N-Acetylgalactosamine Block-co-Polycations Form Stable Polyplexes with Plasmids and Promote Liver-Targeted Delivery

Article abstract of DOI:10.1021/acs.biomac.5b01555

The liver is an ideal target for nucleic acid therapeutic applications (i.e., siRNA, gene therapy, and genome editing) due to its ability to secrete proteins into the blood. In this work, we present the first synthesis of a novel monomer derived from N-acetyl-d-galactosamine (GalNAc) and its polymerization as a facile route to create multivalent delivery vehicles with exceptional targeting efficiency to asialoglycoprotein receptors (ASGPRs) on liver hepatocytes. A series of cationic diblock GalNAc glycopolymers composed of a GalNAc-derived block of fixed length (n = 62) and cationic 2-aminoethylmethacrylamide (AEMA) blocks of varying lengths (n = 19, 33, and 80) have been synthesized and characterized. In addition, nontargeted control polymers consisting of either glucose or polyethylene glycol-derived neutral blocks with an AEMA cationic block were also created and examined. All polymeric vehicles were able to bind and encapsulate plasmids (pDNA) into polymer-pDNA complexes (polyplexes). The GalNAc-derived polyplexes were colloidally stable and maintained their size over a period of 4 h in reduced-serum cell culture media. The GalNAc-derived homopolymer effectively inhibited the uptake of Cy5-labeled asialofetuin (a natural ligand of ASGPRs) by cultured hepatocyte (HepG2) cells at lower concentrations (IC₅₀ = 20 nM) than monomeric GalNAc (IC₅₀ = 1 mM) and asialofetuin (IC₅₀ = 1 μM), suggesting highly enhanced ASGPR binding due to multivalency. These polymers also showed cell type-specific gene expression in cultured cells, with higher protein expression in ASGPR-presenting HepG2 than HeLa cells, which lack the receptor. Biodistribution studies in mice show higher accumulation of pDNA and GalNAc-derived polymers in the liver compared with the glucose-derived nontargeted control. This study demonstrates the first facile synthesis of a multivalent GalNAc-derived block copolymer architecture that promotes enhanced delivery to liver and offers insights to improve targeted nanomedicines for a variety of applications.

Full text of DOI:10.1021/acs.biomac.5b01555

Article
pubs.acs.org/Biomac
N‑Acetylgalactosamine Block-co-Polycations Form Stable Polyplexes
with Plasmids and Promote Liver-Targeted Delivery
Yogesh K. Dhande,† Bharat S. Wagh,‡ Bryan C. Hall,§ Dustin Sprouse,‡ Perry B. Hackett,§
and Theresa M. Reineke*,‡
‡
†Department of Chemical Engineering and Materials Science, and Center for Genome Engineering, Department of Chemistry and
§
Center for Genome Engineering, and Department of Genetics, Cell Biology and Development, and Center for Genome
Engineering, University of Minnesota, Minneapolis, Minnesota 55455, United States
S
* Supporting Information
ABSTRACT: The liver is an ideal target for nucleic acid
therapeutic applications (i.e., siRNA, gene therapy, and genome
editing) due to its ability to secrete proteins into the blood. In
this work, we present the first synthesis of a novel monomer
derived from N-acetyl-D-galactosamine (GalNAc) and its
polymerization as a facile route to create multivalent delivery
vehicles with exceptional targeting efficiency to asialoglycopro-
tein receptors (ASGPRs) on liver hepatocytes. A series of
cationic diblock GalNAc glycopolymers composed of a
GalNAc-derived block of fixed length (n = 62) and cationic
2-aminoethylmethacrylamide (AEMA) blocks of varying
lengths (n = 19, 33, and 80) have been synthesized and characterized. In addition, nontargeted control polymers consisting
of either glucose or polyethylene glycol-derived neutral blocks with an AEMA cationic block were also created and examined. All
polymeric vehicles were able to bind and encapsulate plasmids (pDNA) into polymer−pDNA complexes (polyplexes). The
GalNAc-derived polyplexes were colloidally stable and maintained their size over a period of 4 h in reduced-serum cell culture
media. The GalNAc-derived homopolymer effectively inhibited the uptake of Cy5-labeled asialofetuin (a natural ligand of
ASGPRs) by cultured hepatocyte (HepG2) cells at lower concentrations (IC50 = 20 nM) than monomeric GalNAc (IC50 = 1
mM) and asialofetuin (IC50 = 1 μM), suggesting highly enhanced ASGPR binding due to multivalency. These polymers also
showed cell type-specific gene expression in cultured cells, with higher protein expression in ASGPR-presenting HepG2 than
HeLa cells, which lack the receptor. Biodistribution studies in mice show higher accumulation of pDNA and GalNAc-derived
polymers in the liver compared with the glucose-derived nontargeted control. This study demonstrates the first facile synthesis of
a multivalent GalNAc-derived block copolymer architecture that promotes enhanced delivery to liver and offers insights to
improve targeted nanomedicines for a variety of applications.
