Pendant polymer:Amino-β-cyclodextrin:siRNA guest:Host nanoparticles as efficient vectors for gene silencing

Article abstract of DOI:10.1021/ja300690j

A novel siRNA delivery vector has been developed, based on the self-assembly of monosubstituted cationic β-CD derivatives with a poly(vinyl alcohol)MW27kD (PVA) main-chain polymer bearing poly(ethylene glycol)MW2000 (PEG) and acid-labile cholesterol-modified (Chol) grafts through an acid-sensitive benzylidene acetal linkage. These components were investigated for their ability to form nanoparticles with siRNA using two different assembly schemes, involving either precomplexation of the pendant Chol-PVA-PEG polymer with the cationic β-CD derivatives before siRNA condensation or siRNA condensation with the cationic β-CD derivatives prior to addition of Chol-PVA-PEG to engage host:guest complexation. The pendant polymer:amino- β-CD:siRNA complexes were shown to form nanoparticles in the size range of 120-170 nm, with a slightly negative zeta potential. Cell viability studies in CHO-GFP cells shows that these materials have 10³-fold lower cytotoxicities than 25 kD bPEI, while maintaining gene-silencing efficiencies that are comparable to those of benchmark transfection reagents such as bPEI and Lipofectamine 2000. These results suggest that the degradable Chol-PVA-PEG polymer is able to self-assemble in the presence of siRNA and cationic-β-CD to form nanoparticles that are an effective and low-toxicity vehicle for delivering siRNA cargo to target cells.

Full text of DOI:10.1021/ja300690j

Communication
pubs.acs.org/JACS
Pendant Polymer:Amino-β-Cyclodextrin:siRNA Guest:Host
Nanoparticles as Efficient Vectors for Gene Silencing
Aditya Kulkarni, Kyle DeFrees, Seok-Hee Hyun, and David H. Thompson*
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States
S
* Supporting Information
inorganic nanoparticles have been studied for this purpose.7,8
A variety of cationic polymers also have been investigated as
nonviral vectors, including polyethylenimines (PEI),9 poly-(L-
lysine),10 PAMAM dendrimers,11,12 poly(lactic-co-glycolic acid)
(PLGA),13,14 chitosan,15,16 PEI-alginate nanoparticles,17 and
cyclodextrin (CD) oligomers.6,18−22 All these polymer vectors
are capable of condensing siRNA to form positively charged
particles that enter cells via nonspecific uptake mechanisms;
however, most of these materials either display significant
cytotoxicity at the concentrations needed for effecting nucleic
acid cargo bioactivity or suffer from poor efficiency due to
insufficient endosomal escape.
β-CD has well-known host−guest interactions with a vast
array of compounds with binding constants in the 100.5−105
M−1 range in aqueous media.26 This property has led to a wide
variety of biomedical applications ranging from drug solubiliza-
tion to their use as a building block for nonviral vector
construction. Davis and co-workers reported a class of CD
oligomers6,23−26 as vectors for delivery of siRNA in a clinical
trial for melanoma therapy with encouraging results. The fixed
cationic groups on the relatively rigid oligomeric backbone,
however, may be responsible for the high N:P ratios required
for nucleic acid compaction and delivery in this case.
The mechanism of nucleic acid complex disassembly and
escape from the endosome of cells that have internalized them
is still unclear in the case of most nonviral vector systems. A
variety of ion-exchange,27 endosomolytic, and degradative
processes28 have been proposed; however, the diversity of
proposals is likely a reflection of the multiple internalization
pathways29 and vast array of nucleic acid nanoparticle
formulations employed. Zhang et al. have shown that the
presence of cholesterol-conjugated lipids induces conversion of
membrane lipids from the Lα to the HII phase, thereby causing
disruption of the endosomal membrane.31 Since the presence of
amino-cholesterol derivatives may promote disruption of
biological membranes under endosomal pH conditions to
facilitate intracellular siRNA delivery, we designed a delivery
vector for siRNA based on the self-assembly of cationic β-CD
derivatives with a pendant polymer32 comprised of cholesterol-
modified (Chol) poly(ethylene glycol)-poly(vinyl alcohol)
(PEG-PVA), whose Chol units are linked through an acid-
sensitive acetal motif (Figure 1). It was anticipated that siRNA
compaction could be achieved via complexation with self-
assembled Chol-PVA-PEG:amino-β-CD guest:host pendant
polymer complexes via multivalent electrostatic interactions.
