Transdermal delivery of pramipexole dihydrochloride using dissolving polymeric microneedle patches for improved Parkinson's disease management

Abstract

Parkinson's disease (PD) is a progressive neurodegenerative disorder commonly treated with oral pramipexole dihydrochloride monohydrate (PXCl), a dopamine agonist. Oral therapy is often limited by gastrointestinal complications, variable absorption, and poor patient adherence, highlighting the need for alternative delivery strategies. Dissolving microneedles (MNs) offer a minimally invasive, patient-friendly platform for transdermal drug delivery, enabling sustained and controlled release. In this study, PXCl-loaded MNs were developed using three polymer blends: polymethyl-vinyl-ether-co-maleic acid/polyvinyl alcohol (PMVEMA-PVA30K), carboxymethylcellulose sodium/polyvinyl alcohol (CMC-PVA30K), and polyvinylpyrrolidone/polyvinyl alcohol (PVP-PVA70K). Micro-molding produced MNs with sharp, uniform geometries, and adequate mechanical strength to penetrate the skin, achieving insertion efficiencies above 94%. Fourier-transform infrared spectroscopy confirmed intermolecular hydrogen bonding and ionic interactions between PXCl and the polymer matrices. In vitro release studies across cellulose membranes demonstrated complete PXCl release within 48–72 h, with faster release from CMC- and PVP-based MNs. Notably, PMVEMA-PVA30K MNs, despite slower release, showed the highest PXCl permeation and flux across full-thickness human skin, likely due to stronger drug-polymer interactions, enhanced MN rigidity, and polymer-induced modulation of skin permeability. Biocompatibility assays indicated that all formulations were non-hemolytic and non-cytotoxic. These findings demonstrate that dissolving MNs provide a promising strategy for transdermal PXCl delivery and suggest that PMVEMA-PVA30K matrices may offer an effective, sustained therapeutic approach for PD treatment.

Graphical abstract

Highlights

• •Dissolving microneedle patches were developed for transdermal delivery of PXCl.
• •Polymer composition affected mechanical strength, drug release, and skin permeation.
• •PMVEMA–PVA30K MNs showed the highest transdermal flux across human skin.
• •All MN formulations demonstrated good cytocompatibility and hemocompatibility.

1. Introduction

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by tremor, bradykinesia, rigidity, and postural instability, making it one of the most prevalent neurological conditions globally (Hayes, 2019; Qiu et al., 2025). These motor symptoms primarily arise from the selective loss of dopaminergic neurons in the substantia nigra, a midbrain region critical for dopamine production. Dopamine is a key neurotransmitter that facilitates smooth, coordinated movement. Although PD cannot currently be cured, continuous symptom management is essential to maintain quality of life and functional independence in patients (Connolly and Lang, 2014; Münchau and Bhatia, 2000).

Pharmacological therapy often relies on oral administration of drugs, which can be limited by gastrointestinal side effects, variable absorption, and fluctuating plasma drug concentrations (Hua, 2020). Pramipexole, a non-ergot dopamine agonist, is widely used in early PD to delay the initiation of levodopa therapy and reduce levodopa-related motor complications (Wilson et al., 2020). Pramipexole dihydrochloride monohydrate (PXCl) is a cationic molecule with net positive charge at physiological pH (pKa 5.0 and 9.6) and a low octanol–phosphate buffer partition coefficient of 0.135 (Okura et al., 2007). PXCl is clinically available as immediate-release and extended-release tablets, requiring multiple daily administrations, with doses titrated from 0.375 mg to 4.5 mg to maximize therapeutic effect (Varga et al., 2009). However, many PD patients experience delayed gastric emptying and impaired gut motility, leading to slowed intestinal absorption and reduced oral bioavailability of PXCl (Poirier et al., 2016). In addition, oropharyngeal dysphagia, reported in 11–82% of patients, further complicates oral drug administration (Nascimento et al., 2020). These challenges highlight the clinical need for alternative delivery strategies that bypass the gastrointestinal tract.

Dissolving microneedles (MNs) offer a minimally invasive, patient-friendly approach for transdermal drug delivery, enabling continuous, controlled release while potentially improving adherence and therapeutic outcomes (Prausnitz and Langer, 2008). MNs can be fabricated from a range of polymers, including biocompatible, biodegradable, and water-soluble types, allowing scalable production, tunable mechanical properties, and controlled drug release (Ramadon et al., 2022). In this study, we explore three formulations of dissolving polymeric MNs for PXCl, evaluating their fabrication, mechanical strength, skin penetration, drug release kinetics, and biocompatibility (Fig. 1). This approach aims to provide a novel, effective, and patient-centered strategy for PD treatment, overcoming the limitations of oral therapy while improving the quality of life for patients.

Fig. 1.

Schematic illustration of the experimental workflow for MN patches prepared with different formulations.

2. Material & methods

2.1. Material & skin

PXCl was purchased from Biosynth International, Inc. (Louisville, Kentucky, USA). Polymers used for MN fabrication included polymethyl-vinyl-ether-co-maleic acid (PMVEMA, MW 1,080,000 Da), low-viscosity carboxymethylcellulose sodium (CMC), polyvinyl alcohol (PVA30K, MW 31,000–50,000 Da, 87–89% hydrolyzed), PVA70K (Mowiol®8–88, MW 67,000 Da, 87–89% hydrolyzed), and polyvinylpyrrolidone (PVP, Plasdone K29/32). All polymers were obtained from Sigma-Aldrich (St. Louis, Missouri, USA) or Ashland Inc. (Covington, Kentucky, USA). Sodium azide (NaN₃) was obtained from Sigma-Aldrich. Buffer components, including sodium dihydrogen orthophosphate (NaH₂PO₄), disodium hydrogen orthophosphate (Na₂HPO₄), and sodium chloride (NaCl) were purchased from Ajax Finechem (New South Wales, Australia). High-performance liquid chromatography (HPLC) grade acetonitrile, triethylamine, and phosphoric acid were supplied by Honeywell Burdick and Jackson (Ulsan, Korea), Merck (Hessen, Germany), and J.T.Baker® (Central Valley, Pennsylvania, USA), respectively. The polydimethylsiloxane (PDMS) molds for MN fabrication were purchased from Blueacre Technology Ltd. (Dundalk, Ireland). Unless stated otherwise, all chemicals and solvents were of analytical reagent grade. Phosphate-buffered saline, pH 7.4 (PBS) containing 0.01 M phosphate buffer, 0.142 M NaCl, and 0.02% NaN₃ was used as the medium for the in vitro drug release studies and as the receiver solution for the in vitro skin permeation experiments.

