聚合微膠體眼溶液傳遞角膜專一性啟動子
Cornea Specific Promoter Delivery by In Vivo Eye Drops of Polymeric Micelles
中文摘要
此實驗評估非離子型共聚合物運用於基因傳遞，與眼睛專一性啟動子 (specific promoter): k12 (keratin 12)、 k3.2 (keratocan 3.2)，及轉染效力強之啟動子 CMV (cytomegalovirus) 結合，利用眼溶液的方式局部傳遞至眼睛。
首先評估此聚合物的物化性質，分別利用了一螢光性物質pyrene 來測定聚合物
的臨界微膠體濃度 (Critical Micelle Concentration; CMC)，動態光散射 (Dynamic light scattering; DLS)，的方法來測定其粒徑大小以及界面電位。而在基因表現的 部分，則是利用了CPRG (Chlorophenolred-b-D-galactopyranoside sodium salt) 此 受質來做眼球內基因表現的定量分析，X-gal 此受質來進行染色，確定其基因表
現於眼球中分布的位置。結果發現聚合物濃度在大於0.1%時，會形成微膠體；
而分別帶有CMV、k12、k3.2 三種啟動子的質體 DNA 與聚合物形成 DNA-聚合微 膠體的粒徑大小分別為142.9 ± 8.7，182.1 ± 6.1，187.3 ± 9.1 nm。DNA-聚合微膠
體於室溫、4℃、37℃以及歷經兩次冷凍解凍後都呈現良好的安定性。另外，此聚
合物與質體DNA 形成 DNA-聚合微膠體於 DNase 存在時有一個良好的保護效果。
以兩天六個劑量的眼溶液局部投與眼睛之後，測得k12 的基因表現為 3.58 ± 0.80 mu/mg 與控制組相比較 (3.09 ± 0.39 mu/mg) 有差異性 (P<0.05)。k3.2 的基因 表現於加入EDTA 此一促進劑之後為 3.58 ± 0.80 mu/mg 與控制組相比較 (3.09 ± 0.39 mu/mg) 呈現差異性 (P<0.05)。然而，與沒有加入 EDTA 之 k3.2 相比較 (3.27
± 0.37 mu/mg) 則亦呈現差異性 (P<0.05)。並且利用 RT-PCR 的方式，得到的結果
與上述基因表現有相關聯。而觀察到k12，k3.2 兩啟動子基因表現分別位於角膜
的上皮細胞層 (epithelium) 以及基質層 (stromal layer) 的角膜細胞 (keratocyte) 中。
當為了增加基因傳遞量，利用paracellular enhancer 添加後，使得 tight junction 開啟的這條路徑，DNA-載體複合物之粒徑大小在進行基因傳遞時是一個重要因 子。
綜合上述實驗結果，非離子型共聚合物與質體DNA 所形成的 DNA-聚合微膠體
可運用於眼睛局部投與，為一有效且安全的基因傳遞方式。
英文摘要
The primary objective of this study was to investigate the feasibility of using non- ionic copolymeric micelles as a carrier for eye drops topical gene delivery specific promoter (keratin 12, keratocan 3.2) plasmid DNA with the LacZ gene in vivo.
Using pyrene probe, dynamic light scattering (DLS) test to detect block copolymeric micelles critical micelle concentration (CMC), size distribution and Zeta potential.
Gene expressions were visualized by both the quality of the enzymatic color reaction
using X-gal staining and by quantification of the substrate chlorophenolred-b-D- galactopyranoside (CPRG) in ocular tissues after gene delivery. The CMC of block copolymeric micelle was above 0.1 % copolymer concentration, and micelle size of CMV, k12, k3.2 promoter-plasmids were 142.9 ± 8.7, 182.1 ± 6.1, 187.3 ± 9.1 nm, respectively. DNA-polymeric micelle has good stability in room temperature, 4℃, 37℃ and twice freeze/thaw cycles. In addition, polymer provide plasmid DNA with protect effect to DNase.
After six doses of eye drops topical delivery 3 times a day, it was observed gene expression at cornea area. b-gal expression was detected in k12 promoter delivery (3.58 ± 0.80 mu/mg, compare with control experiment 3.09 ± 0.39 mu/mg, P<0.05).
Furthermore, k3.2 promoter delivery was detected its b-gal expression after addition of EDTA treatment (3.88 ± 1.08 mu/mg, compare with control 3.29 ± 0.41 mu/mg, P<0.05; compare with k3.2 3.27 ± 0.37 mu/mg, P<0.05). And, it was confirmed by RT-PCR reaction, and it had similar results. Gene expression of k12 is restricted to the corneal epithelium; gene expression of k3.2 is restricted to the corneal keratocyte in the stromal layer.
The particle size of DNA-vector complex could succeed delivery into cornea area after using a paracellular enhancer to open cornea’s tight junction.
Taken together, we reported an efficient and stable gene delivery with copolymeric micelles with cornea promoters through eye drops topical delivery can be achieved in mice.
