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Optimization of dip-coating methods for the fabrication of coated microneedles for drug delivery
Journal Pre-proof Optimization of dip-coating methods for the fabrication of coated microneedles for drug delivery Ling Liang, Yang Chen, Bao Li Zhang, Xiao Peng Zhang, Jing Ling Liu, Chang Bing Shen, Yong Cui, Xin Dong Guo PII:
S1773-2247(19)31465-0
DOI:
https://doi.org/10.1016/j.jddst.2019.101464
Reference:
JDDST 101464
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 26 September 2019 Revised Date:
1 December 2019
Accepted Date: 10 December 2019
Please cite this article as: L. Liang, Y. Chen, B.L. Zhang, X.P. Zhang, J.L. Liu, C.B. Shen, Y. Cui, X.D. Guo, Optimization of dip-coating methods for the fabrication of coated microneedles for drug delivery, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/ j.jddst.2019.101464. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Optimization of dip-coating methods for the fabrication of coated microneedles for drug delivery
Ling Liang1, Yang Chen1, Bao Li Zhang1, Xiao Peng Zhang1, Jing Ling Liu1, Chang Bing Shen2, Yong Cui2, Xin Dong Guo1,*
1
Beijing Laboratory of Biomedical Materials, College of Materials Science and
Engineering, Beijing University of Chemical Technology, Beijing, 100029, P.R. China.
2
Department of Dermatology, China-Japan Friendship Hospital, East Street Cherry
Park, Chaoyang District, Beijing, 100029, P.R. China.
*Corresponding author: Xin Dong Guo, Beijing Laboratory of Biomedical Materials, College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing, 100029, China. E-mail: [email protected].
1
Abstract As an approach of transdermal drug delivery, coated microneedles (MNs) have received extensive attention. Dip-coating method is a commonly used method in the preparation of coated MNs, due to its simple fabrication process and low cost. In this work, we proposed four different coating methods based on the dip-coating process, including dip-coating with a dam board, a roller, a fixture and a limit. The drug loading of the single MN prepared from the four methods was not significant (p > 0.05), from 15 to 16 ng. The minimum deviation of drug loading achieved 12.3% from the fixture device method. The in vitro drug delivery was also evaluated and the results revealed that the MNs fabricated from all methods could achieve about 90% drug delivery efficiency. All the results demonstrated that the dip-coating with a fixture method could guarantee a better homogeneous drug loading, which may be potential for future large-scale production of drug coated MNs.
Keywords: microneedle; dip-coating; transdermal drug delivery; coating technology
2
Graphical Abstract
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1. Introduction
Transdermal drug delivery was an attractive and effective method as compared to traditional drug delivery methods, such as oral and injection methods [1-3]. MN has been introduced as a painless method for delivering drugs into the sub-epideral skin layer. Piercing the skin layer can create micro-channels and increase the drug permeability and absorption. In the past decades [4-7], a wide range of active drugs, such as vaccines [8-11], insulin [12], DNA [13] and anticancer drugs [14], were tested for transdermal delivery through MN drug delivery. Currently, four different modes of MNs have been investigated, solid MNs, coated MNs, dissolving MNs and hollow MNs. Among these modes, coated MNs have some advantages as follows. Coated MNs are attractive for rapid delivery of high molecular weight molecules into the skin. The coating drug has a long-term stability in a solid phase [15, 16], even at room temperature. Furthermore, the fabrication process of coated MNs is relatively simple and quick. Therefore, many people have focused on the research of coated MNs, including the fabrication method [17, 18], the coating formulation [19-21] and the application of the coated MNs [22-24]. Nowadays, there are several processes to fabricate coated MNs, including dip-coating [25, 26], gas jet drying [27], spray coating [28], and piezoelectric inkjet printing [29] methods. Among these methods, dip-coating process using a micropositioning dip coater [15, 30] is the most common procedure to coat MNs because of its simplicity and its ability to coat complex shapes [15]. Han Sol Lee et al. [31] used a microstructure well having the same geometry as the MNs to fabricate bleomycin-coated MNs by dipping the MNs into the coating solution three times. Baek et al. [32] used a micropositioning device to dip the MN into the reservoir filled with the coating solution 9 times. Zhang et al. [27] used a dip-coating process that dip the MN into the coating 4
solution once or twice (simply described as dip coating in a reservoir). However, the drug coated on the MN base should be consider a waste of the drug because only MN shafts penetrate into the skin. In addition, due to the elasticity of the skin, the MN shafts could not penetrate the skin completely. The drug coated on the MN shafts near the base of the MNs may also cause a waste of the drug and decrease the drug delivery efficiency. The drug loading and drug delivery were two key factors. To increase them, we are trying to optimize the dip-coating method to address the above issues. As mentioned earlier [33], solid MNs were fabricated from polylactic acid (PLA) due to its biodegradable and biocompatible properties [33] using a hot-compaction process. PLA MNs were formed in the polydimethylsiloxane (PDMS) mold, which was fabricated with laser micro-machining method [34]. To fabricated coated MNs with simple method and less drug waste, four dip-coating methods were proposed and studied in this work, including dip-coating with a dam board, a roller, a fixture and a limit device. The standard deviation of drug loading and drug deliver efficiency of the coated MNs fabricated from the four methods were evaluated. Finally, the in vitro drug delivery was also studied to evaluate the application of the coated MNs.
