TWI688405B - Biodegradable microneedle array - Google Patents
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本發明有關於製造多醣系的可溶性微針系陣列之方法及由此製備的微針陣列。The invention relates to a method for manufacturing a polysaccharide-based soluble microneedle array and a microneedle array prepared thereby.
用於新開發藥物的遞輸系統的發展是富有挑戰性的任務。藥物通過各種常見途徑投予,如口服、腸胃外、眼部、經皮、鼻部、肺部及頰部途徑(Langer,2001)。在這些途徑中,經皮輸藥系統(TDDS)是一種有前景看好的系統,其可在沒有醫務人員的幫助下使用(Prausnitz和Langer,2008;Wokovich等,2006)。TDDS還具有幾個優點,如消除肝臟首渡代謝及口服劑型對胃腸道的副作用(Asbill和Michniak,2000;Hadgraft等,1996)。然而,系統仍存在一些缺點,如低效率。由於各種原因,例如角質層(SC)的皮膚結構及屏障性能,效率可能會降低(Bronaugh和Maibach,2005)。SC屏障的存在意味著只能輸送油溶性分子和低分子量化合物。The development of delivery systems for newly developed drugs is a challenging task. Drugs are administered through various common routes, such as oral, parenteral, ocular, transdermal, nasal, pulmonary, and buccal routes (Langer, 2001). Among these approaches, the transdermal drug delivery system (TDDS) is a promising system that can be used without the help of medical personnel (Prausnitz and Langer, 2008; Wokovich et al., 2006). TDDS also has several advantages, such as eliminating liver first-pass metabolism and the side effects of oral dosage forms on the gastrointestinal tract (Asbill and Michniak, 2000; Hadgraft et al., 1996). However, the system still has some disadvantages, such as low efficiency. For various reasons, such as the skin structure and barrier properties of the stratum corneum (SC), the efficiency may be reduced (Bronaugh and Maibach, 2005). The presence of the SC barrier means that only oil-soluble molecules and low molecular weight compounds can be transported.
為了克服難應付的SC屏障,已經開發了幾種增強皮膚滲透的方法用於藥物輸送。其中一種方法是使用微針陣列,其在通過皮膚屏障輸送治療藥物方面表現出更好的性能,而沒有造成任何損傷(Kaushik等人,2001;McAllister等人,2003;Mikszta等人,2002)。微針陣列(MN)應用於皮膚表面並穿刺表皮而不刺激傷害感受器(Martin等人,2012)。這些MN通過皮膚產生微小的孔,並使得藥物或疫苗擴散到皮膚微循環中(Cheung等,2014;Kim等,2012;Liu等,2012;Sullivan等,2010;Tuan -Mahmood等,2013)。MN陣列係由各種材料製成,例如聚合物、金屬、矽橡膠及多醣(Badran等人,2009;Ding等人,2009;Hafeli等人,2009;Li等人,2009;Lin等人,2001;Martanto等,2004)。其中,聚合物MN陣列越來越有吸引力,因為它們被預期比矽或金屬陣列更便宜地大量生產,並且在應用過程中更安全。當使用溶解聚合物時,藥物與生物分子可結合到MN內部(Chu和Prausnitz,2011;Hirobe等人,2015;Lee等人,2008;Lee等人,2011;Liu等人,2012;Sullivan等人,2010)。釋放藥物後,聚合物溶解在皮膚中,這避免了MN殘留在體內的風險(Ke等人,2012;Sullivan等人,2008)。用於MN應用的聚合物應具有諸如生物相容性、強力學性質及快速溶解速率等特徵。當考慮藥物依賴性釋放時間時,將需要具有不同溶解速率的聚合物來傳遞受控量的藥物。To overcome the difficult SC barrier, several methods have been developed to enhance skin penetration for drug delivery. One method is to use a microneedle array, which shows better performance in delivering therapeutic drugs through the skin barrier without causing any damage (Kaushik et al., 2001; McAllister et al., 2003; Mikszta et al., 2002). The microneedle array (MN) is applied to the skin surface and penetrates the epidermis without stimulating nociceptors (Martin et al., 2012). These MNs create tiny holes through the skin and allow drugs or vaccines to diffuse into the skin's microcirculation (Cheung et al., 2014; Kim et al., 2012; Liu et al., 2012; Sullivan et al., 2010; Tuan-Mahmood et al., 2013). The MN array is made of various materials, such as polymers, metals, silicone rubber, and polysaccharides (Badran et al., 2009; Ding et al., 2009; Hafeli et al., 2009; Li et al., 2009; Lin et al., 2001; Martanto et al., 2004). Among them, polymer MN arrays are becoming more and more attractive because they are expected to be mass-produced cheaper than silicon or metal arrays, and are safer in application. When dissolving polymers are used, drugs and biomolecules can bind inside the MN (Chu and Prausnitz, 2011; Hirobe et al., 2015; Lee et al., 2008; Lee et al., 2011; Liu et al., 2012; Sullivan et al. , 2010). After the drug is released, the polymer dissolves in the skin, which avoids the risk of MN remaining in the body (Ke et al., 2012; Sullivan et al., 2008). Polymers used in MN applications should have characteristics such as biocompatibility, strong mechanical properties, and rapid dissolution rates. When considering the drug-dependent release time, polymers with different dissolution rates will be required to deliver a controlled amount of drug.
固體MN被澆鑄,且係使用可生物降解的聚合物作為兩種類型的結構:「單件」及「逐層」而形成。在「單件」方法中,將含有藥物及聚合物的溶液注射並同時澆鑄在相同模具上;而在「逐層」方法中,模型結構係藉由多個塗佈步驟或與模型的每個薄片的組合而澆鑄。有機固體MN將合成及天然材料的分子量範圍從數百擴大到數千,包括糊精(DEX)、羧甲基纖維素(CMC)、支鏈澱粉(AMP)、聚交酯-共-乙交酯、半乳糖、麥芽糖及聚乙烯吡咯烷酮(Lee等人,2008;Raphael等人,2010)。根據它們不同的分子相容性,可添加許多不同大小的模型化合物;例如,β-半乳糖苷酶、磺醯玫瑰紅(sulforhodamine)、水楊酸鈉、鈣黃綠素及牛血清白蛋白已成功地併入MN中。在過去的十年中,以各種藥物、生物活性蛋白及疫苗的MN遞輸的臨床前評估已顯示有進步(Pettis及Harvey,2012)。動物及人體研究已證明了各種藥物與疫苗的應用。然而,MN臨床研究的發表相對較少(Pettis及Harvey,2012)。包括藥物或生物分子包封在MN中的穩定性、可靠的MN插入皮膚、評估皮膚刺激及皮膚感染、評估藥物動力學及藥效學,以及評估疼痛和其他知覺需要進一步的詳細研究(Kim等人,2012;Pettis和Harvey,2012)。The solid MN is cast and is formed using biodegradable polymers as two types of structures: "single piece" and "layer by layer". In the "single piece" method, the solution containing the drug and polymer is injected and simultaneously cast on the same mold; while in the "layer by layer" method, the model structure is obtained by multiple coating steps or with each of the models The combination of flakes is cast. Organic solid MN extends the molecular weight range of synthetic and natural materials from hundreds to thousands, including dextrin (DEX), carboxymethyl cellulose (CMC), amylopectin (AMP), polylactide-co-glycolide Ester, galactose, maltose and polyvinylpyrrolidone (Lee et al., 2008; Raphael et al., 2010). According to their different molecular compatibility, many model compounds of different sizes can be added; for example, β-galactosidase, sulforhodamine, sodium salicylate, calcein and bovine serum albumin have been successfully Merged into MN. Over the past decade, preclinical evaluation of MN delivery with various drugs, bioactive proteins, and vaccines has shown progress (Pettis and Harvey, 2012). Animal and human studies have proven the application of various drugs and vaccines. However, there are relatively few published MN clinical studies (Pettis and Harvey, 2012). Including the stability of drugs or biomolecules encapsulated in MN, reliable insertion of MN into the skin, evaluation of skin irritation and skin infections, evaluation of pharmacokinetics and pharmacodynamics, and evaluation of pain and other perceptions require further detailed studies (Kim et al. People, 2012; Pettis and Harvey, 2012).
