(M1230-03-14) Quality by Design Guided Development of Hydrogel-Forming Microneedle Array Patches for Enhanced Transdermal Delivery of Biomimetic Peptide Enfuvirtide

Purpose: As the first biomimetic fusion inhibitor of HIV-1, enfuvirtide has been confirmed to be well tolerated and has demonstrated remarkable antiviral activity compared with an optimised background antiretroviral regimen alone[1]. However, a 98% incidence of injection site reactions generated from the 90 mg twice daily subcutaneous injection has often led to the discontinuation of enfuvirtide. Therefore, hydrogel-forming microneedle array patches (HMAPs) were investigated for the sustained transdermal delivery of enfuvirtide in a minimally invasive and painless manner to address the aforementioned issues. In order to establish a systematic and robust approach for the formulation development, quality by design (QbD) and design of experiment were implemented. For the first time, the potential critical material attributes (CMAs) and critical process parameters (CPPs) of hydrogel formulations were identified and were associated with the critical quality attributes (CQAs) of HMAP to ensure the quality targeted product profile (QTPP).

Methods: Biocompatible polymers poly (vinyl alcohol) (PVA) and poly (ethylene glycol) diacid (PEGdiacid) were chemically crosslinked to form an ester bond with each other with poly (vinyl pyrrolidone) (PVP) involved during the esterification process as a mechanical strength enhancer.  Experiments were performed according to a central composite design generated by Design Expert^® to evaluate the effects of 3 independent factors: PEGdiacid ratio (%w/w) in the casting blend, crosslinking temperature (°C) and crosslinking time (minute) on 3 responses swelling percentage (%), gel fraction (%) and in vitro enfuvirtide diffusion (%) of the hydrogels using horizontal diffusion cells. The mathematical relationship was established to demonstrate the effect of the factors on responses and response surface plots were generated to elucidate the interactions between the factors. Additionally, the selected formulation was validated through feasibility and grid searches, with confirmation runs demonstrating robust prediction. The HMAPs fabricated from the optimal and previously reported formulations with different geometries were characterised in terms of mechanical strength and insertion ability and were tested for their feasibility in delivering enfuvirtide in combination with reservoirs in ex vivo studies.

Results: Subject to thermal treatment for a determined time, the hydrogels underwent a colour transformation from transparent to yellowish during crosslinking with varied porosity and network interconnection in cross-section micro-morphology after swelling(Figure 1A and 1B). The swelling property, gel fraction and gel strength of PVA/PVP/PEGdiacid-based hydrogel were tailored by the varied proportion of PEGdiacid crosslinker, crosslinking time and temperature, which affected the diffusion of enfuvirtide across the hydrogel accordingly (Figure 1C, 1D and 1E). Formulation casting from aqueous blends containing 2.25% w/w PEGdiacid, 10% w/w PVP stock, and 15% w/w PVA stock, crosslinked at 150℃ for 20 minutes, demonstrated the highest desirability of 0.871 and was selected for HMAP fabrication. The HMAPs fabricated from the optimal (F2) and previously reported formulation (F1) exhibited well-defined shapes, consistent heights, and precisely controlled interspacing with sufficient hardness and mechanical strength to penetrate the skin without fracturing, evident by a height reduction of less than 20% after compression (Figure 2)^[2]. The needle arrays, neonatal porcine skins and the reservoirs after the ex vivo study in the Franz cell permeation studies were presented in Figure 3A. The cumulative amount of enfuvirtide permeated in 24 hours was presented in Figure 3B. Notably, a substantial increase in the permeation of enfuvirtide within the initial two hours was observed in 14x14 moulded HMAPs. Until the 6-hour mark, a significant disparity existed in the cumulative permeation of enfuvirtide between 11x11 and 14x14 moulded HMAPs for both formulations (p < 0.05). Beyond this time point, enfuvirtide permeation increased gradually, and after 24 hours, the cumulative permeation among all four HMAPs was statistically similar (p >0.05). The amounts were 3764.49 ± 165.40 (34.07% ± 1.50%), 3144.14 ± 533.48 (28.45% ± 4.83%), 3382.24 ± 578.23 (30.61% ± 5.23%), and 4006.345 ± 266.85 (36.26% ± 2.41%) for F1 14x14, F1 11x11, F2 14x14, and F2 11x11, respectively. Less than 0.01% of enfuvirtide from the reservoir was delivered in the control, together with the result of transepidermal water loss (TEWL), highlighting the significance of HMAPs in compromising the integrity of the skin barrier. The distinct permeation kinetics and comparable permeation between formulations in 24h indicated the significance of the geometries of the arrays in governing the drug release.

Conclusion: This study focused on developing HMAPs for transdermal enfuvirtide delivery using a quality by design (QbD) approach. Optimisation relied on three critical material attributes CMA and CPPs of the hydrogel, establishing a design space linked to CQAs to meet the QTPP. The selected optimal formulation underwent validation via feasibility and grid searches, with confirmation runs demonstrating robust prediction. Fabricated HMAPs exhibited promising mechanical strength and insertion ability, indicating feasibility for enfuvirtide delivery in ex vivo permeation studies. This study not only introduced a novel delivery route for enfuvirtide but also established a systematic approach for HMAP development in pharmaceutical applications.

References: [1] Jamjian, M C., McNicholl, I R., 2004. Enfuvirtide: first fusion inhibitor for treatment of HIV infection. American journal of health-system pharmacy, 61(12): 1242-1247.
[2] Anjani, Q K., Permana, A D., Cárcamo-Martínez Á., 2021. Versatility of hydrogel-forming microneedles in in vitro transdermal delivery of tuberculosis drugs. European Journal of Pharmaceutics and Biopharmaceutics, 158, 294-312.

Acknowledgements: This work was supported by the China Scholarship Council.


Figure 1. The morphology (AB), gel strength (C), swelling profile (D), gel fraction (E) of hydrogels, enfuvirtide permeation (F), response surface plots describing the effect of variables on the swelling percentage (a, b, c) and gel fraction (d, e, f) and enfuvirtide permeation across hydrogel (g, h, i), expressed as PEGdiacid amount-temperature-time.


Figure 2. Morphology of the HMAPs made of F1 (a-f) and F2 (g-l) using 11x11 and 14x14 moulds obtained by optical microscopy and SEM. Representative OCT images depicting insertion of HMAPs using 11x11 (m-p) and 14x14 mould (q-t) in Parafilm® (m, n, q, r) and skin (o, p, s, t) for F1 (m, o, q, s) and F2 (n, p, r, t). Results of the HMAPs Parafilm® insertion test from 11x11 moulds (u) and 14x14 moulds (v) (Mean±SD, n=5).


Figure 3. HMAP, skin, and wafer components of 11x11 (a, c) and 14x14 (b, d) HMAPs from formulations F1 (a, b), F2 (c, d), and control (e) after ex vivo permeation. Permeation profiles (B), circular dichroism spectrum (C), and distribution (D) of enfuvirtide and TEWL value (E) of skin in 24 hours ex vivo study (Mean + SD, n=4).