Side Chains for Enhanced siRNA Delivery - American Chemical Society

Chapter 11

Downloaded by UNIV OF NORTH CAROLINA on July 20, 2013 | http://pubs.acs.org Publication Date (Web): July 8, 2013 | doi: 10.1021/bk-2013-1135.ch011

Fine-Tuning of Repeating Aminoethyelene Units in Poly(aspartamide) Side Chains for Enhanced siRNA Delivery Kanjiro Miyata,1 R. James Christie,1 Tomoya Suma,2 Hiroyasu Takemoto,3 Hirokuni Uchida,3 Nobuhiro Nishiyama,1,4 and Kazunori Kataoka*,1,2,3 1Center

for Disease Biology and Integrative Medicine, Graduate School of Medicine; Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunokyo-ku, Tokyo 113-8656, Japan 2Department of Bioengineering and Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunokyo-ku, Tokyo 113-8656, Japan 3Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunokyo-ku, Tokyo 113-8656, Japan 4Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology, R1-11, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan *E-mail: [email protected].

This chapter reviews our strategy for optimization of polycationic structures to improve siRNA delivery and reduce carrier toxicitiy. Improvement in carrier design is realized by modulating the number of repeating aminoethylene (RA) units in the side chain of a poly(aspartamide) derivative (PAsp(R)). Results reveal that the number of RA units in the PAsp(R) side chains affects their protonation behavior between extracellular and endosomal pHs in a distinctive “odd-even” manner. PAsp(R)s with even-numbered RA units induce acidic pH-responsive membrane disruption based on substantially increased protonation at the endosomal pH of 5.5. Ultimately, PAsp(R) with 4 RA units in each side chain demonstrated excellent transfection of siRNA due to accelerated endosomal escape as well as stable siRNA complexation.

© 2013 American Chemical Society In Tailored Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Introduction Over the past two decades, a variety of polycationic materials have been developed to form polyion complexes (PICs) with oppositely charged nucleic acids, such as plasmid DNA (pDNA) and small interfering RNA (siRNA), for delivery into cells. Those efforts have revealed that PIC transfection efficiency is dramatically improved by specific polycationic structures (1–4). Indeed, PICs with poly(ethyleneimine) (PEI) or its derivatives demonstrate high transfection efficiency because of their ability to translocate from the endosome to the cytoplasm, triggered by protonation of their low pKa amines at the reduced endosomal pH (pH ~5.5) (5, 6). In this regard, two possible mechanisms have been proposed to explain the endosomal escape behavior of such polycations. One is the “proton sponge effect”, where endosome disruption occurs by increased osmotic pressure due to an influx of protons and chrolide ions into the endosomal compartment along with amine protonation (5, 6). The other is disruption of endosomal membrane integrity by the binding of polycations to the negatively charged cellular membrane. Thus, polycations with increased cationic charges at the acidic endosomal pH can exert a stronger membrane disruptive effect (4, 6). Despite efficient transfection in cultured cells, the considerable cytotoxicity of PEI has hampered its translation into a pharmaceutical agent, and thus, an optimized polycationic structure that allows more efficient transfection with lower cytotoxicity is needed. In the present study, we demonstrate a systematic strategy for improved endosomal escape of siRNA with reduced carrier-associated toxicity. One of the major causes for the cytotoxicity of PEI is believed to be caused by cytoplasmic membrane disruption even at neutral pH, due to strong positive charges of PEI (7, 8). Thus, it is assumed that less toxic endosomal escape should be realized by a polycation featuring a low degree of protonation (α) (or low density of cationic charges) at neutral pH but greatly increased α at acidic pH. Selective membrane disruption due to a larger change in the α between extracellular pH of 7.4 and endosomal pH of 5.5 (Δα) is targeted in the design of an endosome-escaping polycation. With regard to cationic structures with larger Δα values, we herein focus on the repeating aminoethylene (RA) unit (-NHCH2CH2-), which is the main repeating unit found in PEI. When Δα values of a series of RA compounds are plotted against the number of repeating units in comparison with linear PEI (degree of polymerization (DP) of 520), a distinctive odd-even effect is found as shown in Figure 1, in which the Δα values converge with that of PEI in an up-and-down motion (4, 9). Importantly, it is also found that “smaller” and “even”-numbered RA compounds possess a substantially larger Δα value. To apply the above finding regarding larger Δα values to PIC-based siRNA delivery, we introduce the smaller and even numbered RA units, i.e. 2 and 4, into the side chains of a poly(aspartamide) derivative (PAsp(R)) by an aminolysis reaction of poly(β-benzyl L-aspartate) (PBLA) with diethylenetriamine (DET) and tetraethylenepentamine (TEP), respectively (11). Concurrently, control PAsp(R)s comprising an odd number of RA unit(s), i.e. 1 and 3, in the side chain are 190 In Tailored Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


