-HNS的試驗溶液的白兔；以及實驗組1至3分別表示被處理以含有DF-CeO ₂-HNS1至3的試驗溶液的白兔； 圖6顯示各組白兔的眼表在被滴注以不同氧化鈰中空奈米球體之後的第7天所測得的中心角膜厚度(μm)，其中比較實驗組表示被處理以含有CeO ₂-HNS的試驗溶液的白兔；實驗組1至3分別表示被處理以含有DF-CeO ₂-HNS1至3的試驗溶液的白兔；以及虛線表示不作任何處理的正常對照組白兔所測得的中心角膜厚度； 圖7顯示各組細胞培養物所測得的平均螢光強度(a.u.)，其中正常對照組表示不作任何處理的兔子睫狀體細胞；比較實驗組表示被處理以含有CeO ₂-HNS的試驗溶液的兔子睫狀體細胞；實驗組1至3分別表示被處理以含有DF-CeO ₂-HNS1至3的試驗溶液的兔子睫狀體細胞；以及“*”表示：當與正常對照組作比較， p＜0.05； 圖8顯示各個氧化鈰中空奈米球體的藥物承載量百分比(%)，其中CeO ₂-HNS表示不帶有官能基的氧化鈰中空奈米球體；DF-CeO ₂-HNS1與2表示以不同重量比(亦即1:10以及1:2)的ZM241385與幾丁聚糖來進行官能基化所製得之氧化鈰中空奈米球體；以及“*”表示：當與CeO ₂-HNS作比較， p＜0.05； 圖9顯示各組白兔的眼內壓的相對改變隨著時間的變化，其中病理對照組表示被處理以α-胰凝乳蛋白酶的白兔；比較實驗組表示被處理以α-胰凝乳蛋白酶與硝酸毛果芸香鹼溶液的白兔；實驗組1表示被處理以α-胰凝乳蛋白酶與承載有毛果芸香鹼的CeO ₂-HNS的白兔；實驗組2表示被處理以α-胰凝乳蛋白酶與承載有毛果芸香鹼的DF-CeO ₂-HNS2的白兔；虛線表示眼內壓的基準值(baseline value)；以及“**”表示：當與眼內壓的基準值作比較， p＜0.005；以及 圖10顯示各組白兔的睫狀體組織所測得的平均螢光強度(a.u.)，其中正常對照組表示不作任何處理的白兔；比較實驗組表示被處理以α-胰凝乳蛋白酶與硝酸毛果芸香鹼溶液的白兔；實驗組1表示被處理以α-胰凝乳蛋白酶與承載有毛果芸香鹼的CeO ₂-HNS的白兔；實驗組2表示被處理以α-胰凝乳蛋白酶與承載有毛果芸香鹼的DF-CeO ₂-HNS2的白兔；以及“*”表示：當與正常對照組作比較， p＜0.05。 The above and other objects, features and advantages of the present invention will become apparent upon reference to the following detailed description and preferred embodiments and accompanying drawings in which: Figure 1 shows individual cerium oxide hollow nanospheres Spectrograms obtained by Fourier transform infrared spectroscopy analysis, wherein CeO ₂ -HNS represents cerium oxide hollow nanospheres without functional groups; and DF-CeO ₂ -HNS 1 to 3 represent different weight ratios (ie 1 : 10, 1:2 and 1:1) of ZM241385 and chitosan to carry out functionalization of cerium oxide hollow nanospheres; The results observed by microscopy, in which CeO ₂ -HNS represents cerium oxide hollow nanospheres without functional groups; and DF-CeO ₂ -HNS1 to 3 represent different weight ratios (i.e. 1:10, 1:2 and 1:1) of ZM241385 and chitosan for functionalization of cerium oxide hollow nanospheres; The obtained XRD patterns, in which CeO ₂ -HNS represents cerium oxide hollow nanospheres without functional groups; and DF-CeO ₂ -HNS 1 to 3 represent different weight ratios (i.e. 1:10, 1:2 and 1 : 1) ZM241385 and chitosan to carry out functionalization of the cerium oxide hollow nanospheres; Figure 4 shows the measured zeta potential (mV) of each cerium oxide hollow nanosphere, wherein CeO ₂ - HNS represents cerium oxide hollow nanospheres without functional groups; and DF-CeO ₂ -HNS1 to 3 represent ZM241385 and chitosan in different weight ratios (ie 1:10, 1:2 and 1:1) Cerium oxide hollow nanospheres prepared by functionalization with sugar; Figure 5 shows that the ocular surface of each group of white rabbits was instilled with different cerium oxide hollow nanospheres on the 7th day by hematoxylin. The results of corneal tissue observed by eosin staining, wherein the normal control group represents the white rabbits without any treatment; the comparative experimental group represents the white rabbits treated with the test solution containing CeO ₂ -HNS; and the experimental groups 1 to 3 The white rabbits treated with the test solutions containing DF-CeO ₂ -HNS1 to 3, respectively; Figure 6 shows the ocular surface of each group of white rabbits measured on the 7th day after being instilled with different cerium oxide hollow nanospheres The obtained central corneal thickness (μm), wherein the comparative experimental group represents the white rabbits treated with the test solution containing