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Targeted decationized polyplexes for siRNA delivery Luis Novo, Kaori M. Takeda, Tamara Petteta, George R. Dakwar, Joep B. van den Dikkenberg, Katrien Remaut, Kevin Braeckmans, Cornelus F. van Nostrum, Enrico Mastrobattista, and Wim E. Hennink Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500499x • Publication Date (Web): 10 Nov 2014 Downloaded from http://pubs.acs.org on November 12, 2014
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Molecular Pharmaceutics
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Targeted decationized polyplexes for siRNA
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delivery
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Luís Novoa, Kaori M. Takedab, Tamara Pettetaa, George R. Dakwarc, Joep B. van den
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Dikkenberga, Katrien Remautc, Kevin Braeckmansc,d, Cornelus F. van Nostruma, Enrico
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Mastrobattistaa, Wim E. Henninka*
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a
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University, 3584 CG Utrecht, The Netherlands
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b
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Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht
Department of Materials Engineering, Graduate School of Engineering, The University of
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c
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University, Ghent Research Group on Nanomedicines, Harelbekestraat 72, 9000 Ghent, Belgium
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d
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Belgium
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KEYWORDS siRNA delivery, polymer, targeting, nanoparticle, biocompatibility
Laboratory for General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent
Centre for Nano- and Biophotonics, Ghent University, Harelbekestraat 72, 9000 Ghent,
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Molecular Pharmaceutics
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ABSTRACT
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The applicability of small interfering RNA (siRNA) in future therapies depends on the
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availability of safe and efficient carrier systems. Ideally, siRNA delivery requires a system that is
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stable in the circulation, but upon specific uptake into target cells can rapidly release its cargo
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into the cytoplasm. Previously, we evaluated a novel generation of carrier systems
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(‘decationized’ polyplexes) for DNA delivery and it was shown that folate targeted decationized
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polyplexes had an excellent safety profile and showed intracellular triggered release upon cell
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specific uptake. Targeted decationized polyplexes consist of a core of disulfide crosslinked
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poly(hydroxypropyl methacrylamide) (pHPMA) stably entrapping nucleic acids and a shell of
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poly(ethylene glycol) (PEG) decorated with folate molecules. In the present study the
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applicability of folate targeted decationized polyplexes for siRNA delivery was investigated.
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This required optimization of the carrier system particularly regarding the crosslinking density of
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the core of the polyplexes. Stable and nanosized siRNA decationized polyplexes were
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successfully prepared by optimizing the crosslink density of their core. Upon incubation in
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human plasma, a significant portion of siRNA remained entrapped in the decationized
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polyplexes as determined by fluorescence correlation spectroscopy (FCS). When tested in a
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folate receptor overexpressing cell line stably expressing luciferase, Skov3-luc, sequence specific
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gene silencing was observed. As expected, neither interference on the intrinsic luciferase
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expression nor on the cell metabolic activity (determined by XTT) was induced by the free-
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polymer or the siRNA polyplexes. In conclusion, targeted decationized polyplexes are safe and
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stable carriers that interact with the targeted cells and rapidly disassemble upon cell entry making
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them promising siRNA delivery systems.
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Molecular Pharmaceutics
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INTRODUCTION
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The use of small interfering RNA (siRNA) is currently explored as gene therapy strategy to
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treat several diseases, including viral infections, neurodegenerative disorders and cancer.1,
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siRNA therapies are based on the ability to induce efficient and specific gene silencing to reduce
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or eliminate expression of any target protein of interest. siRNA is composed of a 20–23 double
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strand nucleic acid sequence. siRNA can be synthetically produced and, when introduced in the
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cytoplasm of the cells, it is incorporated into a protein complex called the RNA-induced
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silencing complex (RISC). The siRNA activated RISC catalyzes subsequently the degradation of
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mRNA strands complementary to siRNA, blocking its translation into target proteins.3, 4
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siRNA is especially interesting, because upon release into the cytoplasm, transport into the
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nucleus is not necessary to exert its pharmacological action, unlike therapeutic DNA. However,
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just like DNA, siRNA cannot penetrate cellular membranes by passive diffusion due to its high
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molecular weight and hydrophilic character. Furthermore, siRNA can be easily degraded by
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nucleases present in the bloodstream. Therefore, systemic administration of siRNA requires safe
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and efficient carrier systems.