仍然需要新的經皮遞輸系統。New transdermal delivery systems are still needed.
在本發明中出乎意料地發現,CMC與糊精DEX的組合係使用以製造生物相容性微針陣列(MN)。據此,本發明提供製造多醣系可溶解微針系陣列之方法,其可使用作為侵入性和經皮遞輸系統。由該方法獲得的微針陣列是安全且生物相容的,且可使用於藥物如胰島素的經皮投予。It was unexpectedly discovered in the present invention that the combination of CMC and dextrin DEX is used to manufacture biocompatible microneedle arrays (MN). Accordingly, the present invention provides a method of manufacturing a polysaccharide-based soluble microneedle array, which can be used as an invasive and transdermal delivery system. The microneedle array obtained by this method is safe and biocompatible, and can be used for transdermal administration of drugs such as insulin.
在一方面,本發明提供製造用於經皮遞輸治療劑的微針系陣列之方法,其包含: 混合羧甲基纖維素(CMC)與糊精(DEX),以提供多醣系組合;以及 將該治療劑加入至多醣系組合中。In one aspect, the present invention provides a method of manufacturing a microneedle array for transdermal delivery of a therapeutic agent, comprising: mixing carboxymethyl cellulose (CMC) and dextrin (DEX) to provide a polysaccharide-based combination; and The therapeutic agent is added to the polysaccharide-based combination.
在本發明的一具體實施例中,多醣系組合包含30至70%的CMC及30至70%的DEX。In a specific embodiment of the present invention, the polysaccharide combination comprises 30 to 70% CMC and 30 to 70% DEX.
在本發明的一實例中,多醣系組合包含50%的CMC及50%的DEX。In an example of the present invention, the polysaccharide-based combination includes 50% CMC and 50% DEX.
本文所使用的「治療劑」乙詞是指化合物或物質,其以治療有效量投予哺乳動物時,為哺乳動物提供治療益處,例如藥物。The term "therapeutic agent" as used herein refers to a compound or substance that, when administered to a mammal in a therapeutically effective amount, provides the mammal with a therapeutic benefit, such as a drug.
在本發明的一具體實施例中,治療劑是藥物。In a specific embodiment of the invention, the therapeutic agent is a drug.
在本發明的一具體實施例中,治療劑是胰島素。In a specific embodiment of the invention, the therapeutic agent is insulin.
在另一方面,本發明提供一種用於經皮遞輸治療劑的微針陣列,係由羧甲基纖維素(CMC)與糊精(DEX)的多醣系組合而製成。微針陣列係藉由本發明之方法而製備。In another aspect, the present invention provides a microneedle array for transdermal delivery of therapeutic agents, which is made of a combination of carboxymethyl cellulose (CMC) and dextrin (DEX) polysaccharides. The microneedle array is prepared by the method of the present invention.
用於For MNMN 陣列的模具設計Array mold design
如圖1所示,使用高速切割機及計算機輔助設計(CAD)系統(AutoCAD 2012)在鋁(Al)板上製造模具。根據初步結果將鋁模具進行設計修改。一個鋁模具包括兩個陣列單元,以在鑄造過程中創建兩個微針陣列。在鋁模具中有四個功能區域:(1)針區域(NA);及(2)空白區域(BA);(3)脊區域(RA);及(4)連接溝槽(CT)。模具中心的NA有兩組10×10的錐形針陣列。NA被BA包圍,為模型提供厚度以確保韌性並避免從模具釋放時的應力性破裂。RA用於分離每個單獨的陣列單元,而CT幫助黏性溶液流動較慢,並在快速注射期間避免氣泡。As shown in Figure 1, a high-speed cutting machine and a computer-aided design (CAD) system (AutoCAD 2012) are used to manufacture molds on aluminum (Al) plates. Based on the preliminary results, the aluminum mold was modified. An aluminum mold includes two array units to create two microneedle arrays during the casting process. There are four functional areas in the aluminum mold: (1) the needle area (NA); and (2) the blank area (BA); (3) the ridge area (RA); and (4) the connection groove (CT). The NA in the center of the mold has two sets of 10×10 tapered needle arrays. NA is surrounded by BA, providing thickness to the model to ensure toughness and avoid stress cracking when released from the mold. RA is used to separate each individual array unit, while CT helps the viscous solution flow slower and avoid air bubbles during rapid injection.
為了分析用於MN製造的複合材料,如圖2中所示,該程序被設計為具有兩個測試循環,提供用於製造根據本發明的微針陣列的方案。第一個測試循環是基於含水量與X射線繞射(XRD)及熱重分析(TGA)的結果。此外,第一個檢查點是測試複合混合物是否不能被高達100°C的溫度切割。第二個檢查點測試混合物是否可溶。如果混合溶液可溶,則下一個測試將是生物毒性測試。如果沒有毒性,以5%的混合溶液的良好成膜檢查點測試流動覆蓋範圍。當所有檢查點都經過測試後,展開MN的製造。所有與測試薄膜及MN的製造工作都是在單一「一體化」鑄模具中進行。In order to analyze the composite material used for MN manufacturing, as shown in FIG. 2, the program is designed to have two test cycles, providing a solution for manufacturing the microneedle array according to the present invention. The first test cycle is based on the results of water content and X-ray diffraction (XRD) and thermogravimetric analysis (TGA). In addition, the first checkpoint is to test whether the compound mixture cannot be cut at temperatures up to 100°C. The second checkpoint tests whether the mixture is soluble. If the mixed solution is soluble, the next test will be a biotoxicity test. If there is no toxicity, test the flow coverage with a good film formation checkpoint of 5% mixed solution. After all the inspection points have been tested, the manufacture of MN is started. All manufacturing work with test films and MN is performed in a single "integrated" casting mold.
多醣材料Polysaccharide material
基於它們的生物相容性和水溶性,我們最初選擇四種不同的多醣來分析它們的物理性質,以鑑定合適的分子來製造可溶解的MN。CMC,一種纖維素衍生物,是一種水溶性天然多醣,已廣泛使用於各種食品(Chillo等,2007)及醫療應用。DEX包含藉由澱粉水解所產生的不同大小的水溶性葡萄糖聚合物。DEX在食品產業中使用作為食品添加劑及一些醫藥產品。高度支化的支鏈澱粉(AMP)是水溶性多醣,其為澱粉的兩種主要組分之一。AMP使用於不同的應用,包括食品增稠。海藻糖(TRE)是天然雙醣,其包含兩個藉由α,α-1,1-葡糖苷鍵連接的葡萄糖單元(Higashiyama,2002)。TRE在自然界廣泛分佈,對產業應用而言是一種有吸引力的物質。這種醣在食品產業中使用作為甜味劑(Higashiyama,2002)。Based on their biocompatibility and water solubility, we initially selected four different polysaccharides to analyze their physical properties to identify suitable molecules to make soluble MN. CMC, a cellulose derivative, is a water-soluble natural polysaccharide that has been widely used in various foods (Chillo et al., 2007) and medical applications. DEX contains water-soluble glucose polymers of different sizes produced by the hydrolysis of starch. DEX is used as a food additive and some pharmaceutical products in the food industry. Highly branched amylopectin (AMP) is a water-soluble polysaccharide, which is one of the two main components of starch. AMP is used in different applications, including food thickening. Trehalose (TRE) is a natural disaccharide that contains two glucose units connected by α,α-1,1-glucosidic bonds (Higashiyama, 2002). TRE is widely distributed in nature and is an attractive substance for industrial applications. This sugar is used as a sweetener in the food industry (Higashiyama, 2002).
