Multifunctional pH-Sensitive Amino Lipids for siRNA Delivery

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Review

Multifunctional pH-Sensitive Amino Lipids for siRNA Delivery Maneesh Gujrati, Amita Vaidya, and Zheng-Rong Lu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00538 • Publication Date (Web): 02 Dec 2015 Downloaded from http://pubs.acs.org on December 4, 2015

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Bioconjugate Chemistry

Multifunctional pH-Sensitive Amino Lipids for siRNA Delivery Maneesh Gujrati, Amita Vaidya, Zheng-Rong Lu* Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio 44106, USA

Running title: Multifunctional lipids for siRNA delivery

*To whom correspondence should be addressed Dr. Zheng-Rong Lu M. Frank Rudy and Margaret Domiter Rudy Professor Wickenden 427, Mail Stop 7207 10900 Euclid Avenue Cleveland, OH 44106 Phone: 216-368-0187 Fax: 216-368-4969

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Abstract RNA interference (RNAi) represents a powerful modality for human disease therapy that can regulate gene expression signature using small interfering RNA (siRNA). Successful delivery of siRNA into the cytoplasm of target cells is imperative for efficient RNAi and also constitutes the primary stumbling block in the clinical applicability of RNAi. Significant progress has been made in the development of lipid-based siRNA delivery systems, which have practical advantages like simple chemistry and easy formulation of nanoparticles with siRNA. This review discusses the recent development of pH-sensitive amino lipids, with particular focus on multifunctional pH-sensitive amino lipids for siRNA delivery. The key components of these multifunctional lipids include a protonatable amino head group, distal lipid tails, and two crosslinkable thiol groups, which together facilitate the facile formation of stable siRNAnanoparticles, easy surface modification for target-specific delivery, endosomal escape in response to the pH decrease during subcellular trafficking, and reductive dissociation of the siRNA-nanoparticles for cytoplasmic release of free siRNA. By virtue of these properties, multifunctional pH-sensitive lipids can mediate efficient cytosolic siRNA delivery and gene silencing. Targeted siRNA nanoparticles can be readily formulated with these lipids, without the need for other helper lipids, to promote systemic delivery of therapeutic siRNAs. Such targeted siRNA nanoparticles have been shown to effectively regulate the expression of cancer-related genes, resulting in significant efficacy in the treatment of aggressive tumors, including metastatic triple negative breast cancer. These multifunctional pH-sensitive lipids constitute a promising platform for the systemic and targeted delivery of therapeutic siRNA for the treatment of human diseases. This review summarizes the structure-property relationship of the multifunctional pHsensitive lipids and their efficacy in in vitro and in vivo siRNA delivery and gene silencing.


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INTRODUCTION RNA interference (RNAi) has emerged as one of the most promising platforms for genome modification and a rapidly advancing frontier in medicine. As a therapeutic modality, RNAi represents an entirely novel approach to the treatment paradigms of many diseases (1, 2). Awarded the 2006 Nobel Prize for Physiology or Medicine, the discovery of RNAi is considered as “a major scientific breakthrough that happens once every decade or so”. RNAi using small interfering RNA (siRNA) functions as a posttranslational gene regulation mechanism by inducing mRNA degradation within the cytosol (3, 4). siRNA molecules are typically double stranded, 20–25 nucleotides long, with a molecular weight of approximately 13 kDa. After integration into RNA-induced silencing complex (RISC), siRNA unwinds into single-stranded RNA (ssRNA). The sense strand is degraded, leaving behind the antisense strand, which then guides and enables the RNAi machinery to seek and degrade complementary mRNA targets, allowing for the regulation of target gene expression (3, 4). Although the RNAi approach is generally applicable and relevant to a variety of diseases that exhibit aberrant gene expression, including cancers, genetic disorders, immune diseases, and viral infections (3, 4), RNAi-based clinical therapeutics still remain a distant dream. The primary hurdle in developing siRNA-based therapies is the efficient delivery of siRNA in diseased cells within a target tissue (3). Naked siRNA molecules are anionic, with a relatively large size, and cannot effectively cross the cellular membrane to reach the cytosol and initiate RNAi (5). Additionally, siRNAs are prone to rapid degradation by serum nucleases and clearance from systemic circulation (5, 6). Consequently, safe and efficient delivery of intact siRNA molecules into the cytoplasm of target cells is imperative for effective RNAi functions. To date, numerous delivery systems, including cationic lipids, cationic polymers, siRNA conjugates, and chemically



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modified siRNA, have been designed and evaluated for the systemic delivery of therapeutic siRNA. Several of these delivery systems have also entered different stages of clinical development (7). Compared to other siRNA delivery systems, cationic lipids have several advantages, including simple chemistry and easy formulation of nanoparticles with siRNA. There are two major formulations for cationic lipids and siRNA complexes, i.e., liposomes and lipid nanoparticles (LNPs). LNPs involve complexation with siRNA without forming lipid bilayers and are relatively versatile as compared to liposome formulations. Multiple functionalities can readily be incorporated in the lipid structures to overcome barriers to cytosolic siRNA delivery. In addition, the process of forming LNPs is considerably straightforward, compared to liposomes. For these reasons, LNPs, especially multifunctional lipid/siRNA nanoparticles, have garnered substantial attention in the arena of siRNA delivery. Here, we present an overview of the recent development and role of LNPs in siRNA delivery, with a specific focus on pHsensitive multifunctional amino lipids.

DESIGN CONSIDERATIONS FOR LIPID/siRNA NANOPARTICLES The common goals of siRNA delivery are: i) to protect siRNA from degradation during systemic circulation; ii) to ensure target site accumulation while avoiding non-specific uptake in healthy, non-target tissue; iii) to promote cellular uptake and endosomal escape; and iv) to enable release of siRNA in the cytosol (8, 9). For nanoparticle-based siRNA delivery systems, cationic materials, including cationic polymers and lipids, are commonly used to condensate negatively charged siRNA to form nanoparticles that protect the siRNA from degradation. Biocompatible


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polymers, e.g. polyethylene glycol (PEG), are often used to modify the nanoparticles and minimize non-specific tissue uptake. Nanoparticles can also be decorated with targeting ligands to mediate specific interactions with receptors expressed on the cell-surface to facilitate targeted siRNA delivery and receptor-mediated endocytosis (10, 11). Because RNAi requires the presence of free siRNA in the cytoplasm, developing a foolproof approach that facilitates endosomal escape after internalization and cytosolic release forms a major challenge in the design of efficient siRNA delivery systems. Endosomal escape of nanoparticles is considered as the rate-limiting step in the process of siRNA delivery. siRNAbased nanoparticles are typically internalized via endocytosis, following which they encounter a number of intracellular obstacles. In most cases, the nanoparticles are trafficked through a series of maturing acidic vesicles leading to the lysosomes (12, 13). The siRNA-nanoparticles must therefore be designed to escape these compartments and avoid exposure to the degradative enzymes contained within the harsh acidic environment of the late-endosomes and lysosomes. While the exact mechanism of internalization may vary depending on the physicochemical properties of the siRNA delivery systems, targeted or non-targeted nanoparticles will become localized within endocytotic vesicles (14). The first vesicle in the trafficking pathway, the endosome, can fuse with other sorting vesicles to transport the internalized content back to the cellular membrane and induce exocytosis (15, 16). In fact, recent work using a cationic lipid-siRNA nanoparticle system revealed that up to 70% of the internalized siRNA undergoes exocytosis (17). Endosomal vesicles not involved in recycling will continue trafficking through the endocytotic pathway to the late-endosomes (18), which engage ATPase proton-pump enzymes to rapidly acidify the vesicle environment to a pH of 5–6, before maturing into lysosomes with a pH of 4.5 and