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Recently, several siRNA delivery systems based on polymers, lipids, peptides or proteins have
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been developed.4-6 Cationic polymers form nanoparticles with siRNA via electrostatic
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interactions, referred to as ‘polyplexes’. It has been observed in many studies that polyplexes
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require modification of the carrier surface with a neutral and hydrophilic polymer (e.g.
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poly(ethylene glycol) (PEG)) to shield their charge and minimize unwanted interactions with
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constituents present in extracellular fluids. Furthermore, PEGylation results in polyplexes with
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higher colloidal stability.7,
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The surface decoration of polyplexes with PEG, however, does
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neither avoid unfavorably biodistribution to healthy (nontarget) organs, particularly liver, spleen
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and kidneys, nor premature dissociation of polyplexes resulting in release of free siRNA in the
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circulation.9-13
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Because of the items mentioned above, efficient in vivo delivery of siRNA requires a system
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that is highly stable in the circulation and extracellular milieu, while retaining the ability to
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readily dissociate and release the loaded siRNA within the cell
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introduction of disulfide crosslinks in the core of siRNA polyplexes.17, 18 Disulfide crosslinking
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of nanocarriers is particularly interesting because these linkages are stable in the bloodstream,
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but prone to rapid cleavage in reducing conditions such as the intracellular environment.19
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, which can be achieved by
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Besides the severe effects induced at the systemic level (e.g. embolism or blood coagulation),
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the cationic nature of conventional polyplexes leads to severe toxicity at the cellular level as
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well.4, 20-22 So far, significant improvements have been reported regarding avoidance of systemic
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toxicity of polyplexes by PEgylation, however, cellular toxicity of polycations can only be
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partially reduced (i.e. by the use of biodegradable polymers).23, 24 Polycations can affect the cell
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viability at many levels, since they compromise the cell membrane integrity,25-27 interact with
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crucial cellular polyanions (i.e. cell receptors, enzymes, mRNA or genomic DNA),28 interfere
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with cell expression profile29-31 and activate oncogenes or induce apoptosis.26, 31, 32
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Given the insufficient in vivo stability of siRNA-polyplexes as well as their poor
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biodistribution and their intrinsic toxicity, alternatives for cationic polymers to design polyplexes
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are urgently needed. Decationized polyplexes, based on noncharged and hydrophilic polymers,
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have been developed as interesting plasmid DNA (pDNA) gene delivery systems.33 These
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polyplexes consist of a core of disulfide crosslinked poly(hydroxypropyl methacrylamide)
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(pHPMA), in which pDNA is entrapped, and a PEG shell. The formation and encapsulation of
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Molecular Pharmaceutics
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negatively charged nucleic acid molecules is firstly driven by electrostatic interactions using the
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presence of cationic charges in the polymer. After complex formation, followed by structure
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stabilization exploiting disulfide crosslinking, decationization occurs by hydrolytic cleavage of
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the carbonate ester groups which connect the cationic side groups to the pHPMA backbone.
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These decationized polyplexes showed a high and stable DNA loading, which is exclusively
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based on physical entrapment of the nucleic acid in the disulfide crosslinked core. Therefore,
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decationized polyplexes are intracellularly destabilized resulting in release of their payload by
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the specific cleavage of disulfide crosslinks in the reducing environment of the cytosol.
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Decationized polyplexes are based on neutral polymers which explains their excellent
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cytocompatibility and their low teratogenicity and low mortality potential in a zebrafish toxicity
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assay.33, 34 These particles also have shown a low degree of unspecific uptake, together with very
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high degree of cell specific uptake and transfection upon introduction of a targeting ligand at the
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distal end of the PEG shell.35 The preparation of folic acid (FA) targeted decationized polyplexes
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is particularly interesting because this small targeting molecule selectively binds with high
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affinity to the folate receptor, which is specifically overexpressed in several tumor types,
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including metastatic forms.36 Consequently, folate decorated nanomedicines have been
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developed for tumor targeting.37 FA targeting has also demonstrated its feasibility for siRNA
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delivery to improve the specificity of gene silencing.38-40
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In the present study, the applicability of folate targeted decationized polyplexes siRNA loading
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and release has been investigated. The applicability of decationized polyplexes for formulation
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and delivery of siRNA required optimization of both the polymer and the particle preparation
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process. Given its size,
