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In situ gelling liquid crystalline system as local siRNA delivery system Livia N. Borgheti-Cardoso, Sander A.A. Kooijmans, Marcel H.A.M. Fens, Roy van der Meel, Fabiana T.M.C. Vicentini, Marcia C. A. Fantini, Maria Vitória L.B. Bentley, and Raymond M. Schiffelers Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01141 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017
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In situ gelling liquid crystalline system as local
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siRNA delivery system
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Livia N. Borgheti-Cardoso†, Sander A.A. Kooijmans‡, #, Marcel H.A.M. Fens‡, Roy van der
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Meel‡, §, Fabiana T.M.C. Vicentini†, Marcia C.A. Fantiniǁ, Maria Vitória L.B. Bentley†,*,
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Raymond M. Schiffelers‡
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† School of Pharmaceutical Sciences of Ribeirao Preto, University of São Paulo, Av. do Café,
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s/n, 14040–903 Ribeirão Preto, SP, Brazil.
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‡ Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht,
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Heidelberglaan, 100, 3584 CX Utrecht, The Netherlands.
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# Department of Medical Sciences, University of Torino, The Camussi Laboratory, Via Nizza,
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52, 10126 Torino, Italy.
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§ Department of Biochemistry and Molecular Biology, University of British Columbia, 2350
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Health Sciences Mall, Vancouver, British Columbia, Canada, V6T 1Z3.
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ǁ Instituto de Física, University of São Paulo, R. do Matão, 1371, Butantã, 05508-090 São Paulo,
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SP, Brazil.
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ABSTRACT GRAPHIC.
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KEYWORDS. siRNA, in situ gelling delivery system, liquid crystal, gene silencing
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ABSTRACT. An effective short interfering RNA (siRNA) delivery system protects the siRNA
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from degradation, facilitates its cellular uptake and promotes its release into the cytoplasm. Local
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administration of siRNA presents advantages over systemic administration, such as the
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possibility to use lower doses and allow local and sustained release. In this context, in situ
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solidifying organogels based on monoglycerides (MO), polyethylenimine (PEI), propylene
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glycol (PG) and tris buffer are an attractive strategy for intratumoral delivery of siRNA. In this
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study, precursor fluid formulation (PFF) composed of MO/PEI/PG/tris buffer at
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7.85:0.65:76.5:15 (w/w/w/w) was used to deliver siRNA to tumor cells. The internal structure of
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the gel obtained from PFF was characterized using Small Angle X-Ray Scattering (SAXS). In
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addition, its ability to complex siRNA, protect it from degradation and functionally deliver it to
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tumor cells was investigated. Moreover, in vivo gel formation following intratumoral injection
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was evaluated. The gel formed in excess water from PFF was found to comprise a mixture of
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hexagonal and cubic phases. The system was able to complex high amounts of siRNA, protect it
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from degradation, promote siRNA internalization and induce gene silencing in vitro in a variety
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of tumor cell lines. Moreover, a gel formed in situ following intratumoral injection in a murine
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xenograft model. In conclusion, PFF is a potential delivery system for local and sustained
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delivery of siRNA to tumor tissue after intratumoral administration.
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Introduction RNA interference (RNAi) is a process in which double-stranded RNA molecules (small interfering RNA, siRNA) inhibit gene expression by inducing the degradation of messenger
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RNA (mRNA).1 Since the discovery of RNAi in 1998, siRNA has been evaluated as a
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therapeutic strategy to treat various diseases because, in principle, it ensures potent and specific
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silencing of any desired genetic target.2 In addition, the physicochemical properties of siRNA
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generally do not change with its sequence, and synthetic production of siRNA is relatively
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straightforward.3 Despite these advantages, only a limited number of siRNA-based therapeutics
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have been evaluated in clinical settings.4
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Therapeutic application of siRNA is challenging due to its rapid degradation in the
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circulation and its physicochemical characteristics, including a negative charge and high
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molecular weight (~13 kDa). These prevent cellular uptake, and –even when cellular uptake does
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occur- its escape from the endosome.5 To overcome these challenges, the development of
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appropriate delivery systems is required.