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containing degradative enzymes (19). Any siRNA or nucleic acid load trapped inside these acidic vesicles is presumably degraded. As such, an emphasis on designing siRNA delivery systems capable of escaping the fate of exocytosis and/or lysosomal degradation is the key to achieving efficient target gene knockdown. Proton sponge effect and lamellar phase transition are the two main escape mechanisms that are commonly employed in the design of siRNA delivery systems for mediating endosomallysosomal escape. The proton sponge effect applies to materials that contain amines with pKa values in the range of 5–7, thus matching the pH range encountered during endosomal-lysosomal trafficking (20, 21). Following internalization, the amine groups become protonated as the endosome acidifies. These amines exhibit a buffering capability that results in an influx of protons and chloride ions to create an osmotic imbalance. As water enters the endosomes to counter this effect, the endosomal vesicle inflates, causing it to rupture and ultimately release its contents into the cytosol. As such, primary, secondary, tertiary, and imidazole amines of various pKa’s are often incorporated into nucleic acid delivery systems (22). However, it is important to note that the rupture of endosomes and lysosomes may release their contents into the subcellular compartments causing cell death. Lamellar to reversed hexagonal phase transition mechanism is mainly used in the design of lipid-based siRNA delivery systems. Early reports suggested that lipid-nucleic acid complexes arranged in the inverted hexagonal phase have a greater transfection efficiency compared to those in the lamellar phase (23, 24). This observation was disputed by conflicting reports, which argued that there is a direct correlation between the complex structure and transfection efficiency (25, 26). More recently, it has been suggested that the dynamic evolution of the structure of a complex following its interactions with anionic cellular lipids is the critical factor that


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determines the extent of transfection efficiency (24). The lamellar phase transition occurs with a class of fusogenic cationic-based lipids that form non-bilayer formations under aqueous environments (27-29). These lipids can adopt the inverted hexagonal phase, which is characterized by a long, cylindrical, and inverted micelle structure. Complexes comprised of such lipids that undergo a transition from lamellar to an inverted hexagonal phase (lamellar-tohexagonal inverted phase transition) can fuse with the anionic lipid membranes of intracellular vesicles to release their nucleic acid cargo into the cytosol. To achieve the transition, the lipid must contain unsaturated hydrocarbon chains. Studies have demonstrated that cationic lipidbased systems containing unsaturated lipids have superior gene silencing efficiency compared to systems containing saturated lipids. The unsaturated state of the hydrophobic lipid tails directly influences the fusogenic potential of the delivery system. Dioleoylphosphatidylethanolamine (DOPE) is a lipid with fusogenic capabilities and is commonly incorporated into delivery systems as a helper lipid to mediate lamellar-to-hexagonal transition and improve transfection efficiencies (30, 31). A desirable approach in designing an efficient cytosolic siRNA delivery system is to take advantage of the inherent physiological differences in the subcellular compartments in the path of subcellular trafficking. We have successfully demonstrated that the incorporation of pHsensitive amphiphilicity into lipids can effectively mediate endosomal escape of lipid/siRNA nanoparticles in response to acidification during subcellular trafficking (32-34). Chemical structures that contain both hydrophilic and lipophilic domains are designated as amphiphilic (35). Amphiphiles can self-assemble in an aqueous environment, either alone or with other lipid helpers, and can associate with siRNA to form cationic-lipid/siRNA complexes (36). Amphiphilic materials, e.g. surfactants, can destabilize the lipid bilayer of cell membranes,



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thereby facilitating endosomal escape of the siRNA nanoparticles. pH-sensitive lipid-siRNA carriers should possess minimal amphiphilicity at neutral physiological pH, so as to minimize non-specific cell membrane disruption and cytotoxic effects as the lipid/siRNA nanoparticles travel through systemic circulation. As stated before, nanoparticles encounter an increasingly acidic environment as they progress through the cellular trafficking pathway. Consequently, the lipids acquire specific pH-sensitive amphiphilicity in the acidic environment, resulting in structural changes that enhance interactions with the lipid walls of the vesicles to trigger endosomal membrane destabilization and subsequent escape (37). pH-SENSITIVE LIPIDS FOR siRNA DELIVERY Cationic lipids and pH-sensitive lipids are the two main categories of lipids used in the formation of lipid/siRNA nanoparticles. Early studies reported the use of cationic lipids, such as N-1-[(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), for gene delivery (38-40). These cationic lipids contain a trialkylammonium head group and gemini lipids. The positively charged quaternary ammonium groups in the lipids enable the formation of stable complexes with negatively charged nucleic acids, including siRNA. The cationic lipids possess fusogenic capabilities that mediate high transfection and gene efficiencies (41). However, the cationic lipids are unable to respond to the pH changes that occur in the process of subcellular trafficking, because they already have stable charged head groups. pH-sensitive lipids represent a class of lipids with amino head groups that can be protonated to become positively charged in response to the pH decrease during subcellular trafficking. A small amino head group is used in these lipids to prevent the formation of lipid bilayers from the paired lipid tails, which is critical for cell membrane destabilization. pH-sensitive lipids have several advantageous features over cationic lipids, including low charge density at neutral pH, pH-sensitive amphiphilicity, and pH-


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sensitive cell membrane destabilization. Low positive charge density can minimize potential toxic side effects associated with high charge density during systemic circulation. In this way, pH-sensitive amphiphilicity and membrane destabilization can facilitate the escape of LNPs from the acidic endosomes.

Figure 1. Chemical structure of some pH-sensitive lipids.

