- 學位論文
以微針精準釋放螢光複合物用於皮膚鱗狀上皮細胞癌之治療
Precise release of theranostic fluorescent compound with microneedles for treating squamous cell carcinoma of the skin
摘要
皮膚鱗狀上皮細胞癌為過度暴露於紫外線所導致的皮膚問題，其中日光性角化症為其癌前病變，若不治療則會惡化為鱗狀細胞癌且更嚴重會有癌細胞轉移的風險，並且日光性角化症發展至鱗狀細胞癌為一個連續性的過程，於臨床上較難辨別兩者差異，因此患上此疾病時須及早治療。日光性角化症最主要的治療方式為手術治療、冷凍治療、光動力療法或是擦拭藥膏，但前三種方式不僅價格高昂且有起水泡、發炎等等後遺症的產生；藥膏塗抹則會因皮膚上的角質層而導致吸收不佳的情況且需要數個月的治療。 其中經皮給藥有一種方式為微針系統，可以刺穿角質層直接在皮內給藥，所以給藥效果比塗抹藥膏更佳。本研究使用溶解性微針，材料則選用高分子材料－明膠，明膠在人體內透過組織液即可降解，對人體是不會造成毒害。經皮給藥則有一個缺點為無法確定藥物是否有滲透至皮膚，因此本研究選用螢光藥物改善此缺失。 螢光藥物標靶腫瘤為臨床上常見的方式，但反射光的波長過短，沒有進入紅外線的波段範圍則很難從人體內穿透出來並被接收到。現在臨床已在使用的螢光藥劑為靛青綠（ICG），而ICG的螢光會因為光照、溫度或是水解而被降解，因此利用牛血清白蛋白（BSA）接合ICG可使ICG更加穩定且有效的增加ICG放光波段。 本研究先利用BSA與咪喹莫特（IMQ）複合在一起，而重量比於1:20時有最好的複合效果，並且以BSA複合的方式可以提高IMQ在水中的分散性。接著以1:20此比例合成的複合物與ICG複合，結果BSA與ICG在水中的濃度為4:1時有最佳的螢光效果。合成出的複合藥物以明膠微針搭載後，不論長度或是機械強度都與明膠微針沒有明顯差異。進而比較藥物水溶液以及微針給藥滲透皮膚的效果，也可以明顯發現微針給藥對於皮膚的滲透效果是優於皮上給藥的。之後模擬了IMQ在明膠微針中以及BSA和ICG是否會影響IMQ的作用，因此使用了RAW-BlueTM cell這可以表示類鐸受體的細胞株，結果不論是明膠、BSA或是ICG都不影響IMQ的表現。接下來在小鼠背部建立腫瘤模型，而利用包覆複合物的明膠微針給予治療，經過兩週的治療而腫瘤大小則減小的原本的50%，其餘施打不含IMQ的組別的腫瘤卻逐漸變大，因此再次證明明膠、BSA及ICG不會影響IMQ的作用。而IMQ乳膏給予治療可以發現具有治療效果，但治療效果卻比微針給藥還差，由此更加證明微針給藥至皮內的治療效果比乳膏還要更好。最後利用近紅外光相機追蹤了藥物的動向，可以發現施加ICG-BSA/IMQ微針在小鼠背部後，隨著時間的增加而螢光強度則逐漸減弱，證明了藥物滲透至小鼠體內。本研究突顯了以微針給予IMQ治療皮膚鱗狀上皮細胞癌的可行性，並且明顯證明微針給藥是確實滲透至皮內而非停留於皮膚表面。 關鍵字：皮膚鱗狀上皮細胞癌、咪喹莫特、靛青綠、近紅外光、微針
並列摘要
Squamous cell carcinoma is a skin problem caused by excessive exposure to ultraviolet rays. Actinic keratosis is a precancerous lesion if it is not treated, it will worsen to squamous cell carcinoma or it will cause the risk of cancer metastasis. And the development of actinic keratosis to squamous cell carcinoma is a continuous process, and it is difficult to distinguish the difference between the two clinically. Therefore, early treatment is required for this disease. The main treatment methods for actinic keratosis are surgical treatment, cryotherapy, photodynamic therapy or wipe ointment, but the first three methods are not only expensive but also have sequelae such as blisters, inflammation, etc.; ointment application will cause poor absorption due to the stratum corneum on the skin and require several months of treatment. One way of transdermal administration is the microneedle system, which can pierce the stratum corneum and administer directly in the skin, so the effect is better than applying ointment. In this study, soluble microneedles were used, and gelatin was used as the material. This polymer material can be degraded through tissue fluid in the human body and will not cause harm to the human body. One disadvantage of transdermal administration is that it is impossible to determine whether the drug has penetrated into the skin. Therefore, this study chose fluorescent drugs to improve this deficiency. Fluorescent drugs are a common way to target tumors in clinical practice, but the wavelength of reflected light is too short, and it is difficult to penetrate from the human body and be received without entering the infrared wavelength range. Therefore, combining bovine serum albumin (BSA) with indocyanine green (ICG) effectively increases the wavelength of indocyanine green. This study first used BSA and imiquimod (IMQ) to be compounded together, and the weight ratio of 1:20 has the best compounding effect, and the BSA compounding method can improve the dispersibility of IMQ in water. Then the compound synthesized at a ratio of 1:20 is compounded with ICG. As a result, the concentration of BSA and ICG in water has the best fluorescent effect when the concentration is 4:1. After the synthesized compound drug is loaded with gelatin microneedles, there is no significant difference in length or mechanical strength from gelatin microneedles. Furthermore, comparing the skin penetration effect of the drug solution and the microneedle administration, it can be clearly found that the penetration effect of the microneedle administration on the skin is better than that of the skin administration. After that, this study simulated the effect of IMQ in gelatin microneedles and whether BSA and ICG would affect the effect of IMQ. Therefore, RAW-BlueTM cell was used, which could represent the cell line of toll-like receptors. As a result, neither gelatin, BSA or ICG would affect IMQ's performance. Next, a tumor model was established on the back of the mouse, and the gelatin microneedle loaded with the compound was used for treatment. After two weeks of treatment, the tumor size was reduced by 50%. The tumors of the other groups without IMQ have gradually become larger, so it is once again proved that gelatin, BSA and ICG will not affect the effect of IMQ. The IMQ cream can be found to have a therapeutic effect, but the therapeutic effect is worse than that of microneedle administration, which proves that the therapeutic effect of microneedle administration into the skin is even better than that of cream.Last, a near-infrared camera was used to track the movement of the drug, and it was found that the fluorescence intensity of the ICG-BSA/IMQ microneedle applied to the back of the mouse gradually decreased with the increase of time, which proved the penetration of the drug into the mouse. This study highlights the feasibility of microneedle administration of IMQ to treat squamous cell carcinoma of the skin, and clearly proves that microneedle administration does penetrate into the skin instead of staying on the surface of the skin. Key word : Squamous cell carcinoma of the skin , Indocyanine green , Imiquimod , Near-infrared, Microneedles