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Fabrication and testing analysis of tapered silicon microneedles for drug delivery applications
Microelectronic Engineering 111 (2013) 33–38
Contents lists available at SciVerse ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
Fabrication and testing analysis of tapered silicon microneedles for drug delivery applications Xiao-Xiao Yan, Jing-Quan Liu ⇑, Shui-Dong Jiang, Bin Yang, Chun-Sheng Yang National Key Laboratory of Science and Technology on Nano/Micro-fabrication, Institute of Micro and Nano Science and Technology, Shanghai Jiao Tong University, Shanghai 200240, China
a r t i c l e
i n f o
Article history: Received 15 January 2013 Received in revised form 3 April 2013 Accepted 27 April 2013 Available online 7 May 2013 Keywords: Drug delivery Microneedles Dicing saw Chemical etching
a b s t r a c t Drug delivery plays an important role in the treatment of disease, and the equipment used for drug delivery has a great impact on the delivery efficiency of drugs. Recently developed for drug delivery, the new microneedle apparatus has the potential to improve this efficiency. In this study, an incorporated microfabrication process is proposed for the formation of tapered silicon microneedles. First, a dicing saw with one beveled blade and one right blade is used to directly cut silicon wafers into the basic shape of tapered microneedles. Next, a chemical etching process containing a dynamic step and static step is deployed to refine the basic shape and form sharp and tapered microneedles 400 lm in length and 600 lm in width between the tips of two microneedles. A variety of microneedle array patches are fabricated to investigate their ability to pierce artificial skin with forces ranging from 0.1 N to 10 N. The percutaneous experimental results show that the permeation rate of a 7 7 microneedle array patch through a treated rat skin is 36 times higher than that for intact skins. Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Great strides in medical technology have promoted a number of highly advanced drugs that can be used to easily and rapidly treat a wide range of diseases. Currently, the effective and painless delivery of these drugs into organisms is a hot research topic. Because these drugs belong to the category of macromolecular drugs, certain traditional methods are not sufficient to meet the current requirements, e.g., hypodermic injections create pain, and oral ingestion shows low absorption efficiency. Transdermal drug delivery is an attractive option for addressing this problem by delivering the drugs into the organisms through the skin. However, the extraordinary barrier layers of skin, i.e., the stratum corneum (SC) and the outer 10–15 lm of skin, generally allow only small (<400 Da) and oil-soluble molecules to pass through [1]. However, microneedles are able to penetrate these skin barriers to deliver drugs [2]. Commonly, microneedles 100–500 lm in length are capable of piercing through skin in a minimally invasive and painless manner via self-administration and provide slow delivery over time for disease treatment [3,4]. The action of microneedles opens holes in the skin to provide a path for the subsequent delivery of drugs [4], but the needles can also be connected to storage pools for precisely controlled delivery doses [5]. Additionally, microneedles coated with drugs can directly deliver the treatments [6]. To
⇑ Corresponding author. Tel.: +86 21 34207209; fax: +86 21 34206883. E-mail address: [email protected] (J.-Q. Liu).
treat diseases successfully, the therapeutic infusion rates of drugs vary from micro-liters per hour to several milliliters per hour and depend on the drug formulation and concentration. Drugs conveyed by microneedles that have demonstrated their anticipated effects have been reported, including protein [7], insulin [8,9], DNA [10], and vaccines [11]. Metal, polymer, and silicon materials have been used to fabricate microneedles. Metal microneedles [12] showed good mechanical performance with a higher fracture strength when inserted into skin. Certain polymer microneedles [4,13,14], i.e., PGA, and PLGA, showed good biocompatibility in a long history as resorbable sutures [15]. However, the shapes of metal and polymer microneedles are often limited by their fabrication processes. Silicon microneedles are easily fabricated with various shapes and offer certain biocompatibility [16]. Many sophisticated silicon microneedles have been researched, particularly with the development of MEMS technology. Solid silicon microneedles are commonly produced by a wet etching method [17] and are employed in drug delivery [16]. Hollow silicon microneedles are often fabricated by a dry etching process [5,18–20] and have been applied in auto-drug delivery systems [19]. The large-scale production of microneedles can also reduce the fabrication cost. The cutting process is a promising approach that has been used to fabricate micro-column-shaped Utah electrodes [21] as well as microneedles [22,23]. In this paper, an incorporated process containing a dicing method and a wet etching method is proposed for the fabrication of silicon microneedles. The detailed fabrication process is
0167-9317/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mee.2013.04.039
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Fig. 1. Diagram of the blades: (a) beveled blade; (b) right blade.
Fig. 2. Fabrication process: (a) spin photoresist; (b) cut the basic tapered tip; (c) cut the length; (d) sharpen the tip and smooth the surface.
demonstrated in Section 2, and selected testing experiments are performed in Section 3, including force testing with different microneedle array patches and penetrability and permeation studies using one 7 7 microneedle array patch. Finally, a summary and conclusions are presented in Section 4. 2. Fabrication process and results
Fig. 3. SEM microneedles: (a) cut tapered microneedles; (b) dynamic etch for 3 min, (c) static etch for 5 min.
