The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery

ADR-12935; No of Pages 9 Advanced Drug Delivery Reviews xxx (2016) xxx–xxx

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The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery☆ Xinwei Cheng, Robert J. Lee ⁎ Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, Ohio State University, Columbus, OH 43210, USA

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Article history: Received 5 June 2015 Received in revised form 3 January 2016 Accepted 28 January 2016 Available online xxxx

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Lipid nanoparticles (LNPs) have shown promise as delivery vehicles for therapeutic oligonucleotides, including antisense oligos (ONs), siRNA, and microRNA mimics and inhibitors. In addition to a cationic lipid, LNPs are typically composed of helper lipids that contribute to their stability and delivery efficiency. Helper lipids with cone-shape geometry favoring the formation hexagonal II phase, such as dioleoylphosphatidylethanolamine (DOPE), can promote endosomal release of ONs. Meanwhile, cylindrical-shaped lipid phosphatidylcholine can provide greater bilayer stability, which is important for in vivo application of LNPs. Cholesterol is often included as a helper that improves intracellular delivery as well as LNP stability in vivo. Inclusion of a PEGylating lipid can enhance LNP colloidal stability in vitro and circulation time in vivo but may reduce uptake and inhibit endosomal release at the cellular level. This problem can be addressed by choosing reversible PEGylation in which the PEG moiety is gradually released in blood circulation. pH-sensitive anionic helper lipids, such as fatty acids and cholesteryl hemisuccinate (CHEMS), can trigger low-pH-induced changes in LNP surface charge and destabilization that can facilitate endosomal release of ONs. Generally speaking, there is no correlation between LNP activity in vitro and in vivo because of differences in factors limiting the efficiency of delivery. Designing LNPs requires the striking of a proper balance between the need for particle stability, long systemic circulation time, and the need for LNP destabilization inside the target cell to release the oligonucleotide cargo, which requires the proper selection of both the cationic and helper lipids. Customized design and empirical optimization is needed for specific applications. © 2016 Published by Elsevier B.V.

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Keywords: Nanoparticle Oligonucleotide Drug delivery Antisense siRNA Lipid nanoparticles Endosomal escape Helper lipid

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Oligonucleotide (ON) therapeutics . . . . . . . . . . . . . . . . . . . . . . . 1.2. Chemical modifications on ONs . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Lipid nanoparticles (LNPs) for ON delivery . . . . . . . . . . . . . . . . . . . 1.4. Cationic lipids in LNP formulations. . . . . . . . . . . . . . . . . . . . . . . 1.5. Synthesis of LNP formulations . . . . . . . . . . . . . . . . . . . . . . . . . Helper lipids in LNP formulations. . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Fig. 3), a fusogenic lipid . 2.2. Cholesterol (CHOL) (Fig. 3) . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Phosphatidylcholine (PC) . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. PEGylating lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Anionic lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Other helper lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rational design of LNP composition. . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Optimal LNP composition . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Role of helper lipids in biodistribution and immunogenicity of LNPs. . . . . . . . 3.3. Role of ON cargo in LNP formulation . . . . . . . . . . . . . . . . . . . . . .

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☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Non-Antigenic Regulators-Maiseyeu”. ⁎ Corresponding author. E-mail address: [email protected] (R.J. Lee).

http://dx.doi.org/10.1016/j.addr.2016.01.022 0169-409X/© 2016 Published by Elsevier B.V.

Please cite this article as: X. Cheng, R.J. Lee, The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery, Adv. Drug Deliv. Rev. (2016), http://dx.doi.org/10.1016/j.addr.2016.01.022

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Examples of LNP formulations . . . . . . . . . . 3.4.1. Stable nucleic acid lipid particles (SNALPs) 3.4.2. Smarticles . . . . . . . . . . . . . . . 4. Summary and perspectives. . . . . . . . . . . . . . . Uncited reference . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . .

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1.2. Chemical modifications on ONs

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The problem of poor nuclease stability can be partially addressed by introducing chemical modifications to ONs, such as 2′-O–Me, 2′-F, 2′-O(2-methoxyethyl) (2'MOE), morpholino, and locked-nucleic acid (LNA) nucleoside substitutions, and phosphorothioate, peptide nucleic acid (PNA), and phosphorodiamidate backbone substitutions [10]. It has often been assumed that while antisense ONs typically require the use of a transfection agent for in vitro delivery, they can be delivered in vivo without the use of a delivery vehicle [12]. This argument is supported by the FDA-approval of mipomersen. Mipomersen, trade name Kynamro, is an antisense ON that targets apolipoprotein B. It is given to patients by once a week subcutaneous injection at 200 mg without the use of a delivery system [11]. It is a “gapmer” that contains 2'MOE modified nucleotides at 5′ and 3′ ends and phosphorothioate linkages in the middle [11]. It is possible that the delivery of mipomersen is facilitated by its ability to bind to plasma proteins and by the fact that the liver is the target organ, which is highly accessible from circulation. However, the overall performance of “naked” antisense ONs in clinical trials has been mixed [13]. There is substantial evidence in preclinical studies that delivery vehicles such as lipid nanoparticles (LNPs) can greatly enhance the therapeutic efficacy of antisense ONs in vivo

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1.3. Lipid nanoparticles (LNPs) for ON delivery

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Naturally occurring vesicles, such as enveloped viruses and exosomes, are efficient vehicles of shuttling nucleic acids between different cells. LNPs can be viewed as synthetic versions of these carriers that can be custom-engineered to do the same with therapeutic ONs. Optimized LNPs can simultaneously protect ONs from serum nucleases, extend the systemic circulation time of ONs by preventing renal excretion and reticuloendothelial system (RES) clearance, enhance tumor uptake via the enhanced permeability and retention (EPR) effect, and, at the cellular level, facilitate internalization and endosomal escape of ONs [16]. LNPs for ON delivery typically contain a cationic lipid and other components that are commonly called “helper lipids”. LNPs comprise lipid bilayers that encapsulate ONs inside their aqueous core and between bilayers, typically in a multilamellar structure [16]. Sometimes an additional targeting ligand attached to a lipophilic anchor is also incorporated into the LNPs to enable selective delivery to targeted cells [17]. Non-lipid components, such as polycations (e.g., protamine and cationic polymers), calcium phosphate and membrane lytic peptides can be incorporated into LNPs to generate “hybrid” nanovehicles.

