Cellular environment-responsive nanomaterials for use in gene and siRNA delivery: molecular design for biomembrane destabilization and intracellular collapse.

Expert Opinion on Drug Delivery

ISSN: 1742-5247 (Print) 1744-7593 (Online) Journal homepage: http://www.tandfonline.com/loi/iedd20

Cellular environment-responsive nanomaterials for use in gene and siRNA delivery: molecular design for biomembrane destabilization and intracellular collapse Hiroki Tanaka, Yusuke Sato, Hideyoshi Harashima & Hidetaka Akita To cite this article: Hiroki Tanaka, Yusuke Sato, Hideyoshi Harashima & Hidetaka Akita (2016): Cellular environment-responsive nanomaterials for use in gene and siRNA delivery: molecular design for biomembrane destabilization and intracellular collapse, Expert Opinion on Drug Delivery, DOI: 10.1517/17425247.2016.1154531 To link to this article: http://dx.doi.org/10.1517/17425247.2016.1154531

Accepted author version posted online: 15 Feb 2016.

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Date: 18 February 2016, At: 17:21

Publisher: Taylor & Francis Journal: Expert Opinion on Drug Delivery DOI: 10.1517/17425247.2016.1154531 REVIEW

Cellular environment-responsive nanomaterials for use in gene and siRNA delivery: molecular design for biomembrane destabilization and intracellular collapse

Downloaded by [New York University] at 17:21 18 February 2016

Hiroki Tanaka, Yusuke Sato, Hideyoshi Harashima and Hidetaka Akita*

Faculty of Pharmaceutical Sciences, Hokkaido University Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan

*To whom correspondence should be addressed: Tel: +81-11-706-3735 Fax: +81-11-706-4879 E-mail [email protected]

Abstract

Introduction: The development of gene and nucleic acid-based medication is one of the ultimate strategies in the research field of personalized medicine. For the desired function of a gene or siRNA, these molecules need to be delivered to the appropriate organelle (i.e. nucleus and cytoplasm, respectively). Areas covered: The topics covered herein are rational design in order to control the pharmacokinetics, intracellular trafficking and release (decondensation or decapsulation) of the intended material. Since the endosome and cytoplasm are acidic (endosome) and reducing (cytoplasm) environments, respectively, a large variety of the materials have been developed that induce destabilization of endosome via its protonation, or are spontaneously collapsed in the cytoplasm. Finally, we propose materials (SS-cleavable and pH-activated lipid-like materials: ssPalm) that mount these sensing motifs, i.e., a positive charging unit in response to the acid environment (tertiary amines) and a cleavage unit (disulfide bonding) that is responsive to an reducing Downloaded by [New York University] at 17:21 18 February 2016

environment, respectively. Expert opinion: Currently, the main target of the nanocarrier-mediated siRNA delivery systems is liver. The targeting of non-hepatic tissue is the next challenge. In this case, the design of neutral particle with well-organized intracellular trafficking, as well as an identification of the promising ligand is needed.

Keywords: DNA, intracellular trafficking, siRNA, SS-cleavable pH-activated lipid-like material, ssPalm, pH

Article highlights box •

For successful gene or nucleic acid therapeutics, the cargos must be delivered to the appropriate organelle is necessary

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Functionalized materials that can be responsive to the intracellular environment are essential to control an intracellular trafficking

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Acidic pH-triggered membrane-fusion and/or membrane-disruption represents a rational design for the endosomal escape

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Cleavage of disulfide bonding is a promising trigger for the reductive environment-responsive release of cargo

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Design of neutral particle is beneficial for the in vivo application via intravenous administration

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Neutral particle prepared with ssPalm that mounts positive-charging unit and cleavable unit in response to the


acidic (endosome)- and reductive (cytoplasm)-environment is one of the rational design for the nano DDS platform

1. Introduction In parallel with the rapid progress in post-genome-related “-omics” technologies, gene- or nucleic acid- based therapeutics is now recognized as an attractive approach for developing personalized medications. Gene therapy can complement a hereditary-lacking gene. While the gene therapy approach had not been approved for a long period, largely due to technical and/or regulatory impediments, a viral gene-medicine; Glybera® (UniQure) was first approved the by the European Medicinal Agency (EMA) in 2012. While success is now limited to ultraorphan disease (familial lipoprotein lipase deficiency with pancreatitis attacks), this historical achievement promises to stimulate the scientific community to realize the significance of the gene therapy. Meanwhile, nucleic acids (i.e. short interference RNA; siRNA and micro RNA: miRNA) can be used to knock-down or down-regulate disease-related genes. Phase III trials of nucleic acid therapeutics, such as siRNA against transthyretin familial amyloid (ATTR), and antisense oligonucleotides for muscular dystrophy are currently in progress. For successful gene or nucleic acid therapeutics, a rational technology that can deliver the gene or nucleic Downloaded by [New York University] at 17:21 18 February 2016

acids to the appropriate organelle (nucleus and cytoplasm, respectively) is necessary (Fig.1). Nano-assembled architecture of the artificial materials is a quite key technologies in biomedical application including drug delivery, as well as imaging and scaffold in cell culture[1-3]. From the point of view of nucleic acid delivery, one of the requirements is the ability to overcome biomembranes (endosomal membrane, as well as the nuclear membrane in gene delivery). To date, a large variety of the nanoparticles that are formed via liposomes or polymers have been reported. Especially when a delivery system that can be applied via intravenous administration is designed, we will need to consider an ultimate dilemma: the stability of the nanoparticle in the extracellular domain (i.e. blood circulation), as well as in storage conditions should be maximized, while the particle should be degraded to release the gene and nucleic acids at the target organelle of action. To overcome this dilemma, functionalized materials that can be responsive to the intracellular environment are essential. Nanoparticles are generally taken up via endocytic pathways. Thus, certain kinds of functional devices or units that are capable of inducing endosomal escape are necessary. Otherwise, the particles will be subject to the lysosomal degradation, or will be secreted to the extracellular region via a recycling pathway[4]. The pH in endosomes becomes decreased during the transition from the early endosome to the late one. Thus, the acidic pH-triggered destabilization of the endosomal membrane represents a rational design for the control of intracellular trafficking. In this review, we first summarize strategies for endosomal escape. Meanwhile, the release (decondensation or decapsulation) of the cargo from the carrier is a key rate-limiting process for the efficient transcription of DNA, or the efficiency of gene knockdown of siRNA. In fact, coating a gene or siRNA with a fewer number of lipid envelopes results in a drastic improvement in gene transfection activity or gene knockdown efficiency, in parallel with an extensive decapsulation of the cargo[5, 6]. After endosomal escape, the particles are exposed to a reducing environment (cytoplasm) that is rich in glutathione (GSH)[7, 8]. Thus, reducing environment-triggered collapse is also an attractive design for such biomaterials. Thus, we overviewed materials that are cleaved in the intracellular region. In the final section, we propose the design of materials that mount dual motifs that incorporate the functions of pH-triggered destabilization, and reducing environmentresponsive collapse.

