Development of a Sensory Neuron-Integrated Skin Spheroid Model for the Evaluation of Neuropeptide-Based Topical Delivery Systems

Bianca Aparecida Martin, Juliana Viegas, Luciana Facco Dalmolin, Emerson de Souza Santos, Izabela Pereira Vatanabe, Sabrina Francesca Lisboa, Renata Fonseca Vianna Lopez, Bruno Sarmento

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Abstract

The skin is a complex organ composed of multiple layers and diverse cell types, including keratinocytes, fibroblasts, adipocytes, and sensory neurons, which maintain its structural and functional integrity together. Conventional in vitro and ex vivo models help investigate drug permeation and selected biological effects. However, they are limited in replicating neural interactions critical for assessing the efficacy of neuropeptide-based therapies. To address this limitation, a sensory neuron-integrated skin spheroid (SS) model was established, incorporating key skin cell types and providing a rapid, adaptable, and physiologically relevant platform for screening the biological activity of topical delivery systems targeting neuronal pathways. The model’s responsiveness was demonstrated using acetyl hexapeptide-3 (HEX-3), a neuropeptide that inhibits acetylcholine release. HEX-3 was internalized by spheroid cells, with preferential accumulation around sensory neurons, confirming targeted cellular uptake. In parallel, ex vivo human skin studies confirmed that HEX-3 can traverse the stratum corneum and accumulate in deeper layers. Treatment with this film enhanced skin hydration, reduced scaling, and improved the structural organization of the stratum corneum after 48 h. Functional assays using the SS model showed that HEX-3 treatment suppressed acetylcholine release, upregulated the antioxidant enzyme SOD2, and stimulated type I collagen synthesis. In aged skin samples, the application of HEX-3 significantly increased collagen levels. This effect was mirrored in the spheroid model, which reached collagen levels comparable to those of aged human skin upon treatment. These findings establish the SS model as a robust platform for evaluating the biological activity of neuropeptide-based topical therapies, offering valuable insights for developing advanced strategies for skin rejuvenation and repair.

Keywords: Aged skin; Acetyl hexapeptide-3; Skin spheroids; Neuronal model; Aging products

Enhancing pulmonary delivery and immunomodulation of respiratory diseases through virus-mimicking nanoparticles

Yugo Araújo Martins, Isabel Cristina Guerra-Gomes, Tamara Silva Rodrigues, Caroline Tapparel, Renata Fonseca Vianna Lopez

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Abstract

This study introduces the nanobromhexine lipid particle (NBL) platform designed for effective pulmonary drug delivery. Inspired by respiratory virus transport mechanisms, NBL address challenges associated with mucus permeation and inflammation in pulmonary diseases. Composed of low molecular weight polyethylene glycol-coated lipid nanoparticles with bromhexine hydrochloride, NBL exhibit a size of 118 ± 24 nm, a neutral zeta potential, osmolarity of 358 ± 28 mOsmol/kg, and a pH of 6.5. Nebulizing without leakage and showing no toxicity to epithelial cells, NBL display mucoadhesive properties with a 60% mucin-binding efficiency. They effectively traverse the dense mucus layer of Calu-3 cultures in an air-liquid interface, as supported by a 55% decrease in MUC5AC density and a 29% increase in nanoparticles internalization compared to non-exposed cells. In assessing immunomodulatory effects, NBL treatment in SARS-CoV-2-infected lung cells leads to a 40-fold increase in anti-inflammatory MUC1 gene expression, a proportional reduction in pro-inflammatory IL-6 expression, and elevated anti-inflammatory IL-10 expression. These findings suggest a potential mechanism to regulate the excessive IL-6 expression triggered by virus infection. Therefore, the NBL platform demonstrates promising potential for efficient pulmonary drug delivery and immunomodulation, offering a novel approach to addressing mucus permeation and inflammation in pulmonary diseases.

