Combinatorial synthesis of chemically diverse core-shell
nanoparticles for intracellular delivery
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Citation
Siegwart, D. J. et al. “Combinatorial Synthesis of Chemically
Diverse Core-shell Nanoparticles for Intracellular Delivery.”
Proceedings of the National Academy of Sciences 108.32
(2011): 12996–13001. Web.
As Published
http://dx.doi.org/10.1073/pnas.1106379108
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National Academy of Sciences (U.S.)
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Final published version
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Thu May 26 20:55:39 EDT 2016
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http://hdl.handle.net/1721.1/70069
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Detailed Terms
Combinatorial synthesis of chemically diverse
core-shell nanoparticles for intracellular delivery
Daniel J. Siegwarta,b, Kathryn A. Whiteheada,b, Lutz Nuhna, Gaurav Sahaya, Hao Chenga,b, Shan Jianga,b, Minglin Maa,c,
Abigail Lytton-Jeana, Arturo Vegasa,c, Patrick Fentona,c, Christopher G. Levinsa,c, Kevin T. Loveb, Haeshin Leea,
Christina Corteza, Sean P. Collinsa, Ying Fei Lia, Janice Janga, William Querbesd, Christopher Zurenkod,
Tatiana Novobrantsevad, Robert Langera,b,c,e, and Daniel G. Andersona,b,c,e,1
a
David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; bDepartment of Chemical
Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; cDepartment of Anesthesiology, Children’s Hospital Boston, Harvard Medical
School, Boston, MA 02115; dAlnylam Pharmaceuticals, Inc., 300 Third Street, Cambridge, MA 02142; and eHarvard-MIT Division of Health Sciences and
Technology, Cambridge, MA 02139
Edited* by Alexander M. Klibanov, Massachusetts Institute of Technology, Cambridge, MA, and approved June 20, 2011 (received for review May 2, 2011)
Analogous to an assembly line, we employed a modular design for
the high-throughput study of 1,536 structurally distinct nanoparticles with cationic cores and variable shells. This enabled elucidation
of complexation, internalization, and delivery trends that could
only be learned through evaluation of a large library. Using robotic
automation, epoxide-functionalized block polymers were combinatorially cross-linked with a diverse library of amines, followed
by measurement of molecular weight, diameter, RNA complexation, cellular internalization, and in vitro siRNA and pDNA delivery.
Analysis revealed structure-function relationships and beneficial
design guidelines, including a higher reactive block weight fraction, stoichiometric equivalence between epoxides and amines,
and thin hydrophilic shells. Cross-linkers optimally possessed tertiary dimethylamine or piperazine groups and potential buffering
capacity. Covalent cholesterol attachment allowed for transfection
in vivo to liver hepatocytes in mice. The ability to tune the chemical
nature of the core and shell may afford utility of these materials
in additional applications.
C
hemically diverse nanoparticles form the basis of a number of
emerging applications in materials science (1–3), including
drug and gene delivery vehicles (4, 5). However, it is difficult
to predict the optimal chemical and physical properties for delivery of a specific drug or biomolecule. A key challenge in nanoparticle development is the need to perform distinct synthesis,
characterization, and formulation steps before performance
can be evaluated. Although combinatorial methods have driven
small molecule drug discovery (6), they have been less explored
for the discovery of polymers (7–12) in part due to these inherent
challenges. A less studied area in high-throughput (HT) polymeric
nanoparticle methodology (13–16) is the facile incorporation of
diverse chemical groups into polymers with defined architectures.
By applying HT robotic techniques to the controlled cross-linking
of block copolymers, we were able to prepare a library of core-shell
nanoparticles for nucleic acid complexation and delivery with great
variability in the chemical nature of the protonizable amine-based
core. The evaluation of a large library revealed that internalization
and/or complexation alone is not sufficient for silencing and that
certain chemical functionalities including tertiary dimethylamine
or piperazine groups with potential buffering capacity may be advantageous for polymer-based delivery.