2. Common strategies for realizing endosomal escape

The importance of endosomal escape was also evidenced by the fact that the transfection activity of plasmid DNA (pDNA)-loaded nanoparticles was enhanced in the presence of chloroquine, a lysosomotropic reagent[9, 10]. After endocytosis, the cargos were delivered to lysosomes within 15 min[11]. During this process, the pH in the vesicle decreases to pH6.4 (small and tubule compartment) and pH5.5 (large endosome)[12]. More recent studies indicated that the pH in the vesicular compartment can be decreased to 4.0~4.5[13, 14]. Four categories of devices have been developed for improving endosomal escape (Fig.2): (A) pH-sensitive fusogenic lipids (charge: negative to neutral); (B) pH-sensitive fusogenic lipids (charge: neutral to positive); (C) polycations that have proton sponge characteristics; and (D) pH-sensitive membrane fusogenic peptides.

2.1. Fusogenic lipids (Charge: negative to neutral) The first strategy involves acidic pH-triggered membrane fusion between the lipid bilayer in lipoplexes and endosomal membranes[15]. This event is accompanied by the phase transition of lipids from a lamellar phase to a Downloaded by [New York University] at 17:21 18 February 2016

hexagonal H II phase. 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), a conically shaped lipid, is heavily used as the lipid component of a membrane-fusogenic lipid layer. DOPE seems to be essential to cause this pHdependent membrane fusion, since a similar neutral lipid, DOPC, does not lead to endosomal escape[16]. To allow the liposomes to fuse with endosomes in a pH-dependent manner, a second lipid component is needed. One example of such a material is cholesterol hemisuccinate (CHEMS), an inverted conical shaped lipid[17], whose incorporation (>20mol% of total lipid) can stabilize the DOPE in the lamellar phase with an aid of electrostatic repulsion among the CHEMS[18]. When the pH was decreased to a range close to its pKa (~5.8), the fraction of ionized CHEMS is decreased by the protonation of the carboxyl group. Since the loss of the charge in the headgroup of CHEMS results in a structural change from an inverted cone shape to a cylinder- or cone-shape, the lamellar structure of DOPE/CHEMS is finally transformed into an inverted hexagonal H II phase[19, 20]. This structure is highly unstable and rapidly fuses to and destabilizes the endosomal membrane, releasing the associated DNA into the cytosol (Cullis, P. R. et al. (1986)).

2.2. Fusogenic lipids (Charge: neutral to positive) Other types of pH-sensitive fusogenic liposomes involve the use of an ionizable aminolipid that mounts a tertiary amine that carries a positive (cationic) charge at an acidic pH. An ionizable aminolipid 1,2-dioleoyl-3dimethylammonium propane (DODAP) has been used for nucleic acid[21, 22] or pDNA[23, 24] delivery. Liposomes containing DODAP are neutral at physiological pH, and acquire a positive charge when taken up into the endosomal compartment. Meanwhile, the negatively charged lipid (i.e. phosphatidylserine) content of the endosomal membrane is greater than that of the plasma membrane[25]. Thus, the pH-dependent cationized lipid electrostatically interacts with the anionic phospholipids in the endosomal membrane, and thereby adopt a nonbilayer structure (hexagonal H II )[26]. Presumably, a membrane fusion event accompanies the flip-flop of anionic lipids of the endosomal membranes from the cytoplasmic side to the endosomal lumen side [27]. This lipid mixing displaces the ion-pairing of cationic lipid from DNA or siRNA to the endosome-derived anionic lipids, and thereby allows the cargos to be released into the cytoplasm. Recently, the properties of the ionizable aminolipid, including fusogenic activity and pKa value, has been improved through the rational design of their chemical structures. Heyes et al. demonstrated that unsaturated lipid tails, especially a linoleyl moiety, enhance the activity of the ionizable lipid to form an inverted hexagonal phase

after mixing with negatively charged lipids[28]. One of the most promising siRNA delivery system, which is known as a stable nucleic acid lipid particle (SNALP), contains the optimal ionizable aminolipid containing a linoleyl moiety, DLinDMA. Zimmermann et al. first demonstrated that the injection of the SNALP resulted in a significant silencing of hepatocyte-specific ApoB gene expression in non-human primates[29]. Moreover, Semple et al. introduced a ketal linker in an attempt to emphasize cone shaped structure of the ionizable aminolipid, which significantly enhanced the efficiency of siRNA delivery to hepatocytes[26]. It has been revealed that the pKa value of the ionizable aminolipid increases in response to an increase in the length of the of carbon chain between a linker and a tertiary amine group and of the hydrophilicity of the linker, e.g., ester, ether and ketal bond as hydrophobic linkers and an amide bond as a hydrophilic linker[30]. Moreover, a recent study revealed that potent gene silencing can be achieved only when ionizable aminolipids with ester bonds, but not amide bonds, are used, even though their pKa values were similar [31], which is the optimal range for gene silencing activity in hepatocytes in vivo [30]. The most potent ionizable aminolipid, DLin-MC3-DMA (MC3), which is now being Downloaded by [New York University] at 17:21 18 February 2016

