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Comparison of polymers to enhance mechanical properties of microneedles for bio-medical applications

Comparison of polymers to enhance mechanical properties of microneedles for bio-medical applications To pierce through the skin and interact with the first biofluid available, microneedles should be mechanically strong. However, some polymers used to fabricate microneedles yield insufficient strength for the fabrication of arrays (PDMS, highly porous structures, etc.). To enhance mechanical properties, piercing materials can be used. They aim to pierce the skin evenly and dissolve quickly, clearing the way for underlying microneedles to interact with the interstitial fluid (ISF). Three materials—carboxymethyl cellulose (CMC), alginate, and hyaluronic acid (HA)—are discussed in this article. Low concentrations, for a quick dissolution while keeping enhancing effect, are used ranging from 1–5%(w/w) in deionized water. Their overall aspects, such as geometrical parameters (tip width, height, and width), piercing capabilities, and dissolution time, are measured and discussed. For breaking the skin barrier, two key parameters—a sharp tip and overall mechanical strength—are highlighted. Each material fails the piercing test at a concentration of 1%(w/w). Concentrations of 3%(w/w) and of 5%(w/w) are giving strong arrays able to pierce the skin. For the pur‑ pose of this study, HA at a concentration of 3%(w/w) results in arrays composed of microneedles with a tip width of 48 ± 8 μm and pierced through the foil with a dissolution time of less than 2 min. Keywords: Microneedles, Piercing materials, Enhancing materials, Drug delivery, Carboxymethyl cellulose, Alginate, Hyaluronic acid Introduction the dermis needs to be pierced to reach the blood ves- In a constantly evolving world, due to the increas- sel and nerves. However, the interstitial fluid (ISF) is ing medical efficiency and accessibility demands, the a biofluid that can be accessed without causing pain fabrication and optimization of simpler, less invasive to the subject: ISF is located above pain receptors. and safer devices have to be asserted. For example, ISF is abundantly available among all bodily fluids, standard glucose monitoring devices require blood unlike sweat, urine, or tears, which are linked to bodily for their measurement, and the blood is obtained by responses. Moreover, its composition proportional to repetitively pricking the subject’s finger throughout blood make it a good candidate for continuous moni- the day; as a broader view, drug delivery technologies toring of biomarker as well as its accessibility for local- require specific packaging (smart drug delivery, etc.) ized drug delivery [1–3]. To reach ISF, hypodermal as well as trained medical personnel (vaccine admin- needles can be superfluous, i.e. being too dangerous, istration, etc.). In addition, most biomarker sensing invasive, requiring trained medical staff, heavy waste devices require blood for analyses; for this purpose, management, thus generating anxiety and pain [4–7]. For this purpose, an array of microneedles has been investigated in recent researches. These microneedles *Correspondence: gwenbonf@iis.u‑tokyo.ac.jp can access ISF by causing minimum pain and invasive- LIMMS/CNRS‑IIS UMI 2820, The University of Tokyo, Tokyo, Japan ness [8–12]. Different microneedles serve different Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 2 of 13 purposes, and the four main types of them include Materials and methods plain, hollow, coated and dissolvable [13–17]. Plain Materials microneedles permit to open the way by poking a hole HA has been provided by RAPHAS Co. Ltd., Korea. in the skin for a better penetration of medicine [18]; Sodium CMC was obtained from Merck Japan (Sigma hollow microneedles are used for bio-sensing and Aldrich). Sodium alginate sample has been kindly offered drug delivery [19–21]; coated microneedles are mainly by KIMICA Corporation, Japan. Polydimethylsiloxane TM used for drug delivery [22], and finally, dissolvable (PDMS) (SILPOT 184) was obtained from Dow Corn- microneedles can serve for both bio sensing and drug ing Corp., Japan. Agarose gel was made from Fast Gene delivery purposes [23, 24]. Among the microneedle’s NE-AG01 agarose powder. All chemicals were used as category cited previously, dissolvable microneedles delivered. which are made of biodegradable and bio-compatible polymers meet all the required specifications of a new Fabrication of arrays generation of ISF interacting devices. Specific mor- The arrays were fabricated via micro-molding method. phology like highly porous or sponge like (PDMS) A female PDMS mold was fabricated by casting PDMS microneedles can be used for interacting with ISF [9, on a metal master mold and after annealing at 80  °C for 25]. Drug delivery as well as sensing efficiency relies 1  h; this master mold was designed using an array com- on robust mechanical properties, piercing capabili- prising 169 microneedles with a height of approximately ties, and dissolution time. In highly porous material or 1200  μm and bottom width of approximately 600  μm sponge like material, lack of strength is intrinsically (Fig.  1). The master mold was fabricated by electro-dis - linked to their morphology [26]. Naturally, extremely charge machining. The microneedles were in the shape of high porosity must be attained to interact with ISF, a square based pyramid with a total volume of 850 μL. further leading to a decrease in the overall mechani- For preparing the PDMS mold, a resin base and curing cal properties. To assert this issue, different enhanc- agent were mixed in the ratio of 10:1. The metal mold ing materials can be used. Porous microneedles can was placed in the mixture, degassed under vacuum at be topped with these materials; such material permits around 1  kPa, and annealed at 80  °C for 1  h in an oven. the needles to break through the skin, thereby enabling The resulting mold was peeled and used as it is. access to the ISF. They must be sufficiently strong to In order to prepare solutions of different concentra - evenly pierce the skin, while maintaining their sharp- tions for each material, the required amount of material ness, as well as dissolving rapidly after entering the was dissolved in de-ionized water (1, 3, and 5 in %(w/w) body. The main goal of this article is to help underly- ratios) and vigorously agitated at 1000  rpm using a stir- ing microneedle to have sufficient strength to pierce ring magnet. For CMC and alginate, they required 2 h for the skin without interfering with their desired applica- homogeneous solution as the dissolution was difficult. tion. For this purpose, three materials such as hyalu- u Th s, heating the solution (40  °C) gently was performed ronic acid (HA), carboxymethyl cellulose (CMC) and to help dissolution of the material. The solution of the alginate [24, 27–31], that are widely used in biomedi- material to be cast was poured into the mold, placed in a cal field, were studied. These materials are approved vacuum box for degassing at 1 kPa, and then annealed at by the United States Food and Drug Administration 60 °C for 2 h, until the array was completely dried. (FDA) as well as bio compatible and biodegradable materials [32–34]. This article discusses the com- Methods parison of these three materials by considering their Overall geometry and other aspects were measured using overall aspects, such as geometrical parameters after a 3D digital fine microscope (VC-3000, Omron Co., molding and thermal annealing, piercing capabilities, Japan). The height, width, and tip width were measured and dissolution time, to find the most suitable material and compared. A total of 50 distinct microneedles were for microneedle technology. To rapidly dissolve, and measured within five distinct arrays for each material not interfere with the desired application, the less pos- with different concentrations in order to be statistically sible material should be used; thus, low concentrations representative of each concentration for each material. are used at 1, 3 and 5%(w/w). These concentrations It is essential that the piercing properties are even are considered low regarding the use of HA, CMC and throughout the array. Uneven pressure can lead to alginate as the main material for fabricating micronee- defect while testing, inserting uncontrollable and dles [27, 30, 35]. Indeed, if too much material is used, unwanted parameters in the measurement. However, clogging of the underlying microneedle can occur, nul- apply the same pressure at every point of the array lifying the aimed application such as drug delivery and while measuring the piercing properties is difficult. bio sensing. Therefore, a press composed