Advances in the controlled synthesis of polymers with functional group tolerance [particularly controlled/living radical
polymerization (CRP) (17–20) methods that enable control over
MW and architecture] and in robotic technology (8) motivated
us to explore an experimental design based on modules where
96-well plates are shuffled between different HT instruments
for synthesis, characterization, and screening. This process allows
for examination of key physical and chemical properties in relation to performance. siRNA (21, 22) was selected as an initial
agent for delivery; despite its marked potential to treat various
12996–13001 ∣ PNAS ∣ August 9, 2011 ∣ vol. 108 ∣ no. 32
diseases, safe and effective delivery remains the most significant
barrier to broad clinical use (23). We previously utilized HT
methods for the synthesis and screening of materials for delivery
including lipids (15, 24) for siRNA delivery and linear poly(β-amino ester)s (PBAEs) (14, 25–27) for pDNA delivery. HT synthesis
of star polymers (13) and linear polymers using CRP methods
(10, 11, 19) has been reported but not applied for gene therapy
where cationic charges are required to bind nucleic acids through
electrostatic interactions. Modification of linear polymers for
pDNA delivery has been described (12, 28); however, to our
knowledge such polymers have not been shown to effectively
deliver siRNA. There is a growing interest in sub-100 nm particles
for delivery (4) due to the increased ability of smaller particles to
access capillaries, penetrate tissues, and cross cell membranes.
Because the possible size range for stable liposomes is generally
>50 nm (29), and because star shaped polymers (13, 30, 31) and
dendrimers (32) are considerably smaller, we selected a core-shell
system that allows for greater size range control and the ability to
easily incorporate a multitude of different cationic groups into
the core. Distinct polymer-based nanoparticles may have better
stability than lipid-based systems and greater potential for attachment of targeting ligands (33).
To address these issues, we prepared a library of nanoparticles
composed of cationic cross-linked nanogel cores and variable shells
with precise control over particle size, chemical composition, and
architecture. siRNA can complex to or inside of the core, while the
arms provide a protective shield. To achieve the desired structure,
we employed a method of cross-linking block copolymers (34, 35)
prepared by reversible addition-fragmentation chain transfer
(RAFT) polymerization (18). For cross-linking, we sought an efficient and versatile reaction that would permit library formation.
While other click reactions (36) might have been suitable, we chose
ring-opening of epoxides with amines (37–39) due to the availability
of a large number of amines that can incorporate cationic charge
into the core, and the efficiency of this catalyst-free reaction. By
changing the physical properties of the core and shell, these particles could also find application in the delivery of proteins and drugs,
assembled multilayers (40), electronics (41), as stabilizing particles
Author contributions: D.J.S., R.L., and D.G.A. designed research; D.J.S., K.A.W., L.N., G.S.,
H.C., S.J., M.M., A.L.-J., A.V., P.F., C.G.L., K.T.L., H.L., C.C., S.P.C., Y.L., J.J., W.Q., and C.Z.
performed research; D.J.S., K.A.W., L.N., G.S., H.C., S.J., M.M., A.L.-J., A.V., C.G.L., K.T.L.,
H.L., W.Q., T.N., R.L., and D.G.A. analyzed data; and D.J.S. and D.G.A. wrote the paper.
Conflict of interest statement: R.L. is a shareholder and member of the Scientific Advisory
Board of Alnylam. D.G.A. is a consultant with Alnylam. R.L and D.G.A have sponsored
research grants from Alnylam. Alnylam also has a license to certain intellectual property
invented at MIT. W.Q., C.Z., and T.N. are employed by Alnylam.
*This Direct Submission article had a prearranged editor.
1
To whom correspondence should be addressed. E-mail: dgander@mit.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1106379108/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1106379108
Results and Discussion
Library Design and Synthesis. The rationale for employing the HT
core-shell design was to create a cationic core to facilitate siRNA
complexation, with variation in the nature of the protonizable amine,
and a shell with variation in polymer length and chemical properties.