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1.4. Cationic lipids in LNP formulations

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Cationic lipids can facilitate electrostatic interactions with anionic ONs [16]. This is needed to efficiently incorporate ONs into LNPs during their synthesis. In addition, these lipids can mediate electrostatic interaction between LNPs and the cellular plasma or endosomal membrane and facilitate cellular uptake and endosomal release of ONs [16]. Many cationic lipids have been synthesized for nucleic acid delivery since the initial report by Felgner et al. [18]reported the gene transfer activity of 1-(2, 3-dioleyloxy) propyl]-N,N,N-trimethyl-ammonium (DOTMA). These include lipids with various types of headgroups (tertiary aminebased, quaternary amine based, univalent and multivalent cationic) and lipophilic moieties (typically consisting of unsaturated alkyl or acyl chains or cholesterol) [19]. A few examples of cationic lipids are show in Fig. 1. Cationic lipids when used alone carry a high density of positive charge, can be cytotoxic, and are not optimal for synthesis of LNPs designed for ON delivery in vivo. A number of factors can affect the delivery efficiency of ON-carrying LNPs, including the scheme of chemical modifications on the ON [10], the structure of the cationic lipids, and the choice of helper lipids and their percentages in the formulation [19]. Other important factors include lipid-to-ON ratio and the resulting positive–negative charge ratio and the resulting LNP zeta potential, pH-responsiveness of the zeta potential, degree and reversibility of

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ONs are an emerging therapeutic modality with potential applications in many human diseases, such as metabolic diseases, infectious diseases, cancer and regenerative medicine. Therapeutic ONs can be classified based on their mechanisms of action. Antisense ONs targeting mRNAs are usually DNA-based and can down-regulate gene expression by mechanisms such as RNaseH activation, translational inhibition, and exon skipping [1]. MicroRNAs (miRNAs) are naturally occurring noncoding RNAs that regulate gene expression through RNA interference (RNAi). Anti-microRNAs (antimiRs), usually RNA-based, are ONs that can bind tightly to their corresponding miRNA targets and indirectly upregulate gene expression by inhibiting the activity of the miRNAs [2,3]. Both antisense ONs and antimiRs are single-stranded molecules. Meanwhile, small interfering RNAs (siRNAs) and miRNA mimics are typically RNA ON duplexes, which is a form that can be efficiently loaded into RNA-induced silencing complexes (RISCs) once inside the cytoplasm [4]. Other types of ONs with potential therapeutic application include aptamers [5], ribozymes [6], “CpG” immunostimulatory ONs [7], etc. Antisense ONs and siRNA constitute a majority of therapeutic ONs that have been studied in the clinic. They are relatively straight forward to design and synthesize following established rules. However, their site of action is in the cellular cytoplasm and they often require the use of a transfection agent for delivery in vitro. In vivo therapeutic delivery of ONs faces numerous challenges. First, ONs generally have high molecular weights and are polyanionic, therefore, have very limited cellular membrane permeability on their own [8]. Secondly, ONs can be rapidly cleared from circulation by renal excretion and by the reticuloendothelial system [9]. Finally, ONs are sensitive to degradation by serum exo- and endonucleases while in circulation and following cellular internalization [8]. To adequately address these problems are likely to require a combination of chemical modifications on the ONs and encapsulation into appropriately designed nanoparticles.

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1.1. Oligonucleotide (ON) therapeutics

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[12]. For therapeutic siRNAs, the consensus in the field seems to be that development of an efficient delivery system is the key to their successful clinical translation [14]. End-modification of siRNA with cholesterol or N-acetylgalatosamine (GalNAc) moiety was effective in delivery of these agents into hepatocytes in the liver, facilitated by the low density lipoprotein (LDL) [15] and asialoglycoprotein receptor (ASGR) [15], respectively. It is important to know that liver is a particularly easily accessible organ due to the presence of fenestrated sinusoids allowing easy extravasation of macromolecules and nanoparticles [14]. For delivery to tissues other than the liver, various types of lipid nanoparticles (LNPs) seem to have the greatest success in ON therapeutic delivery [16].

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Please cite this article as: X. Cheng, R.J. Lee, The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery, Adv. Drug Deliv. Rev. (2016), http://dx.doi.org/10.1016/j.addr.2016.01.022

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Fig. 1. Structure of cationic lipids that have been used in LNPs.