Human abdominal skin was obtained from female patients aged 30–60 years undergoing abdominoplasty at the Department of Surgery, Yanhee International Hospital, Thailand. Full-thickness human skin (FTS) was prepared by removing subcutaneous tissue using surgical scissors. Skin samples were rinsed in PBS, dried with lint-free tissue (Scott®, Kimberly-Clark Worldwide, Inc., Irving, Texas, USA), and wrapped in aluminum foil for storage at −20 °C. To ensure tissue integrity, samples were used in MN insertion and permeation studies within a maximum storage period of 6 months. The average thickness of FTS was 0.12 ± 0.01 cm (mean ± SD, n = 4). The use of human tissues was reviewed and approved by the Committee on Human Rights Related to Human Experimentation, Mahidol University, Thailand (COE No. MU-DT/PY-IRB 2022/056.1312).

Fresh human blood was obtained from a healthy adult volunteer using sodium citrate collection tubes for subsequent hemocompatibility evaluations. The procurement and use of human skin and blood specimens were conducted in accordance with ethical guidelines and were approved by the Committee on Human Rights Related to Human Experimentation, Mahidol University, Thailand (COA No. MU-MOU 2024/125.2603).

2.2. Preparation of MN patches

2.2.1. Preparation of nondrug-loaded MN patches

Prior to the fabrication of PXCl-loaded MN patches, nondrug-loaded MN patches were prepared using a micro-molding method (Wang et al., 2016).

2.2.1.1. Nondrug-loaded PMVEMA-PVA30K MN patches

Nondrug-loaded PMVEMA–PVA30K MN patches were prepared following Saepang et al. (Saepang et al., 2021b). Aqueous stock solutions of 20% (w/w) PMVEMA and 20% (w/w) PVA30K were prepared by dissolving pre-weighed amounts of each polymer in deionized water at 70 °C until uniform and clear. The solutions were blended at a 1:2 weight ratio to obtain a 15% (w/w) polymer blend solution. Then, 0.6 g of the polymer blend was added into a 1.5 cm × 1.5 cm PDMS mold. The mold was centrifuged at 4,500 rpm for 15 min to remove trapped air and allowed to dry in a glass desiccator containing anhydrous silica gel under ambient conditions for 24 h.

2.2.1.2. Nondrug-loaded CMC-PVA30K MN patches

Aqueous stock solutions of 15% (w/w) CMC and 20% (w/w) PVA30K were prepared in deionized water at 60 °C until uniform and clear. The solutions were mixed at a 1:1 weight ratio to obtain a 17.5% (w/w) polymer blend. Then, 0.6 g of the blend was added into a 1.5 cm × 1.5 cm PDMS mold, centrifuged at 4,500 rpm for 15 min, and dried in a glass desiccator containing anhydrous silica gel under ambient condition for 72 h.

2.2.1.3. Nondrug-loaded PVP-PVA70K MN patches

Aqueous stock solutions of 60% (w/w) PVP and 20% (w/w) PVA70K were prepared in deionized water at 60 °C. Solutions were blended at a 1:2 weight ratio and supplemented with 4% (w/w) propylene glycol (Vazquez-Muñoz et al., 2019) to prepare the polymer blend. Then, 0.6 g of the blend was added into a 1.5 cm × 1.5 cm PDMS mold, centrifuged at 4,500 rpm for 15 min, and dried in a glass desiccator containing anhydrous silica gel under ambient conditions for 48 h.

Each 1.5 cm × 1.5 cm PDMS mold contained an 11 × 11 array of conical needles over a 0.49 cm² area, with 600 μm needle height, 300 μm base diameter, and 300 μm interspacing. After drying, MN patches were carefully removed, cut into 1 cm × 1 cm squares, and stored in a desiccator until use. The nondrug-loaded MNs were characterized for morphology, mechanical strength, and skin insertion capability.

2.2.2. Preparation of PXCl-loaded MN patches

PXCl-loaded MN patches were prepared by loading 0.6 g of the respective polymer solution containing PXCl at 1.0 mg/cm² into PDMS mold cavities. The fabrication process followed the same procedure as for nondrug-loaded MN patches.

The viscosities of the casting solutions for the nondrug-loaded and drug-loaded PMVEMA-PVA30K, CMC-PVA30K and PVP-PVA70K MN patches in the ranges of 600–800 centipoises (cPs), 11,000–13,000 cPs and 2,000–3,000 cPs, respectively. Within each polymer blend, no statistically significant differences in viscosity were observed between the drug-loaded and non-drug-loaded formulations. The measurements were conducted using a rheometer (Haake RotoVisco 1; Thermo Fisher Scientific, Germany) equipped with a cone and plate (35/2°Ti) model. All tests were performed at a constant temperature of 30 °C and a shear rate of 5.0 s⁻¹ for 5 min.