used in several clinical trials, showed potent gene silencing with a median effective dose (ED 50 ) of 0.005 mg siRNA/kg in mice, and less than 0.03 mg of siRNA/kg in non-human primates[30]. The lipid nanoparticle (LNP) containing MC3, termed Patisiran, showed positive results in clinical studies that are development for the treatment of transthyletin-mediated amyloidosis and hypercholesterolemia[32]. More recently, Maier et al. reported that L319, which is a MC3 analogue and contains biodegradable ester bonds within hydrophobic carbon chains, had a similar activity similar to MC3 in hepatocytes[33]. Another type of ionizable aminolipid is the YSK-lipid. The first generation of the YSK-lipid, YSK05, had a pKa of around 6.4, and better fusogenic activity compared to the conventional ionizable aminolipid, DODAP[34]. Optimized LNPs containing YSK05 (YSK05-LNP) showed an approximately 10-fold higher gene silencing activity than the widely-used transfection reagent, Lipofectamine 2000, in an in vitro experiment[34]. For therapeutic applications, two injections of the YSK05-MEND loaded with siRNAs against the hepatitis C virus (HCV) normalized the inflamed liver tissue and suppressed both HCV genomic RNAs and core proteins in mice with chronic hepatitis C and mice with a persistent HCV infection, respectively[35]. The YSK05-LNP was also applied to the delivery of cyclic diguanosine monophosphate (c-di-GMP), an adjuvant that induces the production of type I interferon through stimulation of the stimulator of interferon genes protein (STING)-tank binding kinase 1 (TBK1) pathway, for cancer immunotherapy[36, 37]. Furthermore, a second generation YSK13-LNP showed an approximately 4-fold higher gene silencing activity in hepatocytes compared to the YSK05-MEND, and achieved better therapeutic effect on chronic hepatitis B virus (HBV) infection compared to the currently available HBV treatment, entecavir, in mice with persistent HBV infections[38]. Taken together, these findings suggest that rational design of ionizable aminolipids is promising strategy for overcoming the endosomal membrane barrier and to improve the cytosolic delivery of nucleic acids, including siRNAs, pDNAs and dinucleotides. In spite of a recent great progress in the efficiency of nucleic acid delivery, it was reported that the efficiency of endosomal escape of siRNAs formulated in the MC3-LNPs was only around 2% in in vivo hepatocytes, as estimated by electron microscopic observations of siRNA-gold nanoparticles [39], which was consistent with the previous finding that only 3% of the total siRNAs were associated with the RNA-induced silencing complex (RISC), as measured by Ago2 immunoprecipitation followed by stem-loop RT-PCR. Xu et al. also reported a similar finding that 4-8% of the injected siRNAs formulated in the LNPs reached the cytosol (S-100 fraction) in

hepatocytes measured by the stem-loop RT-PCR method[40]. Moreover, Wittrup et al. visualized the cytosolic release of siRNAs formulated in L319-LNP from endosomes by quantification of the number of galectin 8 (Gal8)positive vesicles, which are produced in response to the transient damage of endosomal membranes through fusion between LNP and endosomes, and estimated that approximately 3.5% of the internalized siRNAs were released from endosomes[41]. Taken together, based on these detailed investigations, endosomal escape continues to be a severe barrier to delivering nucleic acids into the cytosol. Further development of highly potent fusogenic lipids will clearly be needed if the limitations of endosomal escape are to be overcome.

2.3. Proton-sponge polymer A third material is a polycation composed of secondary or tertiary amines. One of the well-accepted hypotheses of this membrane-destabilization is the “proton sponge effect” [42-44] which is based upon proton-accepting structures, such as secondary amine groups in polyethyleneimine (PEI). The uptake of proton-accepting polymers Downloaded by [New York University] at 17:21 18 February 2016

into the endosome buffers endosomal protons, and subsequently draw in additional protons, as well as chloride ions and water molecules. The influx of ions and water causes swelling and osmotic lysis of the endosome. This hypothesis is supported by the inhibition of PEI-mediated transgene expression by ionophores, which reduce the pH gradient between the endosome and cytosol [45]. However, a very recent study reported that the pH in the lysosome compartment is maintained constant, even when cells were incubated with a pDNA/PEI complex or the free form of PEI [46]. Thus, another mechanism might also be available for PEI-mediated endosomal escape.

2.4. Fusogenic peptides The fourth strategy is to use a pH sensitive membrane fusogenic peptide that was inspired by the mechanism of the endosomal escape of the influenza virus, an envelope-type RNA virus. In this virus, hemagglutinin (HA) on the surface of the lipid envelope plays a key role in membrane fusion; it changes its conformation from a random coil to an α-helix structure in the acidic compartment[47, 48]. Based on this mechanism, Wagner and co-workers used synthetic peptides derived from the N-terminus of HA to enhance transgene expression using DNA/PLL complexes[49-52].

Similarly,

artificial

amphipathic

peptides

such

as

GALA

(WEAALAEALAEALAEHLAEALAEALEALAA)[53, 54], which also undergo structural conversion to the αhelix under acidic conditions, have been synthesized and applied to various gene vectors. These peptides consist of an amphipathic helix motif that is partitioned by acidic residues, such as glutamic acid and aspartic acid. At neutral pH, the negative charge destabilizes the α-helix and, as a result, the peptide forms a random coil structure. However, once the peptide is exposed to the acidic environment in the endosome, the acidic residues (i.e. carboxylic acids) are protonated, resulting in the loss of electrostatic repulsion. As a result, the peptide forms an α-helical structure to destabilize the endosomal membrane. As an alternative fusogenic peptide that can directly bind to the pDNA, a cationic amphipathic peptide, KALA (WEAKLAKALAKALAKHLAKALAKALKACEA) was synthesized[55]. However, the pH-dependency for its structural property is opposite to that for GALA: the peptide forms an amphipathic α-helix at physiological pH, and then shifts to a random coil in an acidic pH. The peptide succeeded in the nuclear delivery of oligonucleotide or pDNA when the molecules were compacted with KALA at a charge (+/-) ratio of 10/1. By conjugating these peptides with a lipid, they can be displayed on the liposomal particle via the incorporation of the lipid moiety into the lipid bilayer. Modification of cholesteryl GALA (Chol-GALA) on the liposomal

