Lívia Neves Borgheti-Cardoso

Delivery Systems and Local Administration Routes for Therapeutic siRNA

ABSTRACTWith the increasing number of studies proposing new and optimal delivery strategies for the efficacious silencing of gene-related diseases by the local administration of siRNAs, the present review aims to provide a broad overview of the most important and latest developments of non-viral siRNA delivery systems for local administration. Moreover, the main disease targets for the local delivery of siRNA to specific tissues or organs, including the skin, the lung, the eye, the nervous system, the digestive system and the vagina, were explored.

Mucoadhesive System Formed by Liquid Crystals for Buccal Administration of Poly(Hexamethylene Biguanide) Hydrochloride

Antimicrobial approaches are valuable in controlling the development of buccal diseases, but some antibacterial agents have a short duration of activity. Therefore, the development of prolonged delivery systems would be advantageous. Liquid crystalline systems comprising monoolein (GMO)/water have been considered to be a potential vehicle to deliver drugs to the buccal mucosa because of the phase properties that allow for controlled drug release as well as its mucoadhesive properties. Therefore, the aim of this study was to develop a GMO/water system for the slow release of poly(hexamethylene biguanide) hydrochloride (PHMB) on the buccal mucosa and test the properties of this system with regard to swelling, release profile, antimicrobial activity, and strength of mucoadhesion, with the overall goal of treating buccal infections. The tested systems were capable of modulating drug release, which is controlled by diffusion of the drug throughout the system. Furthermore, PHMB appeared to improve the mucoadhesive properties of the system and may synergistically act with the drug to promote antimicrobial activity against S. mutas and C. albicans, indicating that liquid crystals may be suitable for the administration of PHMB on the buccal mucosa. Therefore, this system could be proposed as a novel system for mucoadhesive drug delivery.

Self-assembling gelling formulation based on a crystalline-phase liquid as a non-viral vector for siRNA delivery

Liquid crystalline systems (LCSs) form interesting drug delivery systems. These include in situ gelling delivery systems, which present several advantages for use as self-assembling systems for local drug delivery. The aim of this study was to develop and characterize in situ gelling delivery systems for local siRNA delivery. The influence of the components that form the systems was investigated, and the systems were characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), swelling studies, assays of their ability to form a complex with genes and of the stability of the genes in the system, as well as assays of in situ gelling formation and local toxicity using an animal model. The system containing a mixture of monoglycerides (MO), oleylamine (OAM), propylene glycol (PG) and tris buffer (8.16:0.34:76.5:15, w/w/w/w) was considered the most appropriate for local siRNA delivery purposes. The molecular structure was characterized as hexagonal phase; the swelling studies followed a second order kinetic model and the water absorption was a fast process reaching equilibrium at 2 h. The system formed a complex with siRNA and remained in a stable form. The gel was formed in vivo after subcutaneous administration of a precursor fluid formulation in mice and was biodegradable in 30 days. The inflammatory process that took place was considered normal. Therefore, the developed liquid crystalline delivery system shows the appropriate characteristics for use as a local siRNA delivery method for gene therapy.

An in situ gelling liquid crystalline system based on monoglycerides and polyethylenimine for local delivery of siRNAs

The development of delivery systems able to complex and release siRNA into the cytosol is essential for therapeutic use of siRNA. Among the delivery systems, local delivery has advantages over systemic administration. In this study, we developed and characterized non-viral carriers to deliver siRNA locally, based on polyethylenimine (PEI) as gene carrier, and a self-assembling drug delivery system that forms a gel in situ. Liquid crystalline formulations composed of monoglycerides (MO), PEI, propylene glycol (PG) and 0.1M Tris buffer pH 6.5 were developed and characterized by polarized light microscopy, Small Angle X-ray Scattering (SAXS), for their ability to form inverted type liquid crystalline phases (LC2) in contact with excess water, water absorption capacity, ability to complex with siRNA and siRNA release. In addition, gel formation in vivo was determined by subcutaneous injection of the formulations in mice. In water excess, precursor fluid formulations rapidly transformed into a viscous liquid crystalline phase. The presence of PEI influences the liquid crystalline structure of the LC2 formed and was crucial for complexing siRNA. The siRNA was released from the crystalline phase complexed with PEI. The release rate was dependent on the rate of water uptake. The formulation containing MO/PEI/PG/Tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) complexed with 10 μM of siRNA, characterized as a mixture of cubic phase (diamond-type) and inverted hexagonal phase (after contact with excess water), showed sustained release for 7 days in vitro. In mice, in situ gel formation occurred after subcutaneous injection of the formulations, and the gels were degraded in 30 days. Initially a mild inflammatory process occurred in the tissue surrounding the gel; but after 14 days the tissue appeared normal. Taken together, this work demonstrates the rational development of an in situ gelling formulation for local release of siRNA.