of two jaws was used for B onfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 3 of 13 Fig. 1 Picture of a the metal mold used for the fabrication of the PDMS master mold and b a magnified view of one microneedle composing the metal mold (the scale bar corresponds to 1 cm on a and 500 μm on b) this purpose: only the bottom jaw is mobile, allowing almost all the microneedles pierced the aluminum foil to adjust the pressure by a lever. A soft material such as and were still straight after the test; hence, this sample PDMS was placed against the array topped by a sheet of was labeled 1. To better understand the set up for pierc- aluminum foil (10  μm). Here, aluminum foil allows to ing assessment a schematic cross-section of the system separate physically the PDMS plate and the micronee- is presented Fig. 2a. The microneedle array is facing up dles array in order to be easily handled and be used to (microneedle side up), topped by an aluminum foil and simulate the skin for the dissolution properties. Alu- covered with the PDMS sheet. Figure 2b and c shows a minum foil has a shear strength higher than the skin, successful pierced aluminum foil counted as a 1. making aluminum a strict approximation of the skin Finally, the dissolution times of the samples were [36, 37]. Indeed, data from the metals handbook gave assessed by performing an experiment using an aga- a shear strength of 325  MPa for aluminum foil [36] rose gel with a concentration of 2%(w/w), to mimic the against 27  MPa for the human skin presented by Gal- mechanical properties of human skin [38]. The aim of lagher et  al. [37]. To approximate the pressure exerted the material was to pierce the skin, dissolve, and release by a patient through his/her thumb, a pressure of drug or reveal the underlying microneedle as quickly as 15 MPa was applied to each array (1500 g on a surface of possible. Thus, if the array did not have any micronee - 9 cm ) for 2 s. The bottom jaw of the press was adjusted dles left after insertion, it was considered to be dis- before applying pressure. The strength between the solved; in order to be the closest possible to actual skin, jaws was raised up to 15  MPa and maintained for 2  s aluminum foil will not be removed for dissolution test, before releasing it completely. For validation, the sam- indeed, in real life the arrays is not directly in contact of ple should be able to pierce through the aluminum foil the stratum corneum but put against the top surface of and have more than 70% of its microneedles functional the skin. The moisture of the agarose gel will only inter - i.e. still up after the test. Samples fulfilling both criteria act with the microneedle as it is in the expected appli- were labeled as 1, those failing the criteria were labeled cation where ISF and stratum corneum are separated as 0 (Fig.  2). Figure  2a presents the piercing properties with the array by the skin’s top layer. The timer was assessment immediately before applying pressure. The initialized when the array touched the gel, and it was arrays were taped to a glass slide and wrapped using an stopped upon complete dissolution of the micronee- aluminum foil. The system was placed in between the dles. It should be noted that the dissolution times of jaw and surmounted by a sheet of PDMS to simulate samples labeled as 0 in the piercing assessment were soft tissue under the skin. As depicted in Fig. 2b and c, not assessed. Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 4 of 13 Fig. 2 Picture of a the press next to the schematic cross‑section of the assessments, b an array passing the test with visible microneedles sticking out of the aluminum foil and c a piercing assessment top view were well defined and strong enough to withstand the Results and discussion pressure from a finger. Moreover, arrays made of 3 and Overall aspect 5%(w/w) could be bend and folded without breaking Considering the array aspect, the three materials exhib- (Fig.  3a–d). CMC arrays were white to translucent close ited different morphologies without any observation to HA aspect. At a concentration of 1%(w/w), CMC tool (Fig.  3). HA arrays were white to translucent. They arrays were complete and cohesive. They presented were stiff but exhibited flexibility. HA did not stick to no discontinuity of material or broken part but could the PDMS mold during peeling off owing to the oppo - be sheared if manipulated harshly. Both arrays with site hydrophilic character of both materials, PDMS being 3 and 5%(w/w) concentration presented well-defined hydrophobic [39] and HA being hydrophilic [40]. At microneedles structures but seemed less strong than HA 1%(w/w), the HA arrays were incomplete and difficult arrays at the same concentrations. Folding of the array to peel off from the mold without breaking it. At 3 and was possible without breaking for 3 and 5%(w/w). Finally, 5%(w/w), both arrays appeared strong and were detached alginate arrays went from a white to a light brown color from the mold without breaking. Their microneedles Fig. 3 Picture of a, b an array made of HA at 3%(w/w), c, d an array made of CMC at 3%(w/w), e, f an array made of alginate at 3%(w/w) (scale bar corresponds to 1 mm) B onfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 5 of 13 during annealing. However, if the thermal treatment was was measured as approximately 30°, which enables easy stopped before the 2 h the alginate gave a white and flex - penetration into skin. The tip width, height, and width ible array. Still, independent from the annealing time, of the microneedles are measured. The average values these arrays were brittle compared to HA and CMC of each parameter were used to estimate the volume and arrays. A visible shrinkage was noticeable, the array did morphology of each microneedle in the array. Micro- not cover the whole mold after the annealing process. It scopic view of the needle for each material examples are was considered that the color change was caused by such given Fig. 4. shrinkage. In addition, we observed that alginate arrays As presented Fig.  4, microneedles made of HA and shrank during an annealing process and became thicker CMC were very similar, whereas alginate microneedles and denser providing a brittle and brown array. were observed to be shrunk and bent. In Fig.  4a, HA Regarding the microneedles composing the arrays, HA microneedle appeared to be empty in its middle. The and CMC exhibited well-defined microneedles. No miss - same assumption could not be made by the picture given ing microneedles were found. However, alginate array Fig.  4b and c as for CMC and alginate made micronee- showed brittle and bent microneedles structures. They dles. Moreover, HA microneedles were well defined with - appeared to be smaller and bent at their tip (Fig.  3e). out asperities on their side. CMC microneedles came out Moreover, some microneedles had their tips broken less defined. Finally, alginate made microneedle are well (Fig. 3f ). defined but tilted or crooked, more prone to breaking HA and CMC arrays appeared more defined and flex - when inserted into the skin. ible than alginate array, with better looking microneedles Figure  5 shows the tip width as a function of the con- and no broken tips. centration, for each material studied. In the case of HA, CMC, and alginate, the tip width Geometrical consideration was the lowest for the 3%(w/w) solution of each mate- Fifty distinct microneedles were measured for geomet- rial. At a concentration of 1%(w/w), the tip width was rical evaluation. If microneedles had a tip width of less of approximately 72 ± 5  μm for HA and 67 ± 5  μm for than 50  μm and a width of approximately 500  μm, they CMC; however, it was double this value for alginate were generally considered to cause less pain [41]. Actual hence 118 ± 24  μm. This high value, for the solution at specifications on the height were not provided, except 1%(w/w), could be attributed to the low concentration the condition that it should not reach a nerve within of the solution. The entire amount of material had solidi - the body (i.e., it should be less than 1000  μm) [41, 42]. fied onto the walls of the cavities and at the base of the In this study, as stated previously, each microneedle was array; hence, there was insufficient material to maintain shaped from a mold composed of microneedles shaped the sharp tip. The tip was the sharpest at a concentration as a square pyramid, with a tip width of approximately of 3%(w/w). It was less than 48 ± 8 μm for HA, 40 ± 5 μm 30 μm, total length of approximately 1200 μm, and width for CMC, and 57 ± 4  μm for alginate. Finally, the tip of approximately 600 μm (Fig. 1). Moreover, the top angle width increased for each material; indeed, the solution Fig. 4 Optical pictures of a microneedle made