Although many techniques exist for the formation of nanoparticles
(31, 45–48), we required methods that were amenable to parallel HT
synthesis. For this reason, we employed a method of reacting epoxide groups with amines (37–39, 49–51) that avoids the use of salts,
surfactants, and mixed solvents. Instead of using homopolymers and
random copolymers (38), we chose block copolymers that could
form the core-shell morphology. The basic reaction for the synthesis
of core-shell nanoparticles involved block copolymers consisting of
one nonreactive block and one epoxide-functionalized block, such
that the core could be cross-linked with a variety of amines (Fig. 1).
These amines varied greatly in structure (linear, ring, branched),
reactivity (1–4 reactive amine groups per molecule; 1°, 2°, and 3°
amines within the molecule), and MW (small molecule, dendrimer,
and polymer). Each amine could form a distinct and structurally unique nanoparticle starting from one block copolymer.
We synthesized a series of different block copolymers to serve as
precursors for nanoparticle formation (Table S1). Block copolymers possessing poly(oligo(ethylene oxide) methacrylate) (POEOMA) with different lengths of the PEO side chain, may increase
blood circulation time due to the PEO shell of the resulting nanoparticle. Anionic, cationic, zwitterionic, and hydrophobic blocks
were also evaluated as shells. Utilizing the 96-well plate footprint,
each block copolymer was reacted with 96 amines to form a set of
core-shell nanoparticles. The HTapproach enabled randomization
of the core and shell for elucidation of the key chemical and
material properties for intracellular delivery. To facilitate rapid
synthesis, characterization, and screening, we developed a method
where a microtiter plate was passed through a series of modules
(Fig. 2). Periodic analysis by prescreening may lead to an improvement in chemical selection. In this study, we optimized the amine
library by replacing the poorer performing amines with new candidate amines during the process. Automation of this process accelerated not only material handling, but also data manipulations
and reaction calculations. The MW, density, drawn chemical structure, and dissolved concentration were entered into Library Studio
to enable rapid calculation of reaction stoichiometry, taking into account the MW, density, structure (number of amines), and concentration. Through preliminary studies, we found that a mole ratio of
1∶1 of epoxides to 1° or 2° amines and a concentration in dimethyl
sulfoxide (DMSO) of 100 mg∕mL (approximately 300 mM) was optimal for nanoparticle formation with limited macroscopic gelation.
Nanoparticle-Mediated Delivery in Vitro. As an initial screen, we
evaluated the ability of every nanoparticle to complex RNA
(Fig. 3A). Automated methods were developed that allowed for
evaluation of 50 microtiter plates per week. The degree of entrapment is expressed as a percentage based on measured fluorescence, where higher values in the figure indicate stronger
complexation. For nanoparticles with PEO shells (blocks A–J),
a range of complexation was observed that predominantly correlated with the amine. Structurally analogous amines showed that
more protonizable nitrogens resulted in better complexation. For
example, the linear polyamine 301 complexed well in most cases,
whereas the linear aliphatic diamine 112 with similar length did
not. Nanoparticles with anionic shells (blocks K and L) repelled
the anionic phosphate backbone of siRNA, resulting in almost
Fig. 1. Combinatorial synthesis of coreshell nanoparticles. (A) Epoxide-containing
block copolymers and amines were used to
synthesize a combinatorial library of hairy
core-shell nanoparticles. (B) Synthesis occurs
via the ring-opening reaction of epoxides by
amines forming β-hydroxyl groups and crosslinking points separated by the functionality
(X) contained in the amine. Each nanoparticle is named by a letter corresponding to the
starting block copolymer and a number corresponding to an amine.
Siegwart et al.
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in polymer blends (42), and catalytic scaffolds and supramolecular
hosts (43, 44).
