Dissolving Microneedle Array Patches Manufactured By Solvent Casting Technique and Essential Characterization of Microneedle-Based Biomedical Devices

Summary

The present work aims to serve as a practical guide enabling researchers to prepare their own DMAP devices using the solvent-casting method, also known as micromolding, and to perform basic characterization.

Abstract

Microneedle-based technology has revolutionized the field of skin and transdermal delivery research and is considered nowadays as the most straightforward approach to administering drugs through the skin. Microneedle Array Patches (MAPs) success is mainly due to their ability to nullify the skin barrier function that opposes the entrance of topically applied drugs by painless piercing the stratum corneum layer. Dissolving MAPs (DMAPs) stand out among other MAPs-based device types due to the biocompatibility of their constituent materials, the possibility of self-administration by patients, and the absence of sharp waste generated after use. DMAPs are usually made of polymeric materials that redisperse upon insertion into the skin when in touch with interstitial fluids, releasing their cargo. At the bench level, DMAPs are commonly manufactured using the solvent casting method, which involves the use of negative master molds of the desired MAPs shape and length, onto which drug-loaded polymeric dispersions are cast. They are subsequently forced to fill the mold's cavities by the application of a centrifugal force or positive pressure. After drying, DMAPs become solid and are peeled off from the master molds, and they routinely undergo various characterization studies before use. These assays typically comprise the study of the mechanical properties of DMAPs, determination of residual water content, ex vivo capacity to penetrate the skin structure, drug release performance, and in vitro assessment of biocompatibility.

Introduction

Although topical and transdermal administration of pharmacologically active substances through and into the skin is considered a promising alternative to oral drug administration, it presents significant challenges due to the natural skin barrier function exerted by the stratum corneum layer. This limitation especially affects large molecules, particularly those with a molecular weight >500 Da¹. Additionally, passive diffusion through the skin of other molecules with specific physicochemical properties, such as notable hydrophilicity or lipophilicity, is also restricted. Different technological strategies have been proposed to overcome this barrier, including iontophoresis, sonophoresis, electroporation, the use of chemical enhancers, thermal methods, and nanosized systems. Nonetheless, Microneedle Array Patches (MAPs) stand out among them due to several advantages: extreme efficiency in nullifying the stratum corneum function, cost-effectiveness, a painless experience for patients, and the possibility of self-administration without the need for medical personnel².

MAPs are devices that contain micro-sized needle-like projections that pierce the stratum corneum layer and bypass the epidermal barrier. Several MAPs types are described in the literature, namely: solid, coated, dissolving, swelling, and hollow MAPs. Dissolving MAPs (DMAPs) are composed of water-soluble polymeric materials and employ a "poke and release" strategy, which accounts for their extra interesting features. Particularly, they dissolve upon insertion into the skin structure when in contact with interstitial fluids, releasing their content. In comparison with the "poke and patch" strategy performed by solid MAPs, where they are used to create microchannels within the skin structure that allow the passage of a subsequently topically applied formulation^3,4, the "poke and release" approach is not subjected to the time that microchannels remain open. The "poke and flow" strategy requires the preparation of hollow MAPs, whose architecture is considerably more complex and difficult to manufacture. An additional advantage of DMAPs is that they do not generate sharp waste after application, thereby reducing the risk of needle-stick injuries and biological contamination. Moreover, their simple and minimally invasive application enables potential self-administration by patients, supporting their use in resource-limited or geographically remote settings where access to trained healthcare personnel, medical infrastructure, and safe sharps disposal systems is limited². The scientific community focused on dermal and transdermal drug delivery has increasingly turned to MAPs technology, particularly DMAPs, to overcome the limitations of the aforementioned strategies. Although the first publication on MAPs dates back to the 1980s, the technology experienced a major surge in the early 2000s. Since then, the number of publications has grown exponentially, reaching approximately 500 articles per year nowadays⁵.

DMAPs are commonly manufactured by the solvent-casting method or micromolding⁶. This manufacturing technique is based on the use of negative master molds of the desired MAPs shape and length. After casting polymeric dispersions loaded with drugs or even nanosized systems, an external force is applied to fill the microneedle-shaped cavities of the master mold. Once dried, DMAPs can be carefully demolded by peeling them off or dissolving the master mold with a chemical agent. These master molds are made of different materials, with polydimethylsiloxane (PDMS) being the most common, thanks to its flexibility and low adhesivity, which facilitates the demolding of DMAPs. The preparation of master molds is typically carried out using lithographic methods; however, commercial PDMS molds of various shapes, lengths, and densities of microneedles are now available.

