(M1530-07-43) Breaking Through the Barrier: 3D-Printed Microneedles Unlock Superior Transdermal Delivery of 5-Fluorouracil and Genistein for Melanoma Treatment

Purpose: Melanoma, the most aggressive form of skin cancer, poses a significant therapeutic challenge due to its high metastatic potential and resistance to conventional treatments. Although 5-fluorouracil (5-FU) is FDA-approved for the topical treatment of certain skin conditions like actinic keratoses and superficial basal carcinoma, its clinical utility in melanoma is hampered by poor skin permeability and limited bioavailability. Similarly, genistein (GEN), a bioactive flavonoid with anticancer potential, faces similar delivery barriers. To address these limitations, this study proposes the development of an innovative 3D-printed microneedle patch with an integrated reservoir system for the transdermal co-delivery of 5-FU and genistein. This approach is designed to enhance skin permeability, enable localized and sustained drug release, and investigate the synergistic cytotoxic effects of the drug combination against melanoma cells, thereby offering a promising strategy to overcome the drawbacks of conventional topical therapies.

Methods: Conical microneedle arrays (MNAs) were fabricated using high-resolution stereolithography (SLA) 3D printing and UV-cured to enhance mechanical strength. Mechanical integrity was assessed by compression testing. To improve solubility and achieve controlled release, a 5-fluorouracil (5-FU) and hydroxypropyl beta-cyclodextrin (HP-β-CD) inclusion complex was prepared via freeze-drying and confirmed by FTIR and DSC. The complex was incorporated into a thermoresponsive hydrogel using pluronic F-127, and rheological properties were evaluated across 4–50°C. Skin permeation studies were conducted using Franz diffusion with porcine skin. Receptor solution samples were collected over 10 h and analyzed by HPLC for cumulative drug permeation. Skin deposition was similarly quantified by HPLC following layer drug extraction.

Results: Conical hollow MNAs were successfully fabricated using 3D-SLA printing, with each microneedle measuring 1 mm in length, 0.8 mm at the base, and 0.25 mm at the tip (Figure 1E). Compression testing confirmed that the arrays could withstand forces up to 60 N without structural failure. Pork skin penetration studies verified the mechanical integrity of the MNAs, ensuring their suitability for transdermal delivery (Figure 1F). The hydrogel formulation FU-HG#2, composed of 5-FU and genistein complexed with HP-β-CD and incorporated into a 20% F-127 thermoresponsive polymer, exhibited favorable rheological behavior, with a distinct sol–gel transition observed near physiological temperature (~25–28 °C) (Figure 1B). FTIR and DSC analyses confirmed the formation of the 5-FU:HP-β-CD inclusion complex (Figures 1C and 1D), while SEM imaging revealed a porous internal structure of FU-HG#2 suitable for sustained drug release (Figure 3E). When loaded into the MNAs, FU-HG#2 significantly enhanced transdermal delivery. Compared to the marketed 5% 5-FU cream, FU-HG#2 demonstrated a 2.42-fold increase in cumulative skin permeation of 5-FU over 10 hours (Figure 2A). Genistein permeability was also markedly enhanced in FU-HG#2 compared to control and other FU-HG variants (Figure 2B). Drug deposition studies showed that FU-HG#2 resulted in a 305% increase in 5-FU and a 623% increase in genistein accumulation within the dermal layer (Figures 2C and 2D). Correspondingly, skin flux of 5-FU and genistein improved by 3.08-fold and 3.86-fold, respectively, with FU-HG#2 (Figures 2E and 2F). Drug release kinetics were modeled to understand the release mechanism from FU-HG#2. While the First-Order model provided the statistical fit for 5-FU (R² = 0.9565), indicating a concentration-dependent release rate, the Korsmeyer–Peppas model (n = 1.3) revealed a Super Case-II transport mechanism (Figure 3A). A similar pattern was observed for genistein, with a Korsmeyer–Peppas exponent of 2.05 and strong linearity (R² = 0.9665) (Figure 3B), indicating that polymer relaxation, swelling, and erosion governed release in the later stages, beyond simple diffusion. Cytotoxicity assays on B16F10 melanoma cells showed that FU-HG#2 had significantly enhanced anticancer efficacy. At a concentration of 25 µg/mL, FU-HG#2 reduced cell viability to 35.97% ± 6.1%, compared to 53.39% ± 8.05% for 5-FU alone and 66.34% ± 11.02% for genistein alone (Figure 3C). IC₅₀ calculations further supported the synergistic effect: 5-FU alone had an IC₅₀ of 32.3 µg/mL, genistein 649.3 µg/mL, while their combination in FU-HG#2 reduced IC₅₀ to just 3.36 µg/mL (Figure 3D). Western blot analysis revealed that combination treatment effectively downregulated Bcl-2, an anti-apoptotic protein, confirming apoptosis induction and enhanced therapeutic action (Figure 3F). Collectively, these findings demonstrate that the FU-HG#2-loaded MNA system significantly improves transdermal drug delivery, promotes deep dermal deposition, sustains release kinetics via polymer-driven mechanisms, and exerts potent synergistic anticancer effects against melanoma cells.

Conclusion: The 3D-printed microneedle-assisted hydrogel system (FU-HG#2) demonstrated significant improvements in transdermal delivery and anticancer efficacy of 5-fluorouracil and genistein. Enhanced skin permeability, superior dermal drug deposition, and controlled Super Case-II release kinetics were achieved through the integration of thermoresponsive hydrogel and conical microneedle arrays. Notably, the combination therapy exhibited a strong synergistic cytotoxic effect on B16F10 melanoma cells, markedly reducing IC₅₀ and inducing apoptosis via Bcl-2 suppression. These results highlight the potential of FU-HG#2 as a promising transdermal platform for effective localized melanoma treatment.


Figure 1. (A) Composition of FU-HG formulations. (B) Viscosity profiles showing sol–gel transition with temperature. (C) FTIR spectra confirming formation of the 5-FU:HP-β-CD inclusion complex. (D) DSC thermograms of 5-FU, HP-β-CD, and the complex. (E) Microscopic images of 3D-printed microneedle arrays (top,bottom, side, and magnified views). (F) Skin insertion force–time curve showing ~60 N penetration strength.


Figure 2. Transdermal permeation, deposition, and flux of 5-FU and genistein (GEN) through pork skin. (A) Cumulative permeation of 5-FU over 10 h across different formulations, with FU-HG#2 + MNA showing significantly enhanced delivery. (B) Cumulative permeation of GEN over time, with marked improvement in the FU-HG#2 group. (C) Skin deposition of 5-FU at 24 h, indicating highest accumulation with FU-HG#2. (D) GEN deposition in skin, also highest with FU-HG#2. (E) Flux of 5-FU through pork skin, with FU-HG#2 significantly outperforming other groups. (F) GEN flux showing a similar trend. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.


Figure 3. Evaluation of release kinetics, cytotoxicity, and synergistic anticancer effects of FU-HG#2. (A, B) Korsmeyer–Peppas plots showing drug release kinetics of 5-FU (A) and genistein (B) from FU-HG#2. (C) Cell viability of B16F10 melanoma cells treated with 5-FU, genistein, or their combination at various concentrations, demonstrating enhanced cytotoxicity in the combined group. (D) IC₅₀ values calculated from dose–response curves: 5-FU (32.3 µg/mL), genistein (649.3 µg/mL), and the combination (3.36 µg/mL), confirming synergistic activity. (E) SEM image of FU-HG#2 showing a porous network structure. (F) Western blot analysis showing downregulation of anti-apoptotic marker Bcl-2 following combination treatment, indicating apoptosis induction in B16F10 cells.