由Sigma-Aldrich購買CMC鈉鹽(99.5%純度)、DEX(來自馬鈴薯澱粉,具有99%純度)、TRE脫水物(釀酒酵母,適於細胞培養,具有99%純度)及AMP(原始馬鈴薯澱粉)。Purchase CMC sodium salt (99.5% purity), DEX (from potato starch with 99% purity), TRE dehydrate (Saccharomyces cerevisiae, suitable for cell culture, with 99% purity) and AMP (raw potato starch) from Sigma-Aldrich .
用於For MNMN 製造的多醣的物理特徵Physical characteristics of the manufactured polysaccharide
所有多醣的含水量係使用紅外水分分析儀MA150,Sartorius測量。將約0.1g粉末形式的每種糖加熱並分析水分百分比。使用Setaram Labsys-TGDSC DSC131儀器進行TGA,以獲得在澆鑄過程中多醣材料的加熱溫度範圍。將0.008g粉末形式的每種糖樣品從室溫加熱至500℃進行TGA分析,加熱速率為5℃ min-1。粉末形式的材料也使用於XRD分析,使用Rigaku©TTRAXIII X射線粉末繞射儀,以獲得關於材料的結晶與分子排列的資訊。The water content of all polysaccharides was measured using an infrared moisture analyzer MA150, Sartorius. Approximately 0.1 g of each sugar in powder form was heated and analyzed for moisture percentage. TGA was performed using the Setaram Labsys-TGDSC DSC131 instrument to obtain the heating temperature range of the polysaccharide material during casting. Each sugar sample in 0.008g powder form was heated from room temperature to 500°C for TGA analysis at a heating rate of 5°C min-1. Materials in powder form are also used for XRD analysis, using a Rigaku© TTRAXIII X-ray powder diffractometer to obtain information about the crystallization and molecular arrangement of the material.
使用Haake Viscotester 550旋轉黏度計(Thermo Scientific)測量水溶液中多醣的黏度。溶於47.5g溶劑(分別為去離子水、磷酸鹽緩衝液及磷酸鹽緩衝鹽水(PBS))中的2.5g多醣的5%溶液倒入用於測試的黏度計中。The viscosity of the polysaccharide in the aqueous solution was measured using a Haake Viscotester 550 rotary viscometer (Thermo Scientific). A 5% solution of 2.5 g of polysaccharide dissolved in 47.5 g of solvent (deionized water, phosphate buffer, and phosphate buffered saline (PBS), respectively) was poured into the viscometer used for the test.
微針(Microneedles( MNMN )鑄造) Casting
我們在65℃下對MN鑄造使用真空沉積。多醣溶液(5%w / v)係藉由將它們溶解在磷酸鹽緩衝液中並超音震盪60分鐘以完全分散和溶解而製備。為了去除氣泡,將溶液在室溫下以3500rpm離心10分鐘。然後將溶液沿著鋁模具的邊緣緩慢注入。將具有溶液的模具在室溫下保持真空15至20分鐘,以使氣泡向上並重複,直到沒有觀察到可見的氣泡。將模具從室溫加熱至65℃,穩定溫度65℃達24小時。加熱速度設定為1°C min-1。We use vacuum deposition for MN casting at 65°C. The polysaccharide solution (5% w/v) was prepared by dissolving them in phosphate buffer and sonicating for 60 minutes to completely disperse and dissolve. To remove air bubbles, the solution was centrifuged at 3500 rpm for 10 minutes at room temperature. Then the solution was slowly injected along the edge of the aluminum mold. The mold with the solution was kept under vacuum at room temperature for 15 to 20 minutes to make the bubbles upward and repeat until no visible bubbles were observed. The mold was heated from room temperature to 65°C and the stable temperature was 65°C for 24 hours. The heating rate is set to 1°C min-1.
完成的多醣膜或MN產物係藉由手工剝離垂直地從鋁模具中取出。不良成形的產品將不會被完全剝離。選擇標準設定為與模具和無裂紋MN陣列的完全分離。The finished polysaccharide film or MN product is removed vertically from the aluminum mold by manual peeling. Badly formed products will not be completely peeled off. The selection criteria was set to be completely separated from the mold and the crack-free MN array.
製造的made MNMN 的評估evaluation of
在鑄造之後,多醣膜或製造的微針的物理性質係藉由TGA與XRD分析(參見第2.3節)及硬度測試來表徵。After casting, the physical properties of the polysaccharide film or manufactured microneedles were characterized by TGA and XRD analysis (see Section 2.3) and hardness testing.
根據製造商的步驟準則,從模具分離後,使用GARDCO©Wolff-Wilborn硬度鉛筆測試儀(Paul N. Gardner)測試成品的硬度。該刮痕硬度測試確定多醣膜對表面刮傷效應的抵抗性。According to the manufacturer's procedure guidelines, after separation from the mold, use the GARDCO © Wolff-Wilborn hardness pencil tester (Paul N. Gardner) to test the hardness of the finished product. This scratch hardness test determines the resistance of the polysaccharide film to the scratch effect of the surface.
我們在Hitachi©H-7100顯微鏡上使用掃描電子顯微鏡(SEM)和光學顯微鏡(OM)來確定MN的結構。We used Scanning Electron Microscope (SEM) and Optical Microscope (OM) on Hitachi©H-7100 microscope to determine the structure of MN.
細胞毒性Cytotoxicity
使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓溴化物(MTT)測定對正常NIH 3T3(小鼠胚胎細胞)細胞株進行體外細胞毒性試驗。NIH 3T3細胞在含有10%胎牛血清(FBS)的Dulbecco改良伊格爾培養基(DMEM)中生長。對於毒性研究,在加入測試化合物之前,將密度為5,000個細胞/孔的細胞接種在含有10%FBS的DMEM中的24孔盤中,並在37℃,5%的CO2 下培養24小時。將聚合物溶解在PBS中並通過0.22-μM過濾器而過濾。24小時後,加入10μl濃度為0.05%(w / w)的含有測試化合物的培養基,並在37℃,5%的CO2 下培養48小時。實驗重複進行三次,且不含測試化合物的細胞作為對照組。48小時後,將200μl溶於PBS中的MTT溶液(5 mg ml-1)加入到每個孔中,並在37℃培養4小時。除去含有MTT的培養基之後,將形成的甲臢(formazan)結晶溶解於150μL的二甲基亞碸中,並使用微量盤分析儀測量540nm處的吸光度。使用以下公式確定細胞抑制百分比:%細胞抑制= 100-Abs(藥物)/ Abs(對照組)×100,其中Abs是指在540nm處的吸光度。In vitro cytotoxicity of normal NIH 3T3 (mouse embryonic cell) cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay test. NIH 3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). For toxicity studies, before adding test compounds, cells with a density of 5,000 cells/well were seeded in 24-well dishes in DMEM containing 10% FBS and incubated at 37° C., 5% CO 2 for 24 hours. The polymer was dissolved in PBS and filtered through a 0.22-μM filter. After 24 hours, 10 μl of a medium containing the test compound at a concentration of 0.05% (w/w) was added, and cultured at 37°C and 5% CO 2 for 48 hours. The experiment was repeated three times, and cells containing no test compound served as a control group. After 48 hours, 200 μl of MTT solution (5 mg ml-1) dissolved in PBS was added to each well and incubated at 37°C for 4 hours. After removing the MTT-containing medium, the formazan crystals formed were dissolved in 150 μL of dimethyl sulfoxide and the absorbance at 540 nm was measured using a microplate analyzer. Use the following formula to determine the percentage of cell inhibition:% cell inhibition = 100-Abs (drug)/Abs (control group) × 100, where Abs refers to the absorbance at 540 nm.