2.1. Microneedle fabrication process Microneedles with sharp tips and sufficient lengths can easily pierce the epidermis through the SC of skin. However, a single microneedle restricts the drug dose due to its limited volume. Accordingly, a microneedle array with numerous microneedles contained in a patch can be used to increase the dose. In this paper, microneedle arrays are first fabricated by mechanical cutting. One beveled blade (BB) is designed with a bevel angle of h = 20° and a thickness of T1 = 0.6 mm, as shown in Fig. 1(a). The other right blade (RB) with a thickness of T2 = 0.4 mm is shown in Fig. 1(b). Both blades are constructed from resin and have the same internal diameter of d = 88.9 mm and outer diameter of D = 117 mm. The BB is used to form the basic shape of the tapered silicon tip, and the RB controls the length of the microneedles.
A 5-lm layer of photoresist was first spin coated on a silicon wafer with a thickness of 1 mm and a diameter of 75 mm (Fig. 2(a)). This photoresist layer is not only used as the masking layer of the backside wafer during the following wet etching, but it also relieves the vibration of the wafer during the cutting process. Then, a dicing saw (ADT 7000) with the BB was used to cut the silicon wafer along four directions to form the basic tapered tips of the microneedles, as shown in Fig. 2(b). Next, the saw with the RB was used to cut into the middle space of the tips to increase the microneedle length (Fig. 2(c)). The dicing saw is an automatic machine that is able to cut silicon easily and quickly, and the shape of the cut silicon is determined by the blades. In this experiment, the speed of the blades in the dicing saw is approximately
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subsequently immersed into the mixture solution. At the same time, the solution was stirred at a speed of 550 rpm. The etching rate was approximately 30 lm/min. After 3 min, the fixture and solution was maintained in a static state. The etching rate was decreased to 5 lm/min to refine the sharp tips and surfaces of the microneedles. For this step, the etching time was approximately 5 min. The following two equations demonstrate the reaction mechanism of the silicon etching [24]:
Si þ 4HNO3 ! SiO2 þ 2H2 O þ 4NO2
ð1Þ
SiO2 þ 6HF ! H2 SiF6 þ 2H2 O
ð2Þ
2.2. Fabricated microneedles The cut microneedles are 700 lm in length, 400 lm in the bottom-side size and 30 lm in the top-side size, 600 lm in width between the tips of two microneedles, respectively, as shown in Fig. 3(a). The tips of many of the microneedles are not sharp. Therefore, the dynamic etching process smoothes the surface of the cut microneedles and sharpens their tips, and their volumes contract, as shown in Fig. 3(b). Furthermore, the sharper tips and smoother surfaces are formed by the following static etching method, as shown in Fig. 3(c). The length of the microneedles is 400 lm after the final etching process. Finally, the microneedle array patches (MAP) are separated from the fabricated silicon wafer by the dicing saw. 3. Testing and discussion of microneedles Fig. 4. Force test of different array patches: (a) different patches under forces; (b) same force imposed on patches.
14,000 rpm, and its feed speed is 6 mm/s. Therefore, the saw can fabricate a 75-mm diameter silicon wafer to quickly form microneedles for large production. The cut microneedles were placed in a mixed solution (HNO3: HF = 19:2, v:v to etch and create smooth microneedles (Fig. 2(d)). This wet etching method has been reported in detail [21,22]. In brief, the etching process at room temperature consists of a dynamic step and a static step. In the dynamic step, the microneedles were clamped in a fixture with a revolution speed of 30 rpm and
A series of MAP experiments are performed in drug delivery applications, including force testing, a penetrability study (with respect to skin) and a permeation study. 3.1. Force testing The number of microneedles in a patch influences its piercing depth when it contacts the skin. In this study, 3 3, 5 6, 7 7, 9 9 and 11 11 MAPs were selected and tested. These microneedles were used to pierce an artificial skin (polyurethane, PU, 0.35 mm, Shore 85 ± 10) under a piercing speed of 1 mm/min, and the applied force varied from 0.1 N to 10 N. The combination
Fig. 5. Holes left in the artificial skins: (a) 3 3 microneedle array; (b) 5 6 microneedle array; (c) 7 7 microneedle array; (d) 9 9 microneedle array; (e) 11 11 microneedle array; (f) microneedle with RBG. In (e) and (f): 1 is the left hole in the PU; 2 is the intact PU.
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3.2. Penetrability study
Fig. 6. The 7 7 microneedle array patch shown next to a hypodermic needle with a 0.4-mm inner diameter.
of large piercing speed and force can cause the MAP to fracture. Fig. 4(a) shows the relationship between the applied force and the piercing depth. With an increasing number of microneedles assembled in one patch, the piercing force also must be increased to provide the same piercing depth. However, when the number of the microneedles exceeds 49 (7 7), the increasing trend of the piercing force for the same depth flattens out, as shown in Fig. 4(b). Fig. 5(a)–(e) present the optical images of the holes left by the 3 3, 5 6, 7 7, 9 9, and 11 11 MAPs in the PU, respectively, which show that the microneedles insert into the PU quite well. Fig. 6 shows the dimension comparison of the 7 7 MAP and a normal needle.