2. Materials and methods
2.1. Materials The chemicals included polyvinyl alcohol (PVA, MW 6000，ACROS Organics, Geel, Belgium), sucrose (Sigma-Aldrich, St Louis, MO), sulforhodamine B (MW 559 Da, Sigma Aldrich), polylactic acid particles (L-PLA, 1.0 dL/g, Birmingham Polymer, Pelham, AL), and polydimethylsiloxane (PDMS, Sylgard 184, Dow Coring, Midland, MI). Porcine cadaver skin 5
(Pel-Freez Biologicals, Rogers, AR, USA) was used in vitro test. Deionized water with a resistivity of 18.2 MΩ·cm-1 was used for the preparation of coating solution. 2.2. Solid MN fabrication PLA was selected as MN materials because of its biocompatibility and processability [33]. Using the method mentioned previously [30], solid MNs were made of PLA particles. First, the PDMS mold was filled with PLA particles. Then, the mold was heated in vacuum drying oven (Biocool, Pilot1-2LD, China) for 40 min at 200 ℃. Press the molten PLA using a glass slide with approximately 3N force until the mold was filled and cool the mold to room temperature. Finally, the solid MN was removed from the mold for later use. In this work, two different MNs were designed, with and without limit pillars. The height, interspacing distance and the bottom cross section diameter of all MNs was 650 µm, 1000 µm and 300 µm, respectively. The both MNs contained 25 (5 × 5) needles. The only difference was that the diameter of the MN was increased from 8 mm to 16 mm due to the presence of the limit pillars. 2.3. Formulation of the coating solution The coating solution was composed of 21% (w/w) PVA, 26% (w/w) sucrose and 0.5% (w/w) sulforhodamine B (w/w). The PVA was added to increase the viscosity of the solution because of its some advantages of no toxicity, no side effect, a cheap price, good biocompatibility and solubility. The sucrose was used as a stabilizer to reduce the damage of active drugs. In this work, although the model drug (sulforhodamine B) is a model drug, to simulate the fabrication process of MNs for future real drug delivery, the same formulation of polymer matrix was used. To visually observe the results, sulforhodamine B was used as a model drug in this study. First, the 4 g of PVA was dissolved in 10 g of deionized water with stirring at 90 °C until the solution 6
becoming clear and transparent. Then, 5 g of sucrose was added to the PVA solution, which was mixed with shaking until dissolved. Finally, 100 mg of sulforhodamine B was added in the solution and mixed by stirring at room temperature until dissolved. According to demand of coating solution, the added material can be increased and decreased proportionally. 2.4. Fabrication of coated MNs In this work, coated MNs were fabricated with four different dip-coating methods. These fabrication techniques included dip-coating processes with a dam board, a roller, a fixture and a limit device. The draft of the reservoir, dam board, roller and fixture were design by AutoCAD software (Autodesk, USA), and then this device were printed by the 3D printer (Fortus 250mc, Stratasys, USA). 2.4.1. Dip-coating process with a dam board The dip-coating process using a dam board was proposed to fabricate a coated MN, as shown in Fig. 1. First, the dam board with a height of 300 µm was attached to the reservoir. Then, the coating solution was injected into the reservoir with a syringe until the liquid level is level with the upper surface of the reservoir (step ℃). By immersing the PLA MN in the coating solution for 10 s, the coating solution was coated onto the surface of the MN tips (step ℃). Afterwards, the MN was dried under a vacuum and frozen condition (step ℃). First, the temperature was cooled to -40 °C, and the MNs were stored for 1 h. Then, a vacuum condition with a pressure of -99 KPa was applied to MNs at -40 °C for 12 h, 0 °C for 1 h, and 25 °C for another 12 h in a freeze drier (Biocool, Pilot1-2LD, China). Finally, dried coated MNs were fabricated and stored for later use.
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Fig. 1. A schematic diagram of the dip-coating process with a dam board. (℃) The coating solution was injected into the reservoir with a syringe. (℃) Immerse the PLA MN in the coating solution. (℃) The MN was dried under a vacuum and frozen condition for later use.
2.4.2. Dip-coating process with a roller The dip-coating process with a roller was used to fabricate coated MNs, as shown in Fig. 2. First, the coating solution was uniformly distributed to the surface of the roller to produce a thin drug formulation film of a solution layer of about 200 µm (step ℃). The diameter of the roller was 10 mm. Then, PLA MNs were horizontally attached to the cupped device. The height of the MN tips was 50 µm lower than the top of the device. The roller was placed on the top of the device and rotated at a linear speed of 0.3 cm/s (step ℃, ℃). In the rotation process, the coating solution was attached to the surface of the MN tips. The fabricated coated MNs were dried under a vacuum and frozen condition as described in section 2.4.1.
Fig. 2. A schematic diagram of the dip-coating process with a roller. (℃) Drop the coating solution onto the roller with a dropper. (℃) and (℃) The roller was placed on the top of the device and rotated at a linear speed of 0.3 cm/s. 8
2.4.3. Dip-coating process with a fixture Before preparing coated MNs, the fixture device was fabricated. The desired upper part, plate and bottom (Fig. 3A) were first drafted in AutoCAD software (Autodesk, USA). Using this design, the upper part and the bottom were printed by the 3D printer (Fortus 250 mc, Stratasys, USA), as shown in Fig. 3B1 and B3. Besides, a polyformaldehyde (POM) plate (Fig. 3B2) was designed to separate the PLA MNs and the solution. The thickness was 300 µm and the radius was 10 mm. In the middle of the plate, 25 holes (0.15 mm in radius) were cut by the laser cutting machine (VLS3.50, 50 W, Universal Laser System, USA) by adjusting the laser power and speed. These holes corresponded to the MNs one by one. To fabricate coated MNs, the upper part, the PLA MN, the plate and the bottom were combined first, as shown in Fig. 3C1 and C2. The upper part and the bottom were connected by a snap joint. Then, the combined fixture was horizontally connected to a device that can be lifted and lowered. To enable dipping of MN shafts into the reservoir, a microscope connected to computer was assembled on the portable holder (Fig. 3C3). In the fabrication process, the portable holder moved down at a speed of 10 mm/min. At the bottom of the device, the coating solution was poured into a reservoir. When the coating solution submerged the marking line of the fixture device, the portable holder would move up at a speed of 10 mm/min. Afterwards, the fixture was taken down and the coated MN was taken out. All the fabrication process can be observed by the microscope. The fabricated MNs were dried under a vacuum and frozen condition as described in section 2.4.1.
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Fig. 3. Fabrication of coated MNs with a fixture. The images (A) show the CAD draft of the fixture ((A1) The upper part, (A2) The plate, (A3) The bottom). The images (B) show the fixture device ((B1) The upper part, (B2) The plate, (B3) The bottom). The images (C) show the schematic diagram of the dip-coating with a fixture ((C1) Separate parts of the fixture, (C2) The fixture, (C3) A schematic diagram of the fabrication process).
2.4.4. Dip-coating process with a limit A limit device was designed to fabricate coated MNs. Before fabricating coated MNs, the limit plate and limit holes were prepared. First, the desired model shapes and dimensions (Fig. 4A) 10
were drafted in AutoCAD software (Autodesk, USA). Then, the limit plate and limit holes were carved using the laser etching machine. In this work, the limit plate (Fig. 4B1) was made of polymethylmethacrylate (PMMA) plate (2 mm in the thickness) The limit holes (Fig. 4B2) were made of POM plate (300 µm in the thickness). The limit holes had the same function as the plate in the fixture, except that there were four holes for the limit pillars. The limit holes include 25 (5 × 5) holes with a radius of 0.15mm and 4 holes with a radius of 0.25mm. Finally, the limit plate and the limit holes were combined to each other with adhesive (Fig. 4B3, 4C1). In the fabrication process, a device that can be lifted and lowered was chosen, as shown in Fig. 4C3. First, the limit plate and limit holes were fastened to device that can be lifted and lowered. To observe the fabrication, a microscope was also fixed to the device. Then, the PLA MNs were placed on the limit holes (Fig. 4C2). These holes in the limit holes were corresponded to the MNs one by one. The height of the limit pillars was higher than the MNs. Therefore, the position of the patch could be limited and the MNs were not broken. The portable holder moved down at a speed of 10 mm/min. When the limit device touched the solution in the reservoir, the holder would be moved up at the speed of 10 mm/min. Finally, the fabricated MNs were taken out from the limit device and dried under a vacuum and frozen condition as described in section 2.4.1.