體內皮膚滲透性測量In vivo skin permeability measurement
本研究中使用的裸鼠,BALB/cAnN.Cg-Foxn1nu/CrlNarl,係得自台灣國家實驗動物中心。使用繃帶束將MN連接到小鼠。治療後,將皮膚以手術剝離,並準備用於組織切片。The nude mice used in this study, BALB/cAnN.Cg-Foxn1nu/CrlNarl, were obtained from the National Laboratory Animal Center of Taiwan. Bandage bundles were used to connect the MN to the mice. After treatment, the skin is surgically peeled off and prepared for tissue sectioning.
為了追蹤在MN皮膚穿透後蛋白質的釋放,將1%的BSA(牛血清白蛋白,Sigma-Aldrich)粉末混合到多醣(50%CMC + 50%DEX)中用於澆鑄。將皮膚組織片固定在4%多聚甲醛中並包埋在石蠟中。切片用蘇木精與曙紅(H&E)染色。使用針對BSA的抗體(MilliporeTM 07248)進行免疫組織化學(IHC)。To track the release of protein after MN skin penetration, 1% BSA (bovine serum albumin, Sigma-Aldrich) powder was mixed into polysaccharide (50% CMC + 50% DEX) for casting. The skin tissue pieces were fixed in 4% paraformaldehyde and embedded in paraffin. The sections were stained with hematoxylin and eosin (H&E). Immunohistochemistry (IHC) was performed using antibodies against BSA (Millipore TM 07248).
為了追蹤從MN釋放染料及擴散到皮膚中的時間,將0.25%亞甲基藍(MB)粉末與多醣(單獨的CMC,或50%CMC + 50%DEX)混合用於鑄造。將皮膚組織片冷凍,並製備成冷凍切片。In order to track the time of dye release from MN and diffusion into the skin, 0.25% methylene blue (MB) powder was mixed with polysaccharides (CMC alone, or 50% CMC + 50% DEX) for casting. The skin tissue pieces were frozen and prepared into frozen sections.
本研究中動物的使用係經台北醫學大學IACUC所批准。The use of animals in this study was approved by IACUC of Taipei Medical University.
實施例Examples
實施例Examples 11 用於For MNMN 製造的材料調查及選擇Manufacturing materials investigation and selection
在這項研究中,我們提出一種生產用於經皮遞輸藥物的可生物降解的MN之方法。為了選擇合適的材料,分析其粉末形式的多醣CMC、DEX、TRE及AMP,以檢查它們的含水量、結晶度及熱穩定性。將多醣以不同的比例混合,以測試它們在含水緩衝液中的溶解度。隨後,單獨或組合的可溶性多醣被傾倒到模具表面上,以檢查它們的成膜。還測試了單個多醣的細胞毒性。In this study, we propose a method to produce biodegradable MN for transdermal drug delivery. In order to select suitable materials, the polysaccharides CMC, DEX, TRE and AMP in powder form were analyzed to check their water content, crystallinity and thermal stability. The polysaccharides were mixed in different ratios to test their solubility in aqueous buffer. Subsequently, soluble polysaccharides, alone or in combination, were poured onto the mold surface to check their film formation. The cytotoxicity of individual polysaccharides was also tested.
含水量測試的結果顯示在表1中。即使當材料已在55℃下乾燥24小時,水分子存在於所有測試的多醣中。材料的水分含量及其與水分子的親和力避免了在澆鑄過程中材料完全變乾或失去水分,這確保了材料與併入的藥物或蛋白質相容。 表1 各種多醣的含水量
藉由XRD和TGA分析粉末形式的各個醣,以檢查它們的結晶度和熱穩定性。結晶特徵提供關於材料的分子排列及它們的保水能力的資訊。當結合不同的多醣時,這些資訊可能有幫助。使用TGA分析熱性能,確保在澆鑄過程中所使用的乾燥溫度下,材料會很穩定。The individual sugars in powder form were analyzed by XRD and TGA to check their crystallinity and thermal stability. Crystallization features provide information about the molecular arrangement of materials and their ability to retain water. This information may be helpful when combining different polysaccharides. Use TGA to analyze the thermal properties to ensure that the material will be stable at the drying temperature used during the casting process.
所有測試材料顯示顯著的結晶訊號峰(圖3A)。TRE產生最明顯的訊號。DEX是由澱粉水解所產生的一組碳水化合物,因此顯示出與AMP相似的訊號(圖3A)。CMC的較低訊號及較平滑的曲線表明CMC中存在更多的非晶區。All test materials showed significant crystalline signal peaks (Figure 3A). TRE produces the most obvious signal. DEX is a group of carbohydrates produced by the hydrolysis of starch and therefore shows a signal similar to AMP (Figure 3A). The lower signal and smoother curve of CMC indicate that there are more amorphous regions in CMC.
由溫度引起的質量損失情形顯示在圖3B及表2中。第一次質量損失(90-130℃)描繪了水分損失的臨界溫度,而第二次損失(超過240℃)反映了分子分解。CMC和TRE顯示出比AMP和DEX的水分損失更顯著下降的曲線(圖3B)。這結果表明,AMP和DEX在其粉末形式中具有更佳的水分含量。這由含水量測試證實(表1)。TGA資料顯示,在溫度為55與75℃之間的用於鑄造的普通低溫MN製備程序中,多醣具有熱穩定性。降低鑄造溫度增加乾燥時間如下:75℃15小時、65℃24小時、及55℃28小時。因為多醣基質和BSA是穩定的,後續MN澆鑄的溫度設定在65℃。 表2 顯示其熱解溫度的各種多醣的熱重分析(TGA)
糖的溶解度和黏度將直接影響鑄造產品的品質。將多醣以不同的比例混合,以測試它們在含水緩衝液中的溶解度。在所嘗試的各種多醣組合中,支鏈澱粉(AMP)不適合用於製造MN,因為它在含水緩衝液中的溶解度低,且需要連續加熱以溶解它。 糖溶液的黏度在高濃度下增加。在將糖溶液倒入模具之後,材料的黏度影響填充效果,並影響成品。高度黏稠溶液無法填充模具並產生氣泡。具有低黏度的溶液可能無法提供足夠的厚度給成品,導致物理性能降低。因此,找到合適的黏度對於填充模具表面來說是必需的。為了確定用於傾倒的合適黏度,在試管中使用不同濃度的CMC(5-50%w / v),並在試管轉動時目視觀察液體的平穩流動(表3)。只有5%的溶液能完全覆蓋試管,而10%及20%的溶液顯示部分覆蓋。30%及50%的溶液由於其高黏度而緩慢移動,且不能完全覆蓋試管。我們選擇5%的溶液作為進一步研究的基準。 表3 藉由採不同濃度(5-50%)的羧甲基纖維素(CMC)覆蓋試管的流量覆蓋測試綜覽
隨後,我們使用鋁模具背面上的空白表面,以分別使用5%的TRE、DEX或CMC溶液檢查鑄膜(在65℃下乾燥24小時)。TRE產生薄而不規則的半透明膜,其無法固定成片狀結構。DEX產生了一塊相對較厚的塊,其碎成片。因此,TRE及DEX本身不適合用於鑄造成MN陣列。他們被認為使用作為與CMC的混合物。Subsequently, we used the blank surface on the back of the aluminum mold to inspect the cast film using 5% TRE, DEX or CMC solutions (dried at 65°C for 24 hours). TRE produces thin and irregular translucent films that cannot be fixed into a sheet structure. DEX produced a relatively thick block that shattered into pieces. Therefore, TRE and DEX are not suitable for casting into MN array. They are considered to be used as a mixture with CMC.
