Fabrication of Ultra-sharp Polymeric Microneedles via Computer Numerical Control (CNC) Micromachining and Micromolding

Summary

This article introduces a simple, straightforward, and innovative fabrication protocol for producing polymeric microneedles with exceptionally sharp tips through micromolding from master molds created via Computer Numerical Control (CNC) milling. The developed system is cost-effective, easy to construct, and capable of producing microneedles with exceptional sharpness for enhanced penetration, thereby improving delivery performance.

Abstract

Microneedles have recently gained recognition as a promising method in transdermal drug delivery owing to their minimally invasive, painless nature, and they can be flexibly engineered through geometric modifications to meet specific therapeutic requirements. Hence, this study aims to develop microneedle master molds utilizing Computer Numerical Control (CNC) milling technology to create polymeric microneedles with diverse degrees of sharpness to enhance therapeutic efficacy. Previously, our team successfully optimized two critical machining parameters, feed rate, and ramp angle, while analyzing their influence on the obtained needles. Hence, this article presents a comprehensive set of experimental protocols for fabricating and evaluating the performance of the polymeric-based model needles. Furthermore, this work also provides representative results to demonstrate the reproducibility and therapeutic efficacy of the sample needles. Our research team anticipates that this straightforward protocol will enable researchers to create cost-effective, high-precision microneedles, thereby advancing microneedle technology for further biomedical applications, particularly during the pandemic.

Introduction

Microneedles have emerged as a promising candidate for drug delivery in recent years due to their painless, self-administrable nature while circumventing the first-pass liver metabolism¹. Microneedles have captured much attention in many biomedical applications (e.g., vaccination, cancer treatment, diagnostics, cosmetics), which require precise, minimally invasive delivery of therapeutic agents or other substances into the skin or underlying tissues, enhancing treatment efficacy with limited adverse effects².

Depending on the intended applications, microneedles can be made in many forms (i.e., solid, coated, hollow, porous, swellable, and dissolvable), potentially for diagnostic and transdermal drug delivery applications by penetrating the stratum corneum layer³. From an engineering perspective, numerous fabrication techniques are available for microneedle production. Among these, CNC micromachining is a precise and versatile method, widely employed for creating complex three-dimensional structures with high accuracy and adaptability, making it particularly well-suited for microneedle master mold fabrication⁴. In response, our research team has previously proposed an optimized, low-cost, straightforward, and innovative approach to produce microneedle master molds with ultra-sharp tips using the CNC micromachining technique⁵. Furthermore, microneedles can also be made from a wide range of materials (e.g., metals, ceramics, polymers). Among these, polymeric microneedles have emerged as promising due to their biocompatibility, biodegradability, and non-toxicity, making them potentially useful for biomedical applications⁶.

This study introduces an optimized protocol to fabricate microneedle master molds, which are designed to produce needles with high sharpness through CNC micromachining and micromolding. This approach was inspired by our previous research, which explored master mold fabrication via CNC milling. To review the supporting data and figures related to optimizing fabrication parameters (e.g., feed rate, ramp angle), readers are encouraged to refer to the previous publication⁵. Furthermore, this work also evaluates the effectiveness of this proposed protocol by demonstrating the fabrication of a newly developed polymer-based microneedle model. To maximize the benefits of this protocol, readers are encouraged to consider their engineering specifications. For example, critical considerations involve selecting appropriate materials, milling tools, and optimized parameters (e.g., feed rate, spindle speed)⁷. Given the high precision and accuracy required in microneedle fabrication, the CNC milling process must be capable of producing structures that meet specific design criteria-such as tip diameter and diverse geometries-while offering greater flexibility compared to photolithography techniques⁸. In addition, the PDMS negative mold replica in this method demonstrated a high level of reproducibility, which can withstand multiple casting cycles without degradation, making it a potential candidate for a simple, cost-effective fabrication of microneedles with different degrees of sharpness⁹.