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1.5. Synthesis of LNP formulations

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Different synthetic methods for ON-loaded LNPs have been adopted by different investigators. Because ONs are shorter than plasmids and, therefore, have only limited capacity for electrostatic interaction to interact with cationic lipids, they must be encapsulated into LNPs, rather than simple forming an electrostatic complex, to achieve stable incorporation into LNPs. When a low pKa cationic lipid (often with a tertiary amine headgroup) is used as an LNP component, a commonly used method for LNP synthesis is to combine ON with lipid components in 40% ethanol with low ionic strength and at pH 4 as a first step, as illustrated in Fig. 2. Under these conditions the cationic lipid is fully charged and the lipid bilayer structure is highly deformable, allowing them to efficiently interact with negatively charged ON and for the ON to be encapsulated by lipids. Then, ethanol and unencapsulated ON are removed in a second step by dialysis or diafiltration. This stabilizes the LNP structure. In addition, the pH is adjusted to neutral, which lowers the surface charge of the LNP to a level that is compatible to in vivo delivery. At

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PEGylation, and the LNP synthetic protocol, which can exert an influence on the structure of the LNPs [16]. A recent study showed that the percentage of cationic lipids and the processing temperature during LNP synthesis are the most critical factors in determining the in vivo biological activities siRNA LNP [20].

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this point, cationic charge is not needed to prevent ON release since the ON is stably encapsulated in the LNPs. Since there is no particle size reduction during LNP synthesis, the mean particle size of LNPs synthesized is determined by the self-assembly process in step one. It can be regulated by adjusting component ratios, ON concentration and the amount of PEGylating lipid (a helper lipid) present in the lipid composition. Other parameters such as the lipid-to-ON ratio and processing temperature also can be optimized empirically. The LNPs can be sterilized by filtration. For long-term stability, LNPs can be lyophilized with the addition of a disaccharide lyoprotectant.

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2. Helper lipids in LNP formulations

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Helper lipids are typically included as LNP components to provide particle stability, blood compatibility, and to enhance ON delivery efficiency. The remainder of this article will focus on the discussion of the roles of helper lipids in cationic-lipid-based LNPs for ON delivery. Structures of several common helper lipids are shown in Fig. 3.

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2.1. 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (Fig. 3), a 217 fusogenic lipid 218 Early designs of cationic LNPs, alternatively named cationic lipo- 219 somes, were designed for plasmid DNA delivery for gene therapy 220 [21]and have frequently incorporated DOPE as a helper lipid. In gene 221

Please cite this article as: X. Cheng, R.J. Lee, The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery, Adv. Drug Deliv. Rev. (2016), http://dx.doi.org/10.1016/j.addr.2016.01.022

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DOPE is usually described as a fusogenic lipid. It is worth noting that most cationic lipids have dioleyl or dioleoyl elements in their hydrophobic moiety, such as DOTMA, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleyloxy-3-dimethylamino-propane (DODMA), and 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1propanaminium (DOSPA). Interestingly, polyunsaturated alkyl lipid DLinDMA, which has two linoleyl moieties, has shown greater activity

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delivery, getting the DNA molecule across a membrane, typically endosomal, is a critical rate-limiting step. This requires the transient destabilization of the lipid bilayer structure. DOPE has a relatively small headgroup, phosphoethanolamine, and two bulky and unsaturated oleoyl chains, creating a cone-like shape. This lipid geometry can stabilize the non-bilayer hexagonal (HII) phase, which is found in transitional structures during membrane fusion and/or bilayer disruption [22].

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Fig. 2. Synthesis of LNPs encapsulating ONs by ethanol removal and pH adjustment.

Fig. 3. Structure of some commonly used helper lipids.

Please cite this article as: X. Cheng, R.J. Lee, The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery, Adv. Drug Deliv. Rev. (2016), http://dx.doi.org/10.1016/j.addr.2016.01.022

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2.3. Phosphatidylcholine (PC)

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PCs are primary natural components of biological membranes. Due to their cylindrical geometry, PCs favor the formation of the bilayer phase [28]. Saturated PCs, such as distearoylphosphatidylcholine (DSPC) and hydrogenated soybean PC (HSPC), have high melting temperatures (Tms). Therefore, they can be used to construct highly stable liposomes and LNPs. For example, liposomal doxorubicin in the form of Doxil is composed of HSPC/CHOL/mPEG-DSPE [29]. This type of compositional design is optimal for in vivo serum stability. The DSPC/CHOL combination has been used in stabilized nucleic acid lipid particles (SNALPs) developed by Tekmira [4]. When combined with unsaturated cationic lipids, high Tm lipids may form discrete membrane domains separate from the cationic lipids, which have low Tm [30]. A potential concern for highly stable LNPs based on these helper lipids is that they are unable to facilitate endosomal release, which may adversely affect the overall ON delivery efficiency. Unsaturated PCs, such as egg PC and DOPC (Fig. 3), have low Tm values and are in a fluid state at physiological temperatures, making the resulting LNPs highly susceptible to serum protein opsonization [31]. Bilayers formed from these lipids are stabilized by the inclusion of CHOL in the LNP composition. DOPC-based LNPs are more stable than DOPE-based formulations but are less stable than DSPC-based formulations, thus may represent a reasonable

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These lipids can form ion pairs with cationic lipids in LNPs, often to obtain a pH-responsive zeta potential profile [39]. For example, unsaturated fatty acids, such as oleic acid and linoleic acid, and cholesteryl hemisuccinate (CHEMS) (shown in Fig. 4) can be incorporated into cationic LNPs [40]. They can be protonated in the low pH tumor microenvironment or inside an acidic endosome following cellular internalization. This can produce a positive surface charge to the LNPs (due to the cationic lipid component) and/or induce lipid phase transition as a result in headgroup volume reduction for the fatty acid. This in turn promotes tumor cell interaction and endosomal release of the ON cargo [39,41]. The pairing of cationic and anionic lipids in the

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Incorporation of a PEGylating lipid into liposomes and LNPs can increase their colloidal stability on the shelf and resistance to serum protein opsonization and reticuloendothelial clearance in vivo [32]. Methoxypolyethyleneglycol (M.W. 2000)-distearoylphosphatidylethanolamine (mPEG-DSPE), which has a highly stable membrane anchor, is the most commonly used PEGylating lipid in formulations of liposomes and LNPs. PEGylation can result in a longer circulation time for LNPs, greater tumor penetration, and greater tumor accumulation as a result of the enhanced permeability and retention (EPR) effect [33]. In addition, it is easier to prepare LNPs with relatively small particle size and good colloidal stability when a PEGylating lipid is included in the formulation. However, PEGylation can inhibit cellular internalization and intracellular release of ONs by hindering membrane destabilization when the LNPs are taken up by the target cell, resulting in reduced intracellular delivery [34]. Several strategies have been used to resolve this contradiction. First, a low mole percentage of mPEG-DSPE (e.g., 1–2%) can be used. Second, a PEGylating agent with weak (short) bilayer anchor can be used, such as N-[(methoxy poly(ethylene glycol)2000)carbamoyl]-1,2dimyristyloxlpropyl-3-amine (PEG-C-DMA) (Fig. 3), which has relatively short myristyl (14 carbons) moieties as membrane anchors instead of stearoyl (18 carbons) anchors for mPEG-DSPE [35]. As a result, PEG-CDMA is “exchangeable” and is gradually lost while in circulation and the PEGylation density is reduced over time, facilitating LNP interaction with the target cells following endocytosis [35]. Other possible weakly anchored PEGylating agents are D-alpha tocopheryl polyethylene glycol 1000 succinate (TPGS) [36], polysorbate 80 (Tween-80) [37], and PEGCHOL [38]. Finally, reversible PEGylation can be achieved by introducing a cleavable linker sensitive to reduction or interstitial proteases [38].

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CHOL is a commonly used ingredient in liposomes and LNPs as a component that stabilizes lipid bilayers by filling in gaps between phospholipids [23]. Inclusion of CHOL contributes to greater stability of LNPs in the presence of serum proteins. In addition, CHOL has been shown to promote membrane fusion. As a co-lipid in LNP formulations for gene delivery in vivo, CHOL generally outperformed DOPE despite its lower fusogenicity [24]. When present at high percentages, CHOL seems to enhance the activity of cationic lipids and promote gene transfer, possibly by promoting bilayer destabilization [25,26]. The presence of CHOL along with PC results in stable lipid bilayers and is commonly used in ON LNP formulations [27].

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compromise between stability and transfection activity at the cellular 297 level. 298

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than DODMA, which has mono-unsaturated alkyl chains, as a component of LNPs [19]. The higher number of cis-unsaturation in DLinDMA leads to greater volume of the hydrophobic region of this lipid, resulting in a molecular geometry that promotes membrane fusion and bilayer disruption. Therefore, maintaining a high degree of acyl/alkyl chain unsaturation in the lipophilic region of a lipid bilayer is important both for the cationic lipid component and the helper lipid component, possible due to its critical role in intracellular bilayer destabilization [16]. DOPE is a component of Smarticles™, which are being studied in clinical trials for therapeutic delivery of a miR34 mimic. In general, however, DOPE is less frequently used in ON delivery systems compared to in plasmid delivery in the context of nonviral vectors for gene therapy. A reason might be that DOPE-based LNPs have relatively low colloidal stability due to the possibility of size increase due to bilayer fusion during storage and increased interaction with serum proteins while in blood circulation [23]. Compared to the much larger plasmid DNA, ONs are smaller and require less endosome disruption and fusogenic activities for adequate intracellular release. In addition, excessive fusogenicity can lead to unnecessary cytotoxicity. In vivo, opsonization and insertion of plasma proteins tend to render DOPE-based LNPs less fusogenic [23]. It is important to note that electrostatic interactions between cationic lipids and ONs are not as strong as with plasmid DNA. LNP formulations for ONs involve actual encapsulation of the cargo rather than mere electrostatic complexation, as is often the case with plasmid DNA carrier LNPs. Therefore, LNP formulations for ONs are typically based on phosphatidylcholines (PCs) rather than DOPE, and are designed to be more stable and less toxic than those designed for plasmid delivery.

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Fig. 4. pH-sensitive cholesterol derivatives CHEMS (anionic) and MO-CHOL (cationic).

Please cite this article as: X. Cheng, R.J. Lee, The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery, Adv. Drug Deliv. Rev. (2016), http://dx.doi.org/10.1016/j.addr.2016.01.022

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3.1. Optimal LNP composition

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LNP composition should be customized to the specific application and route of administration and optimized and validated based on empirical experimentation. However, there seem to be some general rules that can be used as guidelines for LNP design. For in vitro delivery, when

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The choice of helper lipids is in a way a balancing act between having good stability in blood circulation and ability to destabilize the endosome to release the ON cargo inside the target tissue or cells. In addition, the LNPs should have the ability to extravasate at the target tissue. Extravasation is relatively easy in the liver due to the presence of fenestrated endothelium of sinusoids in that organ. In addition, it is easy to target delivery of ONs to hepatocytes using a ligand of asialoglycoprotein receptor (ASGPR), such as GalNAc and lactosyl moiety attached to the LNP. Delivery to tissues other than the liver is much more challenging due to limitations of endothelial permeability. Delivery to solid tumors can be enabled by the enhanced permeability and retention (EPR) effect. However, this would require the LNPs to have relatively long circulation time and relatively small particle size. Selecting saturated PCs as the primary helper lipids (e.g., DSPC), inclusion of CHOL and PEGylating lipid (e.g., mPEG-DSPE) can reduce plasma protein opsonization of LNPs and maximize the EPR effect. However, as stated above, LNPs that are highly stable would not be able to facilitate ON delivery at the cellular level, which requires LNP destabilization. Incorporation of pH-sensitive lipids, both cationic and anionic, and reversible PEGylation are useful strategies to accommodate these two somewhat contradictory requirements, as illustrated in Fig. 5 [19]. In addition to delivery efficiency, selection of helper lipids must take into consideration potential toxicity and immunological effects in the form of inflammatory cytokine induction [49]. Addition of PEGylating helper lipid can potentially reduce toxicity and immunogenicity. Cytokine induction by ON LNPs should be evaluated empirically and be