與CMC結合的TRE難以與模具表面分離(資料未顯示),導致表面疤痕與重複的洗滌環。與CMC結合的TRE的濃度降低不能產生足夠厚的聚合物膜,黏附在模具上並難以分離,且產生一些碎片(資料未顯示)。XRD分析(圖3A)顯示兩種多醣之間結晶程度的巨大差異。TRE顯示更多的結晶與更少的非晶區域,而CMC顯示更少的結晶與更多的非晶區域(圖3A)。TRE在溶液中作為小分子量分子存在,並在乾燥過程中它顯示出晶體中的大分子裂縫,因此破壞了整個結構。因此,TRE不適合用於與CMC結合來製造MN。TRE combined with CMC is difficult to separate from the mold surface (data not shown), resulting in surface scars and repeated washing rings. The decrease in the concentration of TRE combined with CMC does not produce a sufficiently thick polymer film, adheres to the mold and is difficult to separate, and generates some debris (data not shown). XRD analysis (Figure 3A) shows a huge difference in the degree of crystallinity between the two polysaccharides. TRE shows more crystalline and less amorphous regions, while CMC shows less crystalline and more amorphous regions (Figure 3A). TRE exists as small molecular weight molecules in the solution, and it shows cracks of large molecules in the crystal during the drying process, thus destroying the entire structure. Therefore, TRE is not suitable for manufacturing MN in combination with CMC.
就其結晶學訊號(圖4A)和分子大小而言,DEX相似於CMC;因此,它在鑄造過程中可能與CMC更加相容。CMC單獨或與DEX結合被考慮用於進一步研究。In terms of its crystallographic signal (Figure 4A) and molecular size, DEX is similar to CMC; therefore, it may be more compatible with CMC during the casting process. CMC alone or in combination with DEX is considered for further research.
實施例Examples 22 預澆鑄評估(黏度測試)Pre-cast evaluation (viscosity test)
進行CMC與DEX結合的黏度測試(圖6)。使用三種不同的水性緩衝液(去離子水、磷酸鹽緩衝液,pH7.4和PBS,pH7.4),以不同比率溶解CMC與DEX(100%的CMC、50%的CMC + 50%的DEX、34%的CMC + 66%的DEX、66%的CMC + 34%的DEX、及60%的CMC + 40%的DEX)。混合該材料顯著改變了流體特性。當CMC比率較高時,黏度增加(圖5)。由於緩衝液中存在鹽,溶劑會稍微增加黏度。更換溶劑不能降低黏度。然而,溶劑PBS顯著增加純CMC的黏度。最後,我們選擇PB用於MN鑄造。Conduct a viscosity test for the combination of CMC and DEX (Figure 6). Use three different aqueous buffers (deionized water, phosphate buffer, pH7.4 and PBS, pH7.4) to dissolve CMC and DEX at different ratios (100% CMC, 50% CMC + 50% DEX , 34% CMC + 66% DEX, 66% CMC + 34% DEX, and 60% CMC + 40% DEX). Mixing this material significantly changes the fluid properties. When the CMC ratio is higher, the viscosity increases (Figure 5). Due to the presence of salt in the buffer, the solvent will slightly increase the viscosity. Changing the solvent cannot reduce the viscosity. However, the solvent PBS significantly increased the viscosity of pure CMC. Finally, we chose PB for MN casting.
實施例Examples 33 多醣系Polysaccharides MNMN 陣列的製造Array manufacturing
鋁模具為模型鑄造提供一些優點。它與傳統的MN鑄造方法相容(Lee等人,2008;Migalska等人,2011;Park等人,2016)。Al ASTM 6061(FDA允許用作食品容器)是用於重型結構的高耐腐蝕材料,且在本研究中用於製備可重複使用的模具。為了製造MN,將多醣混合物倒在Al模具上,並在65℃乾燥24小時。我們使用低溫進行鑄造,因為最終產品將與生物分子和藥物一起與多醣生產。在乾燥過程中,考慮了目標分子的熱穩定性(圖3B及表2)。圖5顯示多醣系MN的SEM和OM觀察,其長度為約480μm,基部寬度為370μm,且針中心間距為600μm。Aluminum molds provide some advantages for model casting. It is compatible with traditional MN casting methods (Lee et al., 2008; Migalska et al., 2011; Park et al., 2016). Al ASTM 6061 (FDA allows it to be used as a food container) is a highly corrosion-resistant material for heavy-duty structures, and was used in this study to prepare reusable molds. To make MN, the polysaccharide mixture was poured on an Al mold and dried at 65°C for 24 hours. We use low temperature for casting because the final product will be produced with polysaccharides along with biomolecules and drugs. During the drying process, the thermal stability of the target molecule was considered (Figure 3B and Table 2). Fig. 5 shows SEM and OM observations of the polysaccharide MN, which has a length of about 480 μm, a base width of 370 μm, and a needle-center distance of 600 μm.
在本研究中,我們單獨使用CMC以及CMC/DEX混合物以製造MN。CMC是一種天然可溶解的聚合物,其與生物活性化合物相容,在低溫下處理相對容易,且不昂貴。它已被廣泛使用於醫療應用(Agarwal等人,2015;Chen等人,2015;Pasqui等人,2014),包括MN製造(Kommareddy等人,2012;Lee等人,2017;Lee等人,2008;Park等人, 2016)。In this study, we used CMC and CMC/DEX mixtures alone to make MN. CMC is a naturally soluble polymer that is compatible with biologically active compounds, is relatively easy to handle at low temperatures, and is not expensive. It has been widely used in medical applications (Agarwal et al., 2015; Chen et al., 2015; Pasqui et al., 2014), including MN manufacturing (Kommareddy et al., 2012; Lee et al., 2017; Lee et al., 2008; Park et al., 2016).
實施例Examples 44 製造的made MNMN 之特徵Features
進行鉛筆硬度測試,以確保在與模具分離之後成品的硬度。表4列出了鉛筆硬度測試的細節。CMC與DEX結合顯示出比CMC單獨更佳的硬度。多醣組合在磷酸鹽緩衝液中比在去離子水中顯示出增加的硬度。Carry out a pencil hardness test to ensure the hardness of the finished product after separation from the mold. Table 4 lists the details of the pencil hardness test. The combination of CMC and DEX shows better hardness than CMC alone. The polysaccharide combination showed increased hardness in phosphate buffer than in deionized water.