Here, a guideline for the preparation of DMAPs using the solvent casting technique is provided. In this case, the process is performed using centrifugal force or the application of a positive pressure. This allows an efficient-cost protocol alternative to high-cost manufacturing methods of MAPs-based biomedical devices, enabling a broader group of researchers to incorporate MAPs in their skin-related research work. Additionally, the essential characterization for academic and basic research purposes that precedes in vivo testing includes needle length and geometry, mechanical resistance to compression, ex vivo insertion performance, drug loading and drug release, residual water content, stability, ex vivo drug absorption, and in vitro safety profile.

Protocol

The reagents and equipment used in this study are listed in the Table of Materials.

1. Manufacture of Dissolving Microneedle Arrays by centrifugation

1. Prepare the aqueous colloidal dispersion of the constituent materials of DMAPs by dissolving them in the appropriate amount of water and stirring vigorously. Depending on the selected polymers and concentration, the dispersion time might vary. If needed, warm up the water moderately (40-45 °C) to facilitate the dispersion of the polymer.
   NOTE: The most common polymers used to prepare DMAPs are poly(vinyl pyrrolidone) (PVP), karaya gum, hyaluronic acid (HA), poly(methyl vinyl ether alt-maleic acid) (PMVE-MA), poly(vinyl alcohol) (PVA), carboxymethyl cellulose (CMC), chitosan, etc., however, any other water-soluble material with optimal properties can be used. For most formulations, the typical working concentration ranges from 5% to 30% w/w. Lower concentrations generally produce fast-dissolving DMAPs, while higher concentrations yield slow-dissolving devices. However, the optimal concentration must be determined empirically by preparing different DMAP prototypes, as it depends on the polymer's molecular weight, viscosity, and the desired mechanical properties of the microneedles.
2. Allow the polymers to stir minimally overnight to achieve complete dispersion, while allowing air bubbles to be removed.
3. Centrifuge the polymeric dispersion if air bubbles persist (1000 x g; 5 min; 25 °C).
4. Add the cargo (drug or nanosized particle) at the desired concentration and stir moderately until it is homogeneous without incorporating new air bubbles.
5. Dispose of PDMS molds into 12-well plates. For that, accommodate the molds into the wells with modeling clay, dental cement, or any similar material.
6. Cast 50 µL of the drug-loaded polymeric blend onto the PDMS molds and centrifuge (3000 x g; 5 min; 25 °C). Afterwards, turn the plates 180°, and repeat the centrifugation process to ensure the filling of all needle-like cavities.
7. Carefully remove the excess of drug-loaded polymeric dispersion. Use a pipette to transfer the polymeric dispersion if it is liquid; use a spatula if it is highly viscous.
8. Repeat the double centrifugation process (step 1.6) to put back into the mold any drug-loaded polymeric dispersion that might have overflowed during the previous step.
9. Dry the drug-loaded polymeric dispersion in the molds under vacuum (600 mBar; 30 min; 25 °C).
10. Repeat the protocol at least twice from step 1.6-1.9 to ensure the correct filling of the master molds with the drug-loaded polymeric dispersion.
    NOTE: The centrifugal force can be progressively reduced by means of 500 x g (25 °C) in each repetition.
11. Prepare a highly concentrated polymeric dispersion free of drug to make the baseplate of the DMAPs device. It will ensure that the drug present in the dispersion filled in the mold cavities does not flow back to the baseplate.
12. Cast 50 µL of the free-drug polymeric dispersion onto the PDMS molds and spread by centrifuging twice, including midpoint plates, at a turn of 180° (500 x g; 5 min; 25 °C; 2x). After centrifugation, add 100 µL of the dispersion to each mold, followed by the addition of an additional 50 µL to the baseplate after 12 h.
13. Leave the DMAPs in the PDMS molds to dry for 3-5 days at room temperature in a dry-seal desiccator.
14. Demold the DMAPs from PDMS carefully with forceps or peel them off using scotch cellulose tape (Figure 1).
15. Characterize the manufactured DMAPs (see step 3).