2.3. Characterization of MN patches

2.3.1. MN morphology

The morphology of MN patches was examined using scanning electron microscopy (Abdulaal et al., 2024). MN samples were mounted directly on carbon tape and coated with a thin layer of gold using an auto-fine coater (10 mA, 60 s). The gold-coated samples were then observed under SEM to visualize the microneedle structures and confirm their uniformity and sharpness.

2.3.2. Mechanical testing of MNs

The mechanical strength of MN patches was assessed using a texture analyzer (TA.XTPlus, Stable Microsystems Ltd., Surrey, UK) in compression mode (Pamornpathomkul et al., 2018). Axial compression forces, applied perpendicular to the MN base, were used to simulate insertion forces. Each MN patch was adhered to the movable probe with double-sided adhesive tape and pressed against a stainless-steel plate at 0.5 mm/s until a maximum force of 32 N was reached, corresponding to the average insertion force applied by a human as previously reported (Larrañeta et al., 2014). The trigger force was set at 0.049 N, with pre- and post-test speeds of 1 mm/s. Force-displacement profiles were recorded, and the fracture force of MNs was determined at the point of sudden force drop. MNs were imaged under a light microscope before and after testing to assess changes in needle height.

2.3.3. In vitro model study of MN penetration depth

Parafilm® M (PF) was employed as a model for skin simulation for the MN penetration studies, a method previously validated for the comparative evaluation of MN formulations (Larrañeta et al., 2014; Pamornpathomkul et al., 2018). For the procedure, an MN patch was adhered to the movable probe of a texture analyzer using double-sided adhesive tape. The patch was then inserted into a stack consisting of eight layers of PF at a constant speed of 0.5 mm/s until the force reached 32 N for 30 s. The trigger force was set at 0.049 N (pre-test and post-test speed 1 mm/s). Following the application, the MN patch was carefully withdrawn. The PF layers were separated, and the resulting micro-perforations in each layer were quantified using microscopic imaging. Given the measured thickness of a single PF layer (122 ± 2 μm, n = 12), the depth of MN penetration was determined by calculating the percentage of holes created as a function of membrane depth.

2.3.4. Skin insertion capacity (dye binding study)

The skin insertion capability of MN patches was evaluated using methylene blue staining (Kale et al., 2020). FTS (4 cm × 4 cm) was placed dermis-side down on a 6 mm-thick PDMS sheet and blotted dry with lint-free tissue. MN patches were applied using thumb pressure for 30 s and then removed. Methylene blue (400 μL of 1% w/v solution) was applied to the perforated skin surface and allowed to equilibrate for 30 min. Excess dye was removed, and the skin was rinsed three times with 2 mL PBS. The number of microchannels (MCs) created, indicated by blue spots, was counted. Skin insertion efficiency was calculated as the percentage of successfully created holes relative to the total number of MNs in the array using the following equation (Saepang et al., 2021b):

$$\%\mathit{insertion\; efficiency}=\frac{\mathit{number\; of\; blue\; spot\; observed}}{\mathit{number\; of\; needles}\left(121\right)}x100$$

2.3.5. Fourier transform infrared (FTIR) spectroscopy

FTIR spectra of PXCl, the original polymers (PVA30K, PVA70K, PMVEMA, CMC, and PVP), nondrug-loaded MN patches, and PXCl-loaded MN patches were obtained using a Nicolet™ iS5 FT/IR spectrophotometer (Thermo Fisher Scientific Inc., Massachusetts, USA). Spectra were collected over the range of 4000–400 cm⁻¹, with an average of 32 scans per sample and a resolution of 4 cm⁻¹, to identify characteristic functional groups and potential polymer–polymer interactions within the MN matrices.

2.4. In vitro drug release study

In vitro PXCl release from MN patches was evaluated using an equilibrium cellulose membrane (Cellu Sep® 12-14 K MWCO, USA) placed on a PDMS support. A 5 cm × 5 cm membrane section was used, and MN patches were applied similarly to the skin insertion study. The backside of each patch was sealed with waterproof adhesive tape (Sur-Strap™, New Tac Kasei Co., Ltd., Kagawa, Japan). The membrane and MN patch were then placed in a Franz diffusion cell, with the receptor compartment filled with 5 mL PBS, continuously stirred with a magnetic stir bar and maintained temperature at 32 °C using a thermostat (Safety Thermostat 911 model, PolyScience, Niles, Illinois). Samples (300 μL) were collected from the receptor compartment at predetermined time points: 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 12, 24, 36, and 48 h. For PMVEMA-PVA30K MN patches, additional samples were collected at 60 and 72 h. After each collection, the receptor compartment was replenished with fresh PBS to maintain constant volume. PXCl content in the samples was quantified by HPLC. All experiments were conducted at least in triplicate.

2.5. In vitro skin permeation study

PXCl permeation from MN patches across FTS was performed using Franz diffusion cells with a diffusion area of 1.77 cm². FTS samples were equilibrated in PBS for 1 h, dried with lint-free tissue, and placed dermis-side down on a 6 mm-thick PDMS support to mimic underlying tissue (Kochhar et al., 2013). MN patches were applied as described previously. Treated skin was mounted between the donor and receiver compartments, with the receiver compartment filled with 5 mL PBS and continuously stirred with a magnetic stir bar. Skin temperature was maintained at 32 ± 1 °C using a thermostat. Aliquots of 300 μL were collected at 1, 2, 3, 4, 5, 6, 9, 12, 18, 24, 36, 48, 60, and 72 h and immediately replaced with fresh PBS. PXCl content was determined by HPLC. All experiments were conducted at least in triplicate.