TGA結果顯示,在水性緩衝液中CMC和DEX的不同組合的熱性質在高達200℃時是穩定的(圖5A至5C,表5)。鑄造過程之後的第一質量損失的曲線顯示更溫和及連續的CMC與DEX曲線(對於膜,參見圖7A,與粉末形式比較見圖3B)。然而,當使用PB或PBS作為溶劑時,鑄膜的TGA曲線沒有顯示出顯著差異(圖7A至7C中的曲線CMC、1C1D、1C2D、2C1D),並表明材料與磷酸鹽緩衝液之間沒有形成直接化學反應。鑄膜的質量損失是由於失去水分造成。 表4 各種組合的多醣在水性緩衝液中的鉛筆硬度測試
多醣膜的XRD結果在鑄造程序之前(圖3A)和之後(圖7D)顯示出急遽的差異。結晶訊號出現在18℃至21℃、22℃至24℃、及40℃至42℃(圖7D)。比較訊號面積及間隔顯示,訊號主要由CMC而不是DEX或溶劑所造成。XRD沒有顯示溶劑產物的額外結晶訊號(比較3C2D與PBS 3C2D的曲線,以及圖7D中1C2D與PBS 1C2D的曲線)。與DEX混合只影響訊號峰值的高度。結果證實了鑄造步驟導致CMC緩慢再結晶的假設。再結晶可能起源於加熱過程中逐漸失去水分,導致分子排列整齊有序(Jeong 等人,2016)。結晶是保持水分的材料的重要指標。整齊排列的分子陣列可有效抵抗外部水分侵入晶體結構,並阻止從內部蒸散到外部。The XRD results of the polysaccharide film showed a sharp difference before and after the casting procedure (Figure 3A) and after (Figure 7D). Crystallization signals appear at 18°C to 21°C, 22°C to 24°C, and 40°C to 42°C (Figure 7D). Comparing the signal area and interval shows that the signal is mainly caused by CMC rather than DEX or solvent. XRD showed no additional crystallization signal of the solvent product (compare the curves of 3C2D and PBS 3C2D, and the curves of 1C2D and PBS 1C2D in Figure 7D). Mixing with DEX only affects the height of the signal peak. The results confirmed the hypothesis that the casting step caused the CMC to recrystallize slowly. The recrystallization may originate from the gradual loss of water during heating, which leads to the orderly arrangement of molecules (Jeong et al., 2016). Crystallization is an important indicator of materials that retain moisture. The neatly arranged molecular array can effectively resist the intrusion of external moisture into the crystal structure and prevent evapotranspiration from the inside to the outside.
進行細胞毒性測試以檢查製造的MN的生物相容性(圖8)。在NIH-3T3細胞株上的MTT測定證實MN在製造過程中顯示沒有毒性,並且在MN中沒有積累毒性物質。與對照組相比,單獨的CMC和CMC/DEX混合物的細胞存活力均超過95%。我們使用單獨的CMC和CMC/DEX混合物(50%CMC + 50%DEX)來製備MN用於隨後的體內研究。A cytotoxicity test was conducted to check the biocompatibility of the manufactured MN (Figure 8). MTT assay on NIH-3T3 cell line confirmed that MN showed no toxicity during the manufacturing process, and no toxic substances accumulated in MN. Compared with the control group, the cell viability of CMC alone and CMC/DEX mixture exceeded 95%. We used a separate CMC and CMC/DEX mixture (50% CMC + 50% DEX) to prepare MN for subsequent in vivo studies.
實施例Examples 55 體內皮膚穿透測試及藥物釋放時間測試In vivo skin penetration test and drug release time test
對於體內皮膚穿透測試,與BSA結合的CMC/DEX混合物用於MN製造。MN附著於小鼠24小時。用H&E染色的小鼠皮膚切片的光學顯微鏡觀察清楚地顯示微針在其表面的穿透(圖9A,9B)。IHC + H染色顯示在穿透皮膚後(圖9C,9D)追蹤BSA的釋放(以深褐色來突顯)。結合H&E和IHC + H染色進一步證實並評估了藥物遞輸方法的完整性。製成的MN陣列可使用於裸鼠皮膚,結果顯示BSA成功穿透及釋放。For in vivo skin penetration testing, the CMC/DEX mixture combined with BSA is used for MN manufacturing. MN was attached to the mice for 24 hours. Light microscopic observation of mouse skin sections stained with H&E clearly showed the penetration of microneedles on its surface (Figures 9A, 9B). IHC + H staining showed that after penetrating the skin (Figure 9C, 9D) the release of BSA was followed (highlighted in dark brown). Combining H&E and IHC + H staining further confirmed and evaluated the integrity of the drug delivery method. The fabricated MN array can be applied to the skin of nude mice, and the results show that BSA successfully penetrated and released.
對於藥物溶解和擴散的時間追蹤,將單獨的CMC或與亞甲基藍結合的CMC/DEX混合物使用於MN製造。在通過一系列檢查點將MN插入裸鼠後,代表藥物的亞甲基藍被追蹤到釋放在表面上,並擴散到皮膚中。在處理後的前2小時,小鼠皮膚的冷凍切片顯示沒有顯著的染料訊號(圖10A)。在MN處理4小時後,注意到亞甲基藍的擴散和穿入皮膚(圖10B、10C)。與純CMC相比(圖10D),CMC/DEX混合物顯示更佳的亞甲基藍釋放(圖10C)。For time tracking of drug dissolution and diffusion, CMC alone or a CMC/DEX mixture combined with methylene blue was used for MN manufacturing. After inserting MN into nude mice through a series of checkpoints, the methylene blue representing the drug was tracked to be released on the surface and spread into the skin. In the first 2 hours after treatment, frozen sections of mouse skin showed no significant dye signal (Figure 10A). After 4 hours of MN treatment, the diffusion of methylene blue and penetration into the skin were noticed (Fig. 10B, 10C). Compared to pure CMC (Figure 10D), the CMC/DEX mixture showed better methylene blue release (Figure 10C).
實施例Examples 66 評估Assessment MNMN 溶解Dissolve
為了評估MN的溶解度,我們在插入裸鼠皮膚之前及之後檢查了CMC針的外觀(圖11)。在處理4小時(圖11A)和8小時(圖11B)之後,在針外觀上沒有觀察到顯著的視覺變化。在MN處理24小時之後,針的外觀明顯地變化(圖11C)。由於體液的溶解和吸收,針變得更鬆軟。結果還表明,在處理24小時之後,MN部分溶解。因此,MN可能適合長期釋放在藥物遞輸中。References:
[1] T. Agarwal, T., Narayana, S.N., Pal, K., Pramanik, K., Giri, S., Banerjee, I., 2015. Calcium alginate-carboxymethyl cellulose beads for colon-targeted drug delivery. Int J Biol Macromol 75, 409-417. [2] Asbill, C.S., Michniak, B.B., 2000. Percutaneous penetration enhancers: local versus transdermal activity. Pharm Sci Technolo Today 3, 36-41. [3] Badran, M.M., Kuntsche, J., Fahr, A., 2009. Skin penetration enhancement by a microneedle device (Dermaroller) in vitro: dependency on needle size and applied formulation. Eur J Pharm Sci 36, 511-523. [4] Bronaugh, R.L., Maibach, H.I., 2005. Percutaneous absorption : drugs, cosmetics, mechanisms, methodology, 4th ed. Taylor & Francis, Boca Raton. [5] Chen, Y.C., Ho, H.O., Liu, D.Z., Siow, W.S., Sheu, M.T., 2015. Swelling/floating capability and drug release characterizations of gastroretentive drug delivery system based on a combination of hydroxyethyl cellulose and sodium carboxymethyl cellulose. PLoS One 10, e0116914. [6] Cheung, K., Han, T., Das, D.B., 2014. Effect of force of microneedle insertion on the permeability of insulin in skin. J Diabetes Sci Technol 8, 444-452. [7] Chillo, S., Laverse, J., Falcone, P.M., Del Nobile, M.A., 2007. Effect of carboxymethylcellulose and pregelatinized corn starch on the quality of amaranthus spaghetti. J Food Eng 83, 492-500. [8] Chu, L.Y., Prausnitz, M.R., 2011. Separable arrowhead microneedles. J Control Release 149, 242-249. [9] Ding, Z., Verbaan, F.J., Bivas-Benita, M., Bungener, L., Huckriede, A., van den Berg, D.J., Kersten, G., Bouwstra, J.A., 2009. Microneedle arrays for the transcutaneous immunization of diphtheria and influenza in BALB/c mice. J Control Release 136, 71-78. [10] Hadgraft, J., Peck, J., Williams, D.G., Pugh, W.J., Allan, G., 1996. Mechanisms of action of skin penetration enhancers/retarders: Azone and analogues. International Journal of Pharmaceutics 141, 17-25. [11] Hafeli, U.O., Mokhtari, A., Liepmann, D., Stoeber, B., 2009. In vivo evaluation of a microneedle-based miniature syringe for intradermal drug delivery. Biomed Microdevices 11, 943-950. [12] Higashiyama, T., 2002. Novel functions and applications of trehalose. Pure Appl Chem 74, 