The first reported ionizable or pH-sensitive lipids for siRNA delivery were developed based on the previously reported cationic lipid DOTMA (28). The ionizable lipids had saturated or unsaturated gemini lipids and a tertiary amino head group (pKa = 6.7) capable of protonation in an acidic environment, Figure 1. These lipids, in conjunction with other lipids, including 1,2distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, were used to form stable lipid/siRNA nanoparticles. The lipid nanoparticles facilitated successful structure-dependent RNAi-mediated gene knockdown, with the saturation status of the lipid tails significantly affecting the gene silencing efficiency. The increase of lipid saturation from 2 to 0 double bonds increased lamellar to reversed hexagonal phase transition temperature and decreased fusogenicity. The cis-double bonds within the acyl chains are thought to enhance the cone shape of the lipids to facilitate membrane destabilization. As a result, the amino lipids with unsaturated hydrocarbon chains mediated more efficient gene silencing than the saturated lipids. Specifically,



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DLinDMA, with 2 double bonds in each hydrocarbon chain, resulted in greater transfection efficiency than the other lipids tested, including DLenDMA with 3 double bonds.

DLinDMA was used as a lead pH-sensitive amino lipid for further structural optimization to improve biocompatibility, and siRNA transfection and gene silencing efficiency (42). Chemical or enzymatic labile functional groups with different degradation rates were incorporated into the linker connecting the amino head group and the lipid tails. Introduction of a ketal ring linker domain into DLinDMA (DLin-K-DMA) resulted in the best in vivo silencing efficiency when compared to analogous lipids containing ester, alkoxy, carbamate or thioether linkages. Next, the composition of the amino head group was re-evaluated using DLin-K-DMA by varying the number of ionizable groups to study the downstream effect on overall pKa and functionality. Further structural refinements in the head group design included addition of piperazino, morpholino, trimethylamino, or bis-dimethylamino groups. The dimethylamino group of the original DLin-K-DMA lipid was found to be superior to all structural modifications explored so far. As a final point of optimization, the distance between the amino head group and the dioxolane linker domain was varied through the introduction of methylene groups (Figure 2A). The distance between the cationic head group and the linker domain can influence the overall pKa of the lipids and thus greatly impact the silencing efficiency (Figure 2B). Increasing the distance with a single additional methylene group (DLin-KC2-DMA) was found to produce a fourfold increase in efficiency compared to DLin-K-DMA (Figure 2C). The introduction of additional methylene groups increased the overall pKa of the lipid and subsequently decreased the functional activity, highlighting the need for careful optimization of the structural components of pH-sensitive lipids.


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Figure 2. A. Chemical structures of DLinDMA-derived cationic lipids. B. Biophysical parameters and in vivo silencing activity of the DLinDMA-derived cationic lipids. C. Effect of distance between the cationic head group and linker domain. DLin-K-DMA () has additional methylene groups added between the DMA headgroup and the ketal ring linker to generate DLin-KC2-DMA (), DLin-KC3-DMA (), and DLin-KC4-DMA (). D. Plot of in vivo hepatic gene silencing activity vs. pKa in mice. The cationic lipids were formulated in LNPs and subjected to an ED50 analysis and plotted against their pKa (42). In a follow-up study, a comprehensive evaluation of the role of the amino head groups in a library of ionizable lipid analogues of DLin-KC2-DMA was performed to determine the correlation between the pKa of the head group and median effective dose (ED50) for silencing the hepatic FVII gene in mice (43). Only those lipids with pKa’s ranging from 6.2–6.5 were able to elicit significant in vivo gene silencing (Figure 2D). Dilinoleyl-methyl-4-dimethylaminobutyrate



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(DLin-MC3-DMA, ED50 = 0.03 mg/kg, pKa = 6.44) was identified as the most effective lipid carrier in the library. A pKa value of 6.44 was identified as the optimal pKa for this group of amino lipids. Such findings could serve as valuable design guides for the future development of pH-sensitive amino lipids with similar structural characteristics for siRNA delivery.

Combinatorial chemistry has also been applied to develop amino lipids for siRNA delivery. Large libraries of thousands of amino lipids have significantly expanded the structural variations for comprehensive assessment of the structure-property of the lipids in siRNA delivery via lipid nanoparticles (44-46). Within the library of structures, the following structural parameters of the lipids were systematically adjusted: i) hydrophobic tail chain length and saturation, ii) linkage group between the hydrophobic tail and amino group, iii) primary R group on the amine, iv) the number of hydrocarbon chains, v) the number of amines, and vi) postsynthetic quaternization of the amino group. Structural features of amino lipids, including i) more than two amines per head group; ii) amide bonds between the amine ‘core’ and acyl tails; iii) more than two acyl chains; iv) acyl chains between 8 and 12 carbon atoms; and v) a least one secondary amine, were found to be most ideal for efficient gene silencing. The pKa values also form a key determinant of the in vitro and in vivo function and activity of LNPs. Several lead amino lipids have been identified from these libraries to mediate efficient gene silencing both in vitro and in vivo. In vivo systemic delivery of therapeutic siRNA with lipid-siRNA nanoparticles requires surface modification to avoid rapid clearance by the mononuclear phagocyte system. Hydrophilic polymers, including polyethylene glycol (PEG), are commonly used for the surface modification of nanoparticles to improve their half-life in circulation after systemic administration. PEG with lipids anchors, e.g., PEG-ceramides and PEG-succinoyl-diacylglycerols (PEG-s-DAG), have


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been used to modify the particle surface by physically incorporating the lipid anchors into the lipid LNPs (7). PEGylation of lipid-siRNA nanoparticles significantly reduces non-specific uptake and improves the safety of the nanoparticle, following systemic administration. Recent advancements in the field of siRNA delivery have demonstrated that pH-sensitive amino lipids are a promising class of versatile delivery systems for safe and efficient siRNA delivery for the treatment of human diseases. Currently, five lipid-siRNA nanoparticles with ionizable lipids have advanced to various stages of clinical trials (7). Although most of these nanoparticles target the liver to treat hepatic disorders, this is the significant first step to advance RNAi with siRNA as a targeted therapy in treating human diseases.

MULTIFUNCTIONAL pH-SENSITIVE AMINO LIPIDS FOR siRNA DELIVERY Design and characterization of multifunctional lipids Recently, multifunctional pH-sensitive lipids have emerged as promising carriers for efficient systemic siRNA delivery (32, 47). Multiple functionalities are strategically introduced into a simple and unique lipid framework to overcome the barriers to cytosolic delivery, including endosomal-lysosomal compartments. The key features of these lipids are facile formation of stable siRNA nanoparticles, easy surface modification for target-specific delivery, endosomal escape in response to pH decrease during subcellular trafficking, and reductive dissociation of siRNA nanoparticles to release free siRNA in the cytoplasm. No other lipid components are needed to form stable nanoparticles with siRNA.



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Figure 3. General structure (A) of multifunctional pH-sensitive lipids and schematic description of formation of multifunctional lipid siRNA nanoparticles (B). For efficient systemic in vivo siRNA delivery, features such as i) ability to form stable compact nanoparticle with siRNA, ii) pH-sensitive amphiphilicity-induced endosomal-lysosomal escape, iii) environment-sensitive siRNA release, and iv) facile modification for targeting, have been incorporated in the structural framework of a series of new amino lipids or surfactants with pH-sensitive amphiphilicity. These lipid carriers contain three domains, each playing an integral role in the formation of stable and effective siRNA nanoparticles, namely i) a protonatable amino head group with different pKa’s, ii) two lipid tails, and iii) two crosslinkable thiol groups (Figure 3). The amine groups are comprised of various combinations of primary, secondary, tertiary, and/or aromatic amino groups, which can electrostatically complex with siRNA. The lipids introduce lipophilicity and are intentionally set at distal positions to increase the amphiphilic wedge and to avoid lipid bilayer formation. The lipophilic groups also facilitate the formation of compact nanosized complexes or nanoparticles via hydrophobic interactions in an aqueous solution. In addition, the thiol groups from the cysteinyl residues can form intermolecular disulfide crosslinks via auto-oxidation to further stabilize the nanoparticles. A small portion of the thiol groups can also be used to modify the lipid siRNA nanoparticles with specific moieties for targeted siRNA delivery.


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A key feature of the multifunctional lipid carriers is their pH-sensitive amphiphilicity and the resultant cell membrane disruption, which facilitates endosomal-lysosomal escape of the siRNA nanoparticles at the acidic endosomal-lysosomal pH. Amphiphilic cell membrane disruption is a common phenomenon in nature. The pH-sensitive amphiphilicity allows the lipid carriers to alter their amphiphilic structures when exposed to the acidic environment of the endosomal-lysosomal pathway, resulting in membrane destabilization and subsequent endosomal escape. It is crucial that siRNA delivery carriers have selective amphiphilic behavior so that high membrane disruption is achieved specifically at the endosomal pH and not at the physiological pH. Modification of the amine composition of the head group and the structure of the lipophilic tails serves to further fine-tune the pH-sensitive amphiphilicity, so as to improve the selectivity of endosomal membrane disruption and enhance cytosolic siRNA delivery. Once the multifunctional lipid-siRNA nanoparticles reach the cytoplasm, the disulfide crosslinks in the nanoparticles are reduced by endogenous glutathione to facilitate the dissociation of the nanoparticles, followed by siRNA de-complexion and release. The concept of pH-response multifunctional lipids for siRNA delivery was first demonstrated using a small library of lipids with protonatable amino head groups(32). Ten distinct structures were designed and synthesized with different combinations of protonatable amino head groups, peptide linker groups, and geminal distal lipophilic tails (Figure 4A). Polyamines, differing in the number of amino groups with various pKa’s, including ethylenediamine (E), triethylenetetramine (T), pentaethylenehexamine (P), and spermine (S), were used as the amino head groups to control the pH-sensitive amphiphilic behavior of the carriers. Histidine (H), glycine (G), and cysteine (C) were selected for the peptide linkage. The imidazole group in histidine (pKa = 6.0) was also used to adjust the pH-sensitive amphiphilicity



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of the lipids. Fatty acids of different chain lengths and saturation degrees, including lauric acid (L), oleic acid (O), and steric acid (St), were used as the lipophilic tails. As shown in Figure 4B, the multifunctional lipids demonstrate pH-sensitive amphiphilic cell membrane disruption at pH values corresponding to the environment of the endosomallysosomal trafficking pathway (32). The pH-dependent hemolysis assay reveals that pH-sensitive cell membrane disruption is governed by the structural compositions of the lipids. Generally, all lipids exhibit lower hemolytic activity at pH 7.4, compared to pH 6.5 and 5.4. The inclusion of histidine appears to control the pH-sensitivity better, resulting in low hemolysis at pH 7.4 and 6.5 and high hemolysis at pH 5.4, when comparing THCO with TGCO. The imidazole with pKa of 6.0 also contributes to the pH-sensitivity of the lipids at the endosomal-lysosomal pH. For lipids containing histidinyl residues with the same lipophilic tail groups, the hemolytic activity was found to increase with the number of protonatable amino groups at pH 7.4 and 6.5, suggesting that increasing the number of protonatable amino groups contributes to an increased head group charge and higher amphiphilicity at neutral pH. The hemolytic activity is also dependent on the composition of the lipophilic tail groups. Interestingly, carriers containing the same head group but unsaturated oleoyl groups exhibited less hemolysis than those with saturated lipophilic tails at pH 7.4 and 6.5. The specific pH-sensitive amphiphilicity can be fine-tuned by selecting a combination of the head group and lipophilic tail groups that exhibited low hemolysis at pH 7.4 and 6.5 and high hemolytic activity at 5.4. EHCO, with its combination of primary, tertiary, and aromatic amine groups in tandem with the unsaturated oleic acid lipid tail groups, demonstrated specific hemolytic activity only at pH 5.4 with negligible hemolysis at other pH levels.


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Figure 4. A) Chemical structures and abbreviated names of the pH-sensitive multifunctional amino lipids exhibiting pH-sensitive amphiphilicity for siRNA delivery. B) Hemolytic activity of the multifunctional lipids and DOTAP at pH 7.4, 6.5, and 5.4 with 1% Triton X-100 and PBS as controls. C) Particle size of EHCO/siRNA complexes at different N/P ratios. D) Luciferase



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silencing efficiency and cell viability of the multifunctional lipids complexed with siRNA (100 nM). TransFast and DOTAP were used as controls (32).

It has been well documented that the stoichiometric relationship between the cationic lipid charge and the anionic RNA charge, designated as the lipid-to-siRNA (N/P) ratio, can influence a number of parameters including the particle shape, size, cellular trafficking, and silencing efficiency (48). Initial increase of the N/P ratio led to neutral lipoplexes that were unstable and consequently aggregated into large complexes. Further increase of the N/P ratio resulted in small and stable lipoplexes with a net positive charge. However, such trends heavily depend on the chemical composition of the lipid. The multifunctional lipids, as exemplified with EHCO, also showed similar effect of varying N/P ratios on the formation of the siRNA-lipid nanoparticles (Figure 4C) (32). When mixed and incubated with siRNA for 30 minutes, EHCO formed nanoparticles (of diameter 200 nm) at an N/P ratio as low as 0.5. Increasing the N/P ratio to 4 increased the nanoparticle diameter to 3 µm due to aggregation and lack of electrostatic charge repulsion. Further increase in the N/P ratio to 8 or 10 decreased the nanoparticle size to approximately 200 nm or smaller. All the multifunctional lipids formed stable siRNA nanoparticles with a diameter between 160–210 nm at N/P ratio of 10. The pH-sensitive hemolytic activity of the multifunctional lipids directly correlates with their in vitro silencing efficiency (32). EHCO with better pH-sensitivity resulted in the highest luciferase silencing (88.4 ± 3.1%) compared to the other lipids (48% to 81%) at an N/P ratio of 10 in U87-luc cells (Figure 4D). Two other lipids with oleyl tails, THCO and SHCO, also exhibited higher silencing efficiency than the other lipids. All the multifunctional lipids had high