Keywords: Lipid nanoparticles; Mucus density reduction; Inflammatory response modulation; Viral respiratory diseases; Coronavirus

Iontophoresis impact on corneal properties using an ex vivo bovine eye model

Gabriela Fávero Galvão, Izabella Cristina Bernardo Maríngolo, Yugo Araújo Martins, Janette Bezebeth Villarruel Muñoz, Marina Zilio Fantucci, Ricardo Roberto da Silva, Eduardo Melani Rocha, Eloísa Berbel Manaia, Gilles Ponchel, Renata Fonseca Vianna Lopez

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Abstract

This study addresses the challenge of low drug bioavailability in topical ocular administration by developing and validating an ex vivo bovine eye model chamber to evaluate the effects of iontophoresis on drug delivery and corneal properties. Transepithelial electrical resistance (TEER) was assessed as a predictor of corneal epithelial integrity in dissected bovine eyes. TEER measurements were correlated with methylene blue permeation, confirming a threshold of 4.2 kOhm·cm2 as an indicator of epithelial integrity. The model chamber enabled the application of drug solutions around a defined area of the cornea without leakage, facilitating the placement of electrodes and the application of constant electric currents. Applying iontophoresis at 2 mA/cm2 for 6 min significantly increased rhodamine B penetration into the cornea by nearly sixfold compared to passive diffusion (approximately 1.3 µg/cm2 vs. 0.24 µg/cm2), allowing detectable drug levels in the aqueous humor (27.9 ± 0.5 ng/mL). Morphological analyses revealed temporary changes in the cornea, including a 2.3-fold increase in surface roughness (from 44.6 nm to 105.3 nm) and mild collagen disorganization in the stroma, while Bowman’s membrane remained intact. A significant increase in corneal stiffness was noted, with a 200 % rise in the area under the stress–strain curve after iontophoresis. These findings provide insights into iontophoresis-induced changes and highlight the model’s potential for optimizing ocular drug delivery systems. Additionally, the model aligns with the 3Rs principles and could be instrumental in advancing the understanding of anterior segment diseases driven by structural and biomechanical alterations.

Keywords: Ex vivo model; Iontophoresis; Ocular drug delivery systems

Iontophoresis-driven alterations in nanoparticle uptake pathway and intracellular trafficking in carcinoma skin cancer cells

Gabriela Fávero Galvão, Raquel Petrilli, Vanessa Cristina Arfelli, Andréia Nogueira Carvalho, Yugo Araújo Martins, Roberta Ribeiro Costa Rosales, Leticia Fröhlich Archangelo, Luis Lamberti Pinto daSilva, Renata Fonseca Vianna Lopez

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Abstract

Effective treatment of squamous cell carcinoma (SCC) poses challenges due to intrinsic drug resistance and limited drug penetration into tumor cells. Nanoparticle-based drug delivery systems have emerged as a promising approach to enhance therapeutic efficacy; however, they often face hurdles such as inadequate cellular uptake and rapid lysosomal degradation. This study explores the potential of iontophoresis to augment the efficacy of liposome and immunoliposome-based drug delivery systems for SCC treatment. The study assessed iontophoresis effects on SCC cell line (A431) viability, nanoparticle uptake dynamics, and intracellular distribution patterns. Specific inhibitors were employed to delineate cellular internalization pathways, while fluorescence microscopy and immunohistochemistry examined changes in EGFR expression and lysosomal activity. Results demonstrated that iontophoresis significantly increased cellular uptake of liposomes and immunoliposomes, achieving approximately 50 % uptake compared to 10 % with passive treatment. This enhancement correlated with modifications in endocytic pathways, favoring macropinocytosis and caveolin-mediated endocytosis for liposomes, and macropinocytosis and clathrin-mediated pathways for immunoliposomes. Moreover, iontophoresis induced alterations in EGFR distribution and triggered syncytium-like cellular clustering. It also attenuated lysosomal activity, thereby reducing nanoparticle degradation and prolonging intracellular retention of therapeutic agents. These findings underscore the role of iontophoresis in modulating nanoparticle internalization pathways, offering insights that could advance targeted drug delivery strategies and mitigate therapeutic resistance in SCC and other malignancies.