Protocol

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

1. Fabrication of polymethyl methacrylate (PMMA)-based microneedle master molds

1. Microneedle master mold design
   NOTE: The microneedle master mold design was conducted using Fusion 360 (Autodesk, USA, education license) (see Table of Materials).
   1. Open the software in the Design environment and create a new sketch.
   2. Draw a box using the Extrude function.
   3. Generate stepped cavities using the Extrude Cut tool.
   4. Create a single microneedle body as a cylinder.
   5. Use Chamfer to mill the cylinder to form a conical-shaped microneedle.
   6. Generate a 10 x 10 microneedle master mold using the Array command.
      NOTE: Depending on the intended applications, the dimensions of microneedles can vary. In this protocol, the demonstrated needles are formed with the following dimensions: conical shape, needle height: 600 µm, base diameter: 400 µm, and distance between needles: 1400 µm.
   7. Review and finalize the design for accuracy and consistency.
   8. Save the final designs in a compatible format (e.g., .f3d) for further processing.
2. Computer-aided manufacturing (CAM) setup
   1. Open the mold design in the CAM environment.
   2. Set a PMMA workpiece with stock on the top of the mold.
   3. Perform Face milling to remove the top surface using a flat-end mill¹⁰.
      NOTE: Set up the parameters for the flat end mill, including tool diameter (3 mm), feed rate (300 mm/min), spindle speed (3000 rpm), depth of cut (1 mm), and step over (1 mm).
   4. Use Pocket milling to machine the stepped cavities using a flat-end mill⁵.
      NOTE: Set up the parameters for the flat end mill, including tool diameter (1 mm), feed rate (300 mm/min), spindle speed (4000 rpm), depth of cut (0.5 mm), and step over (0.3 mm).
   5. Apply Pocket milling again to form cylindrical structures.
      NOTE: Set up the parameters for the flat end mill, including tool diameter (0.8 mm), feed rate (200 mm/min), spindle speed (4000 rpm), depth of cut (0.2 mm), and step over (0.2 mm).
   6. Create a PMMA conical-shaped microneedle structure using Bore milling⁵.
      NOTE: Set up the parameters for Bore milling, including tool diameter (0.4 mm), feed rate (100 mm/min), spindle speed (4500 rpm), and ramp angle (1.5°).
   7. To generate the G-code for further processing, right-click on the Setup > Create NC program. From Machine and Post tab, select Tormach PathPilot as the desired post processor. Finally, click on the Post button within the dialog box to generate G-code.
3. Computer Numerical Control (CNC) micromachining setup
   1. Polymethyl methacrylate (PMMA) workpiece preparation
      1. Obtain a PMMA workpiece block with dimensions: width: 30 mm, length: 30 mm, and height: 5 mm.
      2. Secure the PMMA block onto the machining table using clamps to prevent any movement during milling.
   2. CNC machining setup
      1. Use a 3-axis machining center with a maximum spindle speed of 5140 rpm.
      2. Select an appropriate milling tool and insert it into an ER20 collet, ensuring it is properly secured to the spindle.
   3. Cutting process
      1. Clamp the PMMA workpiece on the vice of the CNC machine.
      2. Set up the zero point for accurate positioning between the end mill and workpiece through the x, y, and z axes.
      3. Begin machining with the defined toolpath by a G-code program and cutting parameters.
      4. Start the automated cutting to fabricate the PMMA master mold.
         ​NOTE: Continuously applying an oil lubricant to the workpiece is recommended throughout the cutting process. Reset the zero point (z-axis) after changing the milling tool.
      5. Regularly monitor the process for accuracy and tool stability.
         NOTE: Once the milling is complete, carefully remove the obtained microneedle master mold from the CNC machine.
      6. Clean the machined surface using ultrasound to remove excess lubricant and debris.
4. Negative mold replica
   1. Polydimethylsiloxane (PDMS) casting solution preparation
      1. Mix Elastomer base and curing agent at a ratio of 10:1 (w/w).
      2. Vacuum the mixture for 5 min to remove air bubbles.
   2. PDMS-based negative mold fabrication process
      1. Pour the degassed PDMS mixture over the PMMA master mold with an approximate thickness of 1 mm.
      2. Vacuum the mixture-containing mold for an additional 5 minu to remove air bubbles.
   3. Curing process
      1. Cure the PDMS mold in an oven at 90 °C for 45 min to solidify the structure.
   4. Mold removal and replication
      1. Carefully remove the PDMS-cured mold from the PMMA master mold using a tweezer.
      2. Repeat the replication process to produce multiple PDMS negative molds from a single PMMA master mold.
         NOTE: The PDMS negative molds can be reused for microneedle patch fabrication, demonstrating a high throughput and efficient fabrication approach.

2. Fabrication of polymeric microneedle patches

NOTE: To evaluate the effectiveness of the proposed system, we demonstrated a sample fabrication process to produce polymer-based model microneedles composed of polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), and water-soluble chitosan (WCS), which was previously developed within our laboratory.