2.5.1. Equation in the analysis of permeation data

The total quantity of drug that permeated through the skin was normalized to the surface area of the diffusion cell and plotted as a function of time. The pseudosteady-state flux (J_ps) and the apparent permeability coefficient (P_app) were calculated based on Fick's first law of diffusion (Mitragotri et al., 2011).

$$J_{\mathit{ps}}=\frac{\mathit{dQ}}{\mathit{dt}}\times \frac{1}{A}$$

$$P_{\mathit{app}}=\frac{\mathit{dQ}}{\mathit{dt}}\times \frac{1}{\mathrm{A\Delta }C}=\frac{{\mathit{J}}_{\mathit{ps}}}{\mathit{\Delta }\mathit{C}}$$

where, dQ/dt corresponds to the slope of the linear region of the in vitro permeation profile (plot of cumulative drug permeated amount, Q, versus time, t), and A denotes the diffusion area in contact with the donor formulation (MN patch). ΔC indicates the concentration gradient of the drug across the skin. Under sink conditions, ΔC is typically approximated by the donor concentration.

2.6. HPLC analysis

PXCl quantification was performed using HPLC according to Saepang et al. (Saepang et al., 2021a, Saepang et al., 2021b). The system consisted of an Agilent 1200 series quaternary pump, degasser, autosampler, UV–Vis diode array detector (Agilent Technologies, Santa Clara, CA), and Hypersil™ BDS C18 column with guard column (5 μm particle size, 150 mm × 4.6 mm; Thermo Fisher Scientific, Waltham, MA). The mobile phase consisted of acetonitrile:aqueous phase (3:97, v/v), where the aqueous phase contained 0.5% (v/v) triethylamine with pH adjusted to 6.0 using phosphoric acid. The flow rate was 1.0 mL/min, injection volume 50 μL, and detection wavelength 262 nm. Standard solutions in the mobile phase were prepared for calibration over the range 0.20–6.41 μg/mL (R² = 0.999). The limit of detection (LOD) and limit of quantification (LOQ) were 0.03 and 0.10 μg/mL, respectively.

2.7. Biocompatibility testing

The biocompatibility testing, including cytotoxicity assessment and hemolysis assay, was conducted in accordance with the protocol described in Phetporkha et al. (Phetporkha et al., 2025).

2.7.1. Cytotoxicity assessment

The cytotoxicity of blank MNs and PXCl-loaded MNs fabricated from PMVEMA-PVA30K, CMC-PVA30K, and PVP-PVA70K polymers was evaluated using human dermal fibroblasts (HDFn, ATCC, Manassas, VA, USA) according to ISO 10993-5:2009 guidelines (International Organization for Standardization, 2009). HDFn cells were cultured in the recommended growth medium at 37 °C in a humidified atmosphere with 5% CO₂. Cells were seeded into 96-well plates at a density of 1 × 10⁴ cells/well and incubated overnight to allow adherence.

MN extracts were prepared by incubating each MN patch in complete culture medium at 37 °C for 24 ± 2 h. The extracts were applied to the cells, which were then incubated for an additional 24 ± 2 h. Untreated cells served as a negative control (100% viability), while cells exposed to 1% Triton X-100 served as a positive control. Cell viability was determined using the CCK-8 assay according to the manufacturer's instructions. Absorbance was measured at 450 nm using a microplate reader, and viability was expressed as a percentage relative to the negative control. All experiments were performed in triplicate.

2.7.2. Hemolysis assay

Hemocompatibility of blank and PXCl-loaded MNs was evaluated using a standard hemolysis assay. Lyophilized MN samples were pre-incubated in PBS at 37 °C for 24 ± 2 h to prepare test extracts. Fresh human blood was collected from a healthy volunteer into citrate anticoagulant tubes and centrifuged at 700 xg for 15 min to isolate red blood cells (RBCs). The RBCs were washed three times with PBS and diluted 1:10 for testing.

For the assay, 900 μL of diluted RBC suspension was mixed with 100 μL of each MN extract. PBS served as a negative control (0% hemolysis), and 1% Triton X-100 was used as a positive control (100% hemolysis). Samples were incubated at 37 °C for 3 h, followed by centrifugation to pellet intact RBCs. The supernatant was collected, and hemoglobin release was quantified by measuring absorbance at 540 nm. Hemolysis percentage was calculated relative to the controls. All experiments were conducted in triplicate.

$$\%\mathit{Hemolysis}=\frac{\mathit{OD}\mathit{of\; test}-\mathit{OD}\mathit{of\; Negative\; control}}{\mathit{OD}\mathit{of\; positive\; control}-\mathit{OD}\mathit{of\; negative\; control}}x100$$

3. Result and discussion

3.1. Characterization microneedle patches

3.1.1. Microneedle patch visualization

Blank and PXCl-loaded MNs (1 mg/cm²) fabricated from PMVEMA-PVA30K, CMC-PVA30K, and PVP-PVA70K were successfully obtained using the micro-molding method. The overall morphology of the patches is shown in Fig. 2.

Fig. 2.

SEM and optical images of blank and PXCl-loaded MN arrays fabricated using PMVEMA-PVA30K, CMC-PVA30K, and PVP-PVA70K formulations. All samples exhibit uniform conical geometry, sharp tips, and consistent structural integrity across formulations.

SEM images revealed well-defined conical MNs with smooth, crack-free surfaces across all formulations, both blank and drug-loaded. The consistent geometry and integrity of the MNs indicate that the selected polymer blends possessed sufficient moldability and mechanical robustness for reproducible MN fabrication. These findings confirm that incorporation of PXCl at the tested loading did not adversely affect the structural fidelity of the MN arrays.

The drug loading of PXCl within the MN patches, fabricated via the micro-molding method, was targeted at 1 mg/cm². Drug content was quantified using the validated HPLC method described previously in this manuscript. The drug loading efficiencies for the PMVEMA-PVA30K, CMC-PVA30K, and PVP-PVA70K MN patches were 90.0 ± 2.0%, 92.8 ± 3.8%, and 93.1 ± 4.9% (n = 4), respectively. To compensate for drug loss during the fabrication process, an excess of 15% (relative to the 1 mg/cm² target) was added to the initial casting solutions. Consequently, the final absolute drug contents were 1.04 ± 0.02 mg, 1.07 ± 0.04 mg, and 1.07 ± 0.06 mg per patch (n = 4) for the respective formulations. The consistently high loading efficiencies (>90%) and low variability indicate good content uniformity and robustness of the fabrication process across different polymer formulations.