of a HA at 3%(w/w), b CMC at 3%(w/w) and c alginate at 3%(w/w). (scale bar corresponds to 500 μm) Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 6 of 13 Fig. 5 Tip width as a function of the concentration (%(w/w)) for each material studied (HA (grey), CMC (black) and alginate (white)) could not completely fit the cavity during thermal treat - leads to a significant difference. For 3%(w/w) arrays, ment due to increasing viscosity, resulting in a wider tip given the height of the three materials, alginate appeared of the microneedle. For a concentration of 5%(w/w), the to have less broken tips than for 1%(w/w), raising the tip width was 48 ± 6  μm, 58 ± 7  μm and 81 ± 6  μm for height of the arrays to comparable value with HA and HA, CMC, and alginate, respectively. As stated previ- CMC. Finally, for a concentration of 5%(w/w), HA kept ously, for a painless insertion into the skin, a tip width a high value compared to CMC and alginate arrays. This of less than 50 μm is required, and both solutions of HA gap can be explained by the increasing of the viscosity of and CMC fulfilled these parameters at a concentration the solution for CMC and alginate when increasing the of 3%(w/w). However, alginate did not meet the require- concentration. This effect added to the brittle character - ments for the 1, 3, and 5%(w/w) concentrations. Alginate istic of the alginate array led to more broken tips and a arrays being more brittle than HA and CMC arrays, this lower height. high value could be attributed to broken tips. Figure  7 presents the variation of width for each array The variations in the height of the microneedles are as a function of their concentrations. plotted in Fig. 6. As shown in Fig.  7, a global tendency can be seen. For each material, some variations were noticeable, Indeed, width was lower for alginate for each concen- but microneedle array’s heights gravitated to approxi- tration. It can be attributed to the shrinkage of alginate mately 1000  μm (Fig.  6). The height of the HA arrays arrays. HA array’s width were around 600 μm. More pre- kept increasing from 1012 ± 34  μm for 1%(w/w), to cisely, respectively, a width of 610 ± 32  μm, 600 ± 12  μm, 1033 ± 21  μm at 3%(w/w) to reach 1077 ± 18  μm for and 608 ± 10  μm for 1, 3 and 5%(w/w) for HA. CMC 5%(w/w) concentration. For CMC, height started from array’s width went from 608 ± 16  μm for 1%(w/w), 1086 ± 23  μm at 1%(w/w), decreased to 1074 ± 23  μm at 598 ± 16 at 3%(w/w) to finally reach 539 ± 17  μm for 3%(w/w) and 971 ± 34 μm for 5%(w/w) concentration. For 5%(w/w). For both 1%(w/w) and 3%(w/w) concentra- the alginate arrays, height increased from 930 ± 50  μm tion, the microneedle’s made of HA and CMC appeared to 1026 ± 27  μm before decreasing to 988 ± 23  μm for to be similar. However, for a concentration of 5%(w/w), 1, 3  and 5%(w/w) concentration, respectively. Low value a shrinkage is noticeable through the geometrical meas- for alginate compared to HA and CMC can be explained urement. Indeed, by naked eye, this phenomenon was by the visible shrinkage happening for alginate arrays. not noticeable. Finally, for alginate arrays, a lower width Moreover, broken tips, especially for the 1%(w/w) arrays was expected. It ranges from 574 ± 26  μm for 1%(w/w) B onfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 7 of 13 Fig. 6 Microneedle’s height as a function of the concentration for HA (grey), CMC (black) and alginate (white) Fig. 7 Microneedle’s base width as a function of the concentration for HA (grey), CMC (black) and alginate (white) microscope confirmed the shrinkage of alginate array. to 550 ± 15  μm at 3%(w/w) to reach a final 517 ± 17  μm This phenomenon appeared to be exacerbated by at 5%(w/w) concentration. Besides appearing shrunk by the naked eye, geometrical measurement through a Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 8 of 13 increasing the concentration. To measure shrinkage, width measurement seemed to be the parameters of choice. All the measurements are summarized in Table 1. The relative error given in the table were calculated using a statistical Student’s t-distribution law for a cor- relation coefficient of 0.95. Figure  8 presents the comparative schematic of each average microneedles made in this work. Shrinkage is visible between alginate array and the other materials. Fig. 8 Schematic comparison of each microneedle’s as a function of Regarding the geometrical aspect, for HA arrays, their concentration and material used 3%(w/w) and 5%(w/w) emerged as optimal micronee- dles for helping to break the skin, close to the require- ments specifications stated in previously. CMC arrays where c is the side of the square base and h the total met the criteria introduced for a painless microneedle height. at a concentration of 3%(w/w). Finally, alginate, despite Nevertheless, the total height of the pyramid was not their shrinkage, could not reach the tip size criteria of accessible, and therefore, had to be calculated. Figure 9 less than 50 μm tip size. presents a schematic of the needle, wherein the known Since shrinkage is noticeable for CMC at 5%(w/w) value is indicated by a dotted line. and every alginate array, it had to be determined for By determining the values of the base sides of both each material. The volume of each needles was deter - pyramids (upper and lower pyramid), we can easily mined by calculating the volume using the tip size, calculate the diagonal. Using this value of the diago- height, and width measured previously. To be com- nal, the height of the small pyramid can be calculated, parable and relevant, the volume had to be calculated which in turn yields the volume of the biggest pyramid. by considering the sharpest tip i.e. tip width being the Comparing the complete pyramids for each micronee- smallest possible, tending to 0. To simplify the calcu- dle will enable us to easily compare one microneedle to lation, the microneedles were considered in the shape another. of a regular pyramid. The volume (V ) of a square based Thales theorem provides an equality where the pyramid is given by Eq. (1), half-diagonal of the small pyramid (CD) over the c × h V = (1) Table 1 Recapitulative table of  each array’s microneedle parameters %(w/w) 1 3 5 Hyaluronic acid Tip (µm) 72 ± 5 48 ± 8 48 ± 6 Height (µm) 1012 ± 34 1033 ± 21 1077 ± 18 Width (µm) 610 ± 32 600 ± 12 608 ± 10 Carboxymethyl cellulose Tip (µm) 67 ± 5 40 ± 5 58 ± 7 Height (µm) 1086 ± 23 1074 ± 23 971 ± 34 Width (µm) 608 ± 16 598 ± 16 539 ± 17 Alginate Tip (µm) 118 ± 24 57 ± 4 81 ± 6 Height (µm) 930 ± 50 1026 ± 27 988 ± 23 Width (µm) 574 ± 26 550 ± 15 517 ± 17 Fig. 9 Schematic of a single microneedle for calculating the total The relative error given in the table were calculated using a statistical Student’s pyramid’s volume t-distribution law for a correlation coefficient of 0.95 B onfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 9 of 13 half-diagonal of the whole pyramid (AB) equals the top during geometrical measurement has been confirmed. pyramid height (CE) over the total height (AE), where Finally, alginate arrays showed more shrinkage than HA (AC) is the previously measured height. and CMC. The shrinkage increased from 13 to 21.8 to reach 29.2% for 1, 3 and 5%(w/w), respectively. Previ- CD CE ous studies have also reported the shrinkage of alginate; = where AE = AC + CE (2) AB AE Walker et  al. showed that objects composed of alginate tend to experience shrinkage [44]. A shrinkage of up to When extracting the unknown CE, we end up with the 50% after 30  min of thermal treatment was observed. In Eq. (3) below, this study, the shrinkage was caused by thermal treat- CD × AC ment, as energy was transferred to the matter, enabling CE = (3) CD it to reach its equilibrium state. Moreover, for alginate AB × 1 − AB arrays, it was shown that shrinkage continues even after the completion of thermal treatment [44]. By determining CE, we can calculate AE using Eq. (2); In addition, a high shrinkage can be an obstacle for the thus, the total volume of the pyramid can be obtained. underlying microneedle arrays because it creates ten- Table  2 presents the different volumes and their com - sion, which could lead to potential breakages. A mate- parison with the actual master mold (i.e., the shrinkage). rial design that is viable for mechanical enhancement Globally, shrinkage occurred for every material. For should not exert tension on the system. In this study, the HA, shrinkage was invisible by naked eye but after calcu- best candidates for enhancement were the HA and CMC lation, all arrays shrank. Respectively, HA arrays shrank arrays, with a shrinkage of less than 10% for a concentra- of 3.6, 8.9 and 2.4% of the desired metal mold volume. tion of 3%(w/w). This value stayed under 10% of shrinkage. Low shrink - age of HA has been observed at around 6% in volume by Winter et  al. [43]. CMC arrays shrinkages increased Piercing abilities from 0 to 7.2 and finally 28.6% for respectively 1, 3 and Regarding the piercing properties, Fig.  10 shows 5%(w/w). While CMC array was thermally treated, the microneedle’s piercing as a function of the concentration water content decreased, forming a hydrogel in the mold. for each material. This hydrogel filled the mold and continued to undergo Being a binary test, the array of the microneedles with thermal treatment, leading to a shrinkage. By increas- more than 70% chance of survival were labeled as 1 other- ing the concentration, the hydrogel formation by CMC wise labeled as 0: the results were plotted as a percentage occurred sooner as a network could be formed by the of success. As expected from the geometrical and other presence of more molecules of CMC. Arrays made from overall aspect results, the weakest arrays to pierce the higher concentration led to higher shrinkage. Shrinkage aluminum foil were made of the 1%(w/w) concentration. of CMC array made of 5%(w/w) concentration observed Indeed, no microneedles survived the piercing test for these arrays for every material and every concentration. HA yielded the best results; all the samples passed the Table 2 Volume calculated by  using Eq.  (2) and  shrinkage test at concentrations of 3%(w/w) and 5%(w/w); attribut- percentage, with  standard deviation calculated able to their sharp tips. CMC had seen its arrays pierce at for a student law of 0.95 75% for the concentration at 3%(w/w) and 87.5% for its 6 3 Master mouldVolume (10  µm ) V/V total Shrinkage (%) 5%(w/w) concentration. Even if CMC tips were smaller or close to those of HA, piercing was less efficient. This result can be explained given the picture of micronee- HA dle provided Fig.  4b. CMC microneedles appeared less 1%(w/w) 142 ± 0.02 0.96 3.6 defined than HA microneedles, leading to potential 3%(w/w) 134 ± 0.003 0.91 8.9 breakage and thus less efficiency in piercing capabili - 5%(w/w) 144 ± 0.001 0.98 2.4 ties. The alginate array at a concentration of 3%(w/w) CMC appeared to pierce the aluminum foil more frequently 1%(w/w) 150 ± 0.003 1.02 0.0 than that at a concentration of 5%(w/w). This decreasing 3%(w/w) 137 ± 0.003 0.93 7.2 can be explained by the increasing of tip size for alginate 5%(w/w) 105 ± 0.006 0.71 28.6 arrays from 57  μm to 81  μm. However, even if CMC is Alginate sharper than alginate for a concentration of 3%(w/w), 1%(w/w) 128 ± 0.2 0.87 13.0 alginate pierce more often the aluminum foil: alginate 3%(w/w) 115 ± 0.003 0.78 21.8 arrays are mechanically more stable than CMC arrays 5%(w/w) 105 ± 0.003 0.71 29.2 for 3%(w/w) concentration. To go further, if comparing Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 10 of 13 Fig. 10 Piercing capabilities as a function of the concentration for each material (HA (grey), CMC (black) and alginate (white)) 3%(w/w) CMC and 5%(w/w) alginate, the same number Finally, array made of alginate took approximately 2 min of arrays, successfully penetrated the aluminum foil, with for 3%(w/w) and more than 3 min 30 s for 5%(w/w). a tip size twice bigger for alginate. This corroborate the Figure 12, carbon skeleton of HA, CMC and alginate mechanical strength of the alginate compared to CMC. are presented. HA includes small pendant groups com- Two parameters rose as key features for a microneedles posed of one carboxylic group, one amid group and array: a sharp tip as well as strong mechanical properties. several alcoholic functions by recurring unit (Fig. 12a). Indeed, even if HA appeared less strong than alginate, CMC presents a smaller recurring unit, composed they fully succeeded in piercing the aluminum foil thanks of alcohol terminated group or 2 carbons carboxylic to their sharp tip. CMC resulted in weaker arrays than group (Fig. 12b). Finally, alginate is composed of only 1 alginate but given their sharp tips, succeeded more often carbon carboxylic group and alcohol (Fig. 12c). than alginate array for a concentration of 3%(w/w). The HA by its carbon skeleton allows a quicker dis- converse was true for 5%(w/w) concentration. solution. Indeed, the polymer chain is not sterically Finally, the arrays passing the tests were put through hindered allowing water to penetrate between the the dissolution test. There was no point in testing arrays polymer chain to dissolve it. Less H bonds are notice- that were not strong enough to pierce the aluminum foil. able because of less couple carboxylic/alcoholic group Indeed, if they did not break the skin barrier, they could within the branched function. Regarding, CMC and not dissolve by reaching ISF. alginate, the longer time of dissolution can be explained on a chemical and physical point of view. CMC is com- posed of longer branched group, allowing a better link Dissolution time between the chain polymers. Moreover, H bonds can Figure  11 presents the dissolution time as a function of be found between the carboxylic group and the hydro- the concentration of HA, CMC, and alginate. gen of its branched group. Finally, alginate, being com- As shown in Fig. 11, when the concentration increased posed of only alcoholic group and carboxylic group, from 3  to 5%(w/w), the dissolution time increased for has a higher stability due to a large amount of H bonds. each material. It went from 1  min 30  s for HA arrays of Moreover, its bigger shrinkage, allows water to pen- 3%(w/w) to approximately 2 min for 5%(w/w). For CMC etrate less in between the polymer chain. arrays, the dissolution time was approximately 2  min From a dissolution point of view and an aspect for the against 3 min 20 s for respectively 3%(w/w) and 5%(w/w). application, a successful piercing of the skin followed B onfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 11 of 13 Fig. 11 Dissolution time as a function of the concentration for each material (HA (grey), CMC (black) and alginate (white)) Fig. 12 Molecular chain of a HA, b CMC and c alginate polymers by a fast dissolution, HA arrays at 3%(w/w) are consid- arrays and compared with each other. Considering geo- ered to yield good results. metrical aspect, each material made of 3%(w/w) yields microneedles which are the closest to the require- Conclusion ment specifications. HA arrays gave arrays composed of In this study, three bio-compatible and biodegradable microneedles with a tip size of approximately 48 ± 8  μm, materials which were broadly used, i.e., HA, CMC and a height of approximately 1033 ± 21  μm and a width alginate, were evaluated as the material for microneedle of approximately 600 ± 12  μm, shrinkage was of 8.9%. Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 12 of 13 3. Ventrelli L, Marsilio Strambini L, Barillaro G (2015) Microneedles for Arrays made of CMC yielded microneedles with a tip size transdermal biosensing: current picture and future direction. Adv Healthc of approximately 40 ± 5  μm, a height of approximately Mater 4:2606–2640. https ://doi.org/10.1002/adhm.20150 0450 1074 ± 23 μm and a width of approximately 598 ± 16  μm 4. Kleinknecht RA, Thorndike RM, Walls MM (1996) Factorial dimensions and correlates of blood, injury, injection and related medical fears: cross with a shrinkage of 7.2%. Finally, alginate array’s validation of the medical fear survey. Behav Res Ther 34:323–331. https :// microneedles resulted in a tip size of approximately doi.org/10.1016/0005‑7967(95)00072 ‑0 57 ± 4  μm, a height of approximately 1026 ± 27  μm and 5. Fassler D (1985) The fear of needles in children. Am J Orthopsychiatry 55:371–377. https ://doi.org/10.1111/j.1939‑0025.1985.tb034 52.x a width of approximately 550 ± 15  μm and a shrink- 6. Nir Y, Paz A, Sabo E, Potasman I (2003) Fear of injections in young adults: age of 21.8%. Regarding the piercing capabilities, HA at prevalence and associations. Am J Trop Med Hyg 68:341–344. https ://doi. 3%(w/w) pierced the aluminum foil successfully. Only org/10.4269/ajtmh .2003.68.341 7. Wright S, Yelland M, Heathcote K et al (2009) Fear of needles ‑nature and 87.5% of array made of CMC at 5%(w/w) and alginate at prevalence in general practice. Aust Fam Physician 38:172 3%(w/w) passed the piercing test, being the best result for 8. Samant PP, Prausnitz MR (2018) Mechanisms of sampling interstitial fluid CMC and alginate material. Fastest dissolution time were from skin using a microneedle patch. PNAS 115:4583–4588. https ://doi. org/10.1073/pnas.17167 72115 for the array made of 3%(w/w) for each material under 9. Takeuchi K, Takama N, Kim B et al (2019) Microfluidic chip to interface 2 min. Moreover, HA at 3%(w/w) yielded the fastest dis- porous microneedles for ISF collection. Biomed Microdevices 21:28. https solution with 1 min and 30 s. ://doi.org/10.1007/s1054 4‑019‑0370‑4 10. Hong X, Wei L, Wu F et al (2013) Dissolving and biodegradable micronee‑ Regarding the aspect of applications, i.e. piercing the dle technologies for transdermal sustained delivery of drug and vaccine. skin efficiently and dissolving rapidly to reveal underly - Drug Des Devel Ther 7:945–952. https ://doi.org/10.2147/DDDT.S4440 1 ing microneedles, we concluded that the array made of 11. Kim Y‑ C, Park J‑H, Prausnitz MR (2012) Microneedles for drug and vaccine delivery. Adv Drug Deliv Rev 64:1547–1568. https ://doi.org/10.1016/j. HA at 3%(w/w) as the best material and concentration addr.2012.04.005 considering specified dimensions, piercing efficiency and 12. Sivamani RK, Stoeber B, Wu GC et al (2005) Clinical microneedle injection dissolution time. of methyl nicotinate: stratum corneum penetration. Skin Res Technol 11:152–156. https ://doi.org/10.1111/j.1600‑0846.2005.00107 .x Acknowledgements 13. Nam YS, Park TG (1999) Biodegradable polymeric microcellular foams This work was supported by JSPS standard program (International Research by modified thermally induced phase separation method. Biomaterials Fellow of Japan Society for the Promotion of Science (Postdoctoral Fellow‑ 20:1783–1790. https ://doi.org/10.1016/S0142 ‑9612(99)00073 ‑3 ships for Research in Japan)). 