formation conferred the cytoplasmic delivery of the cargos via membrane fusion with the endosomal membrane[56]. In addition, when the compacted DNA or siRNA nano complex formed with polycations was encapsulated in this liposomal formation (referred to as a multi-functional envelope-type nano device: MEND), they conferred efficient transgene expression [57] or gene knockdown activity [5], depending on the Chol-GALA modification. In the process of the in vivo application of liposomes modified with Chol-GALA, we surprisingly found that the GALA peptide could function as a targeting ligand for the lung endothelium[58]. The cellular uptake of the particle to human lung endothelial cells was completely blocked by pre-incubation with a lectin that specifically binds to the sialic-acid-terminated sugar chain. In contrast, the uptake was not blocked by sialic acid per se. These data collectively indicate that the GALA-modified liposome can target 2 or 3 sugar chains that are terminated with sialic acid similar to an influenza virus. We found that GALA has dual functions; as a ligand for lung endothelial cells that can target sialic acid-terminated sugar chains, and as an inducer of endosomal escape. In fact, the encapsulation of siRNA in this particle resulted in efficient gene knockdown in the lung endothelium Downloaded by [New York University] at 17:21 18 February 2016

after intravenous administration. Therefore, a GALA-modified particle is a highly potent influenza-like carrier of a nucleic acid for the lung endothelium[58]. The other example of a lipid conjugated fusogenic peptide is stearylated KALA (STR-KALA) [59]. This peptide was developed as a functional device for inducing the transfection efficiency in dendritic cells that play a key role in the initiation and regulation of immune responses. For a long period, DNA-encapsulating MENDs were developed as gene carriers, on those stearylated octa-arginines (STR-R8) and/or modified with Chol-GALA[59, 60]. However, they exhibited background level, or only a detectable level of gene expression, while a high gene transfection activity comparable to the adenovirus vector was exhibited in HeLa cells, or other types of dividing cultured cells. Since dendritic cells are non-dividing cells, a strategy designed to overcome the nuclear membrane barrier is necessary. Based on this consideration, we alternatively used a KALA peptide that forms a α−helical structure at physiological pH. We postulated that the KALA would be able to overcome the plasma membrane and the nuclear membrane via step-wise membrane fusion[59]. When GALA was used as a modifier, transfection activity was only slightly increased by 5 fold. In contrast, when KALA was used, transfection activity was expectedly induced by 2 orders of magnitude. However, when the intracellular trafficking of the KALA-modified particle was quantitatively analyzed, we found that the actual mechanism responsible for this drastic enhancement in gene transfection was completely different from our initial concept: the cellular uptake and nuclear transport efficiency of DNA was comparable regardless of whether or not KALA was used in the modification[61]. These data suggested that KALA-modification may also enhance post-nuclear delivery processes such as transcription. To interpret these phenomena, we propose a “switch on” function. In general, the functions of immune-responsive cells remains inactivated to avoid an excess of immune-stimulation. However, when the switches are turned on, cellular functions, as well as transcription and translation are activated. A microarray analysis revealed that the expression levels of the individual mRNAs in dendritic cells were drastically perturbed when the cells were exposed to a KALA-modified MEND. An analysis of the pathway indicated that the expression levels of immune response-related transcription factors (Stats and NF-kB) were induced. This indicates that the KALA-MEND functions as an adjuvant, an immune-activator [61]. While an actual switch has not yet been identified, further mechanism-based analyses strongly support the conclusion that the cytoplasmic delivery of long DNA, followed by the activation of the STING/TBK1 pathway are involved in this adjuvant activity[60].

3. Strategies for the biodegradation of the materials

3.1. SS-cleavage materials After escaping from the endosomal compartment, nucleic acids must be released from the carriers at their site of action. The decoating (decapsulation) of the cargos from the carriers was directly linked to the efficacy of the system, since the pharmacological activity of nucleic acids is exerted only when recognized by the appropriate cellular machinery (i.e. RNA polymerase for pDNA, or RISC for siRNA). The importance of the precise control of nucleic acid dissociation is supported by the fact that the viruses release their genomic DNAs in a specific cellular compartment[62-64]. Therefore, non-viral carriers should be equipped with appropriate machinery for decoating at the final destination. However, the accelerated decoating rate in the endo/lysosomal compartments is negatively correlated with transfection efficiency due to the degradation of the nucleic acid[65]. Taken together, the switching of stability/instability before and after endosomal escape is particularly desirable. One promising Downloaded by [New York University] at 17:21 18 February 2016

answer for the requirement is to utilize the difference in the concentration of glutathione (GSH), a tri-peptide containing the amino acid cysteine: the intracellular GSH concentration is, at most, 1000-fold higher than the extracellular milieu[7]. Thus, by the employment of the disulfide bonding, it can sufficiently stabilize the carriers, while cleavage in the cytoplasm triggers destabilization and decoating. Cationic peptide carriers are convenient for combining this strategy since thiol groups can be readily introduced by incorporating cysteine residues in the peptide sequence. One example is a peptide consisting of a cationic lysine and two cysteine residues (Cys-Trp-Lys 17 -Cys)[66]. The peptide permits the formation of nano-sized particles with DNA aided by inter-molecular cross-linking. The disulfide bonding conferred the resistance of DNA to sonicative share stress, and showed at most a 60-fold higher transfection activity than the non-cleavable large poly-L-lysine (PLL) or a peptides without cross-linking. pDNA, siRNA and mRNA were delivered by means of a peptide containing lysine, cysteine, and histidine (Cys-His 6 -Lys 3 -His 6 -Cys)[67]. In this system, histidine residues buffered the acidification of the endo/lysosomal compartment and enhanced endosomal escape. Other example is linear PEI (molecular weight: 2.3, 3.1 and 4.6 kDa) that is cross-linked by disulfide bonding and an amine reactive linker (ssPEI)[68]. Lowering the intracellular GSH levels by a duroquinone treatment decreased the transfection efficiency. Thus, the dissociation of the polymer that is driven by GSH is a crucial event for successful DNA transfection. Also, when ssPEI was applied to siRNA delivery, the siRNA was delivered to the cytoplasm in a highly dispersed form, while, when branched 25 kDa PEI, a non-cleavable polymer was used, large aggregates were formed in the cells[69]. A block copolymer containing PEG- and thiol- introduced PLL was constructed to form polyion complex micelles[70]. The disulfide bonding in polymers suppressed the anion exchange of antisense DNA against poly(vinyl sulfate) or bovine serum albumin. On the other hand, treatment with DTT triggered the release of the antisense DNA in time- and dose-dependent manner[71]. The polymer, in which the amine groups of lysine were partially substituted with iminothiolate groups also formed a stable complex with siRNA[72]. The complex showed resistance to high salt concentrations up to 600 mM and exhibited a higher knock down efficiency in comparison with a non-cleavable counterpart. The biodegradable nature of disulfide bonding also contributes to biocompatibility. In the case of high molecular weight PEI, the compaction of DNA with a higher charge ratio resulted in prominent transfection activity, while it caused severe cytotoxicity partly because the PEI is not metabolized. In contrast, PEI composed