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Fig. 4. Fabrication of coated MNs with a limit. The (A) images show the CAD graphics of the limit device ((A1) The limit plate. (A2) The limit holes). The (B) images show the limit device ((B1) The limit plate. (B2) The limit holes. (B3) The combined limit device). The images (C) show the schematic diagram of the dip-coating with a limit ((C1) The limit device. (C2) The limit device and MNs with limit pillars. (C3) A schematic diagram of the fabrication process).
2.5. In vitro drug delivery Although coated MNs were fabricated by different methods, the usability was still unknown. In this part, coated MNs (n = 10) in each dip-coating method were inserted into porcine cadaver skin to test the insertion property. First, the skin was pretreated, including removing the hair, scraping the fat and washing the skin. Then, the coated MNs were vertically and manually inserted into the skin for 2 min and then removed. The skin and the MNs were imaged by the stereomicroscope (SZX7, Olympus, Japan). 12
2.6. Drug loading and drug delivery efficiency To test the precision and accuracy of microplate reader (Fluoroskan Ascent®, Thermo Scientific, China), the fluorescence intensity of sulforhodamine B at different concentrations was measured by microplate reader and fluorescence spectrophotometer (Perkinelmer, LS-55, USA). The date was presented in Table S1. For the microplate reader and fluorescence spectrophotometer method, the highest relative standard deviation (RSD) was around 5%, indicating the microplate reader had a good reproducibility. The measurement for sulforhodamine B analysis by the microplate reader was compared to the fluorescence spectrophotometer (Fig. S1). There was a linear relationship (n = 8) between the sulforhodamine B concentrations measured using the two methods, indicating that the sulforhodamine B analysis by the microplate reader had a good agreement with the conventional spectrophotometer. To compare the above fabrication methods, drug loading and drug delivery efficiency of coated MNs (n = 10) in each dip-coating method were tested by a microplate reader. First, prepare the sulforhodamine B solution with a concentration of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml ... (half the concentration one by one) and take 100 µl of them (n = 4) to 96-well plates. Then, measure the fluorescence intensity with a microplate reader. The logarithm of solution concentration and fluorescence intensity was taken for linear fitting, as shown in the Fig. S2. When the solution concentration is between 1.5 × 10-5 mg/ml and 0.0078 mg/ml, the two have a linear relation. The standard curves regression equation is y = 1.0091x – 2.8476, r2 = 0.9977. Limit of detection (LOD) is estimated as three times the standard deviation of the blank. The LOD (n = 10) is 9.5 × 10-6 mg/ml. Finally, mass of the sulforhodamine B in the inserted and non-inserted MNs were determined by dissolving them in deionized water through vigorous mixing, quantifying the 13
concentration of the solution via microplate reader and linear fitting equation and multiplying the measured concentration by the volume of deionized water. The amount of sulforhodamine B delivered into the skin was determined by subtracting the amount remaining on the MNs after insertion from the amount originally on non-inserted MNs. Drug delivery efficiency is the ratio of the amount of sulforhodamine B delivered into the skin to the amount of non-inserted MNs. 2.7 Statistical analysis In this work, all experiments conducted were done at least three replicated samples. All results were statistically analyzed using Origin 8.0 software and presented as mean ± standard deviation (SD). The two-paired Student’s t test was used to compare between two groups. A difference of p < 0.05 was considered to be significant.
3. Results and discussion
3.1. Fabrication of solid MNs Solid MNs were fabricated with PLA via a hot-compaction process. Fig. 5A and 5B show the MNs prepared with or without limit pillars were consisted of 25 MNs (5×5) with a 650 µm height. There were four limited pillars around the MNs in Fig. 5B, which corresponded to several holes in the limit device to achieve fast positioning and provided protection for the MNs.
Fig. 5. Solid MNs. (A) The solid MN without limit pillars. (B) The solid MN with limit pillars. 14
The both MNs contained 25 (5 × 5) needles with a height of 650 µm, an interspacing distance of 1000 µm, and a bottom cross section diameter of single needle of 300 µm. The diameter of the MN was 8 mm and 16 mm, respectively.
3.2. Dip-coating with a dam board Caudill et al. [35] fabricated coated MNs with a mask device (simply described as coating with mask device). Raised edges with a height of 200 - 500 µm were placed on the surface of the mask device. The MN shafts near to the MN base cannot penetrate the skin because of the elastic deformation of the skin, thus causing a waste of the drug coated in this portion. The deformation is approximately 230 µm [33, 36]. Considering the difference of the skin, the height of the dam board was chosen to be 300 µm. The total height of the reservoir and the dam board is higher than the MNs’, which can protect the tip of the MN from damage. The success of coated MNs for drug delivery depends on the dip-coating device. Fig. 6A shows the coated MN made with a dam board device. As can be seen from the arrays, the drug appeared to be uniformly loaded on the upper about 50% of the needles with no coating solution present on the base of the array. The amount drug loaded on the single PLA MN measured by microplate reader was 15.0 ng, which was 3 and 42-fold than that of the Zhang’ and Caudill’s prepared MNs [27, 35], respectively. The delivery efficiency was 90%, which was twice efficiency of the Zhang’s [27]. The different coating methods and drug loading of coated MNs were tabulated in Table 1.
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Fig. 6. Coated MNs fabricated with four methods. (A) The coated MN fabricated using a dam board. (B) The coated MN fabricated using a roller. (C) The coated MN fabricated using a fixture. (D) The coated MN fabricated using a limit.