14. 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Comparison of polymers to enhance mechanical properties of microneedles for bio-medical applications

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Abstract

To pierce through the skin and interact with the first biofluid available, microneedles should be mechanically strong. However, some polymers used to fabricate microneedles yield insufficient strength for the fabrication of arrays (PDMS, highly porous structures, etc.). To enhance mechanical properties, piercing materials can be used. They aim to pierce the skin evenly and dissolve quickly, clearing the way for underlying microneedles to interact with the interstitial fluid (ISF). Three materials—carboxymethyl cellulose (CMC), alginate, and hyaluronic acid (HA)—are discussed in this article. Low concentrations, for a quick dissolution while keeping enhancing effect, are used ranging from 1–5%(w/w) in deionized water. Their overall aspects, such as geometrical parameters (tip width, height, and width), piercing capabilities, and dissolution time, are measured and discussed. For breaking the skin barrier, two key parameters—a sharp tip and overall mechanical strength—are highlighted. Each material fails the piercing test at a concentration of 1%(w/w). Concentrations of 3%(w/w) and of 5%(w/w) are giving strong arrays able to pierce the skin. For the pur‑ pose of this study, HA at a concentration of 3%(w/w) results in arrays composed of microneedles with a tip width of 48 ± 8 μm and pierced through the foil with a dissolution time of less than 2 min. Keywords: Microneedles, Piercing materials, Enhancing materials, Drug delivery, Carboxymethyl cellulose, Alginate, Hyaluronic acid Introduction the dermis needs to be pierced to reach the blood ves- In a constantly evolving world, due to the increas- sel and nerves. However, the interstitial fluid (ISF) is ing medical efficiency and accessibility demands, the a biofluid that can be accessed without causing pain fabrication and optimization of simpler, less invasive to the subject: ISF is located above pain receptors. and safer devices have to be asserted. For example, ISF is abundantly available among all bodily fluids, standard glucose monitoring devices require blood unlike sweat, urine, or tears, which are linked to bodily for their measurement, and the blood is obtained by responses. Moreover, its composition proportional to repetitively pricking the subject’s finger throughout blood make it a good candidate for continuous moni- the day; as a broader view, drug delivery technologies toring of biomarker as well as its accessibility for local- require specific packaging (smart drug delivery, etc.) ized drug delivery [1–3]. To reach ISF, hypodermal as well as trained medical personnel (vaccine admin- needles can be superfluous, i.e. being too dangerous, istration, etc.). In addition, most biomarker sensing invasive, requiring trained medical staff, heavy waste devices require blood for analyses; for this purpose, management, thus generating anxiety and pain [4–7]. For this purpose, an array of microneedles has been investigated in recent researches. These microneedles *Correspondence: gwenbonf@iis.u‑tokyo.ac.jp can access ISF by causing minimum pain and invasive- LIMMS/CNRS‑IIS UMI 2820, The University of Tokyo, Tokyo, Japan ness [8–12]. Different microneedles serve different Full list of author information is available at the end of the article © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 2 of 13 purposes, and the four main types of them include Materials and methods plain, hollow, coated and dissolvable [13–17]. Plain Materials microneedles permit to open the way by poking a hole HA has been provided by RAPHAS Co. Ltd., Korea. in the skin for a better penetration of medicine [18]; Sodium CMC was obtained from Merck Japan (Sigma hollow microneedles are used for bio-sensing and Aldrich). Sodium alginate sample has been kindly offered drug delivery [19–21]; coated microneedles are mainly by KIMICA Corporation, Japan. Polydimethylsiloxane TM used for drug delivery [22], and finally, dissolvable (PDMS) (SILPOT 184) was obtained from Dow Corn- microneedles can serve for both bio sensing and drug ing Corp., Japan. Agarose gel was made from Fast Gene delivery purposes [23, 24]. Among the microneedle’s NE-AG01 agarose powder. All chemicals were used as category cited previously, dissolvable microneedles delivered. which are made of biodegradable and bio-compatible polymers meet all the required specifications of a new Fabrication of arrays generation of ISF interacting devices. Specific mor- The arrays were fabricated via micro-molding method. phology like highly porous or sponge like (PDMS) A female PDMS mold was fabricated by casting PDMS microneedles can be used for interacting with ISF [9, on a metal master mold and after annealing at 80  °C for 25]. Drug delivery as well as sensing efficiency relies 1  h; this master mold was designed using an array com- on robust mechanical properties, piercing capabili- prising 169 microneedles with a height of approximately ties, and dissolution time. In highly porous material or 1200  μm and bottom width of approximately 600  μm sponge like material, lack of strength is intrinsically (Fig.  1). The master mold was fabricated by electro-dis - linked to their morphology [26]. Naturally, extremely charge machining. The microneedles were in the shape of high porosity must be attained to interact with ISF, a square based pyramid with a total volume of 850 μL. further leading to a decrease in the overall mechani- For preparing the PDMS mold, a resin base and curing cal properties. To assert this issue, different enhanc- agent were mixed in the ratio of 10:1. The metal mold ing materials can be used. Porous microneedles can was placed in the mixture, degassed under vacuum at be topped with these materials; such material permits around 1  kPa, and annealed at 80  °C for 1  h in an oven. the needles to break through the skin, thereby enabling The resulting mold was peeled and used as it is. access to the ISF. They must be sufficiently strong to In order to prepare solutions of different concentra - evenly pierce the skin, while maintaining their sharp- tions for each material, the required amount of material ness, as well as dissolving rapidly after entering the was dissolved in de-ionized water (1, 3, and 5 in %(w/w) body. The main goal of this article is to help underly- ratios) and vigorously agitated at 1000  rpm using a stir- ing microneedle to have sufficient strength to pierce ring magnet. For CMC and alginate, they required 2 h for the skin without interfering with their desired applica- homogeneous solution as the dissolution was difficult. tion. For this purpose, three materials such as hyalu- u Th s, heating the solution (40  °C) gently was performed ronic acid (HA), carboxymethyl cellulose (CMC) and to help dissolution of the material. The solution of the alginate [24, 27–31], that are widely used in biomedi- material to be cast was poured into the mold, placed in a cal field, were studied. These materials are approved vacuum box for degassing at 1 kPa, and then annealed at by the United States Food and Drug Administration 60 °C for 2 h, until the array was completely dried. (FDA) as well as bio compatible and biodegradable materials [32–34]. This article discusses the com- Methods parison of these three materials by considering their Overall geometry and other aspects were measured using