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cell viability ranging from 78.6 ± 5.7% to 88.2 ± 1.3%. This high gene silencing efficiency and low cytotoxicity of EHCO is speculated to result from its specific pH-sensitive amphiphilicity and membrane disruption characteristics. New multifunctional lipids, based on structural modifications of the lead lipids EHCO and SHCO, have also been designed and tested. SHCO has been identified as a lead because spermine is a natural polyamine for nucleic acid condensation and also exhibits efficient gene silencing. Spermine naturally complexes with DNA by stabilizing its α-helix structure and is routinely incorporated in non-viral gene carrier systems. Eight new spermine-based multifunctional lipids were synthesized and tested for both plasmid DNA and siRNA delivery (Figure 5A) (49, 50). In these new lipids, L-lysine (K) was introduced as a “Y” joint for the incorporation of the lipid tails to give a wedge-shaped structure. β-Alanine (A) and histidine were used to adjust the distance between the two lipid tails in order to understand the impact of the lipid spacing on the transfection efficiency. The location and number of histidines (H) were varied to allow fine-tuning of the pH-sensitive behavior of the carriers. Cysteine (C) residues were included for stabilization and functionalization of the siRNA nanoparticles. Oleic acid (O) was used for the hydrophobic lipid tail groups to introduce lipophilicity, and to facilitate the pHsensitive endosomal escape. All the spermine-based carriers effectively complexed with siRNA via charge-charge interactions to form stable nanoparticles with sizes ranging between 85–120 nm (Figure 5B) (49, 50). The nucleic acid complexes of all the spermine-based lipids exhibited much greater hemolytic activity at pH 5.5 than at 7.4. Increasing the concentration of the nanoparticles from 16 to 40 µM also resulted in increased hemolytic activities, especially at pH 5.5, for most of the lipids (Figure 5C). Interestingly, SKACO, which has no histidine, demonstrated the most



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selective pH-sensitive behavior with the largest increase in the hemolytic activity as the pH decreased from 7.4 to 5.5. On the other hand, the presence of histidine within all the other structures decreased the hemolytic activity at pH 5.5, as compared to SKACO. The siRNA complexes of these lipids showed low cytotoxicity. It appears that the incorporation of histidine on the branches adjacent to cysteine in the lipids resulted in better gene silencing efficiency than that on the head group adjacent to spermine. SKAHCO (N/P = 12, 50 nM siRNA) exhibited the highest silencing of luciferase in U87 cells (84.6 ± 5.5%) (Figure 5E). The incorporation of histidine in the lipids offered no distinct advantage over the lipids without histidine. Also, the distance between the lipid tails had no clear impact on the gene silencing efficiency with these spermine-based carriers.


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Figure 5. A) Chemical structures of the multifunctional lipid carriers with a spermine head group. B) Particle size of pDNA (top) and siRNA (bottom) complexes at N/P ratio of 12. C) Hemolytic activity of pDNA complexes formed with spermine-based lipids at N/P ratio of 12 at pH 7.4 and 5.5. In vitro transfection efficiency of D) pDNA and E) siRNA complexes at N/P ratio of 12 in U87 cells (49, 50).

On similar lines, the structure of EHCO was modified using L-lysine as the “Y” joint to create EKHCO and EHHKCO (Figure 6A) (51). These two lipids had the same amino acid residues and lipid tail groups, with a difference in the number and position of histidine residues and the distance between the two lipid tails. Both the lipids exhibited concentration-dependent and increased hemolytic activity at pH 5.5. When complexed with siRNA, EKHCO showed better pH-sensitivity compared with EHHKCO. EHHKCO demonstrated concentrationdependent hemolytic activity at pH 7.4, whereas EKHCO had minimal hemolysis, independent of the concentration (Figure 6B). EHHKCO with histidine residues at the head group mediated superior luciferase silencing in U87-luc cells compared to EKHCO with histidines on the branches. This is contrary to the silencing effect observed in the spermine-based lipids (Figure 5), in which the incorporation of histidine on the branches leads to better gene silencing efficiency than that on the head group adjacent to spermine. Spermine has two more secondary amines on the head groups than ethylenediamine. These amines are highly basic, compared to the imidazole group in histidine and can supersede the contribution of the imidazole groups on the head group. In the case of ethylenediamine, the imidazole groups on the head significantly contribute to the basicity of the head group and to the pH-insensitivity. Interestingly, the increased pH-sensitivity of EKHCO to endosomal pH did not translate to better transfection


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efficiency than EHHKCO, presumably due to the large size of the nanoparticles it formed with siRNA (ca. 200 nm), which may have affected cellular uptake (Figure 6C). The results with EKHCO and EHHKCO also suggest that relatively high hemolytic activity at neutral pH may help endosomal escape, thus improving cytosolic siRNA delivery and gene silencing efficiency. Nevertheless, there should be a balance of pH-sensitivity and cell membrane disruptive activity in the pH-sensitive lipids to achieve safe and efficient cytosolic siRNA delivery and gene silencing.

Figure 6. A) Chemical structure of EKHCO and EHHKCO. B) Concentration-dependent hemolytic activity at pH 7.4 and 5.5 of EKHCO and EHHKCO at N/P ratio of 12, respectively. C) Luciferase silencing efficiency of EKHCO and EHHKCO in U87-luc cells at N/P ratio of 12 and 20 nM siRNA concentration (51).



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Based on the above observations, EHCO and SHCO were further modified by removing the histidine and varying the saturation degree of the lipids (Figure 7A)(33). Linoleic acid (Ln) with two cis-double bonds was used to assess the effect of the saturation degree of the lipid tails on siRNA delivery and gene silencing efficiency. The siRNA nanoparticles of all the lipids exhibited good cell viability with N/P ratio as high as 18. The composition of the lipids also had a significant impact on the size and siRNA encapsulation of the nanoparticles. The sperminebased lipids were more efficient at condensing siRNA into nanoparticles, compared to the carriers containing ethylenediamine. The high amine density in the spermine allowed nearly 100% siRNA encapsulation in the nanoparticles formed with the spermine-based lipids at N/P ratios ranging from 2 to 15, while the lipids with ethylenediamine had 90% or higher siRNA encapsulation when the N/P ratio was higher than 10. The lipids with spermine exhibited a faster decrease in size and increase in zeta potential of the siRNA nanoparticles as the N/P ratio was increased from 2 to 15. The lipids with the spermine head group formed smaller nanoparticles than the lipids with ethylenediamine head group and the same composition of histidine and tails at N/P ratios of 5, 8, and 10 with a zeta potential around 25 mV. The lipids without histidine, ECO, ECLn, SCO, and SCLn, formed smaller and compact siRNA-nanoparticles than the corresponding lipids with histidine, EHCO, EHCLn, SHCO, and SHCLn, at N/P ratios of 5, 8, and 10, respectively. The lipid/siRNA nanoparticles showed pH-sensitive cell membrane disruption, as shown by the hemolysis assay at pH 5.4, 6.5, and 7.4 (Figure 7B) (33). The cell membrane disruption activity increased with high N/P ratio at pH 7.4 for all the lipids except EHCO. Increasing the number of amino groups in the head domain resulted in increased hemolytic activity at neutral pH. The presence of histidinyl residues (pKa 6.0 for imidazole) in the lipids reduced the