Table 1. Different coating methods and drug loading of coated MNs
Drug loading(/MN)
Drug loading (ng/needle) （converting to a drug concentration of 0.5%）
Coating method
Drug concentration
Dip coating in a reservoir [27]
30%
90.5±14.5 µg（316 needles）
4.8±0.8 ng
53.3±1.6
Coating with mask devices [35]
0.7%
About 32 µg (64 needles)
0.36 ng
——
Coating with a rotating drum
17%
27.82 µg (1300 needles,
0.63 ng
——
16
Delivery efficiency (%)
[17]
2 dips)
However, it was needed to ensure the reservoir was completely horizontal and the surface of the solution was tangential to the top of the reservoir during the preparation process. In addition, the MNs hit the dam board easily in the process of portable holder moved down due to the reservoir was small, which increased the difficulty of preparation. 3.3. Dip-coating with a roller In previous study, Mahmoud [17] used the rolling of a rotating drum in a drug formulation reservoir to produce a thin drug formulation film (simply described as coating with a rotating drum). The formulation film thickness was controlled by scraping off excess solution from the drum with a doctor blade. MN tips on the array were dipped into the thin film. Inspired by it, we developed a dip-coating method with a roller device. Multiple pieces of MNs can be prepared at one time, improving work efficiency. The image of coated MNs prepared with a roller was shown in Fig. 6B. The amount of the single needle drug loading was 16.2 ng, which was 25 times drug loading of Mahmoud [17] prepared. However, it could be seen that the drug loading capacity of coated MNs located in the middle of the patch was lower than those located in the edge, which might be attributed to the uniform thickness wrapping material of roller. Uneven pressure leaded to the inconsistent thickness, resulting the different drug loading capacity of coated MNs (prepared in the same time). In addition, it was difficult to control the homogeneity of coating solution on the soft material via drop-spread, which directly influenced the homogeneity of drug load. The standard deviation of drug loading was 22.3%, which was related to the dip-coating method. In addition, the soft materials might result in contamination of the MN surface. 17
3.4. Dip-coating with a fixture To decrease the waste of the coating solution and increase the delivery efficiency, the coatings were situated only on a part of the needles. The location of coating was controlled by the micropositioner [32], which increased the preparation cost and difficulty. In this test, the plate for controlling the coating height was used, which not only reduces the cost, but also simplifies the preparation process, only needing to be immersed in the solution. Fig. 6C showed the coated MN prepared with a fixture. It was found that the drug loading of each MN was uniform due to the simple preparation process. When the portable holder moved down, the fixture and reservoir kept on a vertical line, which result in only needing the movement of the holder during the prepared process. In addition, the fixture device not only avoided the damage of the MNs, but also ensured the drug loading and uniformity by completely dipping the MN in the coating solution. The drug loading was 16.3 ng, and the standard deviation of the drug loading was 12.3%. 3.5. Dip-coating with a limit To prepare multiple MNs at one time, a limit hole device was proposed. The design of limit hole made MNs easier to insert the limit plate without damage of the MNs. Fig. 6D showed the coated MN fabricated with a limit hole device. As can be seen from the picture, the coating on the MN is not parallel to the bottom of the patch and the drug loading of the needles on both sides was not uniform, which might be related to the non-leveling of the reservoir. The drug loading was approximately 15.9 ng. The standard deviation of drug loading was 14.0%, which was higher than that prepared with fixture device. During the fabrication process, the larger of limit plate was, the higher of horizontal requirement of the reservoir should be needed. If the container take place a slightly slope, there would be an obvious decrease of the drug loading homogeneity. 18
3.6. Transdermal administration in vitro To evaluate the performance of the coated MNs, in vitro transdermal drug delivery was conducted by inserting the coated MNs into porcine cadaver skin for 2 min. After inserting and removing from the skin, surface examination of the treated skin showed an array (5×5) of clear and red dots corresponding to sites of MN penetration and coating solution deposition from the array (Fig. 7A). The bright-field (Fig. 7AI, II, III, IV), fluorescence (Fig. 7BI, II, III, IV) and the MNs (Fig. 7CI, II, III, IV) images showed that the MNs fabricated with the four methods could completely penetrate the skin and deliver the coated drug formulation into the skin. The 25 spots left the porcine cadaver skin indicated the coated MN could penetrate the skin. After wiping the skin, no residue was observed on the skin surface, indicating the coated solution had been delivered into the skin. In addition, there was few drugs residue on the coated MNs surface after insertion as shown in Fig. 7C, which explained that most of the drug could be delivered into the skin. In general, the coated microneedles prepared by these four methods could insert into the skin and deliver the drug into the skin.
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Fig. 7. Insertion of coated MNs into the porcine cadaver skin. (A) The porcine cadaver skin after the MNs insertion was shown under visible light. The scale bar = 500 µm. (B) The porcine cadaver skin after the MNs insertion was shown under fluorescence. The scale bar = 500 µm. (C) The images of MNs after insertion were taken with a fluorescence microscope (Olympus SZX7, Japan). (I) Test result of the MN fabricated using a dam board. (II) Test result of the MN fabricated using the roller. (III) Test result of the MN fabricated using a fixture. (IV) Test result of the MN fabricated using a limit.
3.7. Drug dose analysis The drug loading and drug delivery efficiency were two key factors in the application of coated MNs. As shown in Fig. 8, the drug loading was approximately 16 ng, and the coated MN prepared by the fixture has the highest drug loading (16.3 ng). All MNs were reserved after the transdermal administration test, and the amount of coated solution remaining on the MNs was 20
measured. Approximately 90% of sulforhodamine B coated on the MNs fabricated with four different methods was delivered into the skin. In conclusion, each of the coated MNs prepared by different methods could great efficiently deliver drugs.
Fig. 8. Drug loading and drug delivery efficiency of the coated MNs fabricated with four methods. (A) The MN fabricated with a dam board. (B) The MN fabricated with the roller. (C) The MN fabricated with a fixture. (D) The MN fabricated with the limit device. Error bar represents SD (n = 10). The two-paired Student’s t test was used to compare between any two groups. There was no statistical difference between any two groups (p > 0.05).
4. Conclusion
In summary, the coated MNs were successfully prepared by the four different methods and the in vitro transdermal delivery, drug loading and drug delivery efficiency were investigated. The coated MNs can penetrate into the skin and leave clear red spots on the skin, indicating that the 21
coated MNs have mechanical properties and the ability to deliver drugs. There was no significant difference (p > 0.05) in drug loading (15-16 ng) and drug delivery efficiency (85-88%) for the different methods. Altogether, the uniformity of the coated MNs drug loading was researched by standard deviation, and the MNs prepared with a fixture device had the most uniform drug loading. This method has the advantages of simple preparation process, easy operation and good homogeneity of the prepared microneedles, which is favorable for popularization and use.
Declaration of competing interest Authors declare that there is no conflict of interest.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (51673019, 51873015), the Fundamental Research Funds for the Central Universities and Research projects on biomedical transformation of China-Japan Friendship Hospital (PYBZ1817), and the long-term subsidy mechanism from the Ministry of Finance and the Ministry of Education of PRC.
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25
CRediT author statement Xin Dong Guo: Conceptualization. Yong Cui: Methodology. Ling Liang and Yang Chen: WritingOriginal draft preparation. Bao Li Zhang and Xiao Peng Zhang: Data curation. Jing Ling Liu: Investigation. Chang Bing Shen: Writing- Reviewing and Editing.
