3D Printing of Microneedle Arrays: Challenges Towards Clinical Translation

Abstract

“3D printing is a viable strategy for production of MNAs with considerably improved design flexibilities compared with conventional manufacturing methods”

Keywords::

• 3D printing
• biomanufacturing
• biomaterials
• COVID-19
• microneedle arrays
• microneedles

Microneedles (MNs) are a rapidly emerging technology for user-friendly and minimally invasive drug delivery and biological fluid sampling applications [1]. Simple, low-cost and reproducible fabrication of microneedle arrays (MNAs) with optimal MN designs is pivotal for their widespread adaptation. Additive manufacturing or 3D printing is a viable strategy for production of MNAs with considerably improved design flexibilities compared with conventional manufacturing methods [2,3]. Here, we discuss 3D printing of MNAs toward enabling several clinical applications.

3D printing for fabrication of MNs

MNAs with diverse MN and array designs can be fabricated via 3D printing to be used for a broad range of medical applications ranging from administration of bioactive molecules (e.g., vaccines and therapeutics) to biosensing. Indeed, different types of 3D-printing technology have already been exploited to either directly manufacture MNAs or to facilitate the fabrication of MNAs through micromolding for painless and bloodless vaccination, treatment and biomarker sensing strategies [2,4,5].

Stereolithography (SLA), one of the prevailing 3D printing methods, has been effectively used for cleanroom-free and inexpensive creation of MNAs without the need for costly and specialized equipment. One study presented SLA 3D printing of MNA master molds that comprise high aspect ratio MNs with the tip radii of between 20 and 40 μm depending on process conditions [6]. These master molds were then used to fabricate silicone negative MNA molds with MN-shaped wells into which either the dissolvable material carboxymethylcellulose (CMC) mixed with RhB or poly(lactic acid) (PLA), a thermoplastic polyester, was cast by solvent-based and thermal micromolding, respectively. As expected, while thermal molding with PLA had a negligible effect on the sharpness of PLA MNs, solvent-based casting considerably reduced the tip radii of CMC MNs (<5 μm) due to shrinkage resulting from evaporation of solvent (e.g., water). Ultimately, SLA 3D printing-enabled PLA MNAs were evaluated on porcine skin, which demonstrated their successful intracutaneous penetration. Additionally, MNs can be used for enhancing the infiltration mechanism for the drugs or remedies that are loaded on the skin. To this end, utilizing SLA 3D-printing technology and various polymers including chitosan and sodium alginate, MNAs were prepared for investigating the effects of different anticellulite herbal products on a guinea pig model in vivo [7]. Several other studies [8–11] used SLA 3D printing for manufacturing of solid (then coated) or hollow MNAs for delivery of a myriad of biocargos, suggesting that SLA 3D printing provides a means for fabrication of diverse MNA types.

Fused deposition modeling (FDM) is a common 3D-printing approach that has been utilized for fabrication of MNs. Specifically, FDM-based 3D printing was used for direct manufacturing of PLA MNAs, followed by a chemical etching strategy to overcome the challenges associated with its limited resolution [12]. While FDM 3D printing of PLA resulted in MNs with tip diameters of between 170 and 220 μm, etching of these relatively blunt PLA MNs with aqueous potassium hydroxide after fabrication rendered PLA MNs with sharper tips ranging between 1 and 55 μm in diameter. Collectively, this etching-enhanced FDM 3D-printing process was capable of creating PLA MNAs that can penetrate porcine skin and deliver coated or embedded cargos into the skin, presenting a flexible technique for cost-effective fabrication of MNAs with different MN and array designs.

Digital light processing (DLP) and continuous liquid interface production (CLIP) methods have been introduced as new photopolymerization-based 3D-printing approaches with improved resolution compared with the aforementioned 3D-printing technologies for fabrication of complex structures [13,14]. While DLP technology employs layer-by-layer fabrication, CLIP uniquely enables the construction of parts or devices in a continuous fashion. One study presented that poly(propylene fumarate) (PPF)-based drug-loaded MNAs can be manufactured through DLP-assisted microstereolithography and suggested that different release profiles can be achieved by altering the molecular weight of PPF [15]. In recent studies, DLP approach was used to create complex MNs with backward-facing barbs to enable stable tissue adhesion [16] or combined with a magnetic field to reinforce the polymer matrix with microbundles of aligned iron-oxide nanoparticles to increase the mechanical rigidity of MNs [17]. Interestingly, CLIP technology enabled rapid manufacturing of MNAs with various MN shapes (e.g., pyramid and turret), dimensions and spacings from different photo-curable materials, such as trimethylolpropane triacrylate (TMPTA) as a model resin and polyethylene glycol (PEG), poly(acrylic acid) (PAA) and poly(ε-caprolactone) (PCL) as biocompatible resins, presenting its high geometric and material capabilities [18]. All materials were mixed with diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (TPO) that serves as a photo-initiator. Geometric evaluation of square pyramidal TMPTA MNs indicated that CLIP could create sharp (tip radii <3.5 μm) MNs with high accuracy (<5% difference between the actual and designed dimensions). Geometric assessment of MNs fabricated from biocompatible materials showed that PEG, PCL and PAA MNs have tip radii of 5 ± 0.4 μm, 4.5 ± 0.6 μm and 6.2 ± 1.0 μm, respectively, further confirming the capabilities of CLIP for fabrication of sharp MNs. Moreover, all MNs from different formulations were able to penetrate murine skin ex vivo without mechanical failure. More recently, spatially controlled coating of MNAs fabricated from TPO-enhanced PEG using CLIP was also demonstrated and these MNAs could deliver the coated cargos to porcine skin ex vivo and murine skin in vivo [19]. Thus, both CLIP and DLP technologies offer attractive alternatives for rapid manufacturing of MNAs with diverse MN shapes, dimensions and densities.