In this experiment, rat skin (RS) was excised from rat abdomen skin after the rat was sacrificed. Next, pads with iso-propanol were used to carefully remove the adhered fat and other visceral debris on the skin until the thickness of the skin was approximately 0.5– 2 mm. The prepared skin was washed in phosphate buffered saline (PBS, PH = 7.2) 3–5 times to obtain clean skin, and each repetition lasted for 5 min. Finally, the skin was wrapped in aluminum foil and stored at 20 °C. Before the animals were killed, they were given free access to food and water ad libitum unless otherwise noted. All procedures were approved by the Laboratory Animal Use Committee of Shanghai Jiao Tong University School of Pharmacy. All possible efforts were carried out to reduce the number of animals used, minimize their suffering, and employ alternatives to in vivo techniques, if available. The experimental study groups were assigned randomly, and the researcher was blinded for the behavior tests. The penetrability of the microneedles can be studied by observing the wounds that the microneedles leave in the RS. These wounds are often observed via confocal microscopy, which is costly. A low-cost method is therefore deployed to evaluate whether this type of microneedle can effectively pierce through skin. In this method, one PU patch is placed under one RS patch to protect the microneedles from fracture when piercing the RS. When the microneedles pierce through the RS, the PU underneath is marked with holes. If the microneedles did not pierce the RS, then dents (and not holes) are observed in the PU. To avoid this phenomenon, Rhodamine B gel (RBG) is coated on the microneedles. When the microneedles pierce through RS, the RBG is left on the PU and can be observed via a fluorescence microscope (Olympus BX61, Olympus, Japan). The RBG is prepared with a mixture of Carbomer (2%) and Rhodamine B (0.042 mg/ml) with a
Fig. 7. Rhodamine B gel on the microneedles and in skins: (a) on microneedles; (b) on the rat skin; (c) on the artificial skin.
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weight ratio of 1:1 [15], and the microneedles are immersed into this gel for 30 s and subsequently dried at room temperature for 24 h. Fig. 5(f) demonstrates how the microneedles with RBG leave holes in the PU. A 7 7 MAP is employed in the piercing test experiment, as shown in Figs. 6 and 7(a). Fig. 7(b) displays an image of the microneedles coated with the RBG. After insertion, the RBG is clearly observed in the holes of the RS treated by the microneedles, as shown in Fig. 7(c). Additionally, the RBG is clearly observed in the PU (Fig. 7(d)). The wounds in the RS are clear, and the holes in the PU are distinct, which demonstrates that the microneedles can easily pierce the skin. 3.3. Permeation study The permeation test is performed to assess the ability of microneedles to deliver drugs [25]. Briefly, Franz-type glass diffusion cells (Shanghai Heqi Glassware Co., Ltd, China)), calcein (622.53 Da, Shanghai Yuanye Bio-Technology Co., Ltd, China) and a fluoro-spectrophotometer (F-2500, Hitachi, Japan) (excitation and emission wavelength set at 495 nm and 513 nm, respectively) are prepared. The Franz cell consists of an upper donor chamber (DC) and a lower receptor chamber (RC) with a 15-ml volume. The sampling port (SP) connected to the RC as an outlet for a pipette is used to conveniently remove the sample solution. After thawing in PBS, the RS is pierced vertically by the microneedles (7 7 array patch) with the force (10 N) and the speed (1 mm/ min). Next, the skin, with its SC side facing upward into the DC, is mounted on a RC filled with PBS solution. It is simultaneously fixed by clamps to the Franz cell, which is immersed into a water bath at a constant temperature of 37 °C. The saturated calcein solution is applied on the skin in the DC, which was subsequently covered with Parafilm to avoid evaporation. The fluid in the RC is continuously stirred with a magnetic bar at 280 rpm to maintain homogeneity. Samples are withdrawn through the SP at predetermined time intervals, and the RC phase is immediately replenished with an equal volume of fresh PBS buffer to maintain a constant volume. At least three replicate experiments are conducted. The standard calcein solution with c = 1 105 mol/l is prepared. Fig. 8 shows the relationship between the fluorescence intensity and the calcein solution concentration. A good linear relationship is observed between them, and the permeation rate of calcein is expressed as
Fig. 9. In vitro transdermal permeation of calcein in the rat skin and pretreatment with or without microneedles. Each value represents the mean (standard deviation) of three measurements.