CeO ₂ -HNS; the experimental groups 1 to 3 represent the experiments treated with DF-CeO ₂ -HNS 1 to 3, respectively The white rabbits in solution; and the dotted line represents the measured central corneal thickness of the normal control white rabbits without any treatment; Figure 7 shows the average fluorescence intensity (au) measured by each group of cell cultures, wherein the normal control group represents Rabbit ciliary body cells without any treatment; the comparative experimental group means treated with Rabbit ciliary cells in test solutions containing CeO ₂ -HNS; experimental groups 1 to 3 represent rabbit ciliary cells treated with test solutions containing DF-CeO ₂ -HNS 1 to 3, respectively; and "*" represents: When compared with the normal control group, p <0.05; Figure 8 shows the drug loading percentage (%) of each cerium oxide hollow nanosphere, wherein CeO ₂ -HNS represents the cerium oxide hollow nanosphere without functional groups; DF-CeO ₂ -HNS1 and 2 represent cerium oxide hollow nanospheres prepared by functionalization with ZM241385 and chitosan in different weight ratios (ie 1:10 and 1:2); and "* " means: when compared with CeO ₂ -HNS, p <0.05; Figure 9 shows the relative changes of intraocular pressure in each group of rabbits with time, wherein the pathological control group was treated with α-chymotrypsin The comparison experimental group represents the white rabbits treated with α-chymotrypsin and pilocarpine nitrate solution; the experimental group 1 represents the white rabbits treated with α-chymotrypsin and CeO ₂ -HNS loaded with pilocarpine ; Experimental group 2 represents white rabbits treated with α-chymotrypsin and DF-CeO ₂ -HNS2 loaded with pilocarpine; dashed line represents the baseline value of intraocular pressure; and "**" represents: when Compared with the reference value of intraocular pressure, p <0.005; and Figure 10 shows the average fluorescence intensity (au) measured in the ciliary body tissue of each group of white rabbits, wherein the normal control group represents the white rabbits without any treatment ;Comparative experimental group represents the rabbits treated with α-chymotrypsin and pilocarpine nitrate solution; Experimental group 1 represents the white rabbits treated with α-chymotrypsin and CeO ₂ -HNS loaded with pilocarpine; experimental group 2 indicates a white rabbit treated with α-chymotrypsin and DF-CeO ₂ -HNS2 loaded with pilocarpine; and "*" indicates: p < 0.05 when compared to normal control.

Claims (13)

一種奈米載體，其包含有： 一金屬氧化物中空奈米球體，其中該金屬氧化物是選自於由下列所構成的群組：CeO ₂、CuO、ZnO、NiO、AuO、Fe ₃O ₄，以及它們的組合；以及 接枝於該金屬氧化物中空奈米球體的複數個腺苷A2A受體拮抗劑(adenosine A2A receptor antagonist)以及複數個緊密型連結調節子(tight junction modulator)。 A nanocarrier comprising: a metal oxide hollow nanosphere, wherein the metal oxide is selected from the group consisting of: CeO ₂ , CuO, ZnO, NiO, AuO, Fe ₃ O ₄ , and their combinations; and a plurality of adenosine A2A receptor antagonists and a plurality of tight junction modulators grafted on the metal oxide hollow nanospheres. 如請求項1的奈米載體，其中該等腺苷A2A受體拮抗劑是選自於由下列所構成的群組：ZM241385、SCH58261、KW6002，以及它們的組合。The nanocarrier of claim 1, wherein the adenosine A2A receptor antagonists are selected from the group consisting of ZM241385, SCH58261, KW6002, and combinations thereof. 