Stability of Free and Liposomal Encapsulated RNA on a Mucoadhesive PVA Polymer for Esophageal RNA Drug Targeting Using the EsoCap System
Abstract
The development of RNA and oligonucleotide-based therapeutics is a longstanding goal and is currently gaining significant attention. Several RNA-based drugs are approved for clinical use. Others are under investigation or in preclinical trials. This study have initiated the development of RNA drugs for localized use in the esophagus, utilizing the EsoCap System. This system’s core component is a mucoadhesive film that carries the RNA drug and is precisely applied to the esophagus. Research into the stability and properties of naked and liposomal-encapsulated RNA on mucoadhesive polymer film reveals that RNAs remain stable in various conditions without degradation, RNA leakage, or liposome fusion observed. The liposome size also remains constant after application on the film, drying, and rehydration. These findings pave the way for RNA drug development for esophageal diseases and their administration via the EsoCap system.
Introduction
The development of innovative advanced therapeutic options to treat, palliate and prevent diseases in our society is dependent on novel and highly effective active substances. For drug therapy in patients, there are various ways to establish innovations which also often lead to, for example, faster recovery or improved compliance. On the pharmacological side, the development of novel active substances improves the therapy of diseases.[1] From a technological perspective, advanced application forms ensure an improved use of medicines, an increase in compliance and often a decrease in duration of treatment.[2] However, even the best and most innovative active substances do not achieve superiority as long as there is no biorelevant possibility of drug delivery and application.[3]
A particularly significant and promising platform technology is, for example, lipid nanoencapsulation in combination with RNA technology, which is already being used worldwide for immunization against the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS Cov-2) virus when applied intramuscularly. An also promising technology is the EsoCap concept for local and long-term therapy of esophagus diseases, which has been investigated in a clinical phase II trial.[3, 4] Innovative active ingredients necessitate innovative dosage forms and advance their further development, as fresh forms of dosing entail a potential for innovative active components.[3, 5] In the field of RNA technology, chemically unmodified nucleic acids have a very short half-life due to their rapid degradation by nucleases and are also eliminated by the body’s own immune system, which is able to recognize foreign ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).[6] Furthermore, solubilized nucleic acids injected into the body were largely ineffective because the nucleic acids must be delivered into the cytoplasm (mRNA, siRNA, antisense oligonucleotides) or the nucleus (DNA, CRISPR-RNA) of the cell, to bind their target or, in the case of mRNA, to be translated to protein.[6] These challenges generated several innovations, such as the modification of nucleic acids to provide resistance to nuclease degradation, reduced immunogenicity and increased interaction with the target cell.[7] In 2018, the first lipid nanoparticle-based siRNA therapeutic, Onpattro, was approved for the treatment of polyneuropathy.[8] In addition to the further development of nucleic acids, it was also possible to draw on decades of development work on lipid-based nanoparticles for use in humans and on PEGylation.[9] The development, emergency use authorization and large-scale use in the USA of the nucleoside-modified mRNA vaccines from BioNTech/Pfizer (Comirnaty/BNT162b2) and Moderna (Spikevax/mRNA-1273), in which the RNA encodes the SARS Cov-2-specific spike protein, can be seen as a breakthrough innovation for this technology.[10] As with Onpattro, lipid nanoparticles with ionisable cationic lipids serve as the delivery platform for BioNTech and Moderna’s vaccines. These lipids have been optimized over decades and make a significant contribution to successful immunization.[10] The development of mRNA vaccines is a particularly topical example of the connection between the success of a therapy, the development of active substances and the use of suitable dosage forms.[3, 5]
In the field of esophageal therapy, the particularly short transit time through the ≈25 cm long muscular tube posed a major challenge for local treatment.[11] In addition, the esophagus has a kind of self-cleaning effect through peristalsis.[12] In studies on the residence time of syrups or other highly viscous preparations, Hefner et al. were able to measure a residence time of only a few minutes on the mucosa of the esophagus by means of scintigraphy.[13] However, the efficacy of local esophageal therapy is directly related to the residence time of the dosage form at the site of application, as shown by Dellon et al. in a study of patients with eosinophilic esophagitis using preparations of different viscosities.[14] The EsoCap concept was developed to address these challenges.[4] It consists of a mucoadhesive, thin polymer film that is rolled up and inserted into a commercially available but slotted hard gelatine capsule.[4] The free end of the film that protrudes from the capsule is connected to a thread called a retainer, which is responsible for the exact delivery of the system as a trigger mechanism.[4] There is also a placebo weight inside the capsule to facilitate the swallowing process.[4] The capsule with the film is placed in a special applicator to which the free end of the retainer is attached. The applicator, which resembles a beak cup, is filled with water so that when the device is taken, the capsule falls out of the applicator into the patient’s throat, where it is swallowed together with the water.[4] Once the retainer is expanded to the maximum, the film is pulled out of the capsule during transport through the esophagus and placed there. Afterwards, the film begins to dissolve and forms a mucoadhesive gel, from which the drug can be easily released.[4] The process resembles an eroding matrix and is not a diffusion-controlled release. Furthermore, mechanical factors such as esophageal peristalsis also contribute to the drug penetrating from the gel into the mucous membrane. The EsoCap system represents a completely new technology for the application of films in the esophagus, although films for local application to buccal or vaginal mucous membranes loaded with small molecules are already available.[4] There are a number of diseases of the esophagus that could potentially be treated depending on the loading of the film. In a Phase II study, the number of eosinophil peaks in inflammatory diseases, particularly eosinophilic esophagitis, was significantly reduced.[3, 4] Other diseases of interest for the device, include Barrett’s esophagus, cancer, spasticity and possibly gastroesophageal reflux disease (GERD).