plasmid delivery in whole animals.14 Indeed, new delivery
vehicles are needed to address these challenges and aid rapid
advancement of new therapies toward the clinic.
INTRODUCTION
■
Tissue-specific delivery offers promise to reduce toxicity and
immunogenicity as well as improve efficacy of numerous
Nonviral vectors, such as cationic polymers, have emerged as
therapeutics ranging from small molecule drugs to proteins and
nucleic acids.1−3 Recent advances in the field of gene therapy
a promising class of gene delivery vehicles because of their
such as the clinical approval of Glybera4 by the European
safety, low immunogenicity, and low cost of production
compared to viruses.15,16 Cationic polymers such as poly-
Commission and numerous ongoing clinical trials5,6 forecast
ethylenimine (PEI),17 poly(L-lysine),18 poly((2-dimethylami-
the great potential of gene and RNAi-based therapeutics, but
no) ethyl methacrylate),19,20 and poly(2-aminoethylmethacry-
efficient delivery of nanomedicines to specific tissues remains a
challenge. The liver is a widely studied target for nucleic acid
therapies due to its large size, regenerative ability, and role in
the production and secretion of serum proteins into the blood,
lamide)21 condense DNA into nanoparticles, called polyplexes,
that are efficiently taken up by cells through either caveolae or
clathrin-mediated endocytic pathways.22−25 However, this
success in vitro has yet to be translated into clinical therapies
which has huge potential for permanent therapeutic inter-
due to several challenges.26 Principally, in the presence of
aqueous ions and other charged species, polyplexes tend to
vention.7 Liver-targeted therapies for the treatment of inherited
disorders such as hemophilia and mucopolysaccharidoses using
viral and nonviral vectors have been promising,8 but are often
limited by either immunogenicity or low efficacy.9−12 Receptor-
mediated targeting has shown encouraging results in delivering
siRNA to the liver,13 but few studies have shown efficient
Received: November 17, 2015
Revised: January 4, 2016
© XXXX American Chemical Society
A
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Figure 1. Schematic of polyplex formation and interactions of GalNAc ligands with ASGPRs on hepatocytes. The GalNAc ligands are drawn as red
circles, and the cationic blocks are drawn in blue. Cationic blocks complex with plasmids to form polyplexes that display multivalent GalNAc chains
on the surface thereby promoting cell type-specific polyplex internalization by binding to ASGPRs.
aggregate with themselves or other biomolecules such as serum
proteins, which severely limits the targeting potential of
polyplexes to specific tissues when delivered through the
circulatory systems of whole animals.18,21,27 For example,
polyplexes formed with PEI become trapped in the lungs
during first-pass circulation due to aggregation upon systemic
injection.28,29 Moreover, PEI polyplexes also suffer from rapid
clearance from the blood by the reticuloendothelial system due
to nonspecific charge-mediated interactions with serum
proteins.30,31 To prevent these detrimental interactions,
polyethylene glycol (PEG) is commonly used as a hydrophilic
outer layer to sterically stabilize polyplexes from aggregation
and prolong circulation times by reducing nonspecific
interactions with the reticuloendothelial system.32,33 However,
PEG has a limited effect on polyplex stability and undergoes
accelerated blood clearance after multiple injections.34,35
Several other structures have been explored as alternatives to
PEG to achieve colloidal stability and biocompatibility. These
include zwitterionic structures like polysulfobetaine, biodegrad-
able polymers such as poly(2-hydroxyethyl methacrylate) and
poly(lactic-co-glycolic acid).36−41 Earlier work from our lab has
demonstrated that diblock polymers containing glucose or
trehalose-substituted monomers copolymerized with a cationic