1263-1269. [13] Hirobe, S., Azukizawa, H., Hanafusa, T., Matsuo, K., Quan, Y.S., Kamiyama, F., Katayama, I., Okada, N., Nakagawa, S., 2015. Clinical study and stability assessment of a novel transcutaneous influenza vaccination using a dissolving microneedle patch. Biomaterials 57, 50-58. [14] Jeong, H., Shepard, K.B., Purdum, G.E., Guo, Y.L., Loo, Y.L., Arnold, C.B., Priestley, R.D., 2016. Additive Growth and Crystallization of Polymer Films. Macromolecules 49, 2860-2867. [15] Kaushik, S., Hord, A.H., Denson, D.D., McAllister, D.V., Smitra, S., Allen, M.G., Prausnitz, M.R., 2001. Lack of pain associated with microfabricated microneedles. Anesth Analg 92, 502-504. [16] Ke, C.J., Lin, Y.J., Hu, Y.C., Chiang, W.L., Chen, K.J., Yang, W.C., Liu, H.L., Fu, C.C., Sung, H.W., 2012. Multidrug release based on microneedle arrays filled with pH-responsive PLGA hollow microspheres. Biomaterials 33, 5156-5165. [17] Kim, Y.C., Park, J.H., Prausnitz, M.R., 2012. Microneedles for drug and vaccine delivery. Adv Drug Deliv Rev 64, 1547-1568. [18] Kommareddy, S., Baudner, B.C., Oh, S., Kwon, S.Y., Singh, M., O'Hagan, D.T., 2012. Dissolvable microneedle patches for the delivery of cell-culture-derived influenza vaccine antigens. J Pharm Sci 101, 1021-1027. [19] Langer, R., 2001. Drug delivery. Drugs on target. Science 293, 58-59. [20] Lee, I.C., Lin, W.M., Shu, J.C., Tsai, S.W., Chen, C.H., Tsai, M.T., 2017. Formulation of two-layer dissolving polymeric microneedle patches for insulin transdermal delivery in diabetic mice. Journal of Biomedical Materials Research Part A 105, 84-93. [21] Lee, J.W., Park, J.H., Prausnitz, M.R., 2008. Dissolving microneedles for transdermal drug delivery. Biomaterials 29, 2113-2124. [22] Lee, K., Lee, C.Y., Jung, H., 2011. Dissolving microneedles for transdermal drug administration prepared by stepwise controlled drawing of maltose. Biomaterials 32, 3134-3140. [23] Li, G., Badkar, A., Nema, S., Kolli, C.S., Banga, A.K., 2009. In vitro transdermal delivery of therapeutic antibodies using maltose microneedles. Int J Pharm 368, 109-115. [24] Lin, W., Cormier, M., Samiee, A., Griffin, A., Johnson, B., Teng, C.L., Hardee, G.E., Daddona, P.E., 2001. Transdermal delivery of antisense oligonucleotides with microprojection patch (Macroflux) technology. Pharm Res 18, 1789-1793. [25] Liu, S., Jin, M.N., Quan, Y.S., Kamiyama, F., Katsumi, H., Sakane, T., Yamamoto, A., 2012. The development and characteristics of novel microneedle arrays fabricated from hyaluronic acid, and their application in the transdermal delivery of insulin. J Control Release 161, 933-941. [26] Martanto, W., Davis, S.P., Holiday, N.R., Wang, J., Gill, H.S., Prausnitz, M.R., 2004. Transdermal delivery of insulin using microneedles in vivo. Pharm Res 21, 947-952. [27] Martin, C.J., Allender, C.J., Brain, K.R., Morrissey, A., Birchall, J.C., 2012. Low temperature fabrication of biodegradable sugar glass microneedles for transdermal drug delivery applications. J Control Release 158, 93-101. [28] McAllister, D.V., Wang, P.M., Davis, S.P., Park, J.H., Canatella, P.J., Allen, M.G., Prausnitz, M.R., 2003. Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: fabrication methods and transport studies. Proc Natl Acad Sci U S A 100, 13755-13760. [29] Migalska, K., Morrow, D.I., Garland, M.J., Thakur, R., Woolfson, A.D., Donnelly, R.F., 2011. Laser-engineered dissolving microneedle arrays for transdermal macromolecular drug delivery. Pharm Res 28, 1919-1930. [30] Mikszta, J.A., Alarcon, J.B., Brittingham, J.M., Sutter, D.E., Pettis, R.J., Harvey, N.G., 2002. Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery. Nat Med 8, 415-419. [31] Park, Y.H., Ha, S.K., Choi, I., Kim, K.S., Park, J., Choi, N., Kim, B., Sung, J.H., 2016. Fabrication of degradable carboxymethyl cellulose (CMC) microneedle with laser writing and replica molding process for enhancement of transdermal drug delivery. Biotechnology and Bioprocess Engineering 21, 110-118. [32] Pasqui, D., Torricelli, P., De Cagna, M., Fini, M., Barbucci, R., 2014. Carboxymethyl cellulose-hydroxyapatite hybrid hydrogel as a composite material for bone tissue engineering applications. J Biomed Mater Res A 102, 1568-1579. [33] Pettis, R.J., Harvey, A.J., 2012. Microneedle delivery: clinical studies and emerging medical applications. Ther Deliv 3, 357-371. [34] Prausnitz, M.R., Langer, R., 2008. Transdermal drug delivery. Nat Biotechnol 26, 1261-1268. [35] Raphael, A.P., Prow, T.W., Crichton, M.L., Chen, X., Fernando, G.J., Kendall, M.A., 2010. Targeted, needle-free vaccinations in skin using multilayered, densely-packed dissolving microprojection arrays. Small 6, 1785-1793. [36] Sullivan, S.P., Koutsonanos, D.G., Del Pilar Martin, M., Lee, J.W., Zarnitsyn, V., Choi, S.O., Murthy, N., Compans, R.W., Skountzou, I., Prausnitz, M.R., 2010. Dissolving polymer microneedle patches for influenza vaccination. Nat Med 16, 915-920. [37] Sullivan, S.P., Murthy, N., Prausnitz, M.R., 2008. Minimally invasive protein delivery with rapidly dissolving polymer microneedles. Adv Mater 20, 933-938. [38] Tuan-Mahmood, T.M., McCrudden, M.T., Torrisi, B.M., McAlister, E., Garland, M.J., Singh, T.R., Donnelly, R.F., 2013. Microneedles for intradermal and transdermal drug delivery. Eur J Pharm Sci 50, 623-637. [39] Wokovich, A.M., Prodduturi, S., Doub, W.H., Hussain, A.S., Buhse, L.F., 2006. Transdermal drug delivery system (TDDS) adhesion as a critical safety, efficacy and quality attribute. Eur J Pharm Biopharm 64, 1-8.To assess the solubility of MN, we examined the appearance of the CMC needle before and after insertion into the skin of nude mice (Figure 11). After 4 hours of treatment (Figure 11A) and 8 hours of treatment (Figure 11B), no significant visual changes were observed in the appearance of the needle. After 24 hours of MN treatment, the appearance of the needle changed significantly (Figure 11C). Due to the dissolution and absorption of body fluids, the needle becomes softer. The results also showed that after 24 hours of treatment, the MN was partially dissolved. Therefore, MN may be suitable for long-term release in drug delivery. References: [1] T. Agarwal, T., Narayana, SN, Pal, K., Pramanik, K., Giri, S., Banerjee, I., 2015. Calcium alginate-carboxymethyl cellulose beads for colon-targeted drug delivery . Int
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當結合附圖閱讀時,將更佳地理解前面的概述以及以下對本發明的詳細描述。The foregoing overview and the following detailed description of the invention will be better understood when reading in conjunction with the drawings.