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Ling Liang, Yang Chen, Bao Li Zhang, Xiao Peng Zhang, Jing Ling Liu, Chang Bing Shen, Yong Cui, Xin Dong Guo
S1773-2247(19)31465-0
DOI:
https://doi.org/10.1016/j.jddst.2019.101464
Reference:
JDDST 101464
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 26 September 2019 Revised Date:
1 December 2019
Accepted Date: 10 December 2019
Please cite this article as: L. Liang, Y. Chen, B.L. Zhang, X.P. Zhang, J.L. Liu, C.B. Shen, Y. Cui, X.D. Guo, Optimization of dip-coating methods for the fabrication of coated microneedles for drug delivery, Journal of Drug Delivery Science and Technology (2020), doi: https://doi.org/10.1016/ j.jddst.2019.101464. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Optimization of dip-coating methods for the fabrication of coated microneedles for drug delivery
Ling Liang1, Yang Chen1, Bao Li Zhang1, Xiao Peng Zhang1, Jing Ling Liu1, Chang Bing Shen2, Yong Cui2, Xin Dong Guo1,*
1
Beijing Laboratory of Biomedical Materials, College of Materials Science and
Engineering, Beijing University of Chemical Technology, Beijing, 100029, P.R. China.
2
Department of Dermatology, China-Japan Friendship Hospital, East Street Cherry
Park, Chaoyang District, Beijing, 100029, P.R. China.
*Corresponding author: Xin Dong Guo, Beijing Laboratory of Biomedical Materials, College of Materials Science and Engineering, Beijing University of Chemical Technology, Beijing, 100029, China. E-mail: [email protected].
1
Abstract As an approach of transdermal drug delivery, coated microneedles (MNs) have received extensive attention. Dip-coating method is a commonly used method in the preparation of coated MNs, due to its simple fabrication process and low cost. In this work, we proposed four different coating methods based on the dip-coating process, including dip-coating with a dam board, a roller, a fixture and a limit. The drug loading of the single MN prepared from the four methods was not significant (p > 0.05), from 15 to 16 ng. The minimum deviation of drug loading achieved 12.3% from the fixture device method. The in vitro drug delivery was also evaluated and the results revealed that the MNs fabricated from all methods could achieve about 90% drug delivery efficiency. All the results demonstrated that the dip-coating with a fixture method could guarantee a better homogeneous drug loading, which may be potential for future large-scale production of drug coated MNs.
Keywords: microneedle; dip-coating; transdermal drug delivery; coating technology
2
Graphical Abstract
3
1. Introduction
Transdermal drug delivery was an attractive and effective method as compared to traditional drug delivery methods, such as oral and injection methods [1-3]. MN has been introduced as a painless method for delivering drugs into the sub-epideral skin layer. Piercing the skin layer can create micro-channels and increase the drug permeability and absorption. In the past decades [4-7], a wide range of active drugs, such as vaccines [8-11], insulin [12], DNA [13] and anticancer drugs [14], were tested for transdermal delivery through MN drug delivery. Currently, four different modes of MNs have been investigated, solid MNs, coated MNs, dissolving MNs and hollow MNs. Among these modes, coated MNs have some advantages as follows. Coated MNs are attractive for rapid delivery of high molecular weight molecules into the skin. The coating drug has a long-term stability in a solid phase [15, 16], even at room temperature. Furthermore, the fabrication process of coated MNs is relatively simple and quick. Therefore, many people have focused on the research of coated MNs, including the fabrication method [17, 18], the coating formulation [19-21] and the application of the coated MNs [22-24]. Nowadays, there are several processes to fabricate coated MNs, including dip-coating [25, 26], gas jet drying [27], spray coating [28], and piezoelectric inkjet printing [29] methods. Among these methods, dip-coating process using a micropositioning dip coater [15, 30] is the most common procedure to coat MNs because of its simplicity and its ability to coat complex shapes [15]. Han Sol Lee et al. [31] used a microstructure well having the same geometry as the MNs to fabricate bleomycin-coated MNs by dipping the MNs into the coating solution three times. Baek et al. [32] used a micropositioning device to dip the MN into the reservoir filled with the coating solution 9 times. Zhang et al. [27] used a dip-coating process that dip the MN into the coating 4
solution once or twice (simply described as dip coating in a reservoir). However, the drug coated on the MN base should be consider a waste of the drug because only MN shafts penetrate into the skin. In addition, due to the elasticity of the skin, the MN shafts could not penetrate the skin completely. The drug coated on the MN shafts near the base of the MNs may also cause a waste of the drug and decrease the drug delivery efficiency. The drug loading and drug delivery were two key factors. To increase them, we are trying to optimize the dip-coating method to address the above issues. As mentioned earlier [33], solid MNs were fabricated from polylactic acid (PLA) due to its biodegradable and biocompatible properties [33] using a hot-compaction process. PLA MNs were formed in the polydimethylsiloxane (PDMS) mold, which was fabricated with laser micro-machining method [34]. To fabricated coated MNs with simple method and less drug waste, four dip-coating methods were proposed and studied in this work, including dip-coating with a dam board, a roller, a fixture and a limit device. The standard deviation of drug loading and drug deliver efficiency of the coated MNs fabricated from the four methods were evaluated. Finally, the in vitro drug delivery was also studied to evaluate the application of the coated MNs.