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Nonionic surfactants, such as diolein [44], monooleoylglycerol (MOG) [45], and SPAN80 [36], have been shown to be effective helper lipids, due to their ability to promote non-bilayer cubic or hexagonal II phase transition. Triolein has been incorporated as a helper lipid for cationic LNPs and has been used in combination with PCs to compose nanoemulsions in gene delivery [46]. As a general rule, a helper lipid should contribute to the stability of LNPs during synthesis and subsequent storage while promote destabilization once inside an endosome (Fig. 1). At the same time, the helper lipid must not be cytotoxic, hemolytic, immunostimulatory, or otherwise possess their own biological activity, which may adversely interfere with the properties of the LNPs. A targeting ligand, such as an antibody or antibody fragment, transferrin, folate, cyclo-RGD, and phage-display derived peptide, can be linked to a lipid anchor via a linker and incorporated into LNPs to target specific cell types based on differential expression of the antigen or receptor [17]. These targeting ligands are typically not considered helper lipids. In addition, non-lipid components, such as cationic polymers, cationic peptides, and cell-penetrating peptides such as HIV Tat can be incorporated into LNPs to facilitate LNP assembly and transmembrane delivery of ONs [47].

347 348

O

353 354

346

R O

2.6. Other helper lipids

344 345

379 380

P

352

342 343

LNPs have direct access to the cells, high zeta potential and fusogenic helper lipids without PEGylation is likely to the best result. Because, for in vitro applications, LNPs can be freshly prepared, formulation colloidal stability is often not that critical. For in vivo applications, factors including colloidal stability and shelf-life, interaction with serum protein and blood cells, RES clearance, circulation time, extravasation rate, and cytokine induction all must be taken into consideration. As a result, there is typically little correlation between in vitro delivery efficiency of a LNP formulation with in vivo efficiency [48]. Cationic LNPs are more efficient in cellular interactions in vitro but highly charged LNPs can be toxic in vivo due to their ability to interact with blood components, resulting in rapid clearance, and to induce cytokine release due to toll-like receptor activation. Therefore, LNPs for in vivo delivery generally need to have low zeta potential and have stabilizing helper lipids such as PC, CHOL, and a PEGylating lipid.

T

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formulation of LNPs has initially been reported in gene delivery [41]. Formation of ion pairs due to the presence of both types of lipids can facilitate pH-responsiveness of the LNP formulation as well as drive endosomal release of ONs by promoting bilayer destabilization. NonpH sensitive anionic lipids, such as phosphatidylglycerol (PG), can provide balancing charges to a pH-sensitive cationic lipid, typically a tertiary amine-based cationic lipid [42]. Alternatively, pH-sensitive anionic lipids (carboxylate-based lipids) and pH-sensitive cationic lipids can be combined to achieve pH-responsiveness, e.g., in the proprietary Smarticles™ formulation [43]. Smarticles™ has been used for the delivery of ON therapeutic in clinical trials in cancer patients, achieving efficient knockdown of a gene target [43].

D

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X. Cheng, R.J. Lee / Advanced Drug Delivery Reviews xxx (2016) xxx–xxx

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6

Fig. 5. Transition of LNPs from systemic circulation to endosomal compartment. A neutral or weakly charged and reversibly PEGylated LNP containing pH-sensitive lipids (left) gradually loses its PEG coating. As it enters the endosome, the PEG coating is lost while the charge is increased due to reduction in endosomal pH.

Please cite this article as: X. Cheng, R.J. Lee, The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery, Adv. Drug Deliv. Rev. (2016), http://dx.doi.org/10.1016/j.addr.2016.01.022

381 382 383 384 385 386 387 388 389 390 391 392 393

397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420

480

[66,77,78,79]

TAT-PEG-DSPE/PEG-DSPE or POD/DOTAP; TCL/DOPE ligand-PEG-DSPE/DDAB/POPC/CHOL; PEG-DSG/DOTAP/POPC/CHOL

PEG-DSPE/CHOL AtuFECT01/PEG-DSPE/CHOL

DOPE, TAT-PEG-DSPE,PEG-DSPE/POD Ligand-PEG-DSPE,PEG-DSG, POPC, CHOL

F

TAT is incorporated as a cell penetrating peptide, enhancing intracellular delivery of oligos. Genosphere™ is prepared from liquid monophase where both lipid and oligonucleotide are soluble as a combination.

Preclinical

[47,66,73,76,77,78]

[74,75]

Phase I, II clinical in advanced solid tumor Preclinical

[53,66,67,68,69,70] [71,72,73]

O

The cationic lipid AtuFECT01 has trivalent positive charges.