overall aspects, such as geometrical parameters after a 3D digital fine microscope (VC-3000, Omron Co., molding and thermal annealing, piercing capabilities, Japan). The height, width, and tip width were measured and dissolution time, to find the most suitable material and compared. A total of 50 distinct microneedles were for microneedle technology. To rapidly dissolve, and measured within five distinct arrays for each material not interfere with the desired application, the less pos- with different concentrations in order to be statistically sible material should be used; thus, low concentrations representative of each concentration for each material. are used at 1, 3 and 5%(w/w). These concentrations It is essential that the piercing properties are even are considered low regarding the use of HA, CMC and throughout the array. Uneven pressure can lead to alginate as the main material for fabricating micronee- defect while testing, inserting uncontrollable and dles [27, 30, 35]. Indeed, if too much material is used, unwanted parameters in the measurement. However, clogging of the underlying microneedle can occur, nul- apply the same pressure at every point of the array lifying the aimed application such as drug delivery and while measuring the piercing properties is difficult. bio sensing. Therefore, a press composed of two jaws was used for B onfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 3 of 13 Fig. 1 Picture of a the metal mold used for the fabrication of the PDMS master mold and b a magnified view of one microneedle composing the metal mold (the scale bar corresponds to 1 cm on a and 500 μm on b) this purpose: only the bottom jaw is mobile, allowing almost all the microneedles pierced the aluminum foil to adjust the pressure by a lever. A soft material such as and were still straight after the test; hence, this sample PDMS was placed against the array topped by a sheet of was labeled 1. To better understand the set up for pierc- aluminum foil (10  μm). Here, aluminum foil allows to ing assessment a schematic cross-section of the system separate physically the PDMS plate and the micronee- is presented Fig. 2a. The microneedle array is facing up dles array in order to be easily handled and be used to (microneedle side up), topped by an aluminum foil and simulate the skin for the dissolution properties. Alu- covered with the PDMS sheet. Figure 2b and c shows a minum foil has a shear strength higher than the skin, successful pierced aluminum foil counted as a 1. making aluminum a strict approximation of the skin Finally, the dissolution times of the samples were [36, 37]. Indeed, data from the metals handbook gave assessed by performing an experiment using an aga- a shear strength of 325  MPa for aluminum foil [36] rose gel with a concentration of 2%(w/w), to mimic the against 27  MPa for the human skin presented by Gal- mechanical properties of human skin [38]. The aim of lagher et  al. [37]. To approximate the pressure exerted the material was to pierce the skin, dissolve, and release by a patient through his/her thumb, a pressure of drug or reveal the underlying microneedle as quickly as 15 MPa was applied to each array (1500 g on a surface of possible. Thus, if the array did not have any micronee - 9 cm ) for 2 s. The bottom jaw of the press was adjusted dles left after insertion, it was considered to be dis- before applying pressure. The strength between the solved; in order to be the closest possible to actual skin, jaws was raised up to 15  MPa and maintained for 2  s aluminum foil will not be removed for dissolution test, before releasing it completely. For validation, the sam- indeed, in real life the arrays is not directly in contact of ple should be able to pierce through the aluminum foil the stratum corneum but put against the top surface of and have more than 70% of its microneedles functional the skin. The moisture of the agarose gel will only inter - i.e. still up after the test. Samples fulfilling both criteria act with the microneedle as it is in the expected appli- were labeled as 1, those failing the criteria were labeled cation where ISF and stratum corneum are separated as 0 (Fig.  2). Figure  2a presents the piercing properties with the array by the skin’s top layer. The timer was assessment immediately before applying pressure. The initialized when the array touched the gel, and it was arrays were taped to a glass slide and wrapped using an stopped upon complete dissolution of the micronee- aluminum foil. The system was placed in between the dles. It should be noted that the dissolution times of jaw and surmounted by a sheet of PDMS to simulate samples labeled as 0 in the piercing assessment were soft tissue under the skin. As depicted in Fig. 2b and c, not assessed. Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 4 of 13 Fig. 2 Picture of a the press next to the schematic cross‑section of the assessments, b an array passing the test with visible microneedles sticking out of the aluminum foil and c a piercing assessment top view were well defined and strong enough to withstand the Results and discussion pressure from a finger. Moreover, arrays made of 3 and Overall aspect 5%(w/w) could be bend and folded without breaking Considering the array aspect, the three materials exhib- (Fig.  3a–d). CMC arrays were white to translucent close ited different morphologies without any observation to HA aspect. At a concentration of 1%(w/w), CMC tool (Fig.  3). HA arrays were white to translucent. They arrays were complete and cohesive. They presented were stiff but exhibited flexibility. HA did not stick to no discontinuity of material or broken part but could the PDMS mold during peeling off owing to the oppo - be sheared if manipulated harshly. Both arrays with site hydrophilic character of both materials, PDMS being 3 and 5%(w/w) concentration presented well-defined hydrophobic [39] and HA being hydrophilic [40]. At microneedles structures but seemed less strong than HA 1%(w/w), the HA arrays were incomplete and difficult arrays at the same concentrations. Folding of the array to peel off from the mold without breaking it. At 3 and was possible without breaking for 3 and 5%(w/w). Finally, 5%(w/w), both arrays appeared strong and were detached alginate arrays went from a white to a light brown color from the mold without breaking. Their microneedles Fig. 3 Picture of a, b an array made of HA at 3%(w/w), c, d an array made of CMC at 3%(w/w), e, f an array made of alginate at 3%(w/w) (scale bar corresponds to 1 mm) B onfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 5 of 13 during annealing. However, if the thermal treatment was was measured as approximately 30°, which enables easy stopped before the 2 h the alginate gave a white and flex - penetration into skin. The tip width, height, and width ible array. Still, independent from the annealing time, of the microneedles are measured. The average values these arrays were brittle compared to HA and CMC of each parameter were used to estimate the volume and arrays. A visible shrinkage was noticeable, the array did morphology of each microneedle in the array. Micro- not cover the whole mold after the annealing process. It scopic view of the needle for each material examples are was considered that the color change was caused by such given Fig. 4. shrinkage. In addition, we observed that alginate arrays As presented Fig.  4, microneedles made of HA and shrank during an annealing process and became thicker CMC were very similar, whereas alginate microneedles and denser providing a brittle and brown array. were observed to be shrunk and bent. In Fig.  4a, HA Regarding the microneedles composing the arrays, HA microneedle appeared to be empty in its middle. The and CMC exhibited well-defined microneedles. No miss - same assumption could not be made by the picture given ing microneedles were found. However, alginate array Fig.  4b and c as for CMC and alginate made micronee- showed brittle and bent microneedles structures. They dles. Moreover, HA microneedles were well defined with - appeared to be smaller and bent at their tip (Fig.  3e). out asperities on their side. CMC microneedles came out Moreover, some microneedles had their tips broken less defined. Finally, alginate made microneedle are well (Fig. 3f ). defined but tilted or crooked, more prone to breaking HA and CMC arrays appeared more defined and flex - when inserted into the skin. ible than alginate array, with better looking microneedles Figure  5 shows the tip width as a function of the con- and no broken tips. centration, for each material studied. In the case of HA, CMC, and alginate, the tip width Geometrical consideration was the lowest for the 3%(w/w) solution of each mate- Fifty distinct microneedles were measured for