Keywords: EGFR; Lysosome degradation; Endocytic routes

Electrostimulable polymeric films with hyaluronic acid and lipid nanoparticles for simultaneous topical delivery of macromolecules and lipophilic drugs

© 2024. Controlled Release Society.

Liquid crystalline nanogel targets skin cancer via low-frequency ultrasound treatment

Tatiana Aparecida Pereira ^a, Danielle Nishida Ramos ^a, Lays Martin Sobral ^a, Yugo Araújo Martins ^a, Raquel Petrilli ^a b, Márcia de Abreu Carvalho Fantini ^c, Andréia Machado Leopoldino ^a, Renata Fonseca Vianna Lopez ^a

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Abstract

The potential of low-frequency ultrasound (LFU) combined with nanotechnology-based formulations in improving skin tumors topical treatment was investigated. The impact of solid lipid nanoparticles (SLN) and hydrophilic nanogels as coupling media on LFU-induced skin localized transport regions (LTR) and the penetration of doxorubicin (DOX) in LFU-pretreated skin was evaluated. SLN were prepared by the microemulsion technique and liquid crystalline nanogels using Poloxamer. In vitro, the skin was pretreated with LFU until skin resistivity of ∼1 KΩ.cm2 using the various coupling media followed by evaluation of DOX penetration from DOX-nanogel and SLN-DOX in skin layers. Squamous cell carcinoma (SCC) induced in mice was LFU-treated using the nanogel with the LFU tip placed 5 mm or 10 mm from the tumor surface, followed by DOX-nanogel application. LFU with nanogel coupling achieved larger LTR areas than LFU with SLN coupling. In LFU-pretreated skin, DOX-nanogel significantly improved drug penetration to the viable epidermis, while SLN-DOX hindered drug transport through LTR. In vivo, LFU-nanogel pretreatment with the 10 mm tip distance induced significant tumor inhibition and reduced tumor cell numbers and necrosis. These findings suggest the importance of optimizing nanoparticle-based formulations and LFU parameters for the clinical application of LFU technology in skin tumor treatment.

Graphical abstract

Keywords: Low frequency ultrasound Doxorubicin Skin cancer therapy Sonophoresis Topical treatment.

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Investigation of the antimicrobial effect of anodic iontophoresis on Gram-positive and Gram-negative bacteria for skin infections treatment

Sofia Gerotto Viola, Luciana Facco Dalmolin, Janette Betzabeth Villarruel Muñoz, Yugo Araújo Martins, Ana Carolina dos Santos Ré, Carolina Patrícia Aires, Renata Fonseca Vianna Lopez

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Highlights

• E. coli is more susceptible to iontophoretic parameter changes than S. epidermidis
• Iontophoresis altered bacteria morphology and induced high rates of E. coli death.
• Anodic iontophoresis can control Gram-negative bacterial proliferation in wounds.

Abstract

Iontophoresis, a non-invasive application of a constant low-intensity electric current, is a promising strategy to accelerate wound healing. Although its mechanisms are not yet fully elucidated, part of its action seems related to inhibiting bacteria growth. This work aimed to investigate the antimicrobial effect of iontophoresis using Staphylococcus epidermidis and Escherichia coli strains, Gram-positive and Gram-negative bacteria, respectively. Anodic iontophoresis was applied to each bacterial suspension using Ag/AgCl electrodes, and bacteria viability was evaluated after 24 h incubation by counting colony-forming units. A Quality-by-Design approach was performed to assess the influence of the iontophoresis’ intensity and application time on bacterial viability. Cell morphology was evaluated by scanning electron microscopy. Iontophoresis showed antimicrobial effects on the Gram-positive bacteria only at 5 mA and 60 min application. However, a linear relationship was observed between intensity and application time for the Gram-negative one, causing drastic morphological changes and up to 98 % death. The cell wall of Gram-negative bacteria seems more susceptible to disorganization triggered by iontophoresis-induced ion transport than Gram-positive ones. Therefore, anodic iontophoresis can be a powerful ally in controlling Gram-negative bacteria proliferation in wounds.

Graphical abstract