Drug stability was also evaluated by monitoring PXCl content using HPLC after storage at 30 °C in a desiccator for 6 months. No noticeable changes in physical appearance were observed for any formulation. The remaining PXCl contents in PMVEMA–PVA30K, CMC–PVA30K, and PVP–PVA70K MN patches were 97.1 ± 3.1%, 92.3 ± 5.4%, and 97.8 ± 4.7% (n = 3), respectively. These results indicate that PMVEMA–PVA30K and PVP–PVA70K formulations exhibited superior stability, and CMC–PVA30K maintained acceptable PXCl retention, supporting the suitability of all three formulations for long-term storage.

3.1.2. Mechanical properties of MN samples

Mechanical integrity is a critical parameter to ensure reliable skin penetration and drug delivery. To evaluate this, MNs were assessed for insertion efficiency using methylene blue staining of FTS and for fracture resistance using a texture analyzer. Both blank and PXCl-loaded formulations were compared to determine whether drug incorporation affected mechanical performance.

3.1.2.1. Insertion efficiency

The insertion efficiency of MNs was determined by counting methylene blue-stained microchannels, which represent successful penetration of the stratum corneum (Fig. 3). Blank MNs showed efficiencies of 100.0 ± 0.0%, 99.0 ± 1.0%, and 95.0 ± 2.0% for PMVEMA-PVA30K, CMC-PVA30K, and PVP-PVA70K, respectively. PXCl-loaded MNs demonstrated slightly reduced efficiencies of 99.0 ± 1.0%, 95.0 ± 2.0%, and 94.0 ± 0.6%, respectively. While drug loading caused a marginal decrease, particularly in CMC-PVA30K and PVP-PVA70K formulations, all patches maintained high (> 94%) insertion efficiencies, confirming their suitability for effective transdermal application. Moreover, Parkinson's disease predominantly affects elderly patients, whose skin exhibits reduced elasticity, hydration, and thickness. However, MNs can still effectively penetrate aged skin due to their micron-scale geometry and stratum corneum bypass, and reduced elasticity may even facilitate insertion.

Fig. 3.

Skin insertion efficiency of blank and PXCl-loaded MNs in different formulation showed high efficiency for all groups. (A) The percentage of insertion efficiency. Data were presented as mean ± SD (n = 3). (B) Representative images of MN insertion into FTS, where blue dots indicate penetration sites (scale bar = 0.2 cm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.1.2.2. Texture analyzer testing

Force–displacement profiles were obtained to evaluate the fracture resistance of the MN patches under axial compression up to 32 N, a force approximating human thumb pressure. Both blank and PXCl-loaded MNs, across all formulations (PMVEMA-PVA30K, CMC-PVA30K, and PVP-PVA70K), exhibited a consistent resistance curves without sudden force drops, indicating the absence of catastrophic fracture (Fig. 4). Post-test microscopy further confirmed that the MNs remained structurally intact, with only minor height reductions observed.

Fig. 4.

Force-displacement curves of (A) blank MNs patch, and (B) PXCl MNs patch after underwent the mechanical analysis which subjected to a 32 N force.

Incorporation of PXCl slightly influenced the deformation behavior of the MNs, as evidenced by subtle variations in the slope of the force–displacement curves compared with their blank counterparts. However, these differences were not statistically significant and did not impair the overall mechanical integrity of the patches. Importantly, the high insertion efficiencies reported in Section 3.1.2.1 corroborate the conclusion that PXCl-loaded MNs retained sufficient robustness to penetrate the stratum corneum effectively. Together, these findings demonstrate that all three polymer blends yielded MNs with adequate mechanical resilience for transdermal application, even after drug incorporation.

The in vitro penetration depth of the MN patches was evaluated using a stacked PF model. This study quantified the percentage of microchannels formed as a function of penetration depth. Given a PF layer thickness of 122 ± 2 μm (n = 12), results confirmed that all tested MN patches successfully bypassed the stratum corneum, reaching a penetration depth of approximately 488–500 μm (corresponding to the fourth PF layer). These findings were corroborated by skin perforation data, which confirmed efficient skin penetration (Fig. S1 A–C). Among the formulations, the PMVEMA–PVA30K MN patch exhibited the highest mechanical performance, achieving 100.0% insertion efficiency at a depth of ∼500 μm. This was followed by the CMC–PVA30K and PVP–PVA70K patches, which showed insertion efficiencies of 94.2% and 91.7%, respectively, at similar depths. These observations were consistent with the insertion efficiency assessments conducted via the quantification of methylene blue-stained microchannels.

3.1.3. FTIR

FTIR analysis of PXCl-loaded MN formulations was carried out to investigate potential interactions between PXCl and the polymer matrices (Fig. 5). The spectra of the individual components were compared with those of the mixtures, allowing assignment of characteristic functional groups and assessment of molecular interactions.

Fig. 5.

FTIR spectra of individual components and mixture of each MN formulation. (A) PMVEMA-PVA30K MN formulation, (B) CMC-PVA30K MN formulation, and (C) PVP-PVA70K MN formulation.

