Abstract
Turner syndrome, a rare chromosomal disorder affecting 1 in 2500 females, is characterized by the partial or complete absence of the second X chromosome. The primary treatment involves oestradiol (E2) hormone replacement therapy, essential for inducing puberty and maintaining secondary sex characteristics. While transdermal E2 patches are preferred due to their controlled release and bypassing of first-pass metabolism, paediatric dosing poses significant challenges since patches cannot be safely cut for adjustment. Although a weight-based nocturnal E2 regimen is recommended, achieving precise, personalised dosing is challenging to achieve. This study explores an innovative approach to obtain personalised transdermal doses using a handheld commercial inkjet printer to apply E2 directly onto the skin, in the form of temporary drug-loaded tattoos. A highly soluble (52.24 ± 8.76 mg/mL) E2-loaded pharma-ink was developed using a propylene glycol and ethanol (2:8) formulation. The ink was successfully printed in various designs on both paper and porcine skin, demonstrating excellent resolution and reproducibility. Dose linearity was confirmed (R2 > 0.96) with 3.8 ± 0.5 µg of E2 delivered per single printing pass using a 2 mg/mL pharma-ink. Personalised dosing is achievable by altering the concentration of E2 in the ink or by adjusting the number of printing passes, ideal for young Turner syndrome patients who require periodic dose adjustments. In vitro Franz Cell permeation studies revealed a first-order extended permeation profile, with 100 % of E2 permeating within 8 h, supporting nocturnal administration to mimic natural hormone fluctuations during puberty. Handheld inkjet printing therefore offers a novel solution by enabling precise, low-dose delivery of E2 directly onto the skin, making it a promising approach for tailored treatment in young patients with Turner syndrome.
Introduction
Turner syndrome is a rare chromosomal disorder affecting approximately 1 in 2500 females [1]. It is characterised by the partial or complete absence of the second X chromosome, first reported as a clinical syndrome in 1938 by Henry Turner [2]. The most common early symptom is short stature, often noticeable by the age of 5, with other features like loss of ovarian function, delayed puberty, and infertility appearing later [3]. Additional manifestations include congenital heart defects in 25–50 % of patients which are the most common cause of death [3,4], a webbed neck, skeletal abnormalities, gastrointestinal disorders, lymphedema, and an increased risk of hypertension, type II diabetes, and other complications [5].
Treatment involves the administration of oestradiol (E2), also known as 17β-oestradiol, the most potent and abundant form of estrogen in the body, in the form of hormonal replacement therapy [6]. E2 is essential in regulating the menstrual cycle, skeletal health, vascular function, and neurological systems and is thus used to induce puberty, maintain secondary sex characteristics, and induce uterine growth for potential future pregnancy [7]. Currently, two main forms of E2 administration exist: oral tablets and transdermal patches. E2 patches come in varying doses (14–100 µg) and release the hormone in a controlled manner. They are a popular platform of drug delivery as they reduce gastrointestinal irritation, avoid first-pass metabolism, are easy to administer or remove, making them especially suitable for patients with dysphagia [8]. Clinical studies show that transdermal E2 provides superior outcomes compared to oral administration for patients with Turner syndrome [[9], [10], [11]]. Transdermal E2 achieves higher therapeutic concentrations, leading to more effective feminization [9], enhanced uterine growth, accelerated spinal bone accumulation, and an overall increase in final height [12]. In contrast, oral E2 has been associated with an increased risk of stroke and thromboembolism and exposes the liver to higher doses than the rest of the body [13].
International guidelines recommend initiating transdermal E2 treatment for puberty induction between the ages of 11 and 12 years, with a starting dose of 3–7 µg/day, gradually increasing every six months over 2–3 years [14,15]. However, the lowest available dose (14 µg/day) is designed for adults [16]. To achieve paediatric doses, patches must be cut, which is discouraged by manufacturers due to concerns about altered adhesive properties, unpredictable drug absorption, and the risk of dose dumping [17,18]. A recent study on cut E2 patches stored at 35 °C for one month reported reductions in E2 content in four commercial brands: Systen/Evorel (−6.9 ± 6 %); Estraderm MX (−15.8 ± 1.5 %); Oesclim (−5.0 ± 3.8 %); Estradot (−57 ± 1 %). Intact patches showed no reduction in E2 content over the same period and under the same conditions [19].