r¼
cv M x st
ð3Þ
where v is the volume of the receptor chamber, M is the molar mass of calcein, s is the area of the microneedle array patch, and t is the sampling time. Fig. 9 shows the cumulative amount of permeated flux of calcein with time, which demonstrates that the permeation rate of calcein through the treated skin is approximately 90 lg/cm2/ h and through the intact skin is 2.5 lg/cm2/h. The experimental results show that the transdermal permeation with microneedles is approximately 36 times greater than that on the intact skins. Thus, these microneedles may improve the efficiency of drug delivery through the skin. 4. Conclusion A low-cost micro-fabrication method is proposed to form tapered silicon microneedles with sharp tips for drug delivery. This method can produce uniformly tapered microneedle array patches in large production. Different array patches subjected to a forcetesting experiment demonstrate that the piercing depth into the skin created by the patches is influenced by the number of microneedles in each patch. A 7 7 microneedle array patch is selected to test the penetrability in terms of piercing rat skins and the permeation weights of calcein through rat skins. The penetrability results show that this type of microneedle can easily insert into rat skins with low force and low speed. The permeation rate of calcein through the skins can be obviously improved with these types of silicon microneedles. Acknowledgments This work is partly supported by National Natural Science Foundation of China (No. 51035005), the Important National Science & Technology Specific Projects (No. 2009ZX09310) and the National Key Scientific Program (2010CB933901). The authors are also grateful to their colleagues for their essential contributions to this work. References
Fig. 8. Relationship between the calcein solution concentrations and their fluorescence intensities. The black dots show the standard calcein solution with 1%, 5%, 9%, 13%, 19%, 25%, 35%, 45%. The red line denotes the fitted straight line of the dots.
[1] H. Schaefer, T.E. Redelmeier, Skin Barrier: Principles of Percutaneous Absorption, Karger, Basel, 1996.
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[2] S. Henry, D.V. Mcallister, M.G. Allen, M.R. Prausnitz, J. Pharm. Sci. 87 (1998) 922–925. [3] S.C. Kuo, Y. Chou, J. Sci. Eng. 7 (2004) 95–98. [4] J.H. Park, M.G. Allen, M.R. Prausnitz, J. Controlled Release 104 (2005) 51–56. [5] B. Stoeber, D. Liepmann, J. Microelectromech. Syst. 14 (2005) 472–479. [6] H.S. Gill, M.R. Prausnitz, Pharm. Res. 7 (2007) 1369–1380. [7] S. Katikaneni, A. Badkar, S. Nema, A.K. Banga, J. Pharm. 378 (2009) 93–100. [8] D.V. McAllister, P.M. Wang, S.P. Davis, J.H. Park, P.J. Canatella, M.G. Allen, M.R. Prausnitz, Proc. Natl. Acad. Sci. USA 100 (2003) 13755–13760. [9] W. Martanto, S.P. Davis, N.R. Holiday, J. Wang, H.S. Gill, M.R. Prausnitz, Pharm. Res. 21 (2004) 947–952. [10] J.A. Mikszta, J.B. Alarcon, J.M. Brittingham, D.E. Sutter, R.J. Pettis, N.G. Harvey, Nat. Med. 8 (2002) 415–419. [11] J.A. Mikszta, V.J. Sullivan, J. Infect. Dis. 191 (2005) 278–288. [12] Y.C. Kim, S. Quan, R.W. Compans, S.M. Kang, M.R. Prausnitz, J. Controlled Release 142 (2010) 187–195. [13] S.P. Sullivan, D.G. Koutsonanos, M.P. Martin, J.W. Lee, V. Zarnitsyn, S.O. Choi, N. Murthy, R.W. Compans, I. Skountzou, M.R. Prausnitz, Nat. Med. 16 (2010) 915–921. [14] T. Shibata, S. Yukizono, T. Kawashima, Microelectron. Eng. 86 (2009) 1439– 1442. [15] C.G. Ambrose, T.O. Clanto, Ann. Biomed. Eng. 32 (2004) 171–177.
[16] Y. Wu, Y. Qiu, S. Zhang, G. Qiu, Y. Gao, Biomed. Microdevices 10 (2008) 601–610. [17] N. Wilke, A. Mulcahy, S.R. Ye, A. Morrissey, Microelectron. J. 36 (2005) 650–656. [18] P. Khanna, K. Luongo, J.A. Strom, S. Bhansali, J. Micromech. Microeng. 20 (2010) 045011. [19] P. Griss, G. Stemme, J. Microelectromech. Syst. 12 (2003) 296–301. [20] E.V. Mukerjee, S.D. Collins, R.R. Isseroff, R.L. Smith, Sens. Actuators A: Phys. 114 (2004) 267–275. [21] P.K. Campbell, K.E. Jones, R.J. Huber, K.W. Horch, R.A. Normann, Biomed. Eng. 38 (1991) 758–768. [22] M. Shikida, M. Ando, Y. Ishihara, T. Ando, J. Micromech. Microeng. 14 (2004) 1462–1467. [23] R. Bhandari, S. Negi, F. Solzbacher. A novel mask-less method of fabrication high aspect ratio microneedles for blood sampling, in: proceedings of IEEE 58th Electronic Components and Technology Conference, Lake Buena Vista, FL, USA, 2008. 1306–1309. [24] H. Robbins, B. Schwartz, J. Electrochem. Soc. 107 (1960) 108–111. [25] Organization for Economic Cooperation and Development (OECD), OECD Guidelines for testing of chemicals, No. 428: Skin Absorption: In vitro Method (OECD, 2004).