The combination of the two existing innovative platform technologies in form of an RNA-loaded mucoadhesive film that can be placed locally in the gastrointestinal tract could have a major impact on the therapy of patients with diseases of the esophagus in the future. The aim of the following study was therefore to apply and stabilize free and liposomal encapsulated RNA onto a mucoadhesive polymer film that can potentially be placed on the mucosa of the esophagus, e.g. by using the EsoCap drug delivery concept. Initial experiments were carried out with free RNA to investigate how drying of the RNA on the EsoCap film affects RNA stability. RNA as a polyanion presents a challenge for cellular transfer due to its negative charges and thus, potentially poor membrane permeability.[15] Therefore, for improved future transfection and cellular delivery, RNA encapsulated in liposomes was included in the study. This should not only facilitate more efficient transfer, but can also provide protection against ubiquitous nucleases present in the human body.
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Materials
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and the instruments for the liposome preparation, extruder set, and polycarbonate membranes with a 100 nm pore size, were purchased from Avanti Polar Lipids inc. (Alabaster, USA). The tested oligonucleotides with and without ATTO633 label (5′-AUUUCGAGUUGGCUGUUGCUU-3′ (RNA A) and 5′-AUUUCGAGUUGGCUGUUGCUU-ATTO633-3′ (RNA B)) were obtained in HPLC grade from biomers.net GmbH (Ulm, Germany). Ethylenediaminetetraacetic acid (EDTA), ethanol (99.9%) and potassium chloride were supplied by Merck KGaA (Darmstadt, Germany), acrylamide/ bisacrylamide (19:1), ammonium persulfate, calcium chloride, sodium chloride, tetramethylethylenediamine and trichloromethane by Carl Roth GmbH (Karlsruhe, Germany), and disodium phosphate, formamide, sucrose and triethylammonium-bicarbonate by Sigma Aldrich (Taufkirchen, Germany). Tris(hydroxymethyl)aminomethane was purchased from Serva (Heidelberg, Germany) and Quant-it RiboGreen reagent from Invitrogen (Carlsbad, USA). Urea was supplied by J.T. Baker (New Jersey, USA).
The gel staining solution SYBR gold was purchased from Thermo Fisher Scientific (Waltham, USA). For the film preparation pharmaceutical broad spectrum polyvinyl alcohol 18–88 (PVA), with a degree of hydrolysis of 88% (Emprove series) and viscosity of 18 mPa*s (4% solution at 20 °C in water), was kindly provided by Merck KGaA (Darmstadt, Germany). Glycerol water free was purchased from Caelo (Germany). TritonX-100 was supplied by Acris Feinchemikalien GmbH (Heidelberg, Germany), Sephadex G-25 and G-50 from Pharmacia (Uppsala, Sweden). Autoclave DX-200 2D was from Systec (Wettenberg, Germany), Laminar air-flow box MS2020 1.8 from Thermo Scientific (Langenselbold, Germany) and Photosystem iBright FL 1500 from Invitrogen (Carlsbad, USA). For measuring the fluorescence of the RiboGreen, a DS-11 spectrofluorimeter from Denovix (Wilmington, USA) was used. For liposome size determination by dynamic light scattering (DLS), the Zetasizer Ultra from Malvern instruments (Herrenberg, Germany) was used. The rheometer DV3T extra was from Brookfield (Firmware V. 1.2.2-9, Hadamar-Steinbach, Germany) and the thermostat for temperature control was a Julabo Labortechnik GmbH system (Seelbach, Germany). The refractometer RX-5000 α-Plus (Atago Co., LTD, Tokyo, Japan) was used to determine the refractive indices.
Aileen Weide, Friederike Brokmann, Bettina Appel, Christoph Rosenbaum, Julius Krause, Una Janke, Mihaela Delcea, Werner Weitschies, Sabine Müller, Stability of Free and Liposomal Encapsulated RNA on a Mucoadhesive PVA Polymer for Esophageal RNA Drug Targeting Using the EsoCap System, First published: 25 June 2024 https://doi.org/10.1002/adtp.202300446
Read also our introduction article on Orally Disintegrating Tablets (ODTs) here:
