Bioorganic & Medicinal Chemistry Letters 23 (2013) 5919–5922

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Chiral cationic polyamines for chiral microcapsules and siRNAdelivery

0960-894X/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.08.083

ã Corresponding author. Tel.: +1 401 874 4243.E-mail address: mlevine@chm.uri.edu (M. Levine).

Scheme 1. General synthetic methods for linear and branched polyethyle(PEI).

Justin Gharavi a, Patrick Marks a, Kelly Moran a, Brett Kingsborough a, Ruchi Verma b, Yuan Chen b,Ruitang Deng b, Mindy Levine a,a

a Department of Chemistry, University of Rhode Island, 51 Lower College Road, Kingston, RI 02881, United Statesb College of Pharmacy, University of Rhode Island, 7 Greenhouse Road, Kingston, RI 02881, United States

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 June 2013Revised 16 August 2013Accepted 19 August 2013Available online 26 August 2013

Keywords:PolyamineChiralitysiRNATransfection

Reported herein is the use of chiral cationic polyamines for two intriguing applications: fabrication of chi-ral covalently-linked microcapsules, and enantiospecific delivery of siRNA to Huh 7 cells. The microcap-sules are easily fabricated from homochiral polymers, and the resulting architectures can be used forsupramolecular chiral catalysis and many other potential applications. Enantiospecific delivery of siRNAto Huh 7 cells is seen by one ã enantiomerã of the polymers delivering siRNA with significantly improvedtransfection efficiency and reduced toxicity compared to the ã enantiomericã polymer and commerciallyavailable transfection reagents. Taken together, the use of these easily accessible polyamine structures fordiverse applications is highlighted in this Letter herein and can lead to numerous future research efforts.

� 2013 Elsevier Ltd. All rights reserved.

Polyethyleneimines (PEIs) are a well-studied class of poly-mers.1,2 These polymers are synthesized commercially via the ringopening of aziridine (Scheme 1, Reaction 2),3ã 5 although this pro-cess leads to highly branched polymers6 with significant polydis-persity indexes. The controlled synthesis of linear PEIs occurs viathe cationic ring opening of oxazolines,7ã 9 followed by hydrolysisof the resulting formamides (Scheme 1, Reaction 1). Using chiraloxazolines as substrates for the polymerization reaction providesstraightforward access to homochiral PEIs,10ã 12 with chiral centersat every polymer repeat unit.

The significant interest in PEIs is driven largely by various appli-cations of PEIs in fields including chiral catalysis,13ã 16 drug deliv-ery,17,18 and oligonucleotide complexation and delivery.19,20 PEIshave also been covalently linked to form PEI-derived microcap-sules,21 which have been used for site-isolated catalysis.22,23 Inone example, the Lewis basic PEI catalyzed a reaction in the samereaction vessel as a Lewis acidic nickel catalyst, which was used tocatalyze the second reaction.24,25

Use of the same PEI scaffold for multiple applications has rarelybeen reported, although such multi-purpose polymers would havesignificant operational advantages. Reported herein is the use of asingle PEI scaffold for two purposes: the fabrication of covalently-linked chiral microcapsules, and the efficient delivery of siRNA toHuh7 cells.26

The chiral PEIs were synthesized via the cationic polymerizationof 4-benzyl-2-oxazoline (1a) (both R and S configurations), fol-lowed by the hydrolysis of the initially formed polyformamide(Scheme 2). The resulting polymers were characterized by 1HNMR spectroscopy, and the results were in agreement with litera-ture-reported spectra.11 Using this methodology, polymers with 13and 30 repeat units were formed, with both R and S configured sidechains.

Once synthesized, the homochiral PEIs were cross-linked toform homochiral microcapsules following the procedure devel-oped by McQuade and co-workers.22 Briefly, polymers 2a were dis-solved in methanol, and added to a solution of 2% Span 85,

neimine

Scheme 2. Synthesis of chiral polymers 2a.

5920 J. Gharavi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5919–5922

followed by the addition of 2,4-tolylene diisocyanate (TDI, com-pound 7) (Equation 1), which cross linked the microcapsules toform a polyurea coating.25 The resulting polyurethane-type struc-tures have been shown to be stable in a variety of aqueousmedia.27,28 After thorough solvent evaporation, chiral microcap-sules were obtained.

The resulting microcapsules were imaged using transmissionelectron microscopy (TEM), and some images are shown in Figure1. The diameters of the particles ranged from 57 to 250 nm, with anaverage diameter of 141 nm (ô35 nm; 62 particles measured).These new supramolecular architectures contain narrow size dis-tributions and uniform structures, in good agreement with litera-ture-reported results for achiral microcapsule analogs.21

The newly formed microcapsules contain a variety of featuresthat make them particularly amenable to supramolecular chiralcatalysis, including: (a) multiple chiral centers, covalently confinedin a small space; (b) multiple amino groups that can be protonatedor deprotonated over a wide pH range;29 and (c) a hydrophobiccore resulting from the hydrophobic benzyl side chains.30

To investigate the effect of capsule formation on the resultingsupramolecular chiral environment, the newly synthesized chiralmicrocapsules were used as catalysts for the transamination reac-tion of ketoacids to amino acids (Equation 2). Obtaining good

Figure 1. TEM images of chiral microcapsules

enantioselectivities in such transamination reactions has been anongoing research problem.10 Preliminary results indicate that themicrocapsule-catalyzed reactions proceeded with significantlyhigher enantioselectivities compared to the polymer-catalyzedreactions (up to 20% enantiomeric excess (ee) obtained for the syn-thesis of L-valine, under conditions where the polymer itselfyielded 4% ee).