在附圖中:In the drawings:
圖1提供顯示提供微針陣列的本發明具體實施例的圖像,包含多醣系微針陣列,其藉由計算機輔助設計(CAD)系統集成了高速切割機而製備,其在該模式中間的鑄造區域;其中NA表示針區域,BA表示空白區域,且RA表示脊區域;而CT表示連接溝槽。FIG. 1 provides an image showing a specific embodiment of the present invention that provides a microneedle array, including a polysaccharide-based microneedle array, which is prepared by a computer-aided design (CAD) system integrated with a high-speed cutting machine, which is cast in the middle of the mode Area; where NA represents the needle area, BA represents the blank area, and RA represents the ridge area; and CT represents the connection groove.
圖2提供根據本發明的用於製造微針陣列的方案。Figure 2 provides a solution for manufacturing a microneedle array according to the present invention.
圖3提供不同多醣分析的圖式,分別包括(A)熱重分析(TGA)的X射線繞射(XRD);及(B)以原始粉末形式的分析。Figure 3 provides diagrams for the analysis of different polysaccharides, including (A) thermogravimetric analysis (TGA) X-ray diffraction (XRD); and (B) analysis in raw powder form.
圖4提供顯示個別的多醣:羧甲基纖維素(CMC)和糊精(DEX)、海藻糖與對照組的細胞毒性測試結果的圖式;其中將NIH 3T3細胞使用於3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓溴化物(MTT)測定,以測試多醣的毒性。Figure 4 provides a graph showing the cytotoxicity test results of individual polysaccharides: carboxymethyl cellulose (CMC) and dextrin (DEX), trehalose, and the control group; NIH 3T3 cells were used in 3-(4,5 -Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was tested to test the toxicity of polysaccharides.
圖5提供顯示在(A)去離子水,(B)磷酸鹽緩衝液,pH 7.4及(C)磷酸鹽緩衝鹽水,pH 7.4中,羧甲基纖維素(CMC)與糊精(DEX)的黏度測量的圖式;其中C表示包含100%的CMC(C)的混合物,1C1D表示包含50%的CMC及50%的DEX的混合物,1C2D表示包含34%的CMC及66%的DEX的混合物,2C1D表示包含66%的CMC及34%的DEX的混合物,且3C2D表示包含60%的CMC及40%的DEX的混合物。Figure 5 provides the carboxymethyl cellulose (CMC) and dextrin (DEX) in (A) deionized water, (B) phosphate buffer, pH 7.4 and (C) phosphate buffered saline, pH 7.4. Viscosity measurement diagram; where C represents a mixture containing 100% CMC (C), 1C1D represents a mixture containing 50% CMC and 50% DEX, 1C2D represents a mixture containing 34% CMC and 66% DEX, 2C1D represents a mixture containing 66% CMC and 34% DEX, and 3C2D represents a mixture containing 60% CMC and 40% DEX.
圖6提供根據本發明的微針陣列的圖像,包括(A)掃描電子顯微鏡(SEM)圖像及(B)光學顯微鏡(OM)觀察。6 provides images of the microneedle array according to the present invention, including (A) scanning electron microscope (SEM) images and (B) optical microscope (OM) observations.
圖7提供顯示以下結果的圖式:提供(A)去離子水作為溶劑;(B)磷酸鹽緩衝液,pH7.4,作為溶劑;(C)磷酸鹽緩衝鹽水,pH 7.4,作為溶劑的熱重分析(TGA)及X射線繞射(XRD);且(D)係由羧甲基纖維素(CMC)與糊精(DEX)所組成的製造的多醣微針之分析,使用去離子水或磷酸鹽緩衝鹽水(PBS)作為溶劑;C表示包含100%的CMC(C)的混合物,1C1D表示包含50%的CMC及50%的DEX的混合物,1C2D表示包含34%的CMC及66%的DEX的混合物,2C1D表示包含66%的CMC及34%的DEX的混合物,且3C2D表示包含60%的CMC及40%的DEX的混合物。Figure 7 provides a graph showing the following results: (A) deionized water as a solvent; (B) phosphate buffer, pH 7.4, as a solvent; (C) phosphate buffered saline, pH 7.4, as a solvent heat Re-analysis (TGA) and X-ray diffraction (XRD); and (D) is an analysis of polysaccharide microneedles made of carboxymethyl cellulose (CMC) and dextrin (DEX), using deionized water or Phosphate buffered saline (PBS) as a solvent; C means a mixture containing 100% CMC (C), 1C1D means a mixture containing 50% CMC and 50% DEX, 1C2D means 34% CMC and 66% DEX 2C1D represents a mixture containing 66% CMC and 34% DEX, and 3C2D represents a mixture containing 60% CMC and 40% DEX.
圖8顯示根據本發明的微針陣列的細胞毒性測試;其中NIH3T3細胞進行3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑鎓溴化物(MTT)測定,以測試多醣的毒性:羧甲基纖維素(CMC);CMC與糊精的混合物(CMC +糊精),及對照組。FIG. 8 shows the cytotoxicity test of the microneedle array according to the present invention; wherein NIH3T3 cells were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT ) Determination to test the toxicity of polysaccharides: carboxymethyl cellulose (CMC); a mixture of CMC and dextrin (CMC + dextrin), and a control group.
圖9顯示在光學顯微鏡下用於體內皮膚穿透測試的組織切片的觀察;其中裸鼠係以根據本發明的微針陣列處理,其中加入牛血清白蛋白(BSA)作為測試藥物:(A)及(B)提供蘇木精與曙紅(hematoxylin and eosin,H&E)染色的兩個代表性圖像,其中箭頭指示微針的穿透;(C)在穿透皮膚後,免疫組織化學與H&E(IHC + H)染色追蹤BSA的釋放後,提供小鼠組織切片的代表性圖像;(D)提供顯示BSA釋放的IHC + H染色組織的特寫視圖(對於(A)及(C),比例尺為50μm,且對(B)及(D)為25μm)。Fig. 9 shows the observation of tissue sections for in vivo skin penetration test under an optical microscope; where nude mice are treated with a microneedle array according to the present invention, and bovine serum albumin (BSA) is added as a test drug: (A) And (B) provide two representative images of hematoxylin and eosin (H&E) staining, where the arrows indicate the penetration of microneedles; (C) after penetrating the skin, immunohistochemistry and H&E (IHC + H) provides a representative image of the mouse tissue section after staining to track the release of BSA; (D) provides a close-up view of the IHC + H stained tissue showing BSA release (for (A) and (C), scale bar Is 50 μm, and 25 μm for (B) and (D)).
圖10顯示從多醣微針釋放並擴散到皮膚中的亞甲基藍(MB)染料的時間追蹤的代表性冷凍切片。小鼠係以含有MB的微針處理(A)2小時,(B)4小時及(C)與(D)8小時。Figure 10 shows a representative frozen section of a time-tracked release of methylene blue (MB) dye released from polysaccharide microneedles and diffused into the skin. The mice were treated with microneedles containing MB for (A) for 2 hours, (B) for 4 hours, and (C) and (D) for 8 hours.
圖11顯示在附著至小鼠之前(上圖)與之後(A)4小時,(B)8小時及(C)24小時(下圖)的微針陣列的代表性圖像。Figure 11 shows representative images of microneedle arrays before (above) and after (A) 4 hours, (B) 8 hours, and (C) 24 hours (lower image) before and after attachment to mice.
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