Contents lists available at SciVerse ScienceDirect
Microelectronic Engineering journal homepage: www.elsevier.com/locate/mee
Fabrication and testing analysis of tapered silicon microneedles for drug delivery applications Xiao-Xiao Yan, Jing-Quan Liu ⇑, Shui-Dong Jiang, Bin Yang, Chun-Sheng Yang National Key Laboratory of Science and Technology on Nano/Micro-fabrication, Institute of Micro and Nano Science and Technology, Shanghai Jiao Tong University, Shanghai 200240, China
a r t i c l e
i n f o
Article history: Received 15 January 2013 Received in revised form 3 April 2013 Accepted 27 April 2013 Available online 7 May 2013 Keywords: Drug delivery Microneedles Dicing saw Chemical etching
a b s t r a c t Drug delivery plays an important role in the treatment of disease, and the equipment used for drug delivery has a great impact on the delivery efficiency of drugs. Recently developed for drug delivery, the new microneedle apparatus has the potential to improve this efficiency. In this study, an incorporated microfabrication process is proposed for the formation of tapered silicon microneedles. First, a dicing saw with one beveled blade and one right blade is used to directly cut silicon wafers into the basic shape of tapered microneedles. Next, a chemical etching process containing a dynamic step and static step is deployed to refine the basic shape and form sharp and tapered microneedles 400 lm in length and 600 lm in width between the tips of two microneedles. A variety of microneedle array patches are fabricated to investigate their ability to pierce artificial skin with forces ranging from 0.1 N to 10 N. The percutaneous experimental results show that the permeation rate of a 7 7 microneedle array patch through a treated rat skin is 36 times higher than that for intact skins. Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Great strides in medical technology have promoted a number of highly advanced drugs that can be used to easily and rapidly treat a wide range of diseases. Currently, the effective and painless delivery of these drugs into organisms is a hot research topic. Because these drugs belong to the category of macromolecular drugs, certain traditional methods are not sufficient to meet the current requirements, e.g., hypodermic injections create pain, and oral ingestion shows low absorption efficiency. Transdermal drug delivery is an attractive option for addressing this problem by delivering the drugs into the organisms through the skin. However, the extraordinary barrier layers of skin, i.e., the stratum corneum (SC) and the outer 10–15 lm of skin, generally allow only small (<400 Da) and oil-soluble molecules to pass through [1]. However, microneedles are able to penetrate these skin barriers to deliver drugs [2]. Commonly, microneedles 100–500 lm in length are capable of piercing through skin in a minimally invasive and painless manner via self-administration and provide slow delivery over time for disease treatment [3,4]. The action of microneedles opens holes in the skin to provide a path for the subsequent delivery of drugs [4], but the needles can also be connected to storage pools for precisely controlled delivery doses [5]. Additionally, microneedles coated with drugs can directly deliver the treatments [6]. To
⇑ Corresponding author. Tel.: +86 21 34207209; fax: +86 21 34206883. E-mail address: [email protected] (J.-Q. Liu).
treat diseases successfully, the therapeutic infusion rates of drugs vary from micro-liters per hour to several milliliters per hour and depend on the drug formulation and concentration. Drugs conveyed by microneedles that have demonstrated their anticipated effects have been reported, including protein [7], insulin [8,9], DNA [10], and vaccines [11]. Metal, polymer, and silicon materials have been used to fabricate microneedles. Metal microneedles [12] showed good mechanical performance with a higher fracture strength when inserted into skin. Certain polymer microneedles [4,13,14], i.e., PGA, and PLGA, showed good biocompatibility in a long history as resorbable sutures [15]. However, the shapes of metal and polymer microneedles are often limited by their fabrication processes. Silicon microneedles are easily fabricated with various shapes and offer certain biocompatibility [16]. Many sophisticated silicon microneedles have been researched, particularly with the development of MEMS technology. Solid silicon microneedles are commonly produced by a wet etching method [17] and are employed in drug delivery [16]. Hollow silicon microneedles are often fabricated by a dry etching process [5,18–20] and have been applied in auto-drug delivery systems [19]. The large-scale production of microneedles can also reduce the fabrication cost. The cutting process is a promising approach that has been used to fabricate micro-column-shaped Utah electrodes [21] as well as microneedles [22,23]. In this paper, an incorporated process containing a dicing method and a wet etching method is proposed for the fabrication of silicon microneedles. The detailed fabrication process is
0167-9317/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mee.2013.04.039
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X.-X. Yan et al. / Microelectronic Engineering 111 (2013) 33–38
Fig. 1. Diagram of the blades: (a) beveled blade; (b) right blade.
Fig. 2. Fabrication process: (a) spin photoresist; (b) cut the basic tapered tip; (c) cut the length; (d) sharpen the tip and smooth the surface.
demonstrated in Section 2, and selected testing experiments are performed in Section 3, including force testing with different microneedle array patches and penetrability and permeation studies using one 7 7 microneedle array patch. Finally, a summary and conclusions are presented in Section 4. 2. Fabrication process and results
Fig. 3. SEM microneedles: (a) cut tapered microneedles; (b) dynamic etch for 3 min, (c) static etch for 5 min.