of short and non-toxic PEI (800 Da) cross-linked by a disulfide linker showed a transfection efficiency comparable to that for the branched 25 kDa material without any measurable cytotoxicity[73]. Similar observations were reported for amphipathic material: the disulfide bonding was placed at the spacer region between the cationic head group and the hydrophobic tail of a lipid. The transfection efficiency of the cleavable lipid was comparable to the non-cleavable one, while the toxicity was diminished[74]. Since the cleavage of the cationic head and hydrophobic tail results in a loss of the driving force for particle formation, it promotes the decoating process in the cell.

3.2. Cleavable PEG From the viewpoint of the in vivo use of a nucleic acid carrier, the particles are exposed to a wide spectrum of biological components. Especially in the blood circulation, high concentrations of salts and serum proteins weaken the colloidal stability of the particles, followed by the disintegration of the carriers. One major obstacle is Downloaded by [New York University] at 17:21 18 February 2016

particle opsonization, and the subsequent clearance by the reticuloendothelial systems (RES). The ability of the carriers to avoid being recognized by RES is an important characteristic for achieving long-term systemic circulation[75, 76]. To confer stealthiness to carriers, grafting of hydrophilic polymers is a gold standard strategy. For example, liposomes equipped with a hydrophilic PEG polymer (PEGylation) prolong the blood circulation time due to the formation of a steric barrier (aqueous layer) on its surface[77]. The long circulation property is closely associated with tumor targeting by a passive targeting strategy that is generally referred as the enhanced permeability and retention (EPR) effect[78]: Particles with long circulation properties can preferentially accumulate in tumor tissue that has a leaky vasculature. However, the PEGylation has a great disadvantage from the point of view of cellular uptake and endosomal escape[79-81], since the hydrophilic layer formed by PEG chains prevents the particle surface from associating with the plasma or endosomal membranes. As a result, the intrinsic in vitro transfection activity is drastically impaired by the PEGylation. In other words, there is a dilemma associated with the use of PEG; it has great merit in terms of stabilizing a particle in vivo, while it impairs the cytoplasmic delivery of the cargo[82, 83]. One of the rational proposals for overcoming this dilemma is to introduce a cleavable linker between the PEG and DNA compacting materials, or liposome-incorporating lipids. The PEGylation of branched or linear PEI via acid-labile acetal[84], or hydrazon linkage[85, 86] was reported. The half-life of p-aminobenzaldehyde methoxy PEG5000 acetal (Fig.4A) varies from 2 hours at physiological pH (pH 7.4) to 3 min at an acidic pH (pH5.5). Also, PEGylation with pyridiylhydrazone Fig.4B), combined with targeting ligands (transferrin or EGF), resulted in a one order magnitude higher transgene expression in comparison with that modified with stable PEG chains, both in vitro and in vivo. In the case of fusogenic liposomes, PEGylated lipids stabilize the lipid bilayer structure and hamper the transition to the inverted hexagonal H II phase. Therefore, PEGylation generally suppresses the fusogenic ability of liposomes containing cone shape lipids such as DOPE (see2.1). In this situation, the cleavage of PEG triggers endosomal escape, as well as the release of the cargo. A thiol-responsive PEG conjugation with p- or odithiobenzyl linkage (Fig.4C)[87] showed that the liposome released an encapsulated fluorescent dye in the presence of cysteine. Similarly, a diorthoester (Fig.4D)[88], a vinyl ether (Fig.4E)[89, 90] and an orthoester (Fig.4F)[91] have been used as acid-labile linkers.

Another class of detachable PEG is composed of an enzymatically cleavable peptide linker. Enzymatic cleavage by the tissue/organ specific protease can suppress non-specific interactions in the circulation and preferable interactions with target cells, once the linker is cleaved by the action of a tissue-selective protease. Matrix metalloproteinase-2 (MMP-2) which contributes to the remodeling of the extracellular matrix represents one such enzyme that is highly expressed in tumors. In combination with a galactose ligand, PEGylated DOPE with an MMP-2 cleavable linker was used for the targeting to hepatocellular carcinoma[92]. The prolonged blood circulation for the MMP-2 cleavable PEG was applied to the gene delivery to solid tumors via the EPR effect[93]. The employment of cleavable PEG resulted in a one order of magnitude higher transfection activity in MMP-2 overexpressing cells in comparison with that of non-cleavable PEG. In addition, the transfection efficiency, normalized by the amount of accumulated particles in the tumor was enhanced by 4-folds in vivo.

