(T0930-07-37) Development of Novel In Vitro Dissolution Assembly for Dissolving Microneedles via 3D Printed Mould - ImprintoGel

Purpose: Microneedles (MNs) are micron-sized needles capable of bypassing the stratum corneum for transdermal drug delivery, improving bioavailability. Various MN types exist, including dissolving MNs (DMNs) that completely dissolve after application, eliminating the need for removal. As MN technology advances, validation of characterization methods is crucial. Literature methods involve inserting MNs into Parafilm^® M and creating a hermetic pouch for dissolution testing but fail to mimic the skin's limited fluid supply and drug solubility gradient. This research aimed to develop an ex vivo skin model for optimizing MN dissolution studies. A 3D printed imprint/mold was developed to provide structure, and different matrix-forming materials were screened and incorporated into the mold to understand factors affecting drug release patterns, mimicking the skin microenvironment.

Methods: The DMNs were prepared using the solvent casting method using custom-made molds: 6x6 Array, H=1,000µm, Base=250µm, Pitch=1,000μm and 10x10 Array measuring H=500µm, Base=150µm, Pitch=500µm, pyramidal in shape obtained from Micropoint Technologies. DMNs were prepared using a rapidly dissolving water-soluble polymer poly(2-ethyl-2-oxazoline) (PeTOx). The MNs were prepared with fluorescein isothiocyanate-dextran (FITC-dextran) as model drug with high molecular weight and water solubility representing biologics. The imprint was designed on the online Tinkercad software, which was further obtained in stereolithographic format(.stl) for printing. The printing parameters were optimized using the different parameters available in the software and the printing temperature was set to 220°C. The dimensions of the final mold were 15mm:15mm:5mm. A novel in vitro dissolution setup involved a 3D-printed imprint with an optimized agarose gel matrix to mimic skin. MNs were inserted into gel molds using finger pressure, covered with Parafilm^® M. The imprint with inserted MNs was floated in 7.4 pH PBS under agitation to study drug release patterns. A full-factorial DOE design was employed to study the effects of three independent factors: microneedle height, agarose concentration, and agarose imprint height on three response variables: % drug release at 1h, 3h, and 6h. ANOVA analysis confirmed the significance of factors (p < 0.05). Responses were transformed using λ=0.5 based on Box-Cox plots. Goals were set to maximize drug release, with varying importance assigned to responses. The three-way interaction model yielded equations with 5 solutions and interaction plots illustrating effects within variables. Further studies would involve investigation of these factors based on model water-insoluble drug, and two different levels of molecular weight ( < 500 daltons, and < 1000 daltons) ensuring validation of the ex vivo set up.

Results: The in vitro dissolution method optimization process involved preparation of MNs at two varying sizes 500 and 1000 µm (Factor B), agarose concentration at two levels 2 and 4% (Factor C) and 3D printed imprint height at 5 and 10mm levels (Factor A). These three independent variables were analyzed for their effect on drug release patterns at three response variables: % drug release at 1h, 3h and 6h. The dissolution method involved sampling at 0.25, 0.5, 1, 2, 3, 4,6 and 24hrs. Figure 1 shows interaction between factors A and B at lower concentration at factor C i.e. 2% agarose. The data suggests that with increasing thickness of 3D imprint, the % drug release tends to decrease at all three timepoints (1, 3 and 6h), whereas with increasing the microneedle height there is substantial increase in the % drug release at 3 and 6h timepoints, however, this effect is not evident at 1h. Additionally, at lower microneedle heights, the effect of thickness on % drug release is more substantial, whereas at higher microneedle heights, the effect of thickness is less pronounced for both the 1-hour and 6-hour timepoints. The parallel contour lines at 3 and 6h interaction suggest parallel i.e. linear relationship between microneedle height and thickness of 3D imprint. Figure 2 suggests the trends in % drug release at higher (4%) agarose concentration, at lower microneedle heights, increasing thickness leads to a more substantial decrease in % drug release at 3 hours. Overall, the analysis suggests that microneedle height, thickness, and concentration significantly influence the drug release behavior, with thickness and concentration having an inverse relationship, while microneedle height has a direct relationship with % drug release at different time points. Figure 3 shows the evident interaction between thickness and concentration, modulating the drug release profiles of the different MN batches. The rationale of the in vitro model was to validate the effect of microneedle properties on % drug release trends, wherein it is evident that increase in microneedle height should confirm increase in % drug release trends physiologically, the pattern is validated by the ex-vivo model.

Conclusion: Current research approaches a novel 3D imprint based in vitro dissolution model for evaluating the drug release in microneedles. Factors such as microneedle height, 3D imprint height and agarose concentration have significant effect on drug release trends, and evident increase in drug release with increase in MN height has been simulated in the model presented.

Figure 1(A) 2D contour interaction plots between factors A. Thickness and B. Microneedle height at lower concentration of agarose (2%), understanding the effect of trends on % drug release at three response variables (1, 3 and 6h) illustrating increasing the MNs height has significant increase in % drug release at 3 and 6h. (B) 3D interaction plots at lower concentration agarose showing increase in microneedle height increases the %drug release

Figure 2 (A, B) 2D and 3D interaction plots for Thickness and MN height at higher concentration of agarose on % drug release indicating lower microneedle height has substantial effect on drug release at lower concentration compared to higher MNs height and higher concentration the effect is less pronounced.

Figure 3 (A) Fluorescence images of FITC-dextran loaded 1000µm microneedles captured using a confocal microscope(B) Graphical representation of in vitro dissolution setup using 3D Printed Imprint (C) In vitro dissolution release patterns of the different MN batches generated from full factorial design via DOE