Fig. 2. Modular design for the synthesis, characterization, and screening of
a library of core-shell nanoparticles. (A) The synthesis of 1,536 core-shell nanoparticles was carried out on a Symyx fluid-handling robot inside of glass
vials in a 96-well plate format. (B) The nanoparticles were purified and filtered through a HT filtering apparatus into a standard 96-well tissue culture
plate. (C) The MW and particle size of the polymers were measured by a HT
GPC system. (D) The same plate format was used for an RNA complexation
assay, carried out on a Tecan cell culture robot. HT in vitro screens for siRNA
and pDNA delivery were performed. (E) Biodistribution of C227 (left two
images) and C80 (right two images) are shown. GPC, siRNA complexation
data, and in vitro siRNA and pDNA delivery results with standard deviation
(s.d.) for nanoparticles C32-C94 (positions A1-A12 in the plate) are presented.
no measurable complexation. In contrast, nanoparticles with a
cationic shell (block M) were able to complex siRNA in every
case, implying that the siRNA is located in the shell (and possibly
in the core) of these nanoparticles, whereas the siRNA is only
complexing with the core of the other nanoparticles.
The libraries were then screened for the ability to productively
deliver siRNA to a HeLa cell line that stably expressed both
firefly and Renilla luciferase (Fig. 3B). Cells were treated with firefly luciferase-targeting siRNA(siLuc)-nanoparticle complexes and
the ratio of firefly to Renilla luciferase expression was measured
after 24 h. Reduction of only firefly luciferase demonstrates noncytotoxic specific silencing, whereas reduction in expression of both
luciferase proteins indicates toxicity or other nonspecific effects.
Following the modular HT approach, the complexes were formed
in 96-well plates by simple mixing. Because the size, shape, and
charge of the nanoparticles vary greatly in this library, we also evaluated them for pDNA delivery. Compared to siRNA, which is rigid
and linear, pDNA is a few hundred times longer, circular, and more
flexible. HeLa cells were transfected with pDNA coding for luciferase, and luciferase activity was measured after 24 h (Fig. 3C).
When considering the maximum siRNA knockdown for one
amine core across all block copolymers, those nanoparticles that
were expected to increase in charge as the pH drops with the transition from outside to inside of the endosome were more likely to
effectively deliver siRNA. We modeled the microspecies pKas of
the GMA-amine products in the core of the nanoparticles, estimating the relative buffering capacity that may be involved in endosomal escape (Table S2). The model system calculations suggest
that 62 of the 102 amines will not gain charge during the expected
drop in pH within the endosomal compartment. The remaining
40 amines are expected to exhibit buffering. Across all block types,
80 different amines enabled >30% luciferase silencing in vitro. Considering only the criteria used to segregate buffering from nonbuffering amines, a significantly higher proportion of the modeled
buffering amines (77%) are represented amongst the effective
group than nonbuffering amines (14%) (Fig. S1).
Analysis of the results also revealed several trends regarding
the block copolymer structure in nanoparticles capable of deliver12998 ∣
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Fig. 3. In vitro screening of core-shell nanoparticles. (A) The degree of RNA
entrapment was quantified using the RiboGreen assay. The average percent
of complexation is shown. (B) HeLa cells stably expressing both firefly and
Renilla luciferases were treated with firefly targeting siRNA-nanoparticles
complexes. The average percent reduction in firefly luciferase activity after
treatment is shown. (C) HeLa cells were treated with pDNA(luc)-nanoparticle
complexes. Activity using LF2000 was standardized to 100. The average percent activity versus LF2000 after treatment is shown. To provide a
picture of the capability of all nanoparticles, the complexation, siRNA delivery, and pDNA experiments were performed in triplicate at a weight ratio of
50∶1 (nanoparticle:siRNA).
ing siRNA in vitro. Considering nanoparticles with POEOMA
shells (blocks A–J), nanoparticles formed from block C showed
the highest knockdown. This was likely due to an optimal block
ratio with a short POEOMA block [degree of polymerization
(DP) 12] and a longer glycidyl methacrylate (GMA) block (DP
135). Block D, which also had a higher weight fraction of PGMA,
demonstrated effective knockdown, although it was less capable
of delivering pDNA. In contrast, nanoparticles formed from
block H, which possessed a lower weight fraction of POEOMA
(DP 48) compared to PGMA (DP 17) were unable to deliver
siRNA or pDNA. This trend of weight fraction is likely related
to resulting charge density, particle formation, and to surface
PEO presentation. J had a long GMA block that resulted in
some macroscopic gelation. F, which was a poorly formed block
copolymer with high polydispersity, was not effective, suggesting
that defined block architecture is important for nanoparticle
formation. Many G nanoparticles could deliver siRNA due to
their less dense core (via copolymerization of 2-hydroxyethyl
Siegwart et al.