ABSTRACT: A novel siRNA delivery vector has been
developed, based on the self-assembly of monosubstituted
cationic β-CD derivatives with a poly(vinyl alcohol)-
MW27kD (PVA) main-chain polymer bearing poly-
(ethylene glycol)MW2000 (PEG) and acid-labile choles-
terol-modified (Chol) grafts through an acid-sensitive
benzylidene acetal linkage. These components were
investigated for their ability to form nanoparticles with
siRNA using two different assembly schemes, involving
either precomplexation of the pendant Chol-PVA-PEG
polymer with the cationic β-CD derivatives before siRNA
condensation or siRNA condensation with the cationic β-
CD derivatives prior to addition of Chol-PVA-PEG to
engage host:guest complexation. The pendant polymer:-
amino-β-CD:siRNA complexes were shown to form
nanoparticles in the size range of 120−170 nm, with a
slightly negative zeta potential. Cell viability studies in
CHO-GFP cells shows that these materials have 103-fold
lower cytotoxicities than 25 kD bPEI, while maintaining
gene-silencing efficiencies that are comparable to those of
benchmark transfection reagents such as bPEI and
Lipofectamine 2000. These results suggest that the
degradable Chol-PVA-PEG polymer is able to self-
assemble in the presence of siRNA and cationic-β-CD to
form nanoparticles that are an effective and low-toxicity
vehicle for delivering siRNA cargo to target cells.
NA interference (RNAi) is a post-transcriptional gene-
Rsilencing mechanism arising from degradation or trans-
lation arrest of target RNA. The ability of 21−23 nucleotide
RNAs (siRNA) to mediate RNAi in mammalian cells has
enormous therapeutic potential for the treatment of viral
infections, cancer, and neurological disorders.1 The use of
siRNA has several advantages over conventional chemotherapy
in that the high specificity nucleic acid drug acts “upstream”
from most conventional chemotherapeutic agents, conferring
the ability to target any protein and the capacity to potentially
evade drug resistance.2 The safe and efficient delivery of siRNA
specifically to target cells, however, remains a major
challenge.3−6 A variety of viral and nonviral vectors have
been developed for this purpose. Although viral vectors have
shown promise, they suffer from scalability, immunogenicity,
and safety issues. Nonviral vectors have attracted considerable
attention due to their modest host immunogenicity and
manufacturability. Many nonviral vectors such as cationic
liposomes, Lipofectamine 2000 (L2k), polypeptides, and
Received: February 5, 2012
Published: April 30, 2012
© 2012 American Chemical Society
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mV). Among the CD variants, particles formulated from 1 had
the lowest observed ζ, followed by 2 and 3, respectively. The
absence of a positive charge on the surface could be due to the
high loading of PEG on the polymer backbone, which is able to
effectively shield the positive charges arising from the cationic
CDs. These results are encouraging since a positive surface
charge is considered to be one of the major reasons for
nanoparticle opsonization or macrophage uptake.34 Gabizon
and Papahadjopoulos have previously shown that liposomes
with a slight negative charge have prolonged circulation times
and enhanced tumor uptake due to RES evasion.35
Dynamic light scattering (DLS) showed that the complex
sizes produced by these different materials and methods were in
the 120−170 nm range, with higher N/P ratios producing
smaller particles (SI). In general, the method of formulation did
not significantly affect the size of the particles. Compound 3
was able to generate smaller particles than 2, which formed
particles smaller than 1, suggesting that an increase in CD
charge leads to smaller particle formation. DLS measurements
as a function of pH revealed that the particles were stable at pH
7.4 for up to 24 h; however, at pH 5.5, the polydispersity of the
particles started increasing after about 4 h, yielding multiple
particle sizes by 48 h. We attribute these observations to
pendant group hydrolysis at low pH, leading to destabilization
of the Chol-PVA-PEG:amino-β-CD:siRNA complexes (SI).
AFM images of Chol-PVA-PEG:amino-β-CD samples
revealed the presence of particles (Figure 2A) of average
Figure 1. Structures of amino-β-CDs 1−3 (left) and Chol-PVA-PEG
(right).
This approach enables the compaction of the siRNA cargo into
stable nanometer-size particles that can then be internalized by
target cells into acidic endosomes. Endosomal degradation of
the polymer acetal linkage should promote release of the
cholesterol pendant groups and decondensation of the cationic
CDs and siRNA cargo (Scheme 1), thereby facilitating
Scheme 1. Conceptual Diagram of Chol-PVA-PEG:Amino-β-
CD:siRNA Complexation and Endosomal Escape
endosomal escape of the cargo. Three cationic β-CD derivatives
(Figure 1) were synthesized to test this concept: mono-6-
(amino)-6-deoxy-β-cyclodextrin (1), mono-6-(N,N′-dimethyl-
ethane-1,2-diamine)-6-deoxy-β-cyclodextrin (2), and mono-6-
(N′-(2-aminoethyl)ethane-1,2-diamine)-6-deoxy-β-cyclodextrin
(3). PVA (27 kD) was used to prepare Chol-PVA-PEG with
13.2 mol% Chol acetal modifications and 26.9 mol% PEG
Figure 2. AFM images of (A) Chol-PVA-PEG:3 and (B) Chol-PVA-
PEG:3:siRNA at N/P = 10 (inset showing high resolution image).