2. Manufacture of Dissolving Microneedle Arrays by applying positive pressure

1. Proceed similarly as described in the previous section until step 1.5.
2. Cast 150 µL of the drug-loaded polymeric blend onto the PDMS molds and place them into a pressure tank.
3. Fill the pressure tank with air until it reaches a pressure of 3-4 bar for at least 15 min.
4. Carefully remove the excess of drug-loaded polymeric dispersion. For that, if the polymeric dispersion is liquid, use a pipette, whereas if it is considerably viscous, remove it with a spatula.
5. Repeat pressure application (steps 2.2-2.3) to put back into the mold any drug-loaded polymeric dispersion that might have overflowed during the previous step.
6. Dry the drug-loaded polymeric dispersion in the molds under vacuum (600 mbar; 30 min; 25 °C).
7. Prepare a highly concentrated polymeric dispersion free of drug to make the baseplate of the DMAPs device. It will ensure that the drug present in the dispersion filled in the mold cavities does not flow back to the baseplate.
8. Cast 0.2 g of the free-drug polymeric dispersion onto the PDMS molds and apply a positive pressure, as described in step 2.3.
9. Proceed similarly as described in the previous section from step 1.13.

3. Characterization of DMAPs

NOTE: In this step, methods involving the use of human and/or animal skin tissues were performed in accordance with institutional guidelines. Additionally, the methods involving the use of animal skin samples were carried out in accordance with the 3Rs principle for animal experimentation.

1. Visualize the DMAPs using a micro-camera with the appropriate magnification to distinguish the needle-like projections⁷.
   NOTE: If they have been loaded with a colored drug or dye, a different color must be observed between the tips and the baseplate.
2. Attach the DMAPs to a support device and visualize the morphology and length of microneedle-like projections by optical microscopy. A sharp tip structure must be observed. Record the length of microneedle-like projections with the appropriate software for your optical microscope.
   NOTE: To measure the microneedle length by optical microscopy, MAPs must be displayed perpendicular to the objectives. Length measurements and the shape of microneedle-like projections must be confirmed by Scanning Electron Microscopy.
3. Evaluate the mechanical properties of DMAPs towards the microneedles' deformation after compression to a solid surface. For that, place the DMAPs device into a commercially available DMAPs applicator and compress it against a stainless-steel flat surface for 30 s. After compressing, record the microneedle-like projections' length and calculate the deformation percentage by comparing it with the initial length according to the following equation⁷:
   
   Where, h_o and h_f stand for microneedles' length before and after the compression, respectively.
   ​NOTE: If available, the use of a texture analyzer standardizes the compression force. In that case, a force of 32 N/array mimics the average thumb force that patients apply when self-administering DMAPs devices.
4. Determine the preliminary insertion capacity of DMAPs by a surrogate artificial skin method based on thermoplastic sheets made of olefin-type material⁸. For that, compress the DMAPs device against eight bound sheets following the conditions described in step 3.4. The number of holes created in each layer is observed by optical microscopy or using a microscope camera, and the percentage of penetration at different depths is calculated.
   NOTE: Commercially available thermoplastic sheets usually present a thickness of 127 µm.
5. Determine the ex vivo insertion capacity of DMAPs using human, porcine, or rodent skin explants previously excised and carefully trimmed. Clean the skin explant with abundant water by soaking for 1-2 min in water and then place it onto an aluminum foil-wrapped foam material with the stratum corneum side facing upwards.
   1. Proceed with the insertion under the conditions described in step 3.4. Leave the DMAPs inserted into the skin until the dissolution of the microneedle tips. If DMAPs are loaded with a dye or colored drug, the coloring of the internal and external sides of the skin reveals a successful DMAPs insertion⁹. Besides, microchannels can be observed by histological analysis of skin sections⁷.
      NOTE: This step, involving ex vivo human or animal skin, was conducted in accordance with the guidelines and approval of the University of Valencia Human and Animal Research Ethics Committee. Informed consent was obtained where applicable, and all experiments adhered to institutional and European ethical standards for the use of human and animal tissues in biomedical research.
6. Determine the residual water content by Thermogravimetric analysis. Weight loss observed until 100-110 °C is attributed to the residual water¹⁰.
   NOTE: Depending on the literature source, the maximum residual water content allowed is 5%-10% of the total weight to ensure optimal drying of DMAPs.
7. Quantify the cargo loading by immersing the DMAPs in 5-10 mL of water (or alternatively, an appropriate aqueous release medium for the analyzed drug) at 32-37 °C and quantifying the cargo amount using an optimal analytical method.
   NOTE: UV-Vis spectrophotometry is commonly used for the analysis of small molecules. For compounds with low detection and quantification limits, high-performance liquid chromatography (HPLC) coupled with UV-Vis, fluorescence, or electrochemical detection is highly recommended. HPLC coupled with mass spectrometry can be employed when exceptional sensitivity and specificity are required. The analytical determination of protein and antibody cargos is typically performed using bicinchoninic acid (BCA) assays and immunoassays, such as enzyme-linked immunosorbent assay (ELISA) or Western blotting, respectively. Drug release profiles are obtained by sampling (0.2-1 mL) at predetermined time points from the release aqueous media¹¹. After every sampling point, the subtracted volume is replaced with the same volume of tempered release media in order to avoid errors related to auto-concentration of samples.
8. Assess the stability of DMAPs by repeating the above-mentioned characterization assays to ensure that DMAPs' features remain constant during the storage period under the designed storage conditions.
   NOTE: If the cargo is thermostable, DMAPs are typically stored at room temperature (25 °C). For thermosensitive active ingredients, DMAPs are stored at 4 °C. The relative humidity should be maintained below 30%. The stability of DMAPs is routinely monitored over a six-month period.
9. Determine the ex vivo drug skin deposition and absorption through the skin using a Franz-diffusion cell (FDC) setup. For this, prepare the skin as described and insert the DMAPs into the skin structure for 30 s, as described in step 3.6.
   1. Afterwards, glue the donor chamber of FDC to the skin with an appropriate amount of cyanoacrylate. When dried, attach the skin-donor chamber complex to the receptor chamber using appropriate clamps¹². Finally, proceed with the sampling protocol as described in subsection 3.5.
      NOTE: This step involving ex vivo human or animal skin was conducted in accordance with the guidelines and approval of the University of Valencia Human and Animal Research Ethics Committee. Informed consent was obtained where applicable, and all experiments adhered to institutional and European ethical standards for the use of human and animal tissues in biomedical research. If DMAPs dissolve slowly, secure them to the skin using surgical adhesive bands to prevent auto-expulsion from the skin structure.
10. Assess in vitro biocompatibility using metabolic activity assays, such as Tetrazolium-based and Resazurin-based assays, or proliferation tests, including 5-Bromo-2'-deoxyuridine bromodeoxyuridine (BrdU) assays, according to the manufacturer's instructions.
    NOTE: Preferred cell lines are keratinocytes, fibroblasts, or melanocytes. Cells unexposed to DMAPs are typically used as negative controls, whereas cells treated with 1% sodium dodecyl sulfate (SDS) serve as positive controls. Biocompatibility assessment depends on the dissolution profile of the DMAPs. For fast-dissolving DMAPs (dissolving within 24 h), the extract-based method is used, where the device is dissolved prior to cell exposure. For slow-dissolving DMAPs, the direct contact method is applied, with the device added directly to the seeded wells. According to the international standards ISO 10993 regulation, cell viability results >70% are considered non-toxic.