Phase I, II clinical in cancer

Various stages of clinical trial DSPC, CHOL, PEG-S-DSG PEG-S-DSG/DLinDMA/CHOL/DSPC; other cationic lipids can be used, e.g., DODMA, DLin-KC2-DMA, and DLin-MC3-DMA

Helper lipids employed

Abbreviations: poly(ethylene glycol) (PEG), palmitoyl-oleoylphosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), (1 ,2-dioleoyloxypropyl)-N,N,N-trimethylammonium (DOTAP), cholesteryl hemisuccinate (CHEMS), 4-(2-aminoethyl)-morpholino-cholesterolhemisuccinate (MO-CHOL), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG-DSG), 3-O-[2-(methoxypolyethyleneglycol)2000]-1,2-distearoyl-sn-glycerol (PEG-S-DSG), 1,2Dioleyloxy-3-dimethylaminopropane (DODMA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), distearoylphosphatidylcholine (DSPC), β-L-arginyl-2,3-Ldiaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride (AtuFECT01), HIV trans-activator of transcription peptide (TAT), PEG-diorthoester (POD), thiocholesterol-based cationic lipids (TCL), dimethyldioctadecylammonium bromide (DDAB).

478 479

t1:14 t1:15 t1:16 t1:17

476 477

Genosphere™

474 475

t1:13

472 473

Nanolipoparticles (NLPs)

470 471

t1:12

468 469

Stable nucleic acid LNPs (SNALPs) examples: ALN-VSP02 (VEGF siRNA), PRO-040201 (ApoB-siRNA) and TKM-080301 (TKM-PLK1 siRNA) DACC lipoplexes or AtuPLEX example: Atu027 (protein kinase 3 siRNA)

466 467

3.4.1. Stable nucleic acid lipid particles (SNALPs) These are LNPs developed by Tekmira [4]. They are synthesized by first preparing a mixture of lipids and siRNA in 40% ethanol at low pH and then removing ethanol and adjusting pH to neutral, resulting in stable LNPs [54]. There are many versions of SNALPs, some of which have been studied in non-human primates or in clinical trials. For example, SNALPs composed of helper lipids CHOL, DSPC, PEG-C-DMA (Fig. 2) and cationic lipid DLinDMA at the molar ratio of 48:20:2:30 [54,55], have been used for the delivery of siRNA for Ebola virus polymerase L gene. In SNALPs, the helper lipid combination of DSPC and CHOL contributes to the stability of the LNPs whereas the PEG-C-DMA (Fig. 2) has relatively short hydrophobic anchors, therefore, is released over time in circulation. The LNPs were 71–84 nm in size and provided complete protection against viremia and death in guinea pigs [55]. However, a phase II clinical trial on a version of these LNPs (TKM-Ebola-Guinea) failed to show an overall therapeutic benefit. Other SNALP-siRNAs for polo-like kinase (PLK1) and kinesin spindle protein (KSP) been given to mice at 2 mg/kg and to greatly increase the survival of tumor-bearing

t1:6 t1:7 t1:8 t1:9 t1:10 t1:11

464 465

Smarticles™ examples: MRX-34 (miR mimic) POPC/DOPE/MO-CHOL/CHEMS and PNT2258 (bcl-2 DNA interference oligo)

463

t1:4 t1:5

461 462

Composition (example)

459 460

Formulation

452 453

t1:3

450 451

Table 1 LNP formulations for ON delivery at various stages of development.

448 449

t1:1 t1:2

446 447

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445

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R

439 440

N C O

437 438

U

435 436

R O

Table 1 shows a list of some LNP formulations in the literature designed for ON delivery. LNP formulations have been the subject of a number of recent review articles [50,51,52,53]. Two formulations in particular, SNALPs and Smarticles, are discussed as examples of the strategic utilization of helper lipids in LNP formulations that resulted in clinical translation.

433 434

7

Particles become positively charged at acidic pH, facilitating intracellular delivery. Both cationic and anionic lipids contribute to pH-sensitivity The cationic lipid is ionizable. The PEGylation is reversible to facilitate both long circulation and siRNA release at the site of target tissue.

457 458

431 432

POPC, DOPE, CHOL, CHEMS

3.4. Examples of LNP formulations

429 430

References

456

427 428

Developmental stage

454 455

Depending on the length, format, and chemical modifications, ONs can have different physical chemical properties, which may in turn affect their incorporation into LNPs. Antisense ONs and antimiRs are single stranded while miRNA mimics and siRNA are double stranded. Double stranded ONs have a helical structure which is more rigid than single-strand ONs. In addition, they have roughly double the number of nucleotides, therefore, have potentially greater electrostatic interactions with the cationic lipids during LNP synthesis. Meanwhile, short LNA-based seed-region targeting antimiRs have much fewer negatively charges, which weakens electrostatic interactions with cationic lipids in LNP formulations. ONs that are end-conjugated to CHOL can interact with lipids by hydrophobic interactions in addition to electrostatic interactions, therefore resulting in much more efficient incorporation into LNPs. However, such modification may reduce the intracellular activity by keeping the ON attached to the membrane, hindering target interaction. Single stranded ONs such as Kynamro has been shown to be active in the clinic as naked oligos. This could be a result of the fact that DNA ONs are less sensitive to nucleases than RNA ONs and can bind to serum proteins such as albumin, which may both increase ON stability in circulation and facilitate cellular delivery of the ON as a protein-ON complex. RNA duplexes such as miRNA mimics and siRNA can potentially benefit more from LNP encapsulation. Although simple GalNAc conjugates have been shown to be active in delivery into the liver, LNP vehicles should be more effective for delivery to non-liver tissues. CpG immunostimulatory ONs and aptamer ONs often act in endosomes or on the cellular surface; therefore, do not necessarily require cytoplasmic delivery. This changes the design requirement for LNP delivery vehicles. Unfortunately there are no generally applicable rules on the type of LNP formulation, including the type of helper lipids that are optimal for the delivery of each ON type. Empirical optimization may be necessary to obtain the best result in vivo.

P

425 426

Mechanism of action

3.3. Role of ON cargo in LNP formulation

D

424

T

423

part of LNP formulation development because it has frequently been identified as the dose-limiting toxicity of LNP formulation in clinical trials.