2. Materials and methods
2.1. Materials The chemicals included polyvinyl alcohol (PVA, MW 6000，ACROS Organics, Geel, Belgium), sucrose (Sigma-Aldrich, St Louis, MO), sulforhodamine B (MW 559 Da, Sigma Aldrich), polylactic acid particles (L-PLA, 1.0 dL/g, Birmingham Polymer, Pelham, AL), and polydimethylsiloxane (PDMS, Sylgard 184, Dow Coring, Midland, MI). Porcine cadaver skin 5
(Pel-Freez Biologicals, Rogers, AR, USA) was used in vitro test. Deionized water with a resistivity of 18.2 MΩ·cm-1 was used for the preparation of coating solution. 2.2. Solid MN fabrication PLA was selected as MN materials because of its biocompatibility and processability [33]. Using the method mentioned previously [30], solid MNs were made of PLA particles. First, the PDMS mold was filled with PLA particles. Then, the mold was heated in vacuum drying oven (Biocool, Pilot1-2LD, China) for 40 min at 200 ℃. Press the molten PLA using a glass slide with approximately 3N force until the mold was filled and cool the mold to room temperature. Finally, the solid MN was removed from the mold for later use. In this work, two different MNs were designed, with and without limit pillars. The height, interspacing distance and the bottom cross section diameter of all MNs was 650 µm, 1000 µm and 300 µm, respectively. The both MNs contained 25 (5 × 5) needles. The only difference was that the diameter of the MN was increased from 8 mm to 16 mm due to the presence of the limit pillars. 2.3. Formulation of the coating solution The coating solution was composed of 21% (w/w) PVA, 26% (w/w) sucrose and 0.5% (w/w) sulforhodamine B (w/w). The PVA was added to increase the viscosity of the solution because of its some advantages of no toxicity, no side effect, a cheap price, good biocompatibility and solubility. The sucrose was used as a stabilizer to reduce the damage of active drugs. In this work, although the model drug (sulforhodamine B) is a model drug, to simulate the fabrication process of MNs for future real drug delivery, the same formulation of polymer matrix was used. To visually observe the results, sulforhodamine B was used as a model drug in this study. First, the 4 g of PVA was dissolved in 10 g of deionized water with stirring at 90 °C until the solution 6
becoming clear and transparent. Then, 5 g of sucrose was added to the PVA solution, which was mixed with shaking until dissolved. Finally, 100 mg of sulforhodamine B was added in the solution and mixed by stirring at room temperature until dissolved. According to demand of coating solution, the added material can be increased and decreased proportionally. 2.4. Fabrication of coated MNs In this work, coated MNs were fabricated with four different dip-coating methods. These fabrication techniques included dip-coating processes with a dam board, a roller, a fixture and a limit device. The draft of the reservoir, dam board, roller and fixture were design by AutoCAD software (Autodesk, USA), and then this device were printed by the 3D printer (Fortus 250mc, Stratasys, USA). 2.4.1. Dip-coating process with a dam board The dip-coating process using a dam board was proposed to fabricate a coated MN, as shown in Fig. 1. First, the dam board with a height of 300 µm was attached to the reservoir. Then, the coating solution was injected into the reservoir with a syringe until the liquid level is level with the upper surface of the reservoir (step ℃). By immersing the PLA MN in the coating solution for 10 s, the coating solution was coated onto the surface of the MN tips (step ℃). Afterwards, the MN was dried under a vacuum and frozen condition (step ℃). First, the temperature was cooled to -40 °C, and the MNs were stored for 1 h. Then, a vacuum condition with a pressure of -99 KPa was applied to MNs at -40 °C for 12 h, 0 °C for 1 h, and 25 °C for another 12 h in a freeze drier (Biocool, Pilot1-2LD, China). Finally, dried coated MNs were fabricated and stored for later use.
7
Fig. 1. A schematic diagram of the dip-coating process with a dam board. (℃) The coating solution was injected into the reservoir with a syringe. (℃) Immerse the PLA MN in the coating solution. (℃) The MN was dried under a vacuum and frozen condition for later use.
2.4.2. Dip-coating process with a roller The dip-coating process with a roller was used to fabricate coated MNs, as shown in Fig. 2. First, the coating solution was uniformly distributed to the surface of the roller to produce a thin drug formulation film of a solution layer of about 200 µm (step ℃). The diameter of the roller was 10 mm. Then, PLA MNs were horizontally attached to the cupped device. The height of the MN tips was 50 µm lower than the top of the device. The roller was placed on the top of the device and rotated at a linear speed of 0.3 cm/s (step ℃, ℃). In the rotation process, the coating solution was attached to the surface of the MN tips. The fabricated coated MNs were dried under a vacuum and frozen condition as described in section 2.4.1.
Fig. 2. A schematic diagram of the dip-coating process with a roller. (℃) Drop the coating solution onto the roller with a dropper. (℃) and (℃) The roller was placed on the top of the device and rotated at a linear speed of 0.3 cm/s. 8
2.4.3. Dip-coating process with a fixture Before preparing coated MNs, the fixture device was fabricated. The desired upper part, plate and bottom (Fig. 3A) were first drafted in AutoCAD software (Autodesk, USA). Using this design, the upper part and the bottom were printed by the 3D printer (Fortus 250 mc, Stratasys, USA), as shown in Fig. 3B1 and B3. Besides, a polyformaldehyde (POM) plate (Fig. 3B2) was designed to separate the PLA MNs and the solution. The thickness was 300 µm and the radius was 10 mm. In the middle of the plate, 25 holes (0.15 mm in radius) were cut by the laser cutting machine (VLS3.50, 50 W, Universal Laser System, USA) by adjusting the laser power and speed. These holes corresponded to the MNs one by one. To fabricate coated MNs, the upper part, the PLA MN, the plate and the bottom were combined first, as shown in Fig. 3C1 and C2. The upper part and the bottom were connected by a snap joint. Then, the combined fixture was horizontally connected to a device that can be lifted and lowered. To enable dipping of MN shafts into the reservoir, a microscope connected to computer was assembled on the portable holder (Fig. 3C3). In the fabrication process, the portable holder moved down at a speed of 10 mm/min. At the bottom of the device, the coating solution was poured into a reservoir. When the coating solution submerged the marking line of the fixture device, the portable holder would move up at a speed of 10 mm/min. Afterwards, the fixture was taken down and the coated MN was taken out. All the fabrication process can be observed by the microscope. The fabricated MNs were dried under a vacuum and frozen condition as described in section 2.4.1.
9
Fig. 3. Fabrication of coated MNs with a fixture. The images (A) show the CAD draft of the fixture ((A1) The upper part, (A2) The plate, (A3) The bottom). The images (B) show the fixture device ((B1) The upper part, (B2) The plate, (B3) The bottom). The images (C) show the schematic diagram of the dip-coating with a fixture ((C1) Separate parts of the fixture, (C2) The fixture, (C3) A schematic diagram of the fabrication process).
2.4.4. Dip-coating process with a limit A limit device was designed to fabricate coated MNs. Before fabricating coated MNs, the limit plate and limit holes were prepared. First, the desired model shapes and dimensions (Fig. 4A) 10
were drafted in AutoCAD software (Autodesk, USA). Then, the limit plate and limit holes were carved using the laser etching machine. In this work, the limit plate (Fig. 4B1) was made of polymethylmethacrylate (PMMA) plate (2 mm in the thickness) The limit holes (Fig. 4B2) were made of POM plate (300 µm in the thickness). The limit holes had the same function as the plate in the fixture, except that there were four holes for the limit pillars. The limit holes include 25 (5 × 5) holes with a radius of 0.15mm and 4 holes with a radius of 0.25mm. Finally, the limit plate and the limit holes were combined to each other with adhesive (Fig. 4B3, 4C1). In the fabrication process, a device that can be lifted and lowered was chosen, as shown in Fig. 4C3. First, the limit plate and limit holes were fastened to device that can be lifted and lowered. To observe the fabrication, a microscope was also fixed to the device. Then, the PLA MNs were placed on the limit holes (Fig. 4C2). These holes in the limit holes were corresponded to the MNs one by one. The height of the limit pillars was higher than the MNs. Therefore, the position of the patch could be limited and the MNs were not broken. The portable holder moved down at a speed of 10 mm/min. When the limit device touched the solution in the reservoir, the holder would be moved up at the speed of 10 mm/min. Finally, the fabricated MNs were taken out from the limit device and dried under a vacuum and frozen condition as described in section 2.4.1.
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Fig. 4. Fabrication of coated MNs with a limit. The (A) images show the CAD graphics of the limit device ((A1) The limit plate. (A2) The limit holes). The (B) images show the limit device ((B1) The limit plate. (B2) The limit holes. (B3) The combined limit device). The images (C) show the schematic diagram of the dip-coating with a limit ((C1) The limit device. (C2) The limit device and MNs with limit pillars. (C3) A schematic diagram of the fabrication process).
2.5. In vitro drug delivery Although coated MNs were fabricated by different methods, the usability was still unknown. In this part, coated MNs (n = 10) in each dip-coating method were inserted into porcine cadaver skin to test the insertion property. First, the skin was pretreated, including removing the hair, scraping the fat and washing the skin. Then, the coated MNs were vertically and manually inserted into the skin for 2 min and then removed. The skin and the MNs were imaged by the stereomicroscope (SZX7, Olympus, Japan). 12
2.6. Drug loading and drug delivery efficiency To test the precision and accuracy of microplate reader (Fluoroskan Ascent®, Thermo Scientific, China), the fluorescence intensity of sulforhodamine B at different concentrations was measured by microplate reader and fluorescence spectrophotometer (Perkinelmer, LS-55, USA). The date was presented in Table S1. For the microplate reader and fluorescence spectrophotometer method, the highest relative standard deviation (RSD) was around 5%, indicating the microplate reader had a good reproducibility. The measurement for sulforhodamine B analysis by the microplate reader was compared to the fluorescence spectrophotometer (Fig. S1). There was a linear relationship (n = 8) between the sulforhodamine B concentrations measured using the two methods, indicating that the sulforhodamine B analysis by the microplate reader had a good agreement with the conventional spectrophotometer. To compare the above fabrication methods, drug loading and drug delivery efficiency of coated MNs (n = 10) in each dip-coating method were tested by a microplate reader. First, prepare the sulforhodamine B solution with a concentration of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml ... (half the concentration one by one) and take 100 µl of them (n = 4) to 96-well plates. Then, measure the fluorescence intensity with a microplate reader. The logarithm of solution concentration and fluorescence intensity was taken for linear fitting, as shown in the Fig. S2. When the solution concentration is between 1.5 × 10-5 mg/ml and 0.0078 mg/ml, the two have a linear relation. The standard curves regression equation is y = 1.0091x – 2.8476, r2 = 0.9977. Limit of detection (LOD) is estimated as three times the standard deviation of the blank. The LOD (n = 10) is 9.5 × 10-6 mg/ml. Finally, mass of the sulforhodamine B in the inserted and non-inserted MNs were determined by dissolving them in deionized water through vigorous mixing, quantifying the 13
concentration of the solution via microplate reader and linear fitting equation and multiplying the measured concentration by the volume of deionized water. The amount of sulforhodamine B delivered into the skin was determined by subtracting the amount remaining on the MNs after insertion from the amount originally on non-inserted MNs. Drug delivery efficiency is the ratio of the amount of sulforhodamine B delivered into the skin to the amount of non-inserted MNs. 2.7 Statistical analysis In this work, all experiments conducted were done at least three replicated samples. All results were statistically analyzed using Origin 8.0 software and presented as mean ± standard deviation (SD). The two-paired Student’s t test was used to compare between two groups. A difference of p < 0.05 was considered to be significant.
3. Results and discussion
3.1. Fabrication of solid MNs Solid MNs were fabricated with PLA via a hot-compaction process. Fig. 5A and 5B show the MNs prepared with or without limit pillars were consisted of 25 MNs (5×5) with a 650 µm height. There were four limited pillars around the MNs in Fig. 5B, which corresponded to several holes in the limit device to achieve fast positioning and provided protection for the MNs.
Fig. 5. Solid MNs. (A) The solid MN without limit pillars. (B) The solid MN with limit pillars. 14
The both MNs contained 25 (5 × 5) needles with a height of 650 µm, an interspacing distance of 1000 µm, and a bottom cross section diameter of single needle of 300 µm. The diameter of the MN was 8 mm and 16 mm, respectively.
3.2. Dip-coating with a dam board Caudill et al. [35] fabricated coated MNs with a mask device (simply described as coating with mask device). Raised edges with a height of 200 - 500 µm were placed on the surface of the mask device. The MN shafts near to the MN base cannot penetrate the skin because of the elastic deformation of the skin, thus causing a waste of the drug coated in this portion. The deformation is approximately 230 µm [33, 36]. Considering the difference of the skin, the height of the dam board was chosen to be 300 µm. The total height of the reservoir and the dam board is higher than the MNs’, which can protect the tip of the MN from damage. The success of coated MNs for drug delivery depends on the dip-coating device. Fig. 6A shows the coated MN made with a dam board device. As can be seen from the arrays, the drug appeared to be uniformly loaded on the upper about 50% of the needles with no coating solution present on the base of the array. The amount drug loaded on the single PLA MN measured by microplate reader was 15.0 ng, which was 3 and 42-fold than that of the Zhang’ and Caudill’s prepared MNs [27, 35], respectively. The delivery efficiency was 90%, which was twice efficiency of the Zhang’s [27]. The different coating methods and drug loading of coated MNs were tabulated in Table 1.
15
Fig. 6. Coated MNs fabricated with four methods. (A) The coated MN fabricated using a dam board. (B) The coated MN fabricated using a roller. (C) The coated MN fabricated using a fixture. (D) The coated MN fabricated using a limit.