RNAi mediated IL-6 in vitro knockdown in psoriasis skin model with topical siRNA delivery system based on liquid crystalline phase

Gene therapy by RNA interference (RNAi) is a post-transcriptional silencing process that can suppress the expression of a particular gene and it is a promising therapeutic approach for the treatment of many severe diseases, including cutaneous disorders. However, difficulties related to administration and body distribution limit the clinical use of small interfering RNA (siRNA) molecules. In this study, we proposed to use nanocarriers to enable siRNA application in the topical treatment of skin disorders. A siRNA nanodispersion based on liquid crystalline phase and composed of monoolein (MO), oleic acid (OA) and polyethylenimine (PEI) was developed and its physicochemical properties, efficiency of complexation and carrier/siRNA stability were assessed. Subsequently, cell viability, cellular uptake, in vitro skin irritation test using reconstructed human epidermis (RHE) and in vitro IL-6 knockdown in psoriasis skin model were evaluated. The results showed that the liquid crystalline nanodispersion is a promising topical delivery system for administration of siRNA, being able to overcome the limitations of the route of administration, as well those resulting from the characteristics of siRNA molecules. The formulation was effective at complexing the siRNA, presented high rate of cell uptake (∼90%), increased the skin penetration of siRNA in vitro, and did not cause skin irritation compared with Triton-X (a moderate irritant), resulting in a 4-fold higher viability of reconstructed human epidermis and a 15.6-fold lower release of IL-1α. A single treatment with the liquid crystalline nanodispersion carrying IL-6 siRNA for 6h was able to reduce the extracellular IL-6 levels by 3.3-fold compared with control treatment in psoriasis skin model. Therefore, liquid crystalline nanodispersion is a suitable nanocarrier for siRNA with therapeutic potential to suppress skin disease-specific genes. This study also highlights the applicability of reconstructed skin models in pharmaceutical field to evaluate the performance of delivery systems without the use of animal models.

Liquid crystalline systems containing Vitamin E TPGS for the controlled transdermal nicotine delivery

Transdermal nicotine patches have been used in smoking cessation therapy, suggested for the treatment of skin disorders with eosinophilic infiltration and have been found to improve attention performance in patients with Alzheimers disease and age-associated memory impairment. However, skin irritation with extended patch use is still a problem. The aim of this work was to develop a simple to prepare liquid crystalline system containing vitamin E TPGS that would be able to control nicotine delivery and reduce irritation and sensitization problems. The liquid crystalline phases were macroscopically characterized by visual analysis and examined microscopically under a polarized light microscope. Topical and transdermal delivery of nicotine were investigated in vitro using porcine ear skin mounted on a Franz diffusion cell. Nicotine skin permeation from the developed cubic phase followed zero-order kinetics (r = 0.993) and was significantly enhanced after 12 h when compared to the control formulation (nicotine solution) (p < 0.05) (138.86 ± 20.44 and 64.91 ± 4.06 μg/cm2, respectively). Cubic phase was also able to target viable skin layers in comparison to control solution (8.18 ± 1.89 and 2.63 ± 2.51 μg/cm2, respectively). Further studies to evaluate skin sensitization and irritation are now necessary.

Topical and Transdermal Delivery of Drug-Loaded Nano/ Microsystems with Application of Physical Enhancement Techniques

Topical and transdermal delivery has been studied over last decades and it presents advantages for the treatment of several disorders, macromolecules delivery and vaccination. The greatest challenge is to overcome the stratum corneum (SC) barrier. Compared to traditional topical formulation strategies, nano /microsystems offer advantages such as increased stability, increased loading dose, coverage of undesired colors, reduced toxicity and prolonged release of active agents. However, there are no conclusive studies demonstrating the ability of such systems to penetrate the skin in relevant therapeutic amounts. The use of physical methods holds great promise for enhancing skin permeation through the SC and for targeting hair follicles. This review discusses the characteristics and feasibility of using a dual approach employing the application of physical methods of permeation enhancement to enable the topical or transdermal delivery of drug-loaded nano/microsystems.

In Situ Gelling Liquid Crystalline System as Local siRNA Delivery System

An effective short interfering RNA (siRNA) delivery system protects the siRNA from degradation, facilitates its cellular uptake, and promotes its release into the cytoplasm. Local administration of siRNA presents advantages over systemic administration, such as the possibility to use lower doses and allow local and sustained release. In this context, in situ solidifying organogels based on monoglycerides (MO), polyethylenimine (PEI), propylene glycol (PG) and tris buffer are an attractive strategy for intratumoral delivery of siRNA. In this study, precursor fluid formulation (PFF) composed of MO/PEI/PG/tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) was used to deliver siRNA to tumor cells. The internal structure of the gel obtained from PFF was characterized using small angle X-ray scattering (SAXS). In addition, its ability to complex siRNA, protect it from degradation, and functionally deliver it to tumor cells was investigated. Moreover, in vivo gel formation following intratumoral injection was evaluated. The gel formed in excess water from PFF was found to comprise a mixture of hexagonal and cubic phases. The system was able to complex high amounts of siRNA, protect it from degradation, promote siRNA internalization, and induce gene silencing in vitro in a variety of tumor cell lines. Moreover, a gel formed in situ following intratumoral injection in a murine xenograft model. In conclusion, PFF is a potential delivery system for local and sustained delivery of siRNA to tumor tissue after intratumoral administration.