Two-photon polymerization (2PP)-based 3D printing has recently emerged as a viable and high-resolution technique for accurate fabrication of micro- and nano-scale features. Researchers have exploited 2PP-directed 3D laser writing to manufacture hollow and open-channel MNs [20,21], as well as to facilitate the production of dissolvable MNAs by creating solid MNA master molds [22] and presented successful skin penetration of the fabricated MNAs. A recent study exploited 2PP-3D printing to fabricate MNA master molds with sharp MNs (tip radii of 2 μm) to enable the production of multicomponent vaccine-loaded dissolving MNAs through additional molding steps [23]. These 2PP-3D printing-enabled dissolving MNAs were then tested for intracutaneous vaccination in mice and results showed that MNA-mediated skin vaccination is more effective than immunization via conventional intramuscular injection in generating antigen-specific cellular and humoral immune responses. As such, 2PP-based 3D laser writing or printing has potential as a versatile technique for accurate and reproducible fabrication of high-quality MNAs with sharp MNs.

Taken together, a growing body of literature suggests that 3D printing paves the way for rapid and simple fabrication of MNAs with a wide range of MN and array design parameters to evaluate and identify optimal MN and MNA designs toward enabling diverse vaccination, treatment and biosensing strategies. MNAs have been commonly used for skin-targeted applications over the past years. Their utilization for noncutaneous sites (e.g., ocular and cardiac tissues) is also starting to emerge [24]. Despite unprecedented progress in 3D printing of MNAs, there are still several hurdles toward clinical translation of 3D-printed MNAs.

Outlook & challenges in clinical translation of 3D printed MNs

MNs promise a simple and potentially self-administered (e.g., for skin applications) approach for safe, painless and effective delivery of a wide range of biomolecules. While scalable fabrication and commercialization of MNAs for cosmetic applications have already been achieved, their widespread adaptation for patient-friendly delivery of therapeutics and vaccines, as well as for biological fluid sampling in the clinic has yet to be realized to exploit the exponentially growing scientific findings strongly supporting the advantages of MNAs over existing established methods. Consistent application and tissue penetration of MNAs is crucial for enabling their reliable clinical application for vaccination, treatment and biomarker sampling and requires optimization of many design parameters. To address this challenge, 3D printing offers unparalleled design flexibilities to rapidly manufacture MNAs with diverse MN and array designs to enable the identification of optimal application-specific MNA designs. However, there are still remaining manufacturing, material and regulatory challenges toward effective clinical translation of 3D-printed MNAs. One barrier in clinical translation is the possible adverse effects caused by contact of the used material in MNs with the living organs. The host immune response to biodegradable materials, the group which is commonly used in implantable systems, mainly depends on their features and mechanism of degradation aside from their intrinsic characteristics [25]. A proposed solution to overcome this challenge is the integration of immunosuppressive compounds with MNs. These anti-inflammatory agents can be incorporated on MNs in a similar way that the drugs are loaded to subside potential inflammatory responses to MN insertion or materials. For instance, 3D printed polydimethylsiloxane implants were reported to be successfully coated with immunomodulatory hydrogels to control immune responses resulting from implantation [26]. Among the polymers that are of high interest in producing MNs, the natural biopolymer silk [27] and synthetic biopolymers such as PCL and PLA [25,28] have been demonstrated to be biocompatible with non-irritating degradation products, manifesting their clinical translation potential.

High-throughput fabrication of sterile MNAs with consistent dimensions and dosage (for drug and vaccine delivery) is essential for their widespread utilization in a safe and efficient manner. Indeed, the COVID-19 pandemic clearly demonstrates this significant unmet need in scalable fabrication of MNAs. Intracutaneous vaccination is an important application of MNAs to exploit the simplicity and user-friendliness of MNAs and efficient immune circuitry of the skin [29,30]. MNAs have been extensively used for the development of skin-targeted vaccines against infectious pathogens and tested in several animal studies and in a small number of clinical studies [31–34]. Recently, MNAs were also tested in mice for intracutaneous vaccination against coronaviruses, including SARS-CoV-2, with promising virus-specific immune responses [35]. Although these preclinical and clinical studies suggest that MNAs could play an important role in facilitating sustainable global immunization programs against COVID-19 and other emerging infectious diseases, scalable fabrication of sterile MNAs in a reproducible manner still needs to be addressed. Thus, it will be necessary to identify favorable (e.g., fast and inexpensive) 3D-printing technologies and postprocessing techniques (e.g., a coating method for coated MNAs or a molding strategy for dissolvable MNAs) for cost-effective mass-production of high-quality MNAs and to establish MNA manufacturing sites using these optimal 3D-printing solutions. Moreover, it will be important to confirm the viability of biocargo integrated with MNAs and the sterility of MNAs via established quality control procedures before widespread distribution of MNAs. To ensure the sterility of MNAs, they can either be fabricated under relatively costly aseptic conditions or sterilized after manufacturing depending on the type of MN and the integrated biocargo. Ultimately, 3D printing of sterile MNAs with high-quality MNs in a reproducible and scalable manner will need to be achieved to enable broad utilization of 3D-printed MNAs for a myriad of applications. Alternatively, 3D printing could be complemented with other fabrication techniques, such as micromolding and injection molding, to facilitate high-throughput manufacturing of MNAs by high-fidelity replication of 3D-printed MNAs.