4. Drawback of cationic particles and a paradigm shift to the use of a neutral particle Downloaded by [New York University] at 17:21 18 February 2016

Since the first successful gene delivery using a cationic liposome, many types of materials have been developed as a DNA carriers[94]. The use of a cationic material has two merits. First, they permit DNA and nucleic acids to be easily compacted into a nano-sized particle. Second, the extensive compaction of DNA and nucleic acids with a high charge (+/-) ratio confers strong electrostatic associations with negatively charged constituents, such as heparan sulfate proteoglycans (HSPGs) on the cellular surface, thus resulting in an enhanced cellular uptake[95]. However, there are several serious drawbacks associated with the use of cationic carriers. First, the intravenous administration of cationic particles results in the formation of large aggregates with erythrocytes or platelets, which are then stacked in the lung microvessels[96-98]. This event may cause tissue ischemia, and possible myocardial damage because of microinfarction[99], and undesired transgene expression in the lung[100]. A second drawback is the short duration of gene expression. When lipoplexes or polyplexes formed with DNA were administered, hepatic gene expression peaked at 6 h after the transfection, and then rapidly decreased within 2 days[101]. The third drawback is cytokine production. Lipoplex or polyplex administration triggers the production of various types of cytokines[101]. From the point of view of intracellular events, the use of the cationic materials is also attended with a demerit, in that the transcription and translation processes are inhibited via electrostatic interactions with DNA and mRNA, respectively[102]. Our quantitative analysis revealed that this inhibition is a dominant mechanism responsible for a 3 orders of magnitude decrease in the transfection efficiency of the lipoplex in comparison with adenovirus[103]. Further quantitative analysis of the mRNA revealed that a 3 orders of magnitude difference in the post-nuclear delivery process was attributed to a 1- and 2- order of magnitude difference in the transcription and translation processes, respectively[102]. Based on these previous findings, the development of a charged neutral particle by excluding cationic materials is one of the next generation particle designs that have great advantages in terms of satisfying biocompatibility in vivo, tissue targeting ability, and the avoidance of electrostatic interactions between the cargos or mRNA. Similar to the above arguments regarding DNA, a neutral design would also be desirable for siRNA delivery since electrostatic interactions between the particles and siRNA could be avoided. In fact, the current strategy for producing siRNA nanoparticles that exhibit a high gene knockdown efficiency (i.e. SNALP) is based on the concept that the particle per se is neutral in physiological pH conditions, and be charged positively in response to the acidic pH in endosomes [26, 30] .

5. Neutral nanoparticle formed at an acidic pH- and a reducing environment-responsive material

5.1. Concept of ssPalm and its in vitro function Based on the above arguments, the best combination of the current designs is theoretically a “neutral” particle that mounts multi-functions of pH-triggered endosomal membrane destabilization, and spontaneous collapse in response to a reducing environment. In an attempt to develop such a carrier, we developed a SS-cleavable ProtonActivated Lipid-like Material (ssPalm) (Fig. 4). This material has two hydrophobic moieties. Thus, similar to the lipid, it can form a liposomal architecture. The material is also functionalized so as to contain dual sensing motifs that can respond to the intracellular environment; tertiary amines as a positively charged unit in response to an acidic compartment (endosome/lysosome) for membrane destabilization, and disulfide bonding as a reducing environment-triggered cleavage unit [104]. Downloaded by [New York University] at 17:21 18 February 2016

As a 1st generation ssPalm, myristic acid (C 14 ) was employed as a hydrophobic scaffold (ssPalmM). The liposomal nanoparticles (LNP) prepared with ssPalmM (LNP ssPalmM ) showed a higher gene transfection activity in the presence of serum, in comparison with the LNP prepared with DODAP, a conventional pH-sensitive lipid with a tertiary amine (LNP DODAP ) or a non-cleavable Palm-derivative (LNP ccPalmM ). More surprisingly, the activity of the LNP ssPalmM was comparable to that for a positively charged LNP prepared with 1,2-dioleoyl-3(trimethylammonium) propane, a cationic lipid with quaternary amines (LNP DOTAP ) in spite of the lower cellular uptake. Thus, well-refined intracellular processes compensated for the poor cellular uptake efficiency. One of the possible reasons for this is that the LNP ssPalmM does not inhibit mRNA translation. In fact, the inhibitory effect of the LNP ssPalmM on the in vitro translation reaction was less than that of the LNP DOTAP . In addition, the cytotoxicity of the material was much less than that for LNP DOTAP . Furthermore, it is noteworthy that the cytotoxicity of LNP ssPalmM remained low even when a high dose of DNA was transfected, while LNP prepared with the noncleavable material (LNP ccPalm ) was quite cytotoxic. Thus, biodegradable characteristics as well as non-cationic features are key factors for low cytotoxicity. As a 2nd generation, fat soluble vitamins (i.e. vitamin A and E) were employed as a hydrophobic scaffold (ssPalmA and ssPalmE, respectively). Three advantages accrue as the result of using these vitamins. First, the vitamins have unique physicochemical properties. For example, vitamin E is more hydrophobic than other vitamins and lipids. Because of this, the stability of the particle can be controlled by selecting the appropriate hydrophobic scaffold since hydrophobic interactions are the main driving force for the self-assembly of such particles. Second, vitamins are processed by a unique transport system. One example is that a nuclear transport system (i.e. cellular retinoic acid-binding proteins: CRABP) is available as nuclear delivery machinery for vitamin A. In fact, LNP ssPalmA exhibited a 1 order of magnitude higher transgene expression in comparison with LNP ssPalmM . Consistent with this gene expression, the ssPalmA particle accumulated extensively at the nuclear periphery. Of note, gene expression and nuclear accumulation was impaired in the presence of an excess amount of retinoic acid. Therefore, the LNP ssPalmA can ride on the endogenous nuclear transport system. Finally, vitamins have unique pharmacological activities. For example, α-tocopherol (vitamin E) succinate has anti-tumor activity[105, 106]. Thus, the pharmacological function of a gene or nucleic acid cargo might be enhanced with the aid of the biological function of vitamins.