1. Polymeric solution preparation
   1. WCS solution preparation
      1. Prepare a 3% (w/v) WCS solution by directly dissolving WCS powder in distilled water.
         NOTE: Use a magnetic stirrer to accelerate the dissolving rate.
   2. PVA/PVP solution preparation
      NOTE: The PVA/PVP solution enhances the structural integrity of the microneedle patch by serving as its supporting base material.
      1. Prepare a PVA/PVP solution at a concentration of 7.5% (w/v) for each substance.
      2. Add the calculated amount of distilled water to the powder mixture.
      3. Mix the solution continuously using a magnetic stirrer while heating at 70 °C for 4 h until it is homogenous.
      4. Cover the solution-containing beaker with a film to prevent evaporation.
2. Microneedle patch fabrication
   NOTE: The fabrication of PVA/PVP/WCS microneedle patches from the obtained PDMS mold using the solvent-casting technique.
   1. Dispense 400 µL of the obtained WCS solution onto the negative PDMS mold using a micropipette.
   2. Place the mold in a vacuum chamber and vacuum for 15 min to remove any air bubbles and ensure the solution fills the mold cavities properly.
   3. Apply another layer of PVA/PVP solution as a base material onto the surface of the mold.
   4. Transfer the mold to an oven and dry at 65 °C for 4 h for complete solidification of the microneedle patch.
   5. Once dried, carefully separate the obtained patch from the PDMS using a tweezer.

3. Evaluation of microneedle patches

1. Mechanical strength evaluation
   1. Experimental setup for microneedle structural integrity measurement
      NOTE: This experiment was conducted using a self-developed system. Briefly, the apparatus comprises (1) a force testing station, (2) a software interface, (3) a USB microscope, and (4) a 10 N load cell.
   2. Measurement software installation
      1. Download three operation files from the software package 'Automatic Needle Injection Configuration' available in different types <.exe>, <config.>, <pdb>, in the Supplementary File 1, Supplementary File 2, and Supplementary File 3. These files correspond to the installation of this mechanical measurement software.
         NOTE: To operate the system, users only open the <.exe> file, while the other files are configuration files and do not require direct interaction.
   3. Measurement procedure
      1. Place a single microneedle on the micro-stage surface, which is attached by double-sided tape.
      2. Adjust the moving speed to 40 mm/h.
      3. Adjust the distance between the sensor and the microneedle tip until the sensor directly contacts the needle tip.
      4. Record the obtained force-displacement graph generated by the measurement software.
      5. Repeat the experiment three times for each sample.
2. Penetration evaluation
   1. Experimental setup for microneedle penetration measurement
      1. Prepare an eight-layered stack of paraffin film (PF) sheets with a thickness of 125 µm each.
      2. Place and secure the PF stack onto the micro-stage surface for stability during the experiment.
   2. Microneedle attachment
      1. Attach a single microneedle to a mechanical load cell for proper vertical insertion.
   3. Insertion procedure
      1. Set the load cell attached with a microneedle to move downward at a constant speed of 40 mm/h into the PF stack.
   4. Data collection and analysis
      1. Remove the microneedle from the PF stack.
      2. Carefully unfold the PF layers to expose each sheet.
      3. Observe the unfolded PF stack under a microscope to count the number of penetration holes in each layer.
      4. Repeat the experiment three times for each sample.

Results

This study introduces a protocol for fabricating microneedle master molds, which are engineered to achieve exceptionally sharp microneedles using CNC micromilling. To examine data about tip sharpness, readers are encouraged to refer to the previous publication⁵. Figure 1 shows the schematic view of the microneedle master mold design with its dimensions. Figure 2 illustrates the transformation of the schematic design into milling instructi...

Discussion

This study developed a unique experimental protocol to fabricate master molds to produce polymeric microneedles with excellent sharpness through CNC micromachining and micromolding. Primarily, this approach includes integrating a schematic design with the fabrication of a master mold via CNC milling, followed by a series of micromolding processes to create microneedle patches. For a long time, micromolding has been widely used due to its ability to produce intricate, reproducible microstructures with high accura...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-axis machining center	Tormach PCNC 1100		CNC mill for cutting materials
Automatic Needle Injection Configuration	Self-developed		Mechanical measurement software
Distilled water	Cole-Parmer WS-200 Series Stuart Water Stills
Fusion 360 software, Education version	Autodesk, USA	Design software (https://www.autodesk.com/asean/products/fusion-360/overview?term=1-YEAR&tab=subscription)
ImageJ software	National Institutes of Health, USA		Image analysis
Polydimethylsiloxane	SYLGARD 184 Silicone Elastomer Kit	1330-20-7	Used for negative molds
Polymethyl methacrylate			Used for master molds
Polyvinyl alcohol	Sigma Aldrich, USA	9003-39-8	Microneedle material
Polyvinyl pyrrolidone	Sigma Aldrich, USA	9002-89-5	Microneedle material
Rhodamine B	Sigma Aldrich, USA	83689-1G	Model drug
Water-soluble chitosan			Microneedle material