Fig. 4. In vitro core-shell nanoparticle-mediated delivery trends. (A) RNA
complexation and luciferase silencing after delivery of nanoparticle:siRNA
complexes (50∶1, wt∕wt) is expressed for the top performing nanoparticles.
(B) siRNA dose response for the top nanoparticles. (C) Complexation and
luciferase activity after delivery of nanoparticle:pDNA complexes (50∶1,
wt∕wt) is expressed for the top performing nanoparticles. (A–C) n ¼ 4; s.d.
is expressed.
Siegwart et al.
formation, siRNA complexation, and high internalization. However,
only C206 exhibited high siRNA silencing ability, possibly due the
higher hydrophobicity and methyl group adjacent to the amines.
Some nanoparticles (e.g., 207) mediate efficient internalization,
but that alone is not sufficient for silencing. In general, incorporation of tertiary amines led to an improvement in complexation,
cellular internalization, and siRNA silencing efficiency.
Characterization of the Core-Shell Nanoparticle Library. The entire
core-shell nanoparticle library was characterized using HT GPC.
In order to decrease the total analysis time from months to weeks,
we utilized a GPC equipped with a single flow injection polymer
analysis column and three detectors capable of measuring both
MW and particle size. GPC traces, absolute MW, Rh , and Rg for
all nanoparticles appear in Table S3. Preliminary studies were carried out before the full library was synthesized. To achieve the greatest flexibility in choice of monomers, we used cumyl dithiobenzoate
as the RAFT chain transfer agent (CTA) in most cases. Conditions
were investigated and optimized for the controlled homopolymerization of OEOMA and GMA (Table S4 and Fig. S2). The semilogarithmic plot of lnð½M0 ∕½MÞ versus time for the polymerization
of GMA was linear, indicating a constant concentration of radicals.
M n also increased linearly with conversion. Chain extension of
OEOMA from PGMA was examined, yielding block copolymers
with a clean GPC shift in MW, and polydispersity below 1.3. GMA
was also polymerized from POEOMA macro-CTAs. Kinetic studies
showed trends of a controlled polymerization. In advance of forming nanoparticles, model ring-opening reactions with small molecules were carried out. At 1∶1 molar equivalents of epoxide to
amine, reaction with piperidine and sodium azide resulted in complete opening of the epoxides (Fig. S2). To examine the effect of
block length on nanoparticle formation, we synthesized a series
of block copolymers that systematically varied in weight % of
OEOMA and GMA blocks (Table S1). These block copolymers
were reacted with different amines, and the particle size of the re-
Fig. 5. In vitro uptake screening. (A) Cellular internalization of selected nanoparticles after 1 hr of incubation is demonstrated by HT automated confocal microscopy. HeLa cells were exposed to all C-based nanoparticles
complexed with Alexa-594 siRNA (50∶1, wt∕wt). Twenty different fields were
imaged and a representative image of nanoparticle/siRNA complex is presented (Alexa 594 is pseudocolored green). Additional images appear in
Fig. S4. (B) The total fluorescence was quantified and plotted. False positives
were identified by visible aggregation (e.g., C208 included for reference) or
significant background and were removed from the graph. Interestingly, C60
nanoparticles interact with the cell membrane but did not enter cells, while
C80 nanoparticles were effectively internalized (see zoom insets).
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methacrylate) but were not superior to the best nanoparticles
formed from C. Nanoparticles formed from cationic blocks (H
and M) and anionic blocks (K and L) did not effectively deliver
siRNA. Some nanoparticles formed from I could deliver siRNA
but not pDNA, possibly due to the shorter lengths of the blocks
and correspondingly smaller size of the nanoparticles. The opposite trend was also observed, where some nanoparticles with a
zwitterionic shell (N) or a hydrophobic shell (O) could deliver
pDNA but not siRNA. To a small extent, a few nanoparticles with
anionic shells (K and L) mediated transfection of pDNA; however, the anionic nanoparticles were largely ineffectual.