block form colloidally stable polyplexes and offer potential as
alternative hydrophilic coating layers.21,42−45 These sugar-
derived neutral blocks form a “stealth” layer that prevents
aggregation while maintaining excellent gene-delivery proper-
ties in cultured cells.21,44
We hypothesized that the highly hydrophilic structure of
sugars can serve a dual purpose: maintain colloidal stability of
polyplexes and promote tissue-specific delivery by selectively
binding to cell surface lectins. To this end, we created a series
of cationic diblock glycopolymers derived from N-acetyl-D-
galactosamine (GalNAc) to target asialoglycoprotein receptors
(ASGPRs) on hepatocytes. GalNAc and galactose have been
studied extensively for targeting ASGPRs on hepatocytes using
mono-, bi-, and triantennary structures to create targeting
functionalities.14,46−53 The triantennary structures have more
than a thousand-fold higher affinities to ASGPR compared to
monovalent structures based on measured half-maximal
inhibitory concentrations (IC50),50,51 and have facilitated the
advancement RNAi therapeutics to clinical trials.13 However,
synthesis of these triantennary structures requires numerous
reaction steps that are time, labor, and yield intensive, making it
desirable to innovate alternative ligands that can be readily
synthesized and applied to a variety of therapeutic cargo.48,51,54
Herein, we demonstrate the synthesis of a novel GalNAc-
based monomer and its incorporation into diblock polycations
exhibiting three cationic block lengths. Complexing the cationic
blocks with anionic plasmids (pDNA) aids formation of a
core−shell structure that displays the GalNAc blocks on the
surface providing a hydrophilic and steric barrier that prevents
polyplex aggregation in salt and serum-containing cell culture
media, and promotes binding to ASGPRs on hepatocytes
(Figure 1). We demonstrate for the first time that this new
multivalent GalNAc motif promotes 5 orders of magnitude
stronger interactions with ASGPRs than monomeric GalNAc.
Preliminary cell culture and mouse biodistribution experiments
reveal selective polyplex localization and delivery to the mouse
liver. The facile synthesis and assembly of these GalNAc-based
block polymers offers a promising new motif to promote both
colloidal stability and selective delivery of a wide range of
nanomedicine formulations to hepatocytes.
MATERIALS AND METHODS
■
Materials and Reagents. All the reagents for polymer synthesis
were purchased from Aldrich (Milwaukee, WI) at the highest available
purity and used as received unless mentioned otherwise. The
monomer 2-aminoethylmethacrylamide (AEMA) was purchased
from Polysciences (Warrington, PA) and used directly. The chain
transfer agent (CTA) 4-cyano-4-(propylsulfanylthiocarbonyl)-
sulfanylpentanoic acid (CPP) was synthesized as previously
reported.27,55 The initiator, 4,4′-azobis(4-cyanopentanoic acid) (V-
501), was recrystallized twice from methanol prior to use.
All cell culture media used in this study were purchased from Life
Technologies (Grand Island, NY): Dulbecco’s modified Eagle medium
(DMEM, high glucose, Glutamax supplement), reduced-serum
medium (Opti-MEM, Glutamax supplement), heat inactivated fetal
bovine serum (HI FBS), phosphate-buffered saline (PBS) pH = 7.4,
trypsin-EDTA (0.25%), and antibiotic−antimycotic (100×). The
MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) was purchased from Invitrogen (Carlsbad, CA). Human
hepatocellular carcinoma (HepG2, ATCC HB-8065) and human
cervical carcinoma (HeLa, ATCC CCL-2) cell lines were purchased
from ATCC (Manassas, VA).
gWiz-luc plasmid DNA, used for the luciferase assay, was purchased
from Aldevron (Fargo, ND). All other in vitro experiments were
performed with pCMV-lacZ (Aldevron). Biodistribution experiments
in mice were performed with pT2/CAL.29 Commercially available
transfection reagents were used as positive controls in this study.