Safe and effective utilization of MNAs in large populations requires identification of materials that are biocompatible, biodegradable and mechanically strong. 3D printing has greatly widened the design space of MNs due to its unique advantages; however, the material capability of 3D-printing technologies is still limited compared with biomaterials that have been used for fabrication of MNAs. For instance, solvent-based biomaterials (e.g., CMC and poly[lactic-co-glycolic acid]), which are commonly used for manufacturing of dissolving or biodegradable MNAs due to their established track record of prior US FDA approval, still pose substantial challenges in 3D printing of high-quality MNs with sharp tips. Mechanical properties of 3D-printed MNs, which could vary depending on the utilized 3D-printing technology and material, should be determined to guarantee failure-free tissue penetration of these MNs. Comprehensive studies evaluating host responses to 3D-printed MNAs manufactured from common or emerging 3D-printing materials in different tissues of animal models will be needed to ensure their safety in large populations. Identification of favorable and inert biomaterials for 3D printing of MNAs will be essential to avoid scar formation due to unexpected breakage of MNs in tissues and long-term implantation of MNAs, while also eliminating the need for surgical removal of MNAs. Moreover, it will be imperative to investigate the effect of process and material conditions on the viability and shelf-life of biocargos integrated with 3D-printed MNAs, as well as the effect of different materials on pharmacokinetics of biocargos delivered by 3D-printed MNAs. Thus, more innovative biomaterials solutions will likely emerge with 3D printing of MNAs in the coming years for both optimizing the target application and satisfying regulatory requirements.

3D-printed or 3D printing-enabled MNAs are rapidly emerging with promising preclinical results, albeit with a smaller number of functional studies in animal models compared with those performed with MNAs fabricated using more conventional techniques. Thus, more comprehensive evaluation of the safety and efficacy of these MNAs for vaccination, treatment and biological fluid sampling approaches in murine and large animal models will be needed in the future. Clinical translation of these MNAs will ultimately require large human studies to demonstrate the efficacy and safety of MNA-delivered therapeutics and vaccines prior to their widespread adaptation. The competitive advantages of MNAs over existing methods for each application will need to be clearly determined to justify initial investment on MNA technology for that particular application. Further, consistent application of MNAs and reproducible cargo delivery by MNAs in humans should be established in large clinical trials in which self-administration potential of 3D-printed MNAs could also be evaluated. Together, clinical translation of 3D-printed MNAs still necessitates overcoming several challenges toward their commercialization.

Collectively, MNAs, enabled by different 3D-printing technologies, offer tremendous potential for minimally invasive drug and vaccine delivery, as well as for simple and patient-friendly sampling of biological fluids. Therefore, it is expected to witness more widespread utilization of 3D-printed MNAs first in preclinical studies and then for clinical applications in the coming years. Addressing the aforementioned fabrication, biomaterials and regulatory challenges with 3D-printed MNAs will be imperative to achieve their effective clinical translation and commercialization.

Financial & competing interests disclosure

S Tasoglu acknowledges Tubitak 2232 International Fellowship for Outstanding Researchers Award (118C391), Alexander von Humboldt Research Fellowship for Experienced Researchers, Marie Skłodowska-Curie Individual Fellowship (101003361) and Royal Academy Newton-Katip Çelebi Transforming Systems Through Partnership award (120N019) for financial support of this research. B Bediz acknowledges the support provided by the Scientific and Technological Research Council of Turkey (TUBITAK) under Grant No. 120R022. Opinions, interpretations, conclusions and recommendations are those of the author and are not necessarily endorsed by the TÜBİTAK. E Korkmaz is supported by a grant from the Institute for Infection, Inflammation and Immunity in Children (i4Kids). E Korkmaz and LD Falo Jr are supported by an NIH fund (UM1-AI106701). LD Falo Jr is supported by NIH grants (R01-AR074285 and R01-AR071277). E Korkmaz is an inventor of an intellectual property with MNAs. LD Falo Jr is an inventor of intellectual properties with MNAs and a Co-founder and scientific advisor of SkinJect, a company that is developing dissolvable MNAs for nonmelanoma skin cancer treatment. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.