Efforts to optimize the reaction conditions are in progress.Interestingly, the chiral PEIs also functioned as efficient siRNA

delivery agents. Although there are many reported examples ofPEIs used for siRNA and DNA delivery,31ã 33 many of these deliveryvehicles suffer from high cytotoxicity.34 The development of genedelivery agents that are both effective and less toxic remains ahighly relevant research objective.

The following 4 polymers were investigated as potential siRNAdelivery agents: R-2a-13; S-2a-13; R-6-13; and S-6-13, where theR/S designation refers to the chirality of the side chain and thenumber 13 refers to the number of repeat units in the polymers.The efficacy of these polymers in transfecting an Alexa488-labeledcontrol siRNA sequence35 to Huh7 cells36 was measured by deter-mining the intracellular fluorescence 24 h post-transfection. Theresults obtained using the chiral polyamines were compared to re-sults obtained using commercially available transfection reagents:Genjet siRNA Transfection Reagent (SignaGen Laboratories); HiPer-Fect Transfection Reagent (Qiagen Laboratories);37 and Lipofect-amine 2000 (Invitrogen Technologies).38

Figure 2 shows a graph of the intracellular fluorescence of Huh7cells following their incubation with Alexa-labeled siRNA with var-ious delivery reagents. The intracellular fluorescence obtained withcompounds S-6-13 and S-2a-13 is substantially higher than thefluorescence observed with positive controls Lipofectamine andGenjet, indicating the polymersã ability to transfect siRNA effi-ciently (Table 1). More interestingly, compounds R-2a-13 and R-6-13, which are identical except for the three-dimensional

8 (Blue line represents a 500 nm scale).

Figure 2. Chart of the intracellular fluorescence of Huh7 cells after transfectionwith siRNA (all PEIs were used at a 1000 nm final concentration).

Table 1Transfection efficiencies of chiral PEIs and commercial transfection agents

Transfection agent Intracellular fluorescence (normalizedto 1.00 for cells alone)

S-6-13 1.26S-2a-13 1.30Lipofectamine 1.05Genjet 1.04R-2a-13 1.06R-6-13 1.05

J. Gharavi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5919–5922 5921

configuration of the benzyl group, transfect siRNA with approxi-mately the same efficiency as Lipofectamine and Genjet, and sub-stantially lower than the ã enantiomericã polymers.

The chirality of the side chains of the PEIs thus has a direct andmeasurable effect on the ability of PEIs to transfect siRNA effi-ciently: S chiral centers (compounds S-6-13 and S-2a-13) transfectsiRNA more efficiently than the R analogs. Such a result may seemintuitive: that the interaction of two chiral macromolecules (chiralPEI and chiral siRNA) depends on the three-dimensional configura-tion of both molecules. This intuition is borne out by the results ofthis study, which is the first direct proof that the chirality of a poly-amine directly impacts its transfection efficiency. Similar effects ofthe chirality on transfection efficiency were recently observed forthe lipid delivery agent 1,2-dioleoyl-3-trimethylammonium-pro-pane (DOTAP).39 In that report, the R enantiomer performed betterthan either the S enantiomer or the racemic DOTAP mixture.

The toxicity of the newly synthesized PEIs was tested using anMTT assay.40 After 24 h of incubation, the absorbance of the cellswas quantified and the cell viability was calculated. Using1000 nM of S-2a-13 reduced the cell viability to 88%, and1000 nM of S-6-13 reduced it to 82%. By comparison, Lipofect-amine reduced cell viability to 89%, and compounds R-2a-13 andR-6-13 reduced viability to 78% and 71%. Thus, the toxicity of thechiral PEIs, like the transfection efficiency, depends on the three-dimensional configuration of the benzylic side chains.

The differences in transfection efficiency and toxicity mean thatthe S- and R-configured PEIs likely have fundamentally differentthree-dimensional architectures. The relationship between the chi-rality of individual stereocenters and the overall polymer configu-ration has been studied for related polymers using circular

dichroism spectroscopy.41ã 43 These differences in chirality affectthe polymersã solubility44 and their interactions with DNA,45

and as shown here, their transfection efficiencies.In summary, chiral polymers 6 and 2a were synthesized via

straightforward, well-precedented procedures. These polymerswere used for two novel applications: the fabrication of chiral,covalently-linked microcapsules, and the transfection of siRNA toHuh7 cells. The chiral microcapsules can be used for a number ofpotential applications in supramolecular chiral catalysis and insupramolecular enantiomer separations. The chirality-dependentsiRNA transfection also provides an intriguing platform for furtherinvestigation. In particular, polymer S-2a-13 demonstrated goodtransfection efficiency and limited toxicity, and will be used forfurther biochemical investigations. The results of these and otherexperiments will be reported in due course.

Acknowledgments

This work was supported by an Institutional DevelopmentAward (IDeA) from the National Institute of General Medical Sci-ences of the National Institutes of Health under grant number 8P20 GM103430-12 to M.L. R.D. is supported by the National Insti-tutes of Health (NIH) (Grant No. R01DK087755).

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2013.08.083.

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