Results

Manufactured DMAPs must denote a homogeneous appearance with a successful formation of microneedle-like projections, as shown in Figure 2A. When a colored cargo is loaded only into the microneedle structure, a small amount of drug can contaminate the baseplate of DMAPs due to drug overflow from the tips or incomplete cleaning before casting the free-drug polymeric dispersion. However, a clear difference is observed between the two structures. When visualized by optical microscopy, microneedl...

Discussion

The protocol described here allows the preparation of DMAPs. However, the versatility and the wide range of polymeric materials that can be used for manufacturing DMAPs allow the production of dissolving devices with different features. The results presented correspond to fast-dissolving DMAPs, as they were produced with PVP, a highly water-dispersible polymer at a low concentration. The dissolution time of DMAPs can be easily tuned by simply substituting the constituent polymer or changing its concentration. Therefore, ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cell culture 12-well plate	N/A	N/A	No specifications. Any commercial cell culture 12-well plate is suitable
Centrifuge	Eppendorf	Centrifuge 5810 G	Replaceable by any other centrifuge adaptable to cell culture plates
Methylene blue	Sigma Aldrich	66720	CAS 122965-43-9. Replaceable by any other dye or brand
Modelling clay	N/A	N/A	No specifications. Any commercial modelling clay is suitable
Olefin-type thermoplastic sheets	Amcor	PM996	Parafilm M
Poly(vinyl alcohol) (PVA) MW: 9-10 kDa	Sigma Aldrich	363146	CAS 9002-89-5. Replaceable by any other brand
Poly(vinyl pyrrolidone) (PVP) MW: 40 kDa	Sigma Aldrich	PVP40	CAS 9003-39-8. Replaceable by any other brand
Polydimethylsiloxane (PDMS) molds	Micropoint Technologies	ST-06	Array needle distribution 15 x 15; Height 600 µm; Base 200 µm; Needle Pitch 500 µm. Replaceable by any other PDMS mold with other cavity shape and length
Pressure Tank	Protrima	AT-10HT	Replaceable by any other pressure tank supporting 4 bar pressure