Abstract
In vitro transcribed messenger ribonucleic acid (mRNA) constitutes an emerging therapeutic class with several clinical applications. This study presents a systematic comparison of different technologies—intradermal injection, microneedle injection, jet injection, and fractional laser ablation—for the topical cutaneous delivery of mRNA. Delivery of Cy5 labeled mRNA and non-labeled enhanced green fluorescent protein (eGFP) expressing mRNA was investigated in a viable ex vivo porcine skin model and monitored for 48 h. Forty 10 µm-thick horizontal sections were prepared from each skin sample and Cy5 labeled mRNA or eGFP expression visualized as a function of depth by confocal laser scanning microscopy and immunohistochemistry. A pixel-based method was used to create a semi-quantitative biodistribution profile. Different spatial distributions of Cy5 labeled mRNA and eGFP expression were observed, depending on the delivery modality; localization of eGFP expression pointed to the cells responsible. Delivery efficiencies and knowledge of delivery sites can facilitate development of efficient, targeted mRNA-based therapeutics.
Highlights
The administration of protein-based therapeutic agents to treat dermatological diseases has brought great relief to patients suffering from severe skin conditions, such as plaque psoriasis, atopic dermatitis, or skin cancer [1,2,3]
To enable a three-dimensional visualization of messenger ribonucleic acid (mRNA) delivery and protein expression, ex vivo porcine skin samples were cryotomed according to the previously described cutaneous biodistribution method (CBM) [47], to produce 40 lamellae, each with a thickness of 10 μm going from the skin surface to the lower dermis (400 μm) (Figure 1a)
Despite the initial sections being somewhat brittle due to skin surface rugosity and lack of tissue cohesion, widefield FM allowed the acquisition of high-resolution images of each complete lamella, enabling a clear visualization of the distribution of the Cy5 labeled mRNA and enhanced green fluorescent protein (eGFP) expression in each skin layer
Summary
The administration of protein-based therapeutic agents to treat dermatological diseases has brought great relief to patients suffering from severe skin conditions, such as plaque psoriasis, atopic dermatitis, or skin cancer [1,2,3]. Their physicochemical properties and susceptibility to degradation necessitate that the administration of such therapeutics must be parenteral, even if the disease is localized, leading to the risk of numerous and serious systemic off-target side effects and limiting more widespread use [3,4]. It consists of the delivery of vectorized genetic material to “modify or manipulate the expression of a gene or to alter
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