geomet- rial. At a concentration of 1%(w/w), the tip width was rical evaluation. If microneedles had a tip width of less of approximately 72 ± 5  μm for HA and 67 ± 5  μm for than 50  μm and a width of approximately 500  μm, they CMC; however, it was double this value for alginate were generally considered to cause less pain [41]. Actual hence 118 ± 24  μm. This high value, for the solution at specifications on the height were not provided, except 1%(w/w), could be attributed to the low concentration the condition that it should not reach a nerve within of the solution. The entire amount of material had solidi - the body (i.e., it should be less than 1000  μm) [41, 42]. fied onto the walls of the cavities and at the base of the In this study, as stated previously, each microneedle was array; hence, there was insufficient material to maintain shaped from a mold composed of microneedles shaped the sharp tip. The tip was the sharpest at a concentration as a square pyramid, with a tip width of approximately of 3%(w/w). It was less than 48 ± 8 μm for HA, 40 ± 5 μm 30 μm, total length of approximately 1200 μm, and width for CMC, and 57 ± 4  μm for alginate. Finally, the tip of approximately 600 μm (Fig. 1). Moreover, the top angle width increased for each material; indeed, the solution Fig. 4 Optical pictures of a microneedle made of a HA at 3%(w/w), b CMC at 3%(w/w) and c alginate at 3%(w/w). (scale bar corresponds to 500 μm) Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 6 of 13 Fig. 5 Tip width as a function of the concentration (%(w/w)) for each material studied (HA (grey), CMC (black) and alginate (white)) could not completely fit the cavity during thermal treat - leads to a significant difference. For 3%(w/w) arrays, ment due to increasing viscosity, resulting in a wider tip given the height of the three materials, alginate appeared of the microneedle. For a concentration of 5%(w/w), the to have less broken tips than for 1%(w/w), raising the tip width was 48 ± 6  μm, 58 ± 7  μm and 81 ± 6  μm for height of the arrays to comparable value with HA and HA, CMC, and alginate, respectively. As stated previ- CMC. Finally, for a concentration of 5%(w/w), HA kept ously, for a painless insertion into the skin, a tip width a high value compared to CMC and alginate arrays. This of less than 50 μm is required, and both solutions of HA gap can be explained by the increasing of the viscosity of and CMC fulfilled these parameters at a concentration the solution for CMC and alginate when increasing the of 3%(w/w). However, alginate did not meet the require- concentration. This effect added to the brittle character - ments for the 1, 3, and 5%(w/w) concentrations. Alginate istic of the alginate array led to more broken tips and a arrays being more brittle than HA and CMC arrays, this lower height. high value could be attributed to broken tips. Figure  7 presents the variation of width for each array The variations in the height of the microneedles are as a function of their concentrations. plotted in Fig. 6. As shown in Fig.  7, a global tendency can be seen. For each material, some variations were noticeable, Indeed, width was lower for alginate for each concen- but microneedle array’s heights gravitated to approxi- tration. It can be attributed to the shrinkage of alginate mately 1000  μm (Fig.  6). The height of the HA arrays arrays. HA array’s width were around 600 μm. More pre- kept increasing from 1012 ± 34  μm for 1%(w/w), to cisely, respectively, a width of 610 ± 32  μm, 600 ± 12  μm, 1033 ± 21  μm at 3%(w/w) to reach 1077 ± 18  μm for and 608 ± 10  μm for 1, 3 and 5%(w/w) for HA. CMC 5%(w/w) concentration. For CMC, height started from array’s width went from 608 ± 16  μm for 1%(w/w), 1086 ± 23  μm at 1%(w/w), decreased to 1074 ± 23  μm at 598 ± 16 at 3%(w/w) to finally reach 539 ± 17  μm for 3%(w/w) and 971 ± 34 μm for 5%(w/w) concentration. For 5%(w/w). For both 1%(w/w) and 3%(w/w) concentra- the alginate arrays, height increased from 930 ± 50  μm tion, the microneedle’s made of HA and CMC appeared to 1026 ± 27  μm before decreasing to 988 ± 23  μm for to be similar. However, for a concentration of 5%(w/w), 1, 3  and 5%(w/w) concentration, respectively. Low value a shrinkage is noticeable through the geometrical meas- for alginate compared to HA and CMC can be explained urement. Indeed, by naked eye, this phenomenon was by the visible shrinkage happening for alginate arrays. not noticeable. Finally, for alginate arrays, a lower width Moreover, broken tips, especially for the 1%(w/w) arrays was expected. It ranges from 574 ± 26  μm for 1%(w/w) B onfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 7 of 13 Fig. 6 Microneedle’s height as a function of the concentration for HA (grey), CMC (black) and alginate (white) Fig. 7 Microneedle’s base width as a function of the concentration for HA (grey), CMC (black) and alginate (white) microscope confirmed the shrinkage of alginate array. to 550 ± 15  μm at 3%(w/w) to reach a final 517 ± 17  μm This phenomenon appeared to be exacerbated by at 5%(w/w) concentration. Besides appearing shrunk by the naked eye, geometrical measurement through a Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 8 of 13 increasing the concentration. To measure shrinkage, width measurement seemed to be the parameters of choice. All the measurements are summarized in Table 1. The relative error given in the table were calculated using a statistical Student’s t-distribution law for a cor- relation coefficient of 0.95. Figure  8 presents the comparative schematic of each average microneedles made in this work. Shrinkage is visible between alginate array and the other materials. Fig. 8 Schematic comparison of each microneedle’s as a function of Regarding the geometrical aspect, for HA arrays, their concentration and material used 3%(w/w) and 5%(w/w) emerged as optimal micronee- dles for helping to break the skin, close to the require- ments specifications stated in previously. CMC arrays where c is the side of the square base and h the total met the criteria introduced for a painless microneedle height. at a concentration of 3%(w/w). Finally, alginate, despite Nevertheless, the total height of the pyramid was not their shrinkage, could not reach the tip size criteria of accessible, and therefore, had to be calculated. Figure 9 less than 50 μm tip size. presents a schematic of the needle, wherein the known Since shrinkage is noticeable for CMC at 5%(w/w) value is indicated by a dotted line. and every alginate array, it had to be determined for By determining the values of the base sides of both each material. The volume of each needles was deter - pyramids (upper and lower pyramid), we can easily mined by calculating the volume using the tip size, calculate the diagonal. Using this value of the diago- height, and width measured previously. To be com- nal, the height of the small pyramid can be calculated, parable and relevant, the volume had to be calculated which in turn yields the volume of the biggest pyramid. by considering the sharpest tip i.e. tip width being the Comparing the complete pyramids for each micronee- smallest possible, tending to 0. To simplify the calcu- dle will enable us to easily compare one microneedle to lation, the microneedles were considered in the shape another. of a regular pyramid. The volume (V ) of a square based Thales theorem provides an equality where the pyramid is given by Eq. (1), half-diagonal of the small pyramid (CD) over the c × h V = (1) Table 1 Recapitulative table of  each array’s microneedle parameters %(w/w) 1 3 5 Hyaluronic acid Tip (µm) 72 ± 5 48 ± 8 48 ± 6 Height (µm) 1012 ± 34 1033 ± 21 1077 ± 18 Width (µm) 610 ± 32 600 ± 12 608 ± 10 Carboxymethyl cellulose Tip (µm) 67 ± 5 40 ± 5 58 ± 7 Height (µm) 1086 ± 23 1074 ± 23 971 ± 34 Width (µm) 608 ± 16 598 ± 16 539 ± 17 Alginate Tip (µm) 118 ± 24 57 ± 4 81 ± 6 Height (µm) 930 ± 50 1026 ± 27 988 ± 23 Width (µm) 574 ± 26 550 ± 15 517 ± 17 Fig. 9 Schematic of a single microneedle for calculating the total The relative error given in the table were calculated using a statistical Student’s pyramid’s volume t-distribution law for a correlation coefficient of 0.95 B onfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 9 of 13 half-diagonal of the whole pyramid (AB) equals the top during geometrical measurement has been confirmed. pyramid height (CE) over the total height (AE), where Finally, alginate arrays showed more shrinkage than HA (AC) is the previously measured height. and CMC. The shrinkage increased from 13 to 21.8 to reach 29.2% for 1, 3 and 5%(w/w), respectively. Previ- CD CE ous studies have also reported the shrinkage of alginate; = where AE = AC + CE (2) AB AE Walker et  al. showed that objects composed of alginate tend to experience shrinkage [44]. A shrinkage