2.1. Microneedle fabrication process Microneedles with sharp tips and sufficient lengths can easily pierce the epidermis through the SC of skin. However, a single microneedle restricts the drug dose due to its limited volume. Accordingly, a microneedle array with numerous microneedles contained in a patch can be used to increase the dose. In this paper, microneedle arrays are first fabricated by mechanical cutting. One beveled blade (BB) is designed with a bevel angle of h = 20° and a thickness of T1 = 0.6 mm, as shown in Fig. 1(a). The other right blade (RB) with a thickness of T2 = 0.4 mm is shown in Fig. 1(b). Both blades are constructed from resin and have the same internal diameter of d = 88.9 mm and outer diameter of D = 117 mm. The BB is used to form the basic shape of the tapered silicon tip, and the RB controls the length of the microneedles.
A 5-lm layer of photoresist was first spin coated on a silicon wafer with a thickness of 1 mm and a diameter of 75 mm (Fig. 2(a)). This photoresist layer is not only used as the masking layer of the backside wafer during the following wet etching, but it also relieves the vibration of the wafer during the cutting process. Then, a dicing saw (ADT 7000) with the BB was used to cut the silicon wafer along four directions to form the basic tapered tips of the microneedles, as shown in Fig. 2(b). Next, the saw with the RB was used to cut into the middle space of the tips to increase the microneedle length (Fig. 2(c)). The dicing saw is an automatic machine that is able to cut silicon easily and quickly, and the shape of the cut silicon is determined by the blades. In this experiment, the speed of the blades in the dicing saw is approximately
X.-X. Yan et al. / Microelectronic Engineering 111 (2013) 33–38
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subsequently immersed into the mixture solution. At the same time, the solution was stirred at a speed of 550 rpm. The etching rate was approximately 30 lm/min. After 3 min, the fixture and solution was maintained in a static state. The etching rate was decreased to 5 lm/min to refine the sharp tips and surfaces of the microneedles. For this step, the etching time was approximately 5 min. The following two equations demonstrate the reaction mechanism of the silicon etching [24]:
Si þ 4HNO3 ! SiO2 þ 2H2 O þ 4NO2
ð1Þ
SiO2 þ 6HF ! H2 SiF6 þ 2H2 O
ð2Þ
2.2. Fabricated microneedles The cut microneedles are 700 lm in length, 400 lm in the bottom-side size and 30 lm in the top-side size, 600 lm in width between the tips of two microneedles, respectively, as shown in Fig. 3(a). The tips of many of the microneedles are not sharp. Therefore, the dynamic etching process smoothes the surface of the cut microneedles and sharpens their tips, and their volumes contract, as shown in Fig. 3(b). Furthermore, the sharper tips and smoother surfaces are formed by the following static etching method, as shown in Fig. 3(c). The length of the microneedles is 400 lm after the final etching process. Finally, the microneedle array patches (MAP) are separated from the fabricated silicon wafer by the dicing saw. 3. Testing and discussion of microneedles Fig. 4. Force test of different array patches: (a) different patches under forces; (b) same force imposed on patches.
14,000 rpm, and its feed speed is 6 mm/s. Therefore, the saw can fabricate a 75-mm diameter silicon wafer to quickly form microneedles for large production. The cut microneedles were placed in a mixed solution (HNO3: HF = 19:2, v:v to etch and create smooth microneedles (Fig. 2(d)). This wet etching method has been reported in detail [21,22]. In brief, the etching process at room temperature consists of a dynamic step and a static step. In the dynamic step, the microneedles were clamped in a fixture with a revolution speed of 30 rpm and
A series of MAP experiments are performed in drug delivery applications, including force testing, a penetrability study (with respect to skin) and a permeation study. 3.1. Force testing The number of microneedles in a patch influences its piercing depth when it contacts the skin. In this study, 3 3, 5 6, 7 7, 9 9 and 11 11 MAPs were selected and tested. These microneedles were used to pierce an artificial skin (polyurethane, PU, 0.35 mm, Shore 85 ± 10) under a piercing speed of 1 mm/min, and the applied force varied from 0.1 N to 10 N. The combination
Fig. 5. Holes left in the artificial skins: (a) 3 3 microneedle array; (b) 5 6 microneedle array; (c) 7 7 microneedle array; (d) 9 9 microneedle array; (e) 11 11 microneedle array; (f) microneedle with RBG. In (e) and (f): 1 is the left hole in the PU; 2 is the intact PU.
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3.2. Penetrability study
Fig. 6. The 7 7 microneedle array patch shown next to a hypodermic needle with a 0.4-mm inner diameter.
of large piercing speed and force can cause the MAP to fracture. Fig. 4(a) shows the relationship between the applied force and the piercing depth. With an increasing number of microneedles assembled in one patch, the piercing force also must be increased to provide the same piercing depth. However, when the number of the microneedles exceeds 49 (7 7), the increasing trend of the piercing force for the same depth flattens out, as shown in Fig. 4(b). Fig. 5(a)–(e) present the optical images of the holes left by the 3 3, 5 6, 7 7, 9 9, and 11 11 MAPs in the PU, respectively, which show that the microneedles insert into the PU quite well. Fig. 6 shows the dimension comparison of the 7 7 MAP and a normal needle.