The Turner syndrome working group of the European Society for Paediatric Endocrinology (ESPE) developed a transdermal E2 treatment protocol for paediatric patients with Turner syndrome [20,21]. The group unanimously recommended the use of 17β-oestradiol, with a weight-based dosage regimen overnight. This approach is supported by several studies, which found that nocturnal administration of E2 more closely mimicked the natural hormone fluctuations observed during spontaneous puberty [21,22]. The protocol starts with doses of 3–7 µg/day for lower-weight females, increasing with weight and treatment duration. Given the need for precise dose personalisation and titration based on the patient’s weight, E2 serum levels, and breast development, a promising solution for delivering these low and individualised doses is two-dimensional (2D) inkjet printing.
Originally developed in the 1950s for office use, inkjet printing has since rapidly expanded into the fields of electronics, biosensors [[23], [24], [25], [26]], medical devices [27], and pharmaceuticals [[28], [29], [30], [31], [32], [33]]. The process involves the precise deposition of small volume (1–100 pL) ink droplets onto 2D or three-dimensional (3D) substrates [34,35]. Formulating the optimal drug loaded ink (pharma-ink) can be challenging, and studies have investigated machine learning to predict ink printability [36]. There are two forms of inkjet printing technologies: thermal and piezoelectric, each differing in their mechanism of ink ejection. In thermal inkjet printing, a heating element generates a bubble that forces the ink droplet out of the printhead. In contrast, piezoelectric inkjet printing uses voltage pulses to eject ink in a controlled stream [37].
In recent years, inkjet printing has been applied to transdermal drug delivery, in the form of coated microneedles [[38], [39], [40], [41], [42]] and drug-releasing patches [43,44]. All transdermal work used in combination with inkjet printing has involved the use of patches or films, but this study evades this and for the first time prints medicines directly onto the patient. To date, no studies have explored the direct printing of drugs onto the skin itself. Unlike patches or films, which require additional adhesive layers and standardized sizes, inkjet printing directly on skin can adapt to individual needs, enabling localized treatment on irregular surfaces and minimising waste from adhesives, backing materials, and packaging associated with traditional patches. Moreover, this technology enhances patient comfort and adherence by creating visually appealing and personalised designs.
The emergence of handheld ‘tattoo printers’ for all ages demonstrates the appeal of inkjet printing for temporary or semi-permanent designs. These handheld printers have been used to print drugs on films [45] and, if used on the skin, they may offer customisation, convenience, and safety, using skin-safe and regulatory approved inks. Inspired by this technology, this study aims to utilise a commercially available handheld inkjet device to print E2 directly onto the skin, offering a personalised treatment approach for young female patients with Turner syndrome. Various low doses in the range of 3–20 µg will be printed to assess the E2 loading within a single print, and challenges associated with E2′s low solubility as a lipophilic drug will be addressed by solubility assessments for ink optimisation. Pharmaceutical printing suitability will be assessed on various surfaces such as paper, wafer paper, and porcine skin for resolution comparison, with porcine skin closely mimicking human skin [46]. In addition, in-vitro permeation studies using Franz diffusion cells will be conducted in triplicate to characterise the release profile and duration of E2 delivery, focusing on its potential for prolonged nocturnal administration of the hormone.
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Patricija Januskaite, Alvaro Goyanes, Mine Orlu, Abdul W. Basit, Inkjet printing of pharmaceutical tattoos for the direct deposition of oestradiol onto skin in turner syndrome, European Journal of Pharmaceutics and Biopharmaceutics, Volume 214, 2025, 114798, ISSN 0939-6411, https://doi.org/10.1016/j.ejpb.2025.114798.
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