E

421 422

[44,45,46,53,63,6,65,66]

X. Cheng, R.J. Lee / Advanced Drug Delivery Reviews xxx (2016) xxx–xxx

Please cite this article as: X. Cheng, R.J. Lee, The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery, Adv. Drug Deliv. Rev. (2016), http://dx.doi.org/10.1016/j.addr.2016.01.022

X. Cheng, R.J. Lee / Advanced Drug Delivery Reviews xxx (2016) xxx–xxx

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ONs are synthetic agents that are molecularly targeted and are relatively easy to design against many biological targets that are not amenable with traditional small-molecule-based strategies. Different ONs are quite similar in terms of physical chemical characteristics, therefore, face common challenges for in vivo delivery. If the delivery problem can be addressed adequately, the therapeutic reach of ONs would be greatly extended. LNP is among the most adopted delivery platform for ONs. Helper lipids play critical roles in the LNP formulations for ON delivery. Depending on the specific application (type of cargo, route of administration) different helper lipids may be selected to address specific challenges to delivery. There is a wide range of components that can be incorporated into LNPs, thus providing a broad space for potential optimization and innovation. While most cationic lipids are proprietary and not widely available, there are many helper lipids that can be obtained as excipients from commercial suppliers. It is likely that delivery efficiency of LNPs is affected by the selection of helper lipids as much as by the cationic component. In addition to composition, synthetic protocol can also affect the structure and characteristics of LNPs. LNPs for in vivo delivery has to account for the need for stability and long circulation time in the blood as well as the need for target cell uptake and endosomal escape of ONs into the cytoplasm. Additional considerations include biodistribution considerations and immunostimulatory effect of LNP formulations, which may limit clinical application. There is no correlation between LNP performance in vitro and in vivo. LNP formulations need to be optimized and validated through empirical experimentation. A LNP formulation can fulfill multiple functions and overcome various biological barriers to facilitate delivery. However, the complexity of LNP formulations is a potential impediment to their clinical translation. Further complicating matters is that changes in ON chemistry

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O

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C

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N

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U

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Uncited reference

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546 547

[64]

549

References

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[1] N. Dias, C.A. Stein, Antisense oligonucleotides: basic concepts and mechanisms, Mol. Cancer Ther. 1 (2002) 347–355 (doi:VL - 1). [2] C.C. Esau, Inhibition of microRNA with antisense oligonucleotides, Methods 44 (2008) 55–60, http://dx.doi.org/10.1016/j.ymeth.2007.11.001. [3] N. Bushati, S.M. Cohen, MicroRNA functions, Annu. Rev. Cell Dev. Biol. 23 (2007) 175–205, http://dx.doi.org/10.1146/annurev.cellbio.23.090506.123406. [4] A. Reynolds, D. Leake, Q. Boese, S. Scaringe, W.S. Marshall, A. Khvorova, Rational siRNA design for RNA interference, Nat. Biotechnol. 22 (2004) 326–330, http://dx. doi.org/10.1038/nbt936. [5] D.H.J. Bunka, O. Platonova, P.G. Stockley, Development of aptamer therapeutics, Curr. Opin. Pharmacol. 10 (2010) 557–562, http://dx.doi.org/10.1016/j.coph.2010. 06.009. [6] J.J. Rossi, Ribozyme diagnostics comes of age, Chem. Biol. 11 (2004) 894–895, http:// dx.doi.org/10.1016/j.chembiol.2004.07.002. [7] J. Vollmer, A.M. Krieg, Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists, Adv. Drug Deliv. Rev. 61 (2009) 195–204, http://dx.doi.org/10.1016/j. addr.2008.12.008. [8] J. Wang, Z. Lu, M.G. Wientjes, J.L.-S. Au, Delivery of siRNA therapeutics: barriers and carriers, AAPS J. 12 (2010) 492–503, http://dx.doi.org/10.1208/s12248-010-9210-4. [9] R.S. Geary, Antisense oligonucleotide pharmacokinetics and metabolism, Expert Opin. Drug Metab. Toxicol. 5 (2009) 381–391, http://dx.doi.org/10.1517/ 17425250902877680. [10] G.F. Deleavey, J.K. Watts, M.J. Damha, Chemical modification of siRNA, Curr. Protoc. Nucleic Acid Chem. (2009), http://dx.doi.org/10.1002/0471142700.nc1603s39. [11] K. Hovingh, J. Besseling, J. Kastelein, Efficacy and safety of mipomersen sodium (Kynamro), Expert Opin. Drug Saf. 12 (2013) 569–579, http://dx.doi.org/10.1517/ 14740338.2013.793670. [12] S. Akhtar, M.D. Hughes, A. Khan, M. Bibby, M. Hussain, Q. Nawaz, et al., The delivery of antisense therapeutics, Adv. Drug Deliv. Rev. 44 (2000) 3–21, http://dx.doi.org/ 10.1016/S0169-409X(00)00080-6. [13] K.T. Flaherty, J.P. Stevenson, P.J. O'Dwyer, Antisense therapeutics: lessons from early clinical trials, Curr. Opin. Oncol. 13 (2001) 499–505, http://dx.doi.org/10.1097/ 00001622-200111000-00013. [14] Y.-K. Oh, T.G. Park, siRNA delivery systems for cancer treatment, Adv. Drug Deliv. Rev. 61 (2009) 850–862, http://dx.doi.org/10.1016/j.addr.2009.04.018. [15] J.H. Jeong, H. Mok, Y.K. Oh, T.G. Park, SiRNA conjugate delivery systems, Bioconjug. Chem. 20 (2009) 5–14, http://dx.doi.org/10.1021/bc800278e. [16] C. Wan, T.M. Allen, P.R. Cullis, Lipid nanoparticle delivery systems for siRNA-based therapeutics, Drug Deliv. Transl. Res. 4 (2014) 74–83, http://dx.doi.org/10.1007/ s13346-013-0161-z. [17] M.S. Shim, Y.J. Kwon, Efficient and targeted delivery of siRNA in vivo, FEBS J. 277 (2010) 4814–4827, http://dx.doi.org/10.1111/j.1742-4658.2010.07904.x. [18] P.L. Felgner, T.R. Gadek, M. Holm, R. Roman, H.W. Chan, M. Wenz, et al., Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 7413–7417, http://dx.doi.org/10.1073/pnas.84.21.7413. [19] S.C. Semple, A. Akinc, J. Chen, A.P. Sandhu, B.L. Mui, C.K. Cho, et al., Rational design of cationic lipids for siRNA delivery, Nat. Biotechnol. 28 (2010) 172–176, http://dx.doi. org/10.1038/nbt.1602. [20] X. Zhao, Y. Liu, J. Song, H. Yong, L. Li, Y. Shen, et al., Cationic lipids percentage and processing temperature are critical in designing siRNA lipid nanoparticles, J. Drug Target. 20 (2012) 281–289, http://dx.doi.org/10.3109/1061186X.2011.645160. [21] L. Ciani, S. Ristori, A. Salvati, L. Calamai, G. Martini, DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery: zeta potential measurements and electron spin resonance spectra, Biochim. Biophys. Acta 1664 (2004) 70–79, http://dx.doi.org/ 10.1016/j.bbamem.2004.04.003. [22] Y. Hattori, S. Suzuki, S. Kawakami, F. Yamashita, M. Hashida, The role of dioleoylphosphatidylethanolamine (DOPE) in targeted gene delivery with mannosylated cationic liposomes via intravenous route, J. Control. Release 108 (2005) 484–495, http://dx.doi.org/10.1016/j.jconrel.2005.08.012. [23] F. Sakurai, T. Nishioka, F. Yamashita, Y. Takakura, M. Hashida, Effects of erythrocytes and serum proteins on lung accumulation of lipoplexes containing cholesterol or DOPE as a helper lipid in the single-pass rat lung perfusion system, Eur. J. Pharm. Biopharm. 52 (2001) 165–172, http://dx.doi.org/10.1016/S0939-6411(01)00165-5. [24] A.P. Dabkowska, D.J. Barlow, A.V. Hughes, R.A. Campbell, P.J. Quinn, M.J. Lawrence, The effect of neutral helper lipids on the structure of cationic lipid monolayers, J. R. Soc. Interface. 9 (2012) 61–548, http://dx.doi.org/10.1098/rsif.2011.0356. [25] T. Yoshioka, S. Yoshida, T. Kurosaki, M. Teshima, K. Nishida, J. 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F