In this experiment, rat skin (RS) was excised from rat abdomen skin after the rat was sacrificed. Next, pads with iso-propanol were used to carefully remove the adhered fat and other visceral debris on the skin until the thickness of the skin was approximately 0.5– 2 mm. The prepared skin was washed in phosphate buffered saline (PBS, PH = 7.2) 3–5 times to obtain clean skin, and each repetition lasted for 5 min. Finally, the skin was wrapped in aluminum foil and stored at 20 °C. Before the animals were killed, they were given free access to food and water ad libitum unless otherwise noted. All procedures were approved by the Laboratory Animal Use Committee of Shanghai Jiao Tong University School of Pharmacy. All possible efforts were carried out to reduce the number of animals used, minimize their suffering, and employ alternatives to in vivo techniques, if available. The experimental study groups were assigned randomly, and the researcher was blinded for the behavior tests. The penetrability of the microneedles can be studied by observing the wounds that the microneedles leave in the RS. These wounds are often observed via confocal microscopy, which is costly. A low-cost method is therefore deployed to evaluate whether this type of microneedle can effectively pierce through skin. In this method, one PU patch is placed under one RS patch to protect the microneedles from fracture when piercing the RS. When the microneedles pierce through the RS, the PU underneath is marked with holes. If the microneedles did not pierce the RS, then dents (and not holes) are observed in the PU. To avoid this phenomenon, Rhodamine B gel (RBG) is coated on the microneedles. When the microneedles pierce through RS, the RBG is left on the PU and can be observed via a fluorescence microscope (Olympus BX61, Olympus, Japan). The RBG is prepared with a mixture of Carbomer (2%) and Rhodamine B (0.042 mg/ml) with a
Fig. 7. Rhodamine B gel on the microneedles and in skins: (a) on microneedles; (b) on the rat skin; (c) on the artificial skin.
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weight ratio of 1:1 [15], and the microneedles are immersed into this gel for 30 s and subsequently dried at room temperature for 24 h. Fig. 5(f) demonstrates how the microneedles with RBG leave holes in the PU. A 7 7 MAP is employed in the piercing test experiment, as shown in Figs. 6 and 7(a). Fig. 7(b) displays an image of the microneedles coated with the RBG. After insertion, the RBG is clearly observed in the holes of the RS treated by the microneedles, as shown in Fig. 7(c). Additionally, the RBG is clearly observed in the PU (Fig. 7(d)). The wounds in the RS are clear, and the holes in the PU are distinct, which demonstrates that the microneedles can easily pierce the skin. 3.3. Permeation study The permeation test is performed to assess the ability of microneedles to deliver drugs [25]. Briefly, Franz-type glass diffusion cells (Shanghai Heqi Glassware Co., Ltd, China)), calcein (622.53 Da, Shanghai Yuanye Bio-Technology Co., Ltd, China) and a fluoro-spectrophotometer (F-2500, Hitachi, Japan) (excitation and emission wavelength set at 495 nm and 513 nm, respectively) are prepared. The Franz cell consists of an upper donor chamber (DC) and a lower receptor chamber (RC) with a 15-ml volume. The sampling port (SP) connected to the RC as an outlet for a pipette is used to conveniently remove the sample solution. After thawing in PBS, the RS is pierced vertically by the microneedles (7 7 array patch) with the force (10 N) and the speed (1 mm/ min). Next, the skin, with its SC side facing upward into the DC, is mounted on a RC filled with PBS solution. It is simultaneously fixed by clamps to the Franz cell, which is immersed into a water bath at a constant temperature of 37 °C. The saturated calcein solution is applied on the skin in the DC, which was subsequently covered with Parafilm to avoid evaporation. The fluid in the RC is continuously stirred with a magnetic bar at 280 rpm to maintain homogeneity. Samples are withdrawn through the SP at predetermined time intervals, and the RC phase is immediately replenished with an equal volume of fresh PBS buffer to maintain a constant volume. At least three replicate experiments are conducted. The standard calcein solution with c = 1 105 mol/l is prepared. Fig. 8 shows the relationship between the fluorescence intensity and the calcein solution concentration. A good linear relationship is observed between them, and the permeation rate of calcein is expressed as
Fig. 9. In vitro transdermal permeation of calcein in the rat skin and pretreatment with or without microneedles. Each value represents the mean (standard deviation) of three measurements.