5.2. in vivo application as a gene carrier. For in vivo applications, the stability of a particle in serum is an important feature. When naked pDNA or core particles are incubated with mouse serum, they are rapidly degraded within 30 min. In contrast, once the pDNA is encapsulated in this particle, the pDNA remains intact for at least 24 h[104]. These data strongly suggest that the encapsulation of a material in the lipid envelope provides protection from enzymatic degradation in the blood circulation. Furthermore, the visualization of the dynamic flow of a particle in sinusoidal capillaries by intravital confocal laser scanning microscopy revealed that the LNP ssPalmM flowed in the sinusoidal capillary as a highly dispersed form, while cationic particles flow as large aggregates and then partially perturb the bloodstream[107]. Moreover, the LNP ssPalmM rapidly accumulated in hepatocytes within 10 min. When the neutral LNP ssPalmM was administered, sustained, liver specific gene expression (>2 weeks) was achieved. Of note, such a prolonged gene expression was achieved only when the pDNA that was free from the unmethylated CpG-motif was used. In contrast, the gene expression of the cationic carrier peaked at 6h, and then dropped under the detection level Downloaded by [New York University] at 17:21 18 February 2016

within 3 days. Regarding the immune response, when pDNA containing unmethylated CpG motifs was used, it stimulated the production of IL12 and TNFα. Cationic particles also trigger the induction of TNFα and IFNγ. When the LNP ssPalmM particles and CpG-free pDNA were combined, the production of all cytokine production was completely negative. Therefore, the poor immune-stimulatory properties are is closely related to long-lasting gene expression. For tumor targeting, surface modification with PEG is the gold standard strategy, since stability in the blood circulation is a key factor for tumor accumulation via the enhanced permeability and retention (EPR) effect [83]. Modification with 5 mol% PEG (average M.W.: 2000) prolonged the blood circulation of LNP ssPalmM . Furthermore, prolongation of the PEG chain from 2000 to 5000 further improved the stability in blood circulation, and thereby induced transgene expression in tumor tissue[108]. By delivering pDNA encoding a solute form of VEGFR (fms-like tyrosine kinase-1: sFlt-1), an anti-angiogenic factor, tumor growth was suppressed in comparison with a PBS-treated group, while the therapeutic effect of the corresponding particles encapsulating luciferase-encoding pDNA showed little or only marginal effects. It is noteworthy that the anti-tumor effect of LNP ssPlamE was significantly higher than that for LNP ssPalmM . The transgene expression of the sFlt-1 and the intrinsic function of α-tocopherol succinate (an inducer of apoptosis)[102, 103] that are employed as a hydrophobic scaffold in ssPalmE might synergistically function to achieve an extensive anti-tumor effect.

5.3. in vivo applications as an siRNA carrier. The most promising in vivo targeting organ is the liver since it is a major clearance organ for nanoparticles as well as low molecular weight compounds and macromolecules. Also, the active targeting of the hepatocyte is possible by using certain kinds of ligands such as acetylgalactosamine (GalNac)[109, 110] or endogenous ApoE[111-113]. To develop a siRNA carrier that is applicable in vivo, the structure of the ssPalm molecule was further remodeled[112]. As the result of an initial screening, an adequate hydrophobic scaffold structure was identified. As a result, the gene knockdown activity against a hepatocyte-specific marker (factor VII) was highest in ssPalmE in comparison with those prepared with other types of ssPalms (ssPalmM and ssPalmA). Consistent with this result, florescence-labeled siRNA accumulated more extensively in the liver than others. To improve gene knockdown efficiency, the structure of the tertiary amine was tuned so as to allow the particle to detect a slight change in endosomal acidification, and then became charged positively for rapid endosomal escape at an

earlier stage. A key parameter that reflects the activity of pH-triggered positive charging is the apparent pKa value. Current studies provided maximum gene knockdown activity when the apparent pKa values were adjusted to approximately ~6.4[30]. The ternary amine structure of ssPalmE was originally flexible. To allow the tertiary amines to accept protons more efficiently, an amine in the form of a piperidine structure was used. Also, the distance from the vitamin E (surface of the particle) molecule to the tertiary amine was systematically extended. By this optimization, we successfully identified the chemical structure of ssPalmE-P4-C2, that was needed for achieving a high membrane destabilization activity (hemolysis activity), and extensive hepatic gene knockdown activity (Fig. 5). Importantly, the rank-order for hemolysis activity and gene knockdown efficiency in the ssPalmE-series derivatives were completely the same to that for the pKa values. Finally, the hepatic uptake mechanism was investigated. When ssPalmE particles were administered to ApoEknockout mice, hepatic accumulation decreased drastically, resulting in a prolonged blood circulation time for the Downloaded by [New York University] at 17:21 18 February 2016

particles. Therefore, LNP ssPalmE-P4-C2 is recognized by ApoE, and is then taken up via LRL receptor families [112]. The character of the ApoE-dependent uptake is useful for the targeting of brain associated cells such as neurons or astrocytes, since these cells also express LDL receptor families[114, 115], and plays a key role in the mutual transfer of lipoproteins[116]. When the pDNA encapsulating LNPs were administered via intracerebroventricular injection, the uptake of LNP prepared with the ssPalmM was under the detection limit, most probably because the particles were washed away by spinal fluid. In contrast, LNP ssPalmA or LNP ssPalmE were taken up by a certain type of cells, and exhibited a transfection activity. Immunofluorescence revealed that gene expression was quite high in astrocytes. [117]

6. CONCLUSION In summary, a neutral particle is one of the important aspects for the successful intravenous administration of nanoparticles, since it can avoid the undesired stacking in lung capillaries via the formation of large aggregates, and it can also minimize immune-responses. In addition, once the particles are taken up by cells, the intracellular environment-triggered activation of the endosomal membrane-destabilization and/or collapse is a rational design for maximizing the function of cargos (DNA and siRNA). ssPalm technology is one of the approaches that satisfy these criteria.