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Glycofect (a donation from Techulon, Inc., Blacksburg, VA) and
JetPEI (Polyplus-Transfection Inc., Illkirch, France) were used as
standards and positive controls for transfections. Unless specified
otherwise, all biological experiments were performed in triplicate, and
the mean and standard deviation of data are reported in all figures and
tables.
was added to a 25 mL round-bottom flask equipped with a magnetic
stir bar. The stirred solution was then sparged with nitrogen for 30
min, and the flask was placed in a preheated oil bath at 70 °C. The
reaction was terminated after 6 h by quenching the reaction flask in
liquid nitrogen, followed by exposure to air. After purification by
dialysis against water (pH 4−5) and lyophilization, the PMAGalNAc62
macroCTA was chain extended with AEMA to yield three diblock
polymers following a similar procedure, as previously published.45 In
general, AEMA (0.132 g, 0.799 mmol), PMAGalNAc62 (0.100 g), and
V-501 (2.60 × 10−4 g, 9.40 × 10−4 mmol) were dissolved in 7.9 mL of
1 M acetate buffer (pH 5.2) and added to a 25 mL round-bottom flask
equipped with a magnetic stir bar. After sparging with nitrogen for 30
min, the stirred reaction was allowed to proceed at 70 °C for 7.5 h.
The reaction mixture was then quenched by cooling the reaction vessel
in liquid nitrogen and exposing to air. The product was purified by
dialysis against DI water (pH adjusted to between 4 and 5 by addition
of HCl) and lyophilized to dryness.
Size exclusion chromatography (SEC) was used to determine the
molecular weight (number-average, Mn and weight-average, Mw) and
dispersity (Đ) for the PMAGalNAc62 macroCTA and the block
copolymers of MAGalNAc and AEMA using an aqueous eluent of 1.0
wt % acetic acid/0.1 M Na2SO4. A flow rate of 0.4 mL/min, Eprogen
(Downers Grove, IL) columns [CATSEC1000 (7 μm, 50 × 4.6),
CATSEC100 (5 μm, 250 × 4.6), CATSEC300 (5 μm, 250 × 4.6), and
CATSEC1000 (7 μm, 250 × 4.6)], a Wyatt HELEOS II light
scattering detector (λ = 662 nm), and an Optilab rEX refractometer (λ
= 658 nm) were used. Astra V (version 5.3.4.18, Wyatt Technologies,
CA) was utilized for the determination of Mn, Đ, and dn/dc of the
(co)polymers. 1H NMR measurements were performed with a
temperature-controlled Varian 400-MR (Palo Alto, CA) spectrometer
operating at a frequency of 399.7 MHz in D2O (HOD internal
standard). Block polymer compositions were determined by
comparing resonances of the MAGalNAc block with those associated
with the AEMA block.
Synthesis of the Methacrylamido N-Acetyl-D-galactosamine
Monomer. Synthesis of Acetylated GalNAc (2). To a solution of
GalNAc (1; 5.15 g, 23.9 mmol) in dry pyridine (240 mL) at room
temperature was added acetic anhydride (20.0 g, 196 mmol), DMAP
(0.0280 g, 0.238 mmol), and stirred for 12 h. After 12 h, the reaction
mixture was diluted with ethyl acetate (240 mL), and washed with 1 N
HCl solution (3 × 100 mL). The organic layer was then washed with
satd NaHCO3 (1 × 100 mL), distilled water (1 × 100 mL), and brine
(1 × 100 mL), dried with Na2SO4, and filtered. The solvent was
concentrated under reduced pressure and dried under high vacuum to
afford 7.20 g (18.8 mmol, 78% yield) of acetylated GalNAc (2) as
white foam. IR (film) 3286, 1741, 1677, 1657, 1558, 1540, 1369, 1321,
1
1211, 1108, 1009, 937; H NMR (300 MHz) δ 6.21 (d, J = 6.0 Hz,
1H), 5.43 (m, 2H), 5.22 (dd, J = 15.0, 3 Hz, 1H), 4.73 (m, 1H), 4.24
(m, 1H), 4.1 (m, 2H), 2.17 (bs, 6H), 2.03 (bs, 6H), 1.95 (s, 3H); 13C
NMR (125 MHz) δ 171.09, 170.39, 170.35, 170.22, 168.91, 91.24,
68.49, 67.74, 66.68, 61.29, 46.94, 23.07, 20.94, 20.73, 20.64, 20.62;
HRMS (ESI) Calcd for C16H23NO10 + Na, 412.1220; found, 412.1214.
Synthesis of 1,2-Oxazoline (3). To a solution of 2 (4.87 g, 12.5
mmol) in 1,2-dichloroethane (130 mL) under a nitrogen atmosphere
was added trimethylsilyl trifluoromethanesulfonate (TMSOTf) (3.06
g, 13.8 mmol), and the solution was heated to 50 °