r¼
cv M x st
ð3Þ
where v is the volume of the receptor chamber, M is the molar mass of calcein, s is the area of the microneedle array patch, and t is the sampling time. Fig. 9 shows the cumulative amount of permeated flux of calcein with time, which demonstrates that the permeation rate of calcein through the treated skin is approximately 90 lg/cm2/ h and through the intact skin is 2.5 lg/cm2/h. The experimental results show that the transdermal permeation with microneedles is approximately 36 times greater than that on the intact skins. Thus, these microneedles may improve the efficiency of drug delivery through the skin. 4. Conclusion A low-cost micro-fabrication method is proposed to form tapered silicon microneedles with sharp tips for drug delivery. This method can produce uniformly tapered microneedle array patches in large production. Different array patches subjected to a forcetesting experiment demonstrate that the piercing depth into the skin created by the patches is influenced by the number of microneedles in each patch. A 7 7 microneedle array patch is selected to test the penetrability in terms of piercing rat skins and the permeation weights of calcein through rat skins. The penetrability results show that this type of microneedle can easily insert into rat skins with low force and low speed. The permeation rate of calcein through the skins can be obviously improved with these types of silicon microneedles. Acknowledgments This work is partly supported by National Natural Science Foundation of China (No. 51035005), the Important National Science & Technology Specific Projects (No. 2009ZX09310) and the National Key Scientific Program (2010CB933901). The authors are also grateful to their colleagues for their essential contributions to this work. References
Fig. 8. Relationship between the calcein solution concentrations and their fluorescence intensities. The black dots show the standard calcein solution with 1%, 5%, 9%, 13%, 19%, 25%, 35%, 45%. The red line denotes the fitted straight line of the dots.
[1] H. Schaefer, T.E. Redelmeier, Skin Barrier: Principles of Percutaneous Absorption, Karger, Basel, 1996.
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[2] S. Henry, D.V. Mcallister, M.G. Allen, M.R. Prausnitz, J. Pharm. Sci. 87 (1998) 922–925. [3] S.C. Kuo, Y. Chou, J. Sci. Eng. 7 (2004) 95–98. [4] J.H. Park, M.G. Allen, M.R. Prausnitz, J. Controlled Release 104 (2005) 51–56. [5] B. Stoeber, D. Liepmann, J. Microelectromech. Syst. 14 (2005) 472–479. [6] H.S. Gill, M.R. Prausnitz, Pharm. Res. 7 (2007) 1369–1380. [7] S. Katikaneni, A. Badkar, S. Nema, A.K. Banga, J. Pharm. 378 (2009) 93–100. [8] D.V. McAllister, P.M. Wang, S.P. Davis, J.H. Park, P.J. Canatella, M.G. Allen, M.R. Prausnitz, Proc. Natl. Acad. Sci. USA 100 (2003) 13755–13760. [9] W. Martanto, S.P. Davis, N.R. Holiday, J. Wang, H.S. Gill, M.R. Prausnitz, Pharm. Res. 21 (2004) 947–952. [10] J.A. Mikszta, J.B. Alarcon, J.M. Brittingham, D.E. Sutter, R.J. Pettis, N.G. Harvey, Nat. Med. 8 (2002) 415–419. [11] J.A. Mikszta, V.J. Sullivan, J. Infect. Dis. 191 (2005) 278–288. [12] Y.C. Kim, S. Quan, R.W. Compans, S.M. Kang, M.R. Prausnitz, J. Controlled Release 142 (2010) 187–195. [13] S.P. Sullivan, D.G. Koutsonanos, M.P. Martin, J.W. Lee, V. Zarnitsyn, S.O. Choi, N. Murthy, R.W. Compans, I. Skountzou, M.R. Prausnitz, Nat. Med. 16 (2010) 915–921. [14] T. Shibata, S. Yukizono, T. Kawashima, Microelectron. Eng. 86 (2009) 1439– 1442. [15] C.G. Ambrose, T.O. Clanto, Ann. Biomed. Eng. 32 (2004) 171–177.
[16] Y. Wu, Y. Qiu, S. Zhang, G. Qiu, Y. Gao, Biomed. Microdevices 10 (2008) 601–610. [17] N. Wilke, A. Mulcahy, S.R. Ye, A. Morrissey, Microelectron. J. 36 (2005) 650–656. [18] P. Khanna, K. Luongo, J.A. Strom, S. Bhansali, J. Micromech. Microeng. 20 (2010) 045011. [19] P. Griss, G. Stemme, J. Microelectromech. Syst. 12 (2003) 296–301. [20] E.V. Mukerjee, S.D. Collins, R.R. Isseroff, R.L. Smith, Sens. Actuators A: Phys. 114 (2004) 267–275. [21] P.K. Campbell, K.E. Jones, R.J. Huber, K.W. Horch, R.A. Normann, Biomed. Eng. 38 (1991) 758–768. [22] M. Shikida, M. Ando, Y. Ishihara, T. Ando, J. Micromech. Microeng. 14 (2004) 1462–1467. [23] R. Bhandari, S. Negi, F. Solzbacher. A novel mask-less method of fabrication high aspect ratio microneedles for blood sampling, in: proceedings of IEEE 58th Electronic Components and Technology Conference, Lake Buena Vista, FL, USA, 2008. 1306–1309. [24] H. Robbins, B. Schwartz, J. Electrochem. Soc. 107 (1960) 108–111. [25] Organization for Economic Cooperation and Development (OECD), OECD Guidelines for testing of chemicals, No. 428: Skin Absorption: In vitro Method (OECD, 2004).