7. Expert opinion Currently, the development of nucleic acid-based medication is a hot topic in pharmaceutical companies and bio-venture companies world-wide. Since the liver is the main clearance organ of nanoparticles, hepatocyte is the first target for the LNP-mediated siRNA delivery. In this area, SNALP has been one of the pioneer technologies. The Alnylam pharmaceuticals reported the positive results of Phase II open-label extension (OLE) study with Patisiran, a LNP-based siRNA delivery system targeting transthyretin (TTR) for the treatment of TTR-mediated amyloidosis (ATTR amyloidosis) (http://www.alnylam.com/product-pipeline/ttr-amyloidosis-fac/). However, LNP is not necessarily ideal for the hepatic delivery of siRNA. Alnylam Pharmaceuticals has reported a Phase II OLE study with Revusiran in parallel, and further started phase III trial, in those chemically modified anti-TTR siRNA was further conjugated with tri-GalNac as a ligand. In this case, the siRNA were applied via subcutaneously administration. As a next generation nucleic acid-mediated medication, mRNA delivery will see

the light of day. In this case, liver-targeting LNP will be still a key technology since the stability of mRNA per se is quite poor, and shielding against the serum components is necessary. As to the LNP-mediated siRNA delivery, the targeting of non-hepatic tissue is the next challenge. In past decades, it has been considered that the nanoparticle and macromolecules spontaneously accumulate in certain kinds of tumor tissues via the EPR effect: particles that have long blood circulation properties gradually accumulate in the tumor via its leaky neovasculature. However, accumulative evidence in clinical trials of the nanoparticles have identified a disturbing trend: the efficiencies of the EPR effect in mouse tumor model are overestimated compared to the human case[118]. Therefore, the importance of the active-targeting strategy will increase even more. In this case, the use of a neutral particle would be highly desirable, since cationic charges in LNP contribute to non-specific cellular binding. In the future, more precise control of intracellular trafficking or organelle targeting will be also needed. For the nuclear targeting of DNA, a neutral particle might also be an important characteristic since cationic materials (such as triphenylphosphonium: TPP+[119]) tend to accumulate in Downloaded by [New York University] at 17:21 18 February 2016

mitochondria. In this review, the current in vivo application of LNP ssPalm for DNA and siRNA delivery systems was introduced. By monitoring hepatic gene silencing or gene expression, the basic chemical structure and/or the proper combination of the helper lipids that are necessary for achieving an in vivo function is now clarified. Based on this formulation, tissue targeting is also ongoing. As to the DNA, a DNA vaccine via subcutaneously administration is one possible application. The application of the LNP ssPalm in mRNA delivery promise to be a future direction. Very recently we found that the ssPalm can form a nano-dispersion of water-insoluble drugs based on co-assembly. Since this particle can overcome the endosomal membrane barrier, and thereafter collapse, this system will be useful for the cytoplasmic delivery of low-molecular compounds. Collectively, ssPalm can be a novel DDS platform that can be used to deliver a large variety of cargoes from low molecular drugs to DNA. The application of ssPalm is now becoming versatile.

Acknowledgement The authors would like to thank Dr M. S. Feather for his helpful advice in writing the English manuscript.

Declaration of Interest This work was supported by JSPS KAKENHI Grant Numbers 15H01806 and 15K14934. H Akita is supported by The Asahi Glass Foundation. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials


discussed in the manuscript apart from those disclosed.

Figure legends: Fig. 1. Biological barriers for DNA/siRNA delivery For the delivery of DNA/siRNA, the pharmacokinetics (stability in blood circulation and tissue targeting) and intracellular trafficking (cellular uptake, endosomal escape, nuclear delivery and dissociation from the carrier)


need to be taken into consideration.

Fig. 2. Strategies of endosomal escape The strategies for the endosomal escape can be divided into 4 categories: (A) pH-dependent formation of a hexagonal H II structure; (B) pH-dependent positive charging; (C) Proton-sponge polymer; (D) Fusogenic peptides. (A) Cholesterol hemisuccinate (CHEMS), an inverted conical shaped lipid can stabilize a conically shaped lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPE) in forming a lamellar phase with an aid of mutual electrostatic repulsion in CHEMS. In response to the acidic pH, electrostatic repulsion is decreased by the protonation of carboxyl group in CHEMS, and thereby results in a formation into an inverted hexagonal H II phase. (B) Neutral liposomes formed with ionizable lipids acquire a positive charge when taken up into the endosomal compartment by the protonation of tertiary amine. This positive charging trigger the interaction of the liposomal surface with negatively charged endosomal membrane. Electrostatic interaction of positively charged lipids with the anionic phospholipids in the endosomal membrane induced a formation of nonbilayer structure (hexagonal H II ). (C) The proton-accepting polymers such as polyethyleneimine buffers endosomal protons, and subsequently draw in additional protons, as well as chloride ions and water molecules. This event causes swelling and osmotic lysis of the endosome. (D) Artificial amphipathic peptides such as GALA consists of an amphipathic helix motif that is partitioned by acidic residues, such as glutamic acid and aspartic acid. At neutral pH, the peptide forms a

random coil structure since the negative charge destabilizes the α-helix formation. In endosome, the acidic residues are protonated, and then lose an electrostatic repulsion. Thereby, the peptide forms an α-helical structure


to destabilize the endosomal membrane.

Fig. 3. Cleavable polyethyleneglycol (PEG)-lipids in response to the acidic environment in endosome Surface modification by PEG improved the stability of a particle in the blood circulation, while it diminished endosomal escape mainly because the hydrophobic PEG layer perturbs the association of the particle with the endosomal membrane. To avoid this PEG dilemma, the linker between the PEG and lipids are designed to be


cleaved in response to an acidic pH.

Fig. 4. Concept of SS-cleavable proton-activated lipid-like material (ssPalm) The ssPalm mounts dual sensing motifs that can respond to the intracellular environment; a proton-sponge unit (tertiary amines) that function in response to an acidic environment (endosome/lysosome), and disulfide bonding


that can be cleaved in a reducing environment (cytosol).

Fig. 5. Tuning of the tertiary amine structure in vitamin E-scaffold SS-cleavable Proton-Activated Lipidlike Material (ssPalmE) An amine with a flexible structure in the ssPalmE was inserted into the form of a piperidine structure. The O-N distance was also optimized so that the ternary amines would be located a proper distance from the liposomal


surface.


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