The performance of the top nanoparticles for siRNA delivery
is detailed in Fig. 4A. In parallel experiments, these nanoparticles
were as effective as Lipofectamine 2000 (LF2000), including C77,
C80, C206, and C227, which were more effective at the given dose
in the presence of serum. The best performing nanoparticles were
able to effectively complex siRNA. Dose response data (Fig. 4B)
revealed an increase in gene silencing as the dose increased. In
the top 40 nanoparticles that silenced >30% of firefly expression,
five amines appeared in more than one nanoparticle (77, 80, 94,
208, and 222). The top 20 nanoparticles all had amines with 1–2
reactive groups. Having more than two reactive amines possibly
led to interparticle cross-linking. E206 was the best nanoparticle
for pDNA due to long block lengths and large size, where the cells
expressed 625% more luciferase compared to cells transfected
with LF2000 (Fig. 4C).
To examine the effect of amine structure on cellular internalization, we quantified the uptake after 1 h of incubation with each of
the C-based nanoparticles complexed with fluorescent siRNA
(Fig. 5) and used image analysis to identify key structural trends.
Nanoparticles containing tertiary amines were more effectively
internalized than nanoparticles with secondary amines. Within tertiary alkyl amines, dimethyl (80) > diethyl (222) > dibutyl (223),
and within secondary alkyl amines, methyl (211) > ethyl (210). In
addition to uptake, siRNA complexation and silencing efficiency
was also improved for nanoparticles with pendant dimethyl tertiary
amines. Similarly, nanoparticles with piperazine (227) and pyrrolidine (77) structures showed enhanced complexation, delivery, and
internalization, as compared to morpholine (224) and imidazole
(94) groups. Although complexation remained high for C224, the
internalization decreased slightly and the siRNA silencing ability
decreased greatly compared to optimal C227. Increased cellular uptake was observed for linear polyamines with internal methyl substitutions (e.g., 235 versus 234; 127 versus 125). PEG-based diamine
cross-linkers (103-1, 206, 207, 208) enabled efficient nanoparticle
Fig. 6. Characterization of core-shell nanoparticles.
(A) Dried FF110 nanoparticles on mica showed uniform
particle size around 34 nm by AFM. (B) TEM was utilized
to visualize the core-shell structure of R126 nanoparticles. The insert clearly showed an electron dense crosslinked core, and a more electron loose shell. These TEM
images are unstained. Additional AFM and TEM images
appear in Fig. S3. Cell internalization of FITC-C80 (C)
and FITC-C227 (D) nanoparticles after 1 hr of incubation
was demonstrated by confocal microscopy. Punctate
green fluorescence was observed within the cell.
sulting nanoparticles was measured by DLS. Desired particle size
(10–100 nm) was obtained for most block copolymers (15–85
weight % GMA). Atomic force microscopy (AFM) images of nanoparticles prepared by the reaction of FF with the linear aliphatic
diamine 110 showed uniform particles with a diameter of 34 nm
(Fig. 6A). Polyamines were also able to efficiently cross-link the
block copolymers to form the hairy nanoparticle shape. The reaction of R or S with 126 formed particles with a diameter of about
21 nm, as measured by DLS, with the diameter measured by AFM
slightly smaller (22.9 nm) for R126 than for S126 (25.4 nm) due to
the slightly shorter GMA block (Fig. S3). To verify the core-shell
structure, transmission electron microscopy (TEM) was employed.
An examination of R126 (Fig. 6B) and C227 nanoparticles
(Fig. S3E) revealed a higher electron density in the core, compared
with the shell, indicating the core-cross-linked morphology. The
measured diameter of C80 and C227 nanoparticles by DLS and
TEM was in agreement (Table S5 and Fig. S3). In addition to the
HT internalization studies, we examined cellular internalization
by synthesizing FITC-labeled nanoparticles via reaction of the isothiocyanate group with dangling amines to covalently label the
core. At higher magnification, C80-FITC and C227-FITC can be
clearly visualized inside endosomes that are the hallmark for endocytic trafficking (Fig. 6 C and D). Because size and surface charge
can also affect cellular uptake, the diameter and zeta potential of
top performing nanoparticles were also measured (Table S5).