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hemolytic activity at neutral pH and improved the pH-sensitivity, as compared to the corresponding lipids without histidine. These observations suggest that the two additional secondary amino groups within spermine facilitate stronger electrostatic interactions with siRNA and enhance protonation of the head group in weakly acidic environment to facilitate greater membrane disruption. The incorporation of histidine decreased the overall pKa of the cationic carrier, thereby reducing its membrane disrupting ability at neutral pH. The saturation degree of lipid tails did not significantly affect the properties of the siRNA-nanoparticles when comparing the lipids with same head groups and lipid tails with different saturation degree. Accordingly, by altering the number, composition, and pKa of the amino head groups, the overall pKa, degree of protonation, and pH-sensitive behavior of the multifunctional lipids can be tuned.



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Figure 7. A) Chemical structures of pH-sensitive multifunctional amino lipid siRNA carriers. B) pH-dependent hemolytic activities of siRNA carriers formulated at an N/P ratio of 10 at pH 7.4, 6.5, and 5.5. C) Luciferase silencing efficiency in HT29-luc cancer cells at 100 nM siRNA concentration for all siRNA carriers formulated at an N/P ratio of 12 (33).

The multifunctional lipids ECO and ECLn were found to have the greatest gene silencing efficiency in both HT29 and CHO cells among these series of lipids (Figure 7C)(33). ECO was also effective in mediating high in vitro gene silencing efficiency (ca. 80%) in U87-Luc cells in culture medium containing 50% serum (37). Interestingly, EHCO and EHCLn with fine-tuned cell membrane disruption at pH 5.4, which is the pH in the late endosomes, did not necessarily correlate with better gene silencing efficiency. Better cell membrane disruption of ECO and ECLn at relatively weak acidic pH might promote rapid endosomal escape after endocytosis of the nanoparticles. Although the spermine-based lipids exhibited greater hemolysis, superior ability of siRNA complexation, and high cell membrane disruption at weak acidic pH, these lipids mediated relatively low gene silencing efficiency, possibly because their strong complexation with siRNA inhibited the cytosolic release of the siRNA cargo from the nanoparticles. In summary, the multifunctional pH-sensitive amino lipids are designed to have distal lipid tails to avoid the formation of lipid bilayers, varying the amino head group to fine-tune the pH-sensitivity and gene silencing, and incorporating dual thiol groups to further stabilize the siRNA nanoparticles. These lipids form stable nanoparticles without the help of other lipids and hydrophobic components. Some structure-property correlations have been observed for


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designing better siRNA delivery systems with this class of multifunctional lipids. Although amino head groups with more protonatable aliphatic amines can enhance siRNA encapsulation and nanoparticle formation, they may result in poor siRNA release from the nanoparticles and low gene silencing efficiency. Lipids with one double bond show improved gene silencing efficiency and a further increase of the unsaturation may not directly increase the efficiency of gene silencing. Similarly, incorporation of histidinyl imidazole groups improves the pHsensitivity of the lipids, but does not necessarily translate into better gene silencing efficiency.

Endosomal escape mediated by multifunctional pH-sensitive lipids Endosomal escape and cytosolic siRNA release are the key steps of siRNA delivery systems for efficient RNAi. ECO is one of the lead multifunctional lipids exhibiting superior gene silencing efficiency (33, 37). ECO/siRNA nanoparticles at N/P = 10 have been demonstrated to effectively protect siRNA degradation in culture medium containing 50% serum, suggesting good stability of the nanoparticles during circulation. The ability of the ECO/siRNA nanoparticles to promote endosomal escape was confirmed with confocal microscopy using an Alexa Fluor 647-labeled siRNA and LAMP1, which is a specific marker for late endosomes and lysosomes (Alexa Fluor 488-labeled anti-LAMP), in U87 human glioma cells (Figure 8) (37). ECO/siRNA nanoparticles were found to interact with the cell membrane as early as 5 minutes post-transfection, as evident by the strong fluorescent siRNA signal seen along the periphery of the cells. Between 30 min and 2 h, the siRNA signal colocalized with the LAMP1-stained vesicles, indicating that the ECO/siRNA nanoparticles were trafficked into the late endosomes. After 4 h, a dispersed siRNA signal appeared throughout the cytosol and the



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colocalization between siRNA and LAMP1-stained vesicles diminished, suggesting that ECO facilitates endosomal escape and cytosolic release of the siRNA cargo.

Figure 8. Illustration of pH sensitive endosome escape and pH-sensitive membrane disruption of ECO/siRNA nanoparticles (A) and confocal images showing subcellular trafficking, endosomal escape, and cytoplasmic siRNA release of ECO/siRNA nanoparticles (AF 647) using LAMP1antibody (AF 488) (B).

The multifunctional lipids are designed to stabilize the nanoparticles through electrostatic charge interactions, hydrophobic condensation, and disulfide cross-links. It is also expected that once the siRNA/lipid nanoparticles escape from the endosomes, a high concentration of


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glutathione in the cytosol will reduce the disulfide bonds to facilitate the dissociation of the nanoparticles and release the siRNA cargo. This hypothesis was demonstrated by decomplexation and release of the siRNA cargo from ECO when ECO/siRNA nanoparticles was incubated with glutathione (Figure 9A and B) (37). Furthermore, depletion of intracellular glutathione by inhibiting glutathione production with buthionine sulfoximine (BSO) substantially reduced the luciferase silencing efficiency and cytosolic distribution of fluorescent siRNA in U87-Luc cells (Figure 9C and D). These results support the design concept that the inclusion of cysteine residues within the multifunctional lipid is a key feature for stabilization of the ECO/siRNA nanoparticles in circulation and to facilitate cytosolic disulfide bond reduction and siRNA release for cytosol-specific siRNA delivery.



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Figure 9. Release of siRNA from ECO/siRNA nanoparticles by glutathione-mediated disulfide bond reduction and siRNA release (A and B). Luciferase silencing efficiency (C) and cytosolic siRNA release (D) are diminished upon intracellular inhibition of glutathione by buthionine sulfoximine (37).