The spectrum of PMVEMA as in Fig. 5A displayed characteristic absorption bands at 1773 cm⁻¹ (C O stretching of carboxylic acid groups) and 1217 cm⁻¹ (C—O stretching) (Torres-Figueroa et al., 2021). PVA30K exhibited a broad –OH stretching band at 3200–3600 cm⁻¹ and C—H aliphatic stretching vibrations at 2908 and 2940 cm⁻¹(Bulut et al., 2025). In PXCl-loaded MNs, the broad –OH stretching band shifted from approximately 3340 cm⁻¹ to 3290 cm⁻¹, accompanied by band broadening and reduced intensity, indicating hydrogen bonding between hydroxyl groups of PMVEMA–PVA30K and amine groups of PXCl. In addition, the C O stretching band of PMVEMA shifted from 1773 cm⁻¹ to around 1701 cm⁻¹ with decreased intensity, suggesting ionic interactions between the protonated amine of PXCl and the carboxyl groups of PMVEMA.

The spectrum of CMC (Fig. 5B) exhibited characteristic peak at 2881 cm⁻¹ is based on -CH₂ asymmetric stretching, and the two peaks at 1588 and 1409 cm⁻¹ are based on asymmetric COO⁻ and symmetric COO⁻ stretching, respectively, along with a broad –OH stretching band at 3000–3600 cm⁻¹(Bulut et al., 2025). In the CMC–PVA30K formulation, incorporation of PXCl resulted in a shift of the asymmetric COO⁻ stretching band from 1588 to approximately 1575 cm⁻¹ and the symmetric COO⁻ band from 1409 to around 1398 cm⁻¹, together with broadening of the –OH stretching region (3000–3600 cm⁻¹), consistent with ionic interactions between PXCl and the carboxylate groups of CMC.

For PVP (Fig. 5C), a distinct carbonyl band at 1640 cm⁻¹ (C O stretching of the pyrrolidone group) was observed (Marin-Silva et al., 2025), while PVA70K contributed broad O—H stretching (3200–3600 cm⁻¹) and C—H stretching (2908 and 2940 cm⁻¹). Following PXCl loading, the characteristic carbonyl band of PVP shifted from 1640 to approximately 1628 cm⁻¹ with reduced intensity, while the –OH stretching band broadened, suggesting hydrogen bond formation between PXCl amine groups and the carbonyl moieties of PVP.

Taken together, the FTIR results, including quantified peak shifts of key functional groups, confirmed the presence of intermolecular interactions—primarily hydrogen bonding and, in the case of PMVEMA and CMC, additional ionic interactions—between PXCl and the polymer matrices. These interactions indicate that each polymer system establishes a distinct interaction profile with the drug, stabilizing PXCl within the MN structures.

3.2. Release of the PXCl

In vitro PXCl release from MN patches was evaluated using an equilibrium cellulose membrane mounted on a PDMS support in Franz diffusion cells, with PBS as the receptor medium at 32 °C. Samples were collected up to 72 h and analyzed by HPLC. Fig. 6A illustrates the in vitro cumulative drug release profiles of PXCl from the three dissolving MN patch formulations. Within the first 12 h, the amounts of PXCl released from the PMVEMA-PVA30K, CMC-PVA30K, and PVP-PVA70K MN patches (each loaded with 1.0 mg/cm² of drug) were 0.58 ± 0.11, 0.72 ± 0.06, and 0.79 ± 0.08 mg, corresponding to 57.6 ± 10.7%, 72.4 ± 5.8%, and 79.3 ± 8.4% of the cumulative drug load, respectively. At later time points, nearly complete drug release was achieved: 0.96 ± 0.04 mg from PMVEMA-PVA30K at 72 h, 0.96 ± 0.10 mg from CMC-PVA30K at 48 h, and 0.97 ± 0.05 mg from PVP-PVA70K at 48 h. These values represent almost 100% release of the drug incorporated into the MN patches (Fig. 6B).

Fig. 6.

In vitro release profile of PXCl from different MN patch formulations (A) cumulative released amount of PXCl (B) percentage release of PXCl. Data are presented as mean ± SD (n = 4–5).

The complete release of PXCl from all formulations can be primarily attributed to the hydrophilic nature of both the drug and the polymer matrices. PXCl is a highly water-soluble dopamine agonist (Cotton, 2008; Friedl and Eisenreich, 2011), which facilitates its rapid diffusion once the polymeric matrix dissolves. Similarly, the hydrophilic polymers employed (including PMVEMA, CMC, PVP, and PVA) are water-soluble and capable of rapid hydration and dissolution in aqueous environments (Oh et al., 2022; Pamornpathomkul et al., 2018; Qiang et al., 2023; Rojas et al., 2022). When the MNs were exposed to the release medium, the polymer matrices absorbed water, swelled, and dissolved, releasing the encapsulated PXCl. These results confirm that all three systems function effectively as dissolving MN patches for PXCl delivery.

The release kinetics of all MN formulations during the initial 6 h phase were evaluated using several kinetic models, including zero-order, first-order, Hixson–Crowell, Higuchi, and Korsmeyer–Peppas models, as shown in Table 1. Among these, the Higuchi model exhibited the highest correlation coefficients (R² = 0.9620–0.9957), indicating that drug release is predominantly governed by diffusion from the polymeric matrix. This suggests that the release mechanism is primarily controlled by Fickian diffusion through the hydrated MN structure. The Korsmeyer–Peppas model also demonstrated good agreement with the experimental data (R² = 0.9753–0.9947), particularly within the initial release phase (<60%). The calculated release exponent (n) values ranged from 0.2699 to 0.5124, indicating different transport mechanisms depending on the formulation. Specifically, PMVEMA-PVA30K (n = 0.3394) and PVP-PVA70K (n = 0.2699) exhibited Fickian diffusion behavior (n ≤ 0.45), suggesting that drug release is primarily controlled by diffusion through the polymer matrix. In contrast, CMC-PVA30K (n = 0.5124) showed non-Fickian (anomalous) transport, indicating a combination of diffusion and polymer relaxation or swelling mechanisms. The kinetic plots for all formulations fitted to Higuchi and Korsmeyer–Peppas models are illustrated in Fig. S2. The relatively lower correlation observed for zero-order and Hixson–Crowell models suggests that the drug release is neither constant over time nor significantly governed by matrix erosion or geometrical changes. Overall, these results confirm that diffusion is the dominant mechanism governing drug release during the initial phase, with formulation-dependent contributions from polymer swelling or relaxation.