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An ideal delivery system should fulfill two contradictory requirements: it should stably complex siRNA to protect it from the extracellular environment and to promote its cellular
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uptake and, once in the intracellular environment, it should effectively release the siRNA into the
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cytoplasm. The balance between protection and release of siRNA in the cytoplasm has been the
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biggest challenge of non-viral delivery systems.6–8
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To design an efficient siRNA delivery system, it is also important to consider the target
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tissue and the appropriate route of administration. Each route of administration has its own
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characteristics and obstacles that should be overcome to deliver siRNA. Systemic administration
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faces several challenges including low bioavailability, rapid clearance by macrophages of the
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mononuclear phagocyte system, systemic toxicity and -for extra hepatic targeting- inefficient
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targeting to the desired organ or cell type.2,9 Such issues are generally avoided when drug
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delivery systems are administered locally. Furthermore, this route of administration presents
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other advantages, such as the possibility to use lower doses, allow local and sustained release and
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decrease immunostimulation.9
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Injectable depot formulations such as in situ gelling delivery systems are an appealing
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approach to release siRNA locally 8,10–15 because they are minimally invasive and painful when
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compared to implants. These formulations are injected into the body, and form a gel locally
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which can promote a sustained release of the drug.16
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The first in situ gelling delivery systems for siRNA release were obtained by in situ
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crosslinking or precipitation method.12,14 These systems locally release the siRNA, but not in a
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controllable way. Furthermore, the siRNA was released without being complexed to a carrier,
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which resulted in a short half-life and low transfection efficiency.13,15 In addition, the in situ
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crosslinking and precipitation method can harm tissues and drug cargo.16
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Such side effects can be avoided by formulating the gelling delivery systems from
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biocompatible and safe materials.17 In this context, in situ solidifying organogels offer an
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attractive approach.10,11 These can be obtained from amphiphilic lipids such as monoglycerides
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(MO) which absorb water from the environment and self-assemble in inverted liquid crystalline
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structures.17,18 The change of a fluid formulation containing MO into a viscous liquid crystalline
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structure upon contact with excess water can be explained based on transformations of the
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critical packing parameter (cpp), which is described by the equation cpp = vs/aolc (where vs is the
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hydrophobic chain volume, ao is the polar head group area and lc is the chain length).19 The
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increase in the water content favors the polar head group of the MO to move more freely causing
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a disorder of the MO hydrophobic chain which increases vs. The ao tends to be constant because
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the interaction of the polar head groups is strong due to hydrogen bonds. Hence, the cpp
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increases because vs increases while ao and lc are constant, favoring the transformation from a
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lamellar to a cubic phase.11,20 These fluid MO-based systems that are formulated for in situ
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gelling have several advantages such as the well-defined internal nanostructures that are capable
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of solubilizing and protecting hydrophilic, hydrophobic and amphiphilic drugs. In addition, the
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favorable toxicity profile and the potential to control the release of different drugs renders these
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systems highly attractive for drug release applications.17,20–23
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In previous work, we developed in situ solidifying organogels based on MO.10,11
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Propylene glycol (PG) and tris buffer were added to guarantee fluidity and polyethylenimine
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(PEI) was incorporated to complex the siRNA. It was shown this fluid system, or precursor fluid
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formulation (PFF), forms a gel in the presence of excess water. The incorporation of PEI was
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essential to complex the siRNA, which was released from this system in a controlled manner and
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complexed with PEI.11 In the present study, the PFF was evaluated for its ability to effectively
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deliver siRNA to tumor cells. The internal structure of the gel in a range of PFF/siRNA ratios,
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obtained in excess of water, was evaluated by Small Angle X-Ray Scattering (SAXS). The
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capability of the PFF to complex siRNA and to protect it from degradation in serum was also
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analyzed. In addition, it was evaluated whether the system could promote cellular uptake of
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siRNA and induce subsequent gene silencing in vitro. Finally, a study to evaluate gel formation
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in vivo after intratumoral (i.t.) injection was performed.
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Materials and Methods
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Materials
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Monoglyceride (MO, Myverol 18–92 K consisting of 93% of monoglycerides (containing
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65% glyceryl monolinoleate, 23% glyceryl monooleate, 6% monoglyceride (C16), 4%
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monoglyceride (C18), 1% monoglyceride (C20) and 1% monoglyceride (C18:3)), 6%
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diglycerides and triglycerides following the same fatty acid profile as the MO and other minor
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components (