Surface modification of siRNA nanoparticles of the multifunctional lipids Surface modification of siRNA nanoparticles is a commonly used approach to impart biocompatibility, minimize non-specific uptake by the reticuloendothelial system (RES), and improve target-specific delivery for systemic in vivo delivery applications (52, 53). Polyethylene glycol is a hydrophilic and biocompatible polymer, and is commonly used to modify nano-sized drug delivery systems to reduce non-specific tissue uptake (53). The presence of thiol groups in the multifunctional lipids allows for facile and in situ modification of siRNA nanoparticles with PEG using simple thiol-maleimide chemistry (Figure 10A) (54). PEG with a maleimido functional group is added to achieve 2.5 mol% of the lipids and thus reacts with a small portion of the lipids for a short period of time (ca. 30 min.). The mixture is then complexed with siRNA to form stable PEGylated siRNA nanoparticles. PEGylation of the EHCO/siRNA nanoparticles significantly altered intracellular uptake and gene silencing, but not the nanoparticle size. The cellular uptake and gene silencing of the PEGylated nanoparticles was evaluated in the Chinese Hamster Ovary (CHO)-d1EGFP cells, which over-expressed bombesin (BN) receptors using a rhodamine-red labeled anti-GFP siRNA (Figure 10B) (54). BN receptors are often overexpressed on the surface of various types of cancers, including ovarian cancer and glioblastoma cells (55). Unmodified EHCO/siRNA nanoparticles were readily internalized


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through non-specific interactions between the cationic nanoparticle and the anionic cell membrane, resulting in a strong intracellular red fluorescent signal and reduced green fluorescence. Modification of the EHCO/siRNA nanoparticles with mPEG (5,000 Da, 2.5 mol% of EHCO) significantly reduced the internalization of the nanoparticles and inhibited the gene silencing, because the PEG layer on the surface prevents the non-specific interaction of nanoparticles with the cells. To promote target-specific siRNA delivery and enhance gene silencing efficiency with the PEGylated siRNA nanoparticles, targeting ligands can be chemically conjugated to the PEG moieties to facilitate receptor-mediated endocytosis (10). A targeting agent can be incorporated at the end of PEG3400-maleimide (3,400 Da) to prepare targeted siRNA nanoparticles. A BN peptide with high affinity towards BN receptors was conjugated to hetero-bifunctional NHSPEG3400-maleimide by reacting with active ester to give BN-PEG-Mal. The peptide moiety was then conjugated to EHCO through the reaction of the maleimide group of BN-PEG-Mal with the thiol groups of EHCO (PEG/EHCO = 2.5 %) for 30 min. Next, the BN-targeted EHCO/siRNA nanoparticles were formed by self-assembly of the EHCO and PEG-EHCO mixture with siRNA for an additional 30 min (Figure 10 A) (54). The BN-targeted EHCO/siRNA nanoparticles were found to significantly enhance cell uptake via receptor-mediated endocytosis and exhibit high gene silencing efficiency in CHO-GFP cells that highly express BN receptors, as evident by the strong intracellular rhodamine signal and a significant reduction of the GFP signal (Figure 10 B).



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Figure 10. A) Surface functionalization and self-assembly of EHCO to form targeted siRNA nanoparticles. B) Confocal images of various EHCO/siRNA formulations with rhodamine redlabeled anti-GFP siRNA to demonstrate (top) GFP silencing efficiency and (bottom) cellular uptake (54).

The multifunctional lipids enable facile surface modification and incorporation of targeting agents to minimize non-specific tissue uptake and to promote target-specific siRNA delivery and gene silencing. Targeted multifunctional lipid-siRNA nanoparticles have the potential for specific siRNA delivery via systemic administration. It is important to note that


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although PEG is commonly used in surface modifications, repeated systemic administrations may induce an immune response to PEG, facilitating rapid clearance of PEGylated nanoparticles (7). A better modification approach is necessary to circumvent this hurdle in future designs of targeted siRNA nanoparticles.

In vivo siRNA delivery with multifunctional pH-sensitive lipids for cancer therapy The effectiveness of the multifunctional lipids in in vivo siRNA delivery and gene silencing has been demonstrated using ECO and EHCO in animal models. Both ECO and EHCO have shown effective in vitro silencing of reporter genes. They have been tested for delivering siRNAs specific to cancer genes for cancer therapy in mice through local and systemic administrations.

ECO/siSAT1

nanoparticles

with

siRNA

(siSAT1)

specific

to

spermidine/spermine-N1-acetyltransferase 1 (SAT1), an enzyme involved in polyamine catabolism, were tested in a subcutaneous U87MG glioblastoma tumor model after single intratumoral injection to sensitize tumors to radiation therapy (56). Silencing of SAT1 with the nanoparticles sensitized the tumor to radiation and significantly attenuated tumor growth in combination with radiation therapy (Figure 11A). The mice treated with a combination of ECO/siSAT1 and radiation had an improved survival rate over radiation alone (Figure 11B). This data suggests that ECO can facilitate localized siRNA delivery, achieve target gene knockdown, and thus sensitize cancer cells to radiation therapy.



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Figure 11. A) Tumor response curves of U87MG tumors following intratumoral delivery of anti-SAT1 (siSAT1) or non-specific (siCTRL) siRNA by ECO and radiation. Briefly, mice with subcutaneous U87MG tumors of 100-200 mm3 were injected intratumorally with 500 pmol/L of siRNA packed in ECO. After 48 hours, the tumors of mice receiving irradiation were irradiated with 8 Gray (Gy). B) Kaplan-Meier survival curves following combination siRNA and radiation therapy (56). In addition, the EHCO/siRNA nanoparticles were administered through intracranial stereotactic injections for the treatment of orthotopic U87-LucNeo xenografts in a mouse model (57). The ability of the EHCO/siRNA nanoparticles to diffuse into the tissue after injection was demonstrated using a Cy3.5-labeled siRNA. Fluorescent histology revealed that the EHCO/siRNA nanoparticles readily permeated throughout the brain tissue, 5 min following the injection. Intratumoral injections of EHCO/siRNA nanoparticles delivering anti-HIF-1α siRNA (designated siHIF-1589 or siHIF-1124) showed significantly decreased tumor growth than the non-specific control groups (siNeg or siGFP) (Figure 12A). PEGylated EHCO/siRNA nanoparticles administered via an osmotic pump for 14 days significantly increased the survival of the cohort treated with PEGylated EHCO/HIF-1α (PEG1589), as compared to the control


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group treated with non-specific PEGylated EHCO/siGFP nanoparticles (PEGGFP) and nonPEGylated EHCO/HIF-1α (1589) (Figure 12B). The PEGylated EHCO/HIF-1α nanoparticles were more effective than EHCO/HIF-1α nanoparticles because the former were still functional for up to 2 weeks during storage in cell culture medium with 10% bovine serum at 37ºC. These results indicate that PEGylation can significantly improve the stability of EHCO/siRNA nanoparticles during storage in solution, possibly by preventing aggregation of the nanoparticles and providing better protection to the siRNA cargo from degradation. Postmortem analysis of the tumor tissues showed that the EHCO/siHIF-1α nanoparticles resulted in robust down-regulation of HIF-1α and its downstream transcriptional targets, including vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT-1), c-MET, and carbonic anhydrase-IX (CA-IX). The in vivo down-regulation of HIF-1α and its downstream cancer-related genes with EHCO/siRNA and PEGylated EHCO/siRNA nanoparticles suppressed tumor growth and prolonged the survival of the tumor-bearing mice.