Table 1. Different coating methods and drug loading of coated MNs
Drug loading(/MN)
Drug loading (ng/needle) （converting to a drug concentration of 0.5%）
Coating method
Drug concentration
Dip coating in a reservoir [27]
30%
90.5±14.5 µg（316 needles）
4.8±0.8 ng
53.3±1.6
Coating with mask devices [35]
0.7%
About 32 µg (64 needles)
0.36 ng
——
Coating with a rotating drum
17%
27.82 µg (1300 needles,
0.63 ng
——
16
Delivery efficiency (%)
[17]
2 dips)
However, it was needed to ensure the reservoir was completely horizontal and the surface of the solution was tangential to the top of the reservoir during the preparation process. In addition, the MNs hit the dam board easily in the process of portable holder moved down due to the reservoir was small, which increased the difficulty of preparation. 3.3. Dip-coating with a roller In previous study, Mahmoud [17] used the rolling of a rotating drum in a drug formulation reservoir to produce a thin drug formulation film (simply described as coating with a rotating drum). The formulation film thickness was controlled by scraping off excess solution from the drum with a doctor blade. MN tips on the array were dipped into the thin film. Inspired by it, we developed a dip-coating method with a roller device. Multiple pieces of MNs can be prepared at one time, improving work efficiency. The image of coated MNs prepared with a roller was shown in Fig. 6B. The amount of the single needle drug loading was 16.2 ng, which was 25 times drug loading of Mahmoud [17] prepared. However, it could be seen that the drug loading capacity of coated MNs located in the middle of the patch was lower than those located in the edge, which might be attributed to the uniform thickness wrapping material of roller. Uneven pressure leaded to the inconsistent thickness, resulting the different drug loading capacity of coated MNs (prepared in the same time). In addition, it was difficult to control the homogeneity of coating solution on the soft material via drop-spread, which directly influenced the homogeneity of drug load. The standard deviation of drug loading was 22.3%, which was related to the dip-coating method. In addition, the soft materials might result in contamination of the MN surface. 17
3.4. Dip-coating with a fixture To decrease the waste of the coating solution and increase the delivery efficiency, the coatings were situated only on a part of the needles. The location of coating was controlled by the micropositioner [32], which increased the preparation cost and difficulty. In this test, the plate for controlling the coating height was used, which not only reduces the cost, but also simplifies the preparation process, only needing to be immersed in the solution. Fig. 6C showed the coated MN prepared with a fixture. It was found that the drug loading of each MN was uniform due to the simple preparation process. When the portable holder moved down, the fixture and reservoir kept on a vertical line, which result in only needing the movement of the holder during the prepared process. In addition, the fixture device not only avoided the damage of the MNs, but also ensured the drug loading and uniformity by completely dipping the MN in the coating solution. The drug loading was 16.3 ng, and the standard deviation of the drug loading was 12.3%. 3.5. Dip-coating with a limit To prepare multiple MNs at one time, a limit hole device was proposed. The design of limit hole made MNs easier to insert the limit plate without damage of the MNs. Fig. 6D showed the coated MN fabricated with a limit hole device. As can be seen from the picture, the coating on the MN is not parallel to the bottom of the patch and the drug loading of the needles on both sides was not uniform, which might be related to the non-leveling of the reservoir. The drug loading was approximately 15.9 ng. The standard deviation of drug loading was 14.0%, which was higher than that prepared with fixture device. During the fabrication process, the larger of limit plate was, the higher of horizontal requirement of the reservoir should be needed. If the container take place a slightly slope, there would be an obvious decrease of the drug loading homogeneity. 18
3.6. Transdermal administration in vitro To evaluate the performance of the coated MNs, in vitro transdermal drug delivery was conducted by inserting the coated MNs into porcine cadaver skin for 2 min. After inserting and removing from the skin, surface examination of the treated skin showed an array (5×5) of clear and red dots corresponding to sites of MN penetration and coating solution deposition from the array (Fig. 7A). The bright-field (Fig. 7AI, II, III, IV), fluorescence (Fig. 7BI, II, III, IV) and the MNs (Fig. 7CI, II, III, IV) images showed that the MNs fabricated with the four methods could completely penetrate the skin and deliver the coated drug formulation into the skin. The 25 spots left the porcine cadaver skin indicated the coated MN could penetrate the skin. After wiping the skin, no residue was observed on the skin surface, indicating the coated solution had been delivered into the skin. In addition, there was few drugs residue on the coated MNs surface after insertion as shown in Fig. 7C, which explained that most of the drug could be delivered into the skin. In general, the coated microneedles prepared by these four methods could insert into the skin and deliver the drug into the skin.
19
Fig. 7. Insertion of coated MNs into the porcine cadaver skin. (A) The porcine cadaver skin after the MNs insertion was shown under visible light. The scale bar = 500 µm. (B) The porcine cadaver skin after the MNs insertion was shown under fluorescence. The scale bar = 500 µm. (C) The images of MNs after insertion were taken with a fluorescence microscope (Olympus SZX7, Japan). (I) Test result of the MN fabricated using a dam board. (II) Test result of the MN fabricated using the roller. (III) Test result of the MN fabricated using a fixture. (IV) Test result of the MN fabricated using a limit.
3.7. Drug dose analysis The drug loading and drug delivery efficiency were two key factors in the application of coated MNs. As shown in Fig. 8, the drug loading was approximately 16 ng, and the coated MN prepared by the fixture has the highest drug loading (16.3 ng). All MNs were reserved after the transdermal administration test, and the amount of coated solution remaining on the MNs was 20
measured. Approximately 90% of sulforhodamine B coated on the MNs fabricated with four different methods was delivered into the skin. In conclusion, each of the coated MNs prepared by different methods could great efficiently deliver drugs.
Fig. 8. Drug loading and drug delivery efficiency of the coated MNs fabricated with four methods. (A) The MN fabricated with a dam board. (B) The MN fabricated with the roller. (C) The MN fabricated with a fixture. (D) The MN fabricated with the limit device. Error bar represents SD (n = 10). The two-paired Student’s t test was used to compare between any two groups. There was no statistical difference between any two groups (p > 0.05).
4. Conclusion
In summary, the coated MNs were successfully prepared by the four different methods and the in vitro transdermal delivery, drug loading and drug delivery efficiency were investigated. The coated MNs can penetrate into the skin and leave clear red spots on the skin, indicating that the 21
coated MNs have mechanical properties and the ability to deliver drugs. There was no significant difference (p > 0.05) in drug loading (15-16 ng) and drug delivery efficiency (85-88%) for the different methods. Altogether, the uniformity of the coated MNs drug loading was researched by standard deviation, and the MNs prepared with a fixture device had the most uniform drug loading. This method has the advantages of simple preparation process, easy operation and good homogeneity of the prepared microneedles, which is favorable for popularization and use.
Declaration of competing interest Authors declare that there is no conflict of interest.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (51673019, 51873015), the Fundamental Research Funds for the Central Universities and Research projects on biomedical transformation of China-Japan Friendship Hospital (PYBZ1817), and the long-term subsidy mechanism from the Ministry of Finance and the Ministry of Education of PRC.
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25
CRediT author statement Xin Dong Guo: Conceptualization. Yong Cui: Methodology. Ling Liang and Yang Chen: WritingOriginal draft preparation. Bao Li Zhang and Xiao Peng Zhang: Data curation. Jing Ling Liu: Investigation. Chang Bing Shen: Writing- Reviewing and Editing.
Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
Ling Liang, Yang Chen, Bao Li Zhang, Xiao Peng Zhang, Jing Ling Liu, Chang Bing Shen, Yong Cui, Xin Dong Guo