4. Summary and perspectives

487 Q6 488

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485 486

R O

512

3.4.2. Smarticles These are LNPs developed by Marina Biotech. A key feature for these LNPs is there pH-sensitivity and incorporation of both a cationic and an anionic lipid. Smarticles composed of POPC, DOPE, CHEMS, and MO-CHOL at molar ratios of 6:24:23:47 was used in a formulation designated PNT2258, which incorporates a 24-nucleotide DNA oligo targeting a region upstream of the bcl-2 gene [57]. In this formulation, DOPE can potentially promote membrane fusion. MO-CHOL (structure shown in Fig. 4) is a cationic cholesterol-derivative with a morpholino-headgroup with a pKa of ~ 6.5. CHEMS has a pKa value in a similar range [58]. Therefore, both MOCHOL and CHEMS contributed to the overall pH sensitivity of Smarticles, resulting in a greater change in zeta potential in response to pH changes. The Smarticles were synthesized under transient acidic ethanolic conditions where there was an overall positive charge to the lipid mixture. This facilitates the efficient encapsulation of the negatively charged ON PNT100 [57]. The pH was then changed to 7.5, which deprotonated MOCHOL and CHEMS, resulting in a negative zeta potential of −40 mV. These LNPs did not include a PEGylating lipid since the negative charge provides electrostatic stabilization [58,59]. LNPs with negative zeta potential are less toxic and immunostimulatory than those with cationic zeta potential. At the cellular level, the LNPs will be protonated again upon exposure to endosomal pH and facilitate endosomal escape of the ON. The PNT2258 Smarticles have shown antitumor activity against several xenograft tumors and is currently in clinical trial in patients with recurrent and refractory non-Hodgkin's lymphoma [60]. In addition, Smarticle-based MRX34, which carries a double-stranded miRNA-34 mimic, is currently in Phase I clinical trial in solid tumors and hematological malignancies. MRX34 is the first miRNA mimic to enter clinical trial [61,62].

544 545

P

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are likely to affect the optimal LNP formulation for delivery. So optimization of LNPs requires integration of many considerations. It would be interesting to study the interplay among the various factors, such as ON conformation/chemistry and LNP composition.

D

mice. SNALP formulations, such as TKM-HBV and TKM-PLK1 have been evaluated in clinical trials with promising results [56].

T

481 482

E

8

Please cite this article as: X. Cheng, R.J. Lee, The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery, Adv. Drug Deliv. Rev. (2016), http://dx.doi.org/10.1016/j.addr.2016.01.022

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Please cite this article as: X. Cheng, R.J. Lee, The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery, Adv. Drug Deliv. Rev. (2016), http://dx.doi.org/10.1016/j.addr.2016.01.022

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