Figure 12. A) Normalized average radiance denoting the tumor growth in mice injected intracranially with EHCO formulated with various siRNAs. B) Survival curves of mice treated



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with Alzet osmotic pumps delivering various formulations of EHCO/siRNA and PEGylated EHCO/siRNA nanoparticles 58.

Targeted multifunctional lipid-siRNA nanoparticles modified with peptides via a PEG spacer are also effective in silencing cancer-related genes for cancer therapy via systemic administration. The EHCO/siRNA nanoparticles were modified with BN and cyclic RGD peptides with a PEG (3,700 Da) spacer in a similar manner as described in Figure 10A, and were used to deliver anti-HIF-1α siRNA (2.5 mg/kg) to regulate tumor hypoxia in nude mice bearing human U87 glioblastoma xenografts (58). RGD is commonly exploited as a targeting peptide due to its high affinity and specificity towards αvβ3 integrin overexpressed in angiogenic blood vessels and endothelial cancer cells. Intravenous injections of both BN- and RGD-targeted EHCO/siRNA nanoparticles (RGD and BN) significantly suppressed tumor growth in mice, when compared to free siRNA (siRNA only) and non-targeted PEGylated (mPEG) control groups (Figure 13A and B). PEI/siRNA complexes were also used in a control group but the treated mice died immediately following intravenous injection due to the high systemic toxicity of PEI. These results demonstrate the potential of the peptide-targeted EHCO/siRNA nanoparticles for systemic therapy against cancer.


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Figure 13. A) Normalized tumor growth curve and B) normalized tumor volume of mice treated intravenously with various formulations of targeted (RGD and BN) and non-targeted (mPEG) EHCO/anti-HIF-1α siRNA nanoparticles (58).

Recently, RGD-targeted ECO/siRNA nanoparticles have also been used to silence β3integrin for the treatment of metastatic triple-negative breast cancer (59). Down-regulation of β3integrin has been demonstrated to inhibit epithelial-mesenchymal transition (EMT), the seminal event of metastasis by which epithelial cancer cells acquire the ability to invade and disseminate to secondary sites. In vitro silencing of β3 integrin with ECO/siRNA nanoparticles was sustained for least 7 days following a single transfection in two breast cancer cell lines (NME and MDAMB-231). The effectiveness of RGD-targeted ECO/siRNA nanoparticles (RGD-ECO/siβ3) was demonstrated after systemic administration in two mouse metastatic breast cancer models. In the first, female nude mice were inoculated in the lateral tail vein with post-EMT NME cells to induce metastatic tumor outgrowth within the pulmonary environment. Systemic administration of RGD-targeted ECO/siβ3 nanoparticles significantly inhibited pulmonary outgrowth of the metastatic breast cancer cells, compared to the non-targeted and non-specific control groups (Figure 14A). In the second model, post-EMT MDA-MB-231 cells were engrafted in the



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mammary fat pads of female nude mice. Treatment with RGD-ECO/siβ3 nanoparticles significantly reduced primary tumor burden, as compared to the control groups (Figure 14B), and inhibited the expression of β3-integrin, as shown after primary tumor resection (Figure 14C). Following the resection, further treatment with RGD-ECO/siβ3 nanoparticles resulted in robust inhibition of both tumor metastases and recurrence of the primary tumor. Importantly, after release from the nanoparticle treatment, mice treated with RGD-ECO/siβ3 nanoparticles remained tumor-free while the tumor burden of mice in the control groups continued to increase (Figure 14D).

Figure 14. A) Pulmonary outgrowth of metastatic NME cells treated with RGD-ECO/siNS, RGD-ECO/siβ3, or RAD-ECO/siβ3 nanoparticles. B) Growth of primary MDA-MB-231 tumors measured by digital calipers. C) Semi-quantitative real-time quantification of β3 integrin mRNA


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from resected primary MDA-MB-231 tumors. D) Representative bioluminescent images of mice following their release from the nanoparticle therapy (59).

The multifunctional lipids have shown several advantages for use in in vivo siRNA delivery, including simple and reproducible formulation of siRNA nanoparticles, no need of helper lipids, convenient surface modification of the nanoparticles, highly efficient cytosolic siRNA delivery, high intracellular gene silencing efficiency, and effective in vivo gene silencing via systemic administration. The capability of the targeted multifunctional lipid/siRNA nanoparticles to allow long-term repeated intravenous injections demonstrates the superior safety profile of the lipids. Safe and efficient systemic siRNA delivery using these multifunctional lipids may open up avenues in creating new therapies for cancer and other life-threatening diseases that cannot be treated with conventional therapies. These multifunctional lipids also have the potential to treat viral infections, e.g., Ebola infections. Besides siRNA delivery, the multifunctional lipids can also be used for the delivery of other therapeutic RNA species, including miRNA and its mimics.

2.6 Concluding Remarks Significant progress has been made in the field of pH-sensitive amino lipids for siRNA delivery. Compared to other siRNA delivery systems, including cationic polymers and inorganic nanoparticles, amino lipid-siRNA nanoparticles represent advanced delivery systems for human use. Several siRNA nanoparticles with amino lipids have progressed to clinical trials. The multifunctional pH-sensitive lipids have been designed to overcome the hurdles in the systemic



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delivery of therapeutic RNA into target tissues and cells. The structure and property relationship of the multifunctional pH-sensitive lipids have been systematically investigated by varying the composition and structure of the lipids. The knowledge obtained with these multifunctional pHsensitive lipids may provide a useful guide for design and development of more efficient and safer siRNA delivery systems. From our experience, the most important parameters for the development of multifunctional pH sensitive lipids are the structure of the amino head groups, careful tuning of the p of the head group for pH-sensitive cell membrane destabilization and endosomal escape, distant location of unsaturated lipid tails to avoid lipid bilayer formation, disulfide crosslinks to stabilize siRNA nanoparticles during systemic delivery and to facilitate siRNA release in cytosol, and facile surface modification to minimize non-specific tissue uptake and specific siRNA delivery. Several lipids with high siRNA delivery efficiency have been identified as lead carriers for effective systemic siRNA delivery. Comprehensive preclinical assessments are needed to further demonstrate the safety and efficacy of the multifunctional lipids for clinical translational development. The multifunctional pH-sensitive lipids are promising for targeted systemic delivery of therapeutic siRNA in treating human diseases with RNAi.

Acknowledgement Research support was provided, in part, by grants R01 EB00489 and R01 CA194518 from the National Institutes of Health.


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(16)

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Multifunctional pH-Sensitive Amino Lipids for siRNA Delivery

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