Scale bar = 100 nm.
diameters 33
6 nm and heights of 1.5
0.6 nm. Upon
1
carbamate modifications based on H NMR analysis (Support-
ing Information (SI)).
addition of siRNA at N/P = 10, larger particles were formed
that were of an average diameter of 51 8 nm and height of 5
1.7 nm (Figure 2B) (SI). The low heights may be due to
deformation of the particles during the sample preparation for
AFM. The sizes determined by AFM are smaller than those
measured by DLS due to the dry nature of the AFM samples
(i.e., polymer solvent swelling is absent). These results support
the conclusion that supramolecular complexation of Chol-PVA-
PEG with amino-β-CD produces a non-covalent assembly that
is capable of condensing siRNA into compact and unimodal
particles.
The in vitro cytotoxicity of amino-β-CDs, Chol-PVA-PEG,
and their host:guest complexes are an extremely important
factor for their consideration as a safe nonviral vector. Figure 3
shows that Chol-PVA-PEG, all of the amino-β-CDs, and the
Chol-PVA-PEG:1 host:guest complex were nearly at least 3
orders of magnitude less cytotoxic than bPEI (i.e., the LD50’s
of bPEI, Chol-PVA-PEG, and 1:1 Chol-PVA-PEG:1 were 0.01,
The ability of these non-covalent pendant polymer
assemblies to condense siRNA was then evaluated. Two
different complexation methods (Scheme 1) were used to
evaluate the relative capacity of Chol-PVA-PEG:amino-β-CDs
guest:host polymer assemblies toward siRNA condensation. In
method A, Chol-PVA-PEG was pre-associated with amino-β-
CDs before addition to the siRNA solution. In method B, the
siRNA was first complexed with amino-β-CDs, followed by
addition of Chol-PVA-PEG. Zeta potentials were measured for
both types of complexes to determine the surface charge of the
resulting transfection particles (SI). We observed that
complexes formed by both methods had slightly negative zeta
potentials (ζ < −8 mV). As the N/P ratio increases from 10 to
20, the ζ-potential approaches neutrality. Method B (ζ = −16
to −12 mV) particles were shown to be more negatively
charged than those produced by method A (ζ = −10 to −8
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Journal of the American Chemical Society
Communication
attribute this enhancement to the effect that Chol has on
membrane phase behavior such that endosomal escape is
promoted by the pendant Chol group.
In conclusion, a novel and efficient siRNA delivery system
has been developed based on the self-assembly of cationic CD
derivatives with cholesterol-modified PEG-PVA. PVA, linked to
Chol via a pH-sensitive acetal linkage, provides a scaffold for
binding of cationic CD amines that are capable of condensing
siRNA into nanoparticles less than 200 nm in size. These
complexes are capable of achieving gene knockdown
efficiencies in the same range as 25 kDa bPEI, L2k, while
being 3−4 orders of magnitude less toxic.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures; synthesis and characterization of
amino-β-CDs and Chol-PVA-PEG; DLS; zeta potential data;
and FACS raw data. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
Figure 3. Cell viabilities of 1−3, Chol-PVA-PEG, and Chol-PVA-PEG
+1 host:guest pendant polymer complexes in CHO-GFP cells using
25kD bPEI as control. The cells were treated with increasing amine
concentrations of amino-β-CDs, Chol-PVA-PEG, Chol-PVA-PEG+1,
and bPEI for 24 h in serum-free media before analysis by MTS assay.
S
9.5, and 7.9 mM, respectively, while those of 1−3 were all >10
mM and had negligible effect on the cell viability).
AUTHOR INFORMATION
Corresponding Author
davethom@purdue.edu
■
The in vitro gene knockdown efficiency of the complexes
formed between the anti-GFP siRNA and the Chol-PVA-
PEG:amino-β-CD guest:host pendant polymer system was
assessed in CHO-GFP cells at N/P = 20 in the presence of
serum relative to control vectors (bPEI and L2k) and negative
control siRNA (Figure 4). Method A and B complexes both
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We express our special thanks for the support of this work by
the Purdue Department of Chemistry. We also thank Prof. Z.
R. Lu from Case Western University for kindly providing us
with the CHO-GFP cells.
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