近年研究中發現超音波結合吸附 Minoxidil 之白蛋白微氣泡可加強藥物促生髮效果，但白蛋白微氣泡對於皮膚過敏反應是個隱憂，且白蛋白微氣泡市面上也較少用於經皮穿透，而本研究中，提供了一種新型超音波微氣泡對比劑之藥物釋放載體，利用超音波導入結合溶菌酶微氣泡對比劑，將溶菌酶微氣泡對比劑表面吸附一層 Minoxidil (Mx)，為了減少Mx對於皮膚副作用，也將溶劑改良成水溶液製劑，而溶菌酶是一種知名的抗菌劑，在近期文獻上發現其具有促進毛髮生長的功效，且其抗菌效果可抑制頭皮分泌油脂所滋生的細菌，又相較於白蛋白為較弱過敏原，然而超音波特殊的穴蝕效應不僅可以打開表皮通透度，進而增加表皮與皮下組織細胞之藥物吸收，能有效加速生長期毛囊生長。
以正負電荷吸附使Mx吸附於溶菌酶微氣泡 (LyzMBs) 表面形成Mx之溶菌酶微氣泡 (Mx-LyzMBs)，透過 DLS 量測粒徑與電位、in vitro與 in vivo (n=5)，動物實驗組別主要以 (1) 控制組 (Control)、(2) 單純施打超音波能量 (US)、(3) 施打超音波能量於溶菌酶微氣泡 (US+LyzMBs)、(4) 施打超音波能量於溶菌酶微氣泡混合Mx溶液 (US+LyzMBs+Mx)、(5) 施打超音波能量於吸附Mx之溶菌酶微氣泡 (US+Mx-LyzMB)。
經吸附Mx之溶菌酶微氣泡，其粒徑大小隨吸附時間及比例不同而改變，單純溶菌酶微氣泡與吸附 Mx之溶菌酶微氣泡 (比例2：1，吸附時間 2 hr) 依序為 2.4 ± 0.14 µm、4.3 ± 0.70 µm；表面電位則依序為 58.6 ± 2.65 mV、39.0 ± 1.09 mV；溶菌酶微氣泡吸附Mx的吸附率為 20.3 %；以 FITC 觀察經皮穿透由施打超音波能量於溶菌酶為氣泡後經 6 hr 深度可達 1267.3 µm，利用透析方式觀察藥物釋放，在 1 hr 時釋放效果可達 37.9 %；利用 Activity Assay Kit 及免疫組織化學染色 (IHC) 相互檢測溶菌酶經皮穿透效果，在超音波能量施打於溶菌酶微氣泡經 6 hr 測得活性有 65.8 µU/ml 而 18 hr 後皮膚內的溶菌酶活性達到 118.5 µU/ml；而毛囊組織培養中發現 US+Mx-LyzMBs 組在培養第 2 天成長幅度為最大 (108.18 µm)，有最快促使生長之顯著效果 (p<0.001)；動物實驗中 US+Mx-LyzMBs 組相較 C 組提早 2 天進入生長期，在第 12 天也有明顯的生長效果，綜合以上結果超音波結合吸附 Mx 之溶菌酶微氣泡的方法能改善局部脫髮症狀 (Alopecias)，促進毛髮生長及提前進入生長期。

In our previous study, a new type of Minoxidil (Mx) coated albumin shelled microbubbles (AMBs) and combined with ultrasound energy in the water phase was demonstrated to enhance hair growth. However, skin allergy was concerned by using AMBs. Lysozyme (Lyz) is a safe adjunct to antifungals and could be used to improve acne treatment due to its antibactericidal effect. Recently, Lyz was found to stimulate hair follicle growth. In this study, a new minoxidil coated Lyz shelled microbubble was produced to inhibit bacterium or the allergy caused in oil scalp. Moreover, the potential of Mx coated Lyz shelled MBs combine with US either to enhance hair follicle growth or to promote transdermal drug delivery was investigated.
The in vitro and in vivo experimental parameters was randomly divided into five groups (n=5 animals per group): (1) no treatment (Control), (2) US treatment alone (US), (3) US combines with LyzMBs (US+LyzMBs), (4) US combines with LyzMBs and penetrating Mx (US+LyzMBs+Mx), (5) US combines with Mx-LyzMBs (US+Mx-LyzMBs).
Mx grafted with LyzMBs (mean diameter of 2400 ± 140 nm) were synthesized into self-assembled complexes of Mx-LyzMBs that had mean diameters of 4300 ± 700 nm, respectively. The zeta potentials of LyzMBs and Mx-LyzMBs were 58.6 ± 2.65 mV and 39.0 ± 1.09 mV, respectively. The loading efficiency of Mx on cationic LyzMBs was 20.3%. In vitro skin permeation studies have demonstrated the ability of US-mediated Mx-LyzMBs cavitation to act on FITC was 1267.3 µm when the FITC solution was left for 6 hours in the histology sections. In Franz diffusion experiments performed in vitro, the release rates increased 37.9 % at 1 hour. Biological activity of lysozyme in skin were determined by activity assay kit and immunohistochemistry (IHC) and expression, and activities in group US+Mx-LyzMBs were 65.8 µU/ml and 118.5 µU/ml at 6 hr and 18 hrs, respectively. In hair follicle cell culture experiments, the growth of hair follicle cells were enhanced significantly in US+Mx-LyzMBs group at 2th day (p<0.001). For the in vivo experiments, the Mx-LyzMBs would be expected to promote hair growth more rapidly than control group and reduced the treatment dose and side effects. The results confirm that US combined with Mx-LyzMBs can significantly enhance hair growth in water without requiring the use of any chemical enhancement method to increase the penetrating of Mx and Lyz in skin.

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