of up to When extracting the unknown CE, we end up with the 50% after 30  min of thermal treatment was observed. In Eq. (3) below, this study, the shrinkage was caused by thermal treat- CD × AC ment, as energy was transferred to the matter, enabling CE = (3) CD it to reach its equilibrium state. Moreover, for alginate AB × 1 − AB arrays, it was shown that shrinkage continues even after the completion of thermal treatment [44]. By determining CE, we can calculate AE using Eq. (2); In addition, a high shrinkage can be an obstacle for the thus, the total volume of the pyramid can be obtained. underlying microneedle arrays because it creates ten- Table  2 presents the different volumes and their com - sion, which could lead to potential breakages. A mate- parison with the actual master mold (i.e., the shrinkage). rial design that is viable for mechanical enhancement Globally, shrinkage occurred for every material. For should not exert tension on the system. In this study, the HA, shrinkage was invisible by naked eye but after calcu- best candidates for enhancement were the HA and CMC lation, all arrays shrank. Respectively, HA arrays shrank arrays, with a shrinkage of less than 10% for a concentra- of 3.6, 8.9 and 2.4% of the desired metal mold volume. tion of 3%(w/w). This value stayed under 10% of shrinkage. Low shrink - age of HA has been observed at around 6% in volume by Winter et  al. [43]. CMC arrays shrinkages increased Piercing abilities from 0 to 7.2 and finally 28.6% for respectively 1, 3 and Regarding the piercing properties, Fig.  10 shows 5%(w/w). While CMC array was thermally treated, the microneedle’s piercing as a function of the concentration water content decreased, forming a hydrogel in the mold. for each material. This hydrogel filled the mold and continued to undergo Being a binary test, the array of the microneedles with thermal treatment, leading to a shrinkage. By increas- more than 70% chance of survival were labeled as 1 other- ing the concentration, the hydrogel formation by CMC wise labeled as 0: the results were plotted as a percentage occurred sooner as a network could be formed by the of success. As expected from the geometrical and other presence of more molecules of CMC. Arrays made from overall aspect results, the weakest arrays to pierce the higher concentration led to higher shrinkage. Shrinkage aluminum foil were made of the 1%(w/w) concentration. of CMC array made of 5%(w/w) concentration observed Indeed, no microneedles survived the piercing test for these arrays for every material and every concentration. HA yielded the best results; all the samples passed the Table 2 Volume calculated by  using Eq.  (2) and  shrinkage test at concentrations of 3%(w/w) and 5%(w/w); attribut- percentage, with  standard deviation calculated able to their sharp tips. CMC had seen its arrays pierce at for a student law of 0.95 75% for the concentration at 3%(w/w) and 87.5% for its 6 3 Master mouldVolume (10  µm ) V/V total Shrinkage (%) 5%(w/w) concentration. Even if CMC tips were smaller or close to those of HA, piercing was less efficient. This result can be explained given the picture of micronee- HA dle provided Fig.  4b. CMC microneedles appeared less 1%(w/w) 142 ± 0.02 0.96 3.6 defined than HA microneedles, leading to potential 3%(w/w) 134 ± 0.003 0.91 8.9 breakage and thus less efficiency in piercing capabili - 5%(w/w) 144 ± 0.001 0.98 2.4 ties. The alginate array at a concentration of 3%(w/w) CMC appeared to pierce the aluminum foil more frequently 1%(w/w) 150 ± 0.003 1.02 0.0 than that at a concentration of 5%(w/w). This decreasing 3%(w/w) 137 ± 0.003 0.93 7.2 can be explained by the increasing of tip size for alginate 5%(w/w) 105 ± 0.006 0.71 28.6 arrays from 57  μm to 81  μm. However, even if CMC is Alginate sharper than alginate for a concentration of 3%(w/w), 1%(w/w) 128 ± 0.2 0.87 13.0 alginate pierce more often the aluminum foil: alginate 3%(w/w) 115 ± 0.003 0.78 21.8 arrays are mechanically more stable than CMC arrays 5%(w/w) 105 ± 0.003 0.71 29.2 for 3%(w/w) concentration. To go further, if comparing Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 10 of 13 Fig. 10 Piercing capabilities as a function of the concentration for each material (HA (grey), CMC (black) and alginate (white)) 3%(w/w) CMC and 5%(w/w) alginate, the same number Finally, array made of alginate took approximately 2 min of arrays, successfully penetrated the aluminum foil, with for 3%(w/w) and more than 3 min 30 s for 5%(w/w). a tip size twice bigger for alginate. This corroborate the Figure 12, carbon skeleton of HA, CMC and alginate mechanical strength of the alginate compared to CMC. are presented. HA includes small pendant groups com- Two parameters rose as key features for a microneedles posed of one carboxylic group, one amid group and array: a sharp tip as well as strong mechanical properties. several alcoholic functions by recurring unit (Fig. 12a). Indeed, even if HA appeared less strong than alginate, CMC presents a smaller recurring unit, composed they fully succeeded in piercing the aluminum foil thanks of alcohol terminated group or 2 carbons carboxylic to their sharp tip. CMC resulted in weaker arrays than group (Fig. 12b). Finally, alginate is composed of only 1 alginate but given their sharp tips, succeeded more often carbon carboxylic group and alcohol (Fig. 12c). than alginate array for a concentration of 3%(w/w). The HA by its carbon skeleton allows a quicker dis- converse was true for 5%(w/w) concentration. solution. Indeed, the polymer chain is not sterically Finally, the arrays passing the tests were put through hindered allowing water to penetrate between the the dissolution test. There was no point in testing arrays polymer chain to dissolve it. Less H bonds are notice- that were not strong enough to pierce the aluminum foil. able because of less couple carboxylic/alcoholic group Indeed, if they did not break the skin barrier, they could within the branched function. Regarding, CMC and not dissolve by reaching ISF. alginate, the longer time of dissolution can be explained on a chemical and physical point of view. CMC is com- posed of longer branched group, allowing a better link Dissolution time between the chain polymers. Moreover, H bonds can Figure  11 presents the dissolution time as a function of be found between the carboxylic group and the hydro- the concentration of HA, CMC, and alginate. gen of its branched group. Finally, alginate, being com- As shown in Fig. 11, when the concentration increased posed of only alcoholic group and carboxylic group, from 3  to 5%(w/w), the dissolution time increased for has a higher stability due to a large amount of H bonds. each material. It went from 1  min 30  s for HA arrays of Moreover, its bigger shrinkage, allows water to pen- 3%(w/w) to approximately 2 min for 5%(w/w). For CMC etrate less in between the polymer chain. arrays, the dissolution time was approximately 2  min From a dissolution point of view and an aspect for the against 3 min 20 s for respectively 3%(w/w) and 5%(w/w). application, a successful piercing of the skin followed B onfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 11 of 13 Fig. 11 Dissolution time as a function of the concentration for each material (HA (grey), CMC (black) and alginate (white)) Fig. 12 Molecular chain of a HA, b CMC and c alginate polymers by a fast dissolution, HA arrays at 3%(w/w) are consid- arrays and compared with each other. Considering geo- ered to yield good results. metrical aspect, each material made of 3%(w/w) yields microneedles which are the closest to the require- Conclusion ment specifications. HA arrays gave arrays composed of In this study, three bio-compatible and biodegradable microneedles with a tip size of approximately 48 ± 8  μm, materials which were broadly used, i.e., HA, CMC and a height of approximately 1033 ± 21  μm and a width alginate, were evaluated as the material for microneedle of approximately 600 ± 12  μm, shrinkage was of 8.9%. Bonfante et al. Micro and Nano Syst Lett (2020) 8:13 Page 12 of 13 3. Ventrelli L, Marsilio Strambini L, Barillaro G (2015) Microneedles for Arrays made of CMC yielded microneedles with a tip size transdermal biosensing: current picture and future direction. Adv Healthc of approximately 40 ± 5  μm, a height of approximately Mater 4:2606–2640. https ://doi.org/10.1002/adhm.20150 0450 1074 ± 23 μm and a width of approximately 598 ± 16  μm 4. Kleinknecht RA, Thorndike RM, Walls MM (1996) Factorial dimensions and correlates of blood, injury, injection and related medical fears: cross with a shrinkage of 7.2%. 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Micro and Nano Systems Letters – Springer Journals

Published: Jul 25, 2020

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