Table 1.

The coefficient of determination (R²) and kinetics of PXCl release from MN formulations during the initial 6 h phase.

MN Formulations	Zero-order	First-order	Hixson-Crowell	Higuchi	Korsmeyer-Peppas
	R2	R2	R2	R2	R2	n
PMVEMA-PVA30K	0.9650	0.9859	0.9805	0.9957	0.9947	0.3394
CMC-PVA30K	0.9094	0.9686	0.9520	0.9777	0.9819	0.5124
PVP-PVA70K	0.8950	0.9642	0.9469	0.9620	0.9753	0.2699

3.3. Permeation of the PXCl

The permeation profiles of PXCl from 1.0 mg/cm² drug-loaded MN patches are presented in Fig. 7. Over a 72 h period, the cumulative amounts delivered were 583 ± 75 μg/cm² for PMVEMA-PVA30K, 361 ± 80 μg/cm² for CMC-PVA30K, and 444 ± 38 μg/cm² for PVP-PVA70K. The initial J_ps calculated between 1 and 8 h (Table 2, Column 2) were 21.1 ± 9.8, 4.4 ± 1.6, and 6.1 ± 1.2 μg/cm²·h, respectively. Correspondingly, the P_app (Table 2, Column 3) were 9.4 ± 4.4 × 10⁻⁸, 1.9 ± 0.7 × 10⁻⁸, and 2.7 ± 0.6 × 10⁻⁸ cm/s. Statistical analysis was conducted using the non-parametric Kruskal–Wallis test followed by Dunn's post-hoc test to compare the initial J_ps among the three MN groups. The statistic results indicated no significant differences in the initial J_ps between PMVEMA-PVA30K and PVP-PVA70K (p > 0.05), or between CMC-PVA30K and PVP-PVA70K (p > 0.05). However, the overall results for initial J_ps and P_app demonstrated that PXCl delivery from PMVEMA-PVA30K was significantly greater than that of PVP-PVA70K, followed by CMC-PVA30K (p < 0.05).

Fig. 7.

Permeation profile of PXCl across full-thickness human skin from different MN patch formulations. Data represent the mean ± SD (n = 3–4).

Table 2.

Pseudo-steady state flux (1–8 h), apparent permeability of PXCl across FTS using 1 mg/cm² PXCl-loaded MNs from different formulations.

1 mg/cm2 PXCl-loaded MN formulations	Pseudo-steady state flux (1–8 h) (μg/cm2h)	Apparent permeability coefficient (cm/s)
PMVEMA-PVA30K	21.1 ± 9.8	9.4 ± 4.4 × 10−8
CMC-PVA30K	4.4 ± 1.6	1.9 ± 0.7 × 10−8
PVP-PVA70K	6.1 ± 1.2	2.7 ± 0.6 × 10−8

These findings were not aligned with the in vitro drug release profiles. This discrepancy likely arises from the different experimental environments; the in vitro release study utilized a larger volume of PBS (∼5 mL) sufficient to dissolve the drug, whereas the in vitro permeation study involved a limited volume of fluid within the vicinity of the MN in the skin. A probable explanation for the differing profiles is the variation in polymer viscosity. The viscosities of the casting solutions for the drug-loaded PMVEMA-PVA30K, CMC-PVA30K, and PVP-PVA70K were 600–800 cPs, 11,000–13,000 cPs, and 2,000–3,000 cPs, respectively. These values correlate inversely with the permeation data: the formulation with the lowest viscosity exhibited the highest skin permeation, whereas the highest viscosity resulted in the slowest delivery. This suggests that the polymeric MNs likely dissolved and became trapped within the dermis, an effect that hindered drug delivery in the skin but was not captured by in vitro release models.

Moreover, the observed inverse correlation between the in vitro drug release and drug permeation profiles can be attributed to the disparate barrier properties of the two systems. Synthetic cellulose membranes lack of the complex phospholipid and protein architecture characteristic of skin (Flaten et al., 2015), which likely restricts the interaction between the PMVEMA polymer and the skin barrier. In contrast, within FTS, the carboxyl groups of PMVEMA may actively facilitate drug–skin partitioning, particularly when MN insertion bypasses the stratum corneum. The higher mechanical strength of PMVEMA-PVA30K MNs may also promote deeper penetration, increasing drug deposition in viable tissue. These findings indicate that while cellulose membrane assays serve as a reliable tool for initial release screening, they may significantly underestimate the drug delivery efficiency. The superior permeation performance of PMVEMA-PVA30K MNs highlights their potential as the most effective system for PXCl transdermal delivery.

3.4. Biocompatibility

The hemolysis assay was conducted to evaluate the blood compatibility of the three dissolving microneedle formulations (PMVEMA-PVA30K, CMC-PVA30K, and PVP-PVA70K) in both blank and PXCl-loaded MNs. As shown in Fig. 8, all formulations produced hemolysis levels well below the 2% threshold defined by ISO 10993–4, confirming non-hemolytic behavior. The positive control (1% Triton X-100) caused complete lysis of RBCs, while the negative control (PBS) showed non-hemolysis, validating the reliability of the assay. No significant difference was observed between blank and PXCl-loaded MNs, indicating that neither the polymer matrices nor the incorporation of PXCl adversely affected RBC integrity. Visual inspection of the RBC suspensions supported these findings, as clear supernatants were observed for all test samples, in contrast to the intense red coloration seen in the positive control. Overall, these results demonstrate that all MN formulations possess excellent hemocompatibility.