如請求項1的奈米載體，其中該等緊密型連結調節子是選自於由下列所構成的群組：幾丁聚糖、聚-L-離胺酸、聚-L-精胺酸、魚精蛋白、聚乙烯亞胺、胺端基聚醯胺樹枝狀聚合物(amine terminated polyamidoamine dendrimer, PAMAM-NH ₂)、聚(2-胺乙基丙烯醯胺)、四級化的聚(甲基丙烯酸二甲胺乙酯)[quaternized poly(dimethylaminoethyl methacrylate)]，以及它們的組合。 The nanocarrier of claim 1, wherein the tight junction regulators are selected from the group consisting of: chitosan, poly-L-lysine, poly-L-arginine, Protamine, polyethyleneimine, amine terminated polyamidoamine dendrimer (PAMAM-NH ₂ ), poly(2-amine ethyl acrylamide), quaternized poly(methyl amide) quaternized poly(dimethylaminoethyl methacrylate) [quaternized poly(dimethylaminoethyl methacrylate)], and combinations thereof. 如請求項1的奈米載體，其中該金屬氧化物中空奈米球體有被聚乙二醇化以供該等腺苷A2A受體拮抗劑與緊密型連結調節子的接枝。The nanocarrier of claim 1, wherein the metal oxide hollow nanospheres are pegylated for grafting of the adenosine A2A receptor antagonists with tight junction regulators. 如請求項1的奈米載體，其中該金屬氧化物中空奈米球體具有一範圍落在20 nm至150 nm內的粒徑。The nanocarrier of claim 1, wherein the metal oxide hollow nanospheres have a particle size ranging from 20 nm to 150 nm. 如請求項1的奈米載體，其中，該等腺苷A2A受體拮抗劑與緊密型連結調節子的用量比是介於1：1 (w/w)至1：300 (w/w)之間。The nanocarrier of claim 1, wherein the dosage ratio of the adenosine A2A receptor antagonist to the tight junction modulator is between 1:1 (w/w) to 1:300 (w/w) between. 一種藥物遞送系統，其包含有一如請求項1的奈米載體以及一眼睛藥物。A drug delivery system comprising the nanocarrier as claimed in item 1 and an ophthalmic drug. 如請求項7的藥物遞送系統，其中，該奈米載體與該眼睛藥物的用量比是介於1：0.2 (w/w)至1：0.5 (w/w)之間。The drug delivery system of claim 7, wherein the dosage ratio of the nanocarrier to the eye drug is between 1:0.2 (w/w) and 1:0.5 (w/w). 如請求項7的藥物遞送系統，其中該眼睛藥物是選自於由下列所構成的群組：硝酸毛果芸香鹼(pilocarpine nitrate)、Y-27632、蒂目羅(Timolol)、卡特洛(Carteolol)、溴莫尼定(Brimonidine)、多佐胺(Dorzolamide)、布林佐胺(Brinzolamide)、乙醯偶氮胺(Acetazolamide)、拉坦前列素(Latanoprost)、曲伏前列素(Travoprost)、比馬前列素(Bimatoprost)、甘露醇(Mannitol)、異山梨醇(Isosorbide)、環孢素(cyclosporine)、維替泊芬(verteporfin)、維生素A棕櫚酸鹽與維生素E(vitamin A palmitate and vitamin E)、維生素B12 (vitamin B12)、蘭尼單抗(ranibizumab)、柔癌捕(aflibercept)、溴珠單抗(brolucizumab)、貝伐珠單抗(bevacizumab)，以及它們的組合。The drug delivery system of claim 7, wherein the eye drug is selected from the group consisting of: pilocarpine nitrate, Y-27632, Timolol, Carteolol, bromine Brimonidine, Dorzolamide, Brinzolamide, Acetazolamide, Latanoprost, Travoprost, Bimatoprost Bimatoprost, Mannitol, Isosorbide, Cyclosporine, Verteporfin, Vitamin A palmitate and vitamin E, Vitamin B12, ranibizumab, aflibercept, brolucizumab, bevacizumab, and combinations thereof. 一如請求項7至9中任一項的藥物遞送系統供應用於製備一用來治療和/或預防眼睛疾病之醫藥品的用途。A drug delivery system as claimed in any one of claims 7 to 9 provides use for the preparation of a medicament for the treatment and/or prevention of eye diseases. 如請求項10的用途，其中該眼睛疾病是選自於由下列所構成的群組：眼前段疾病(anterior segment eye disease)、眼後段疾病(posterior segment eye disease)，以及它們的組合。The use of claim 10, wherein the eye disease is selected from the group consisting of: anterior segment eye disease, posterior segment eye disease, and combinations thereof. 如請求項10的用途，其中該眼睛疾病是選自於由下列所構成的群組：青光眼、白內障、葡萄膜炎、黃斑退化、眼內炎、糖尿病視網膜病變、乾眼症，以及它們的組合。The use of claim 10, wherein the eye disease is selected from the group consisting of: glaucoma, cataract, uveitis, macular degeneration, endophthalmitis, diabetic retinopathy, dry eye, and combinations thereof . 如請求項10的用途，其中該醫藥品是呈一供眼內投藥的劑型。The use of claim 10, wherein the medicinal product is in a dosage form for intraocular administration.

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