prepared using the same aminolysis reaction with ethylenediamine (EDA) and triethylenetetramine (TET), respectively (11). It is important to note that this aminolysis reaction provides a series of PAsp(R)s with the same DP and molecular weight distribution, as they are prepared from the same parent PBLA stock, which is necessary to accurately compare the effect of varying the RA units in polymer side chains. In the following sections, we describe the correlation between the Δα value of each PAsp(R) and its membrane disruptive effect, and then optimize the number of RA units for successful siRNA delivery with less toxic endosomal escape.

Figure 1. Δα values of RA compounds plotted against the number of repeating aminoethylene units. The Δα values are calculated based on the Henderson-Hasselbalch equation with the pKa values of each RA compound as well as linear PEI with a DP of 520 (4, 9, 10).

Results and Discussion PBLA with a DP of 92 and a Mw/Mn of 1.02 was used for the aminolysis reaction with EDA, DET, TET, or TEP to obtain the corresponding PAsp(R)s (termed PAsp(EDA), PAsp(DET), PAsp(TET), and PAsp(TEP), respectively) (Figure 2) (11, 12). The prepared PAsp(R)s were subjected to a potentiometric titration at physiological ionic strength and temperature (150 mM NaCl and 37 °C) to determine their α values at pH 7.4 and 5.5 and their corresponding Δα values. As summarized in Table I, the obtained Δα values clearly exhibit an odd-even effect in the number of RA units in polymer side chains; PAsp(R)s bearing even-numbered RA units, i.e. PAsp(DET) and PAsp(TEP), showed a significantly larger Δα than those bearing odd-numbered RA units, i.e. PAsp(EDA) and PAsp(TET), as well as PEI. Thus, it is demonstrated that the unique protonation behavior of low molecular weight RA compounds (Figure 1) is maintained in the 191 In Tailored Polymer Architectures for Pharmaceutical and Biomedical Applications; Scholz, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


side chain of PAsp(R) (Table I). Furthermore, the major protonated structures in each PAsp(R) side chain are estimated from the α values at pH 7.4 and 5.5. Corresponding to their large Δα values, PAsp(DET) and PAsp(TEP) distinctly alter their protonation state between pH 7.4 and 5.5 (Table I). It should be noted that the diprotonated diaminoethane structure (-NH2+CH2CH2NH3+) is adopted in PAsp(DET) and PAsp(TEP) at pH 5.5, providing a drastically increased cationic charge density.

Figure 2. Aminolysis reaction of PBLA with amine compounds and chemical structures of poly(aspartamide) derivatives bearing repeating aminoethylene units in the side chain.

Next, the membrane disruptive effect of each PAsp(R) at pH 7.4 and 5.5 was quantitatively evaluated by a hemolysis assay with murine erythrocytes. In this assay, the amount of hemoglobin liberated from erythrocytes following incubation with PAsp(R) indicates the degree of membrane disruption activity (Figure 3) (8, 11). At the extracellular pH of 7.4, all the PAsp(R)s show a low level of hemolysis activity (

Side Chains for Enhanced siRNA Delivery - American Chemical Society

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