Fig. 8.

Hemolysis study of PXCl loaded MN formulations (A) Percentage hemolysis of polymeric formulations after 3 h incubation with human RBCs at 37 °C. triton X-100 (1%) and PBS served as positive (+ve) and negative (−ve) controls, respectively. The dashed line indicates the 2% hemolytic threshold. The data are represented as mean ± SD (n = 3). (B) Representative RBC suspensions post-incubation, showing hemolysis only in the positive control.

The cytocompatibility of the formulations was assessed using a CCK-8 assay on human dermal fibroblasts after 24 h of exposure. As shown in Fig. 9, both blank and PXCl-loaded MNs maintained cell viability levels above 80%, which is widely regarded as the threshold for non-cytotoxicity according to ISO 10993-5. In comparison, the positive control caused a near-complete loss of viability, confirming the sensitivity of the assay. No significant differences were detected between blank and PXCl-loaded formulations, demonstrating that neither the base polymer compositions nor the incorporation of PXCl induced cytotoxic effects. Together, these results confirm that all tested MN formulations are biocompatible and suitable for safe transdermal application.

Fig. 9.

Cytotoxicity of microneedle formulations assessed by CCK-8 assay. Cell viability of fibroblasts after 24 h exposure to blank and PXCl-loaded MNs. All formulations showed >80% viability, indicating good cytocompatibility. All data are presented as mean ± SD (n = 3).

3.5. Clinical relevance

Excised human skin (ex vivo) is a well-established model for predicting in vivo transdermal drug delivery, as the barrier layers—specifically the stratum corneum and dermis—are structurally comparable (Abd et al., 2016). In the present study, in vitro skin permeation data were used to evaluate the potential performance of MN-mediated delivery in humans. The required area (A_r) of MN patch to attain the steady-state plasma concentration (C_ss) of PXCl was estimated using the following Equation (Naik et al., 2000):

A_r × J_ss = C_ss × CL

The C_ss and the clearance (CL) of PXCl from human subjects were 5.09 ng/mL and 419 mL/min, respectively, as reported in the literature (Wright et al., 1997)). Assuming in vivo PXCl steady-state fluxes (J_ss) of 21.1 ± 9.8, 4.4 ± 1.6, and 6.1 ± 1.2 μg/cm²·h for PMVEMA-PVA30K, CMC-PVA30K, and PVP-PVA70K MN patches (each containing 1 mg/cm² PXCl), the required application areas to reach therapeutic levels would be 7.4 ± 4.0, 34.2 ± 18.3, and 21.6 ± 4.5 cm², respectively. These calculated areas are relatively large (7–34 cm²) suggesting that higher drug loading is required to enhance clinical feasibility. However, potential discrepancies between in vitro and in vivo MN-mediated delivery should be considered. Such variances may arise from physiological factors, including the viability of in-skin microchannels under in vivo conditions (Milewski et al., 2010; Nguyen and Banga, 2017). Moreover, PXCl is clinically administered as either an immediate-release formulation taken three times daily or as a once-daily extended-release formulation, with an elimination half-life of approximately 8–12 h (up to ∼ 12–14 h in elderly patients). Oral dosing typically ranges from 0.375 to 4.5 mg/day, producing C_ss in the low ng/mL range (∼5 ng/mL). In this study, the 48–72 h release profile observed from the MN system suggests sustained transdermal drug input following microneedle-mediated bypass of the stratum corneum, which may help maintain therapeutic plasma levels while potentially reducing dosing frequency and minimizing peak–trough fluctuations compared with oral administration. Based on the ex vivo permeation results, the PMVEMA–PVA30K formulation achieved a J_ss of 21.1 ± 9.8 μg/cm²h, corresponding to an estimated patch area of approximately 7.4 ± 4.0 cm² to reach therapeutic levels, indicating that clinically relevant drug delivery may be achievable within a practical patch size. Collectively, these findings highlight the potential of the MN system as an alternative delivery strategy that provides more controlled and sustained systemic drug input, particularly beneficial for patients with dysphagia or gastrointestinal dysfunction. However, in vivo pharmacokinetic studies will be required to confirm systemic exposure, bioavailability, and optimal dosing intervals.

4. Conclusion

Dissolving polymeric MNs were successfully developed for the transdermal delivery of PXCl. All formulations demonstrated favorable mechanical properties, insertion efficiency, complete drug release, and excellent biocompatibility. Among them, PMVEMA-PVA30K MNs showed slower release but achieved the highest PXCl permeation and flux across human skin, owing to strong drug–polymer interactions, enhanced insertion performance, and polymer-induced permeation enhancement. These findings suggest that dissolving MNs, particularly PMVEMA-PVA30K formulations, represent a promising transdermal drug delivery approach for PXCl in the context of Parkinson's disease. Further in vivo pharmacokinetic and long-term safety evaluations are warranted to fully establish their therapeutic relevance.

CRediT authorship contribution statement

Soracha D. Thamphiwatana: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Data curation, Conceptualization. Kankanit Phetporkha: Investigation. Chitinart Thedrattanawong: Investigation. Doungdaw Chantasart: Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Ethics approval and consent to participate

This study was performed in line with the principles of the Declaration of Helsinki. The use of human tissues was reviewed and approved by the Committee on Human Rights Related to Human Experimentation, Mahidol University, Thailand (COE No. MU-DT/PY-IRB 2022/056.1312). All procedures involving human blood samples were approved by the Committee on Human Rights Related to Human Experimentation, Mahidol University, Thailand (COA No. MU-MOU 2024/125.2603).

Funding

This research project is supported by Mahidol University (MU's Strategic Research Fund: 2023) Grant Number MU-SRF-RS-02A/66.

Declaration of competing interest

The authors declare no conflicts of interest.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.