In Vivo Core-Shell Nanoparticle-Mediated Delivery of siRNA to Hepatocytes in Mice. Based on results from the in vitro screen, we
examined the utility of top performing nanoparticles in facilitating siRNA delivery in vivo by employing the mouse Factor VII
gene silencing model (15, 24). Factor VII is a useful gene target
for the evaluation of hepatocyte-specific delivery because it is
only produced in the cells of the liver parenchyma, possesses a
relatively short plasma half-life, and is secreted into the blood,
enabling facile protein quantification. siRNA-directed against
the blood clotting Factor VII was complexed with nanoparticles
at a weight ratio of 10∶1 (NP:siRNA) and delivered intravenously. Mouse body weight was monitored over the duration of
the experiment because body weight loss can indicate toxicity
associated with nanoparticle treatment.
Since in vivo delivery to hepatocytes poses more challenges
than the in vitro delivery to HeLa cells, we investigated the
inclusion of cholesterol because covalent attachment of cholesterol to siRNA has been shown to improve in vivo delivery to
the liver (52, 53). Of all the nanoparticles tested, C80 showed
the most improvement after cholesterol attachment and was focused on for this study. 10 Mole % of the epoxide groups were
reacted with N 0 -cholesteryloxycarbonyl-1,2-diaminoethane (318),
followed by addition of 80 to stoichiometrically cross-link the remaining epoxide groups. Covalent attachment of cholesterol to
C80 nanoparticles enabled 40% silencing of Factor VII (Fig. 7A).
Noncovalent encapsulation of unmodified cholesterol had no
measured effect. Delivery of control siGFP using these nanoparticles did not result in any measurable silencing, suggesting
specific delivery to the liver and silencing of Factor VII. No appreciable weight loss was observed, indicating minimal toxicity.
To probe the biodistribution of these nanoparticles with and
without cholesterol in vivo, a single bolus dose of 1 mg∕kg Cy513000 ∣
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labeled siRNA-nanoparticle complex was administered via tail
vein injection (Fig. 7 B and C). C80-Cy5-siRNA nanoparticles
were observed in the liver, kidneys, and lungs. The attachment of
cholesterol resulted in increased liver accumulation.
We believe that the development of this library of core-shell nanoparticles represents an important step forward for the HT synthesis of polymers for intracellular delivery. We aimed to move
beyond simple linear polymers by exploring CRP for the formation
of complex architectures with great chemical diversity in the core
and shell. This synthetic method enables parallel generation of
large libraries of chemically distinct core-shell materials and decreases the time from design to analysis. The common structural
features of these materials suggest certain design criteria for creating future nanoparticles, including (i) a block weight ratio around
15% nonreactive block and 85% reactive block, (ii) a stoichiometic
balance between epoxides and amines, (iii) thin water-soluble hydrophilic shells, (iv) tertiary amines with 1–2 reactive sites, and (v)
amines with buffering capacity. Covalent attachment of cholesterol
to the nanoparticle was required for in vivo delivery to the liver.
Further studies are warranted to extend this technology for the
broadest applications of RNAi therapy and drug delivery. We also
desire to use what we have learned in this study to aid in the design
of fully degradable nanoparticles that satisfy the aforementioned
general properties and design criteria. Finally, we believe that the
modular design utilized in this report is applicable to many other
systems. By changing the composition of the blocks and crosslinkers, a variety of other nanoparticles could be formed with
wide-ranging applications.
Methods
HT Nanoparticle Synthesis. Robotic synthesis was performed on a customized
Symyx Core Module equipped with four different liquid dispensing elements
(a single tip, a motor driven gripper with a heated piercing tip, a positive
displacement tip, and a heated parallel 4-tip), heating and cooling bay elements, a magnetic stirring element, a mass balance, and imaging equipment.
Library Studio (Symyx Discovery Tools) was used to design the core-shell na-
Fig. 7. In vivo silencing of Factor VII in liver hepatocytes. Nanoparticles were
purified by dialysis, complexed with siRNA at a weight ratio of 10∶1 (nanoparticle:siRNA), and delivered intravenously to C57BL/6 mice. Mice received a
single bolus administration of 5 mg∕kg total siRNA via tail-vein injection and
Factor VII levels were quantified 48 hr post injection. (A) Covalent attachment
of cholesterol to C80 nanoparticles enabled silencing. Noncovalent encapsulation of cholesterol and delivery of control siGFP resulted in no knockdown.
Data points represent group mean s:d. Data points marked with asterisks
are significant relative to control treated groups (***, P < 0.0001; t-test, double-tailed, n ¼ 14). Representative images of isolated organs for Cy5-siRNAC80 (b) and Cy5-siRNA-C80-10 mol% cholesterol (C) nanoparticle complexes
demonstrated nanoparticle accumulation in the liver, kidneys, and lungs
after 120 min. The covalent attachment of cholesterol to C80 resulted in enhanced liver accumulation and reduced lung accumulation.
Siegwart et al.
HT RNA Complexation Assay. The Quant-iT RiboGreen RNA assay (Invitrogen)
was performed on a customized Tecan Freedom EVO 200 with 8-Tip, LiHa,
MCA-96, and Extended RoMa elements. Fluorescence was measured using
a coupled Infinite M200 multimode monochromator-based reader. The assay
was programmed in EVOware Plus.
(2 μg∕mL) for nuclei identification. Cells were then imaged with an automated spinning disk confocal microscope (OPERA, Perkin Elmer). Quantitation of internalized fluorescently labeled siRNA was done using Acapella
software. After identification of cell location and perimeter, intracellular siRNA signal intensity over the whole search region (single field) was calculated.
Data presented are an average of intracellular intensity from 20 different
fields plotted in Excel. The same defined pattern of fields from each
well were acquired to eliminate bias during analysis. Quality control was
performed on all images and large aggregates or nonspecific background
seen in rare fields was excluded from analysis.
Supporting Information. Additional descriptions of materials, instrumentation, modeling, and experimental details, including the synthesis of all block
copolymers, are available in SI Text.
HT Confocal Microscopy. 15,000 HeLa cells were plated in black 96-well plates
(Greiner Bio-one). Cells were starved for 30 min in the presence of PBS/BSA
and pulsed for 1 h at 37 °C with 50 ng of BLOCK-iT Alexa Fluor Red Fluorescent Oligo (Invitrogen) complexed with the nanoparticle at a nanoparticle/
siRNA ratio of 50∶1 (wt∕wt). Cells were chased for 30 min in the presence
of unlabeled nanoparticles and washed with PBS. Cells were then fixed using
4% paraformaldehyde and counterstained in PBS containing Hoescht
ACKNOWLEDGMENTS. The authors thank Boris Klebanov, Manos Karagiannis,
Christian J. Kastrup, Avi Schroeder, Carmen Barnes, and Robert S. Siegwart
for their help. This work was supported by Alnylam Pharmaceuticals and
National Institutes of Health (NIH) Grants R01-EB000244-27 and 5-R01CA132091-04. D.J.S. acknowledges postdoctoral support from NIH NRSA
award F32-EB011867.
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APPLIED PHYSICAL
SCIENCES
noparticle libraries. For each nanoparticle, a solution of amine (100 mg∕mL in
DMSO) was added to a stirring solution of block copolymer (100 mg∕mL in
DMSO) such that the mole ratio of epoxides to amine was 1∶1 and the total
concentration of reactive components was on the order of 300 mM. Reaction
blocks with 96 1 mL glass vials equipped with tumbling stir bars and covered
with a rubber mat were used. The resulting mixtures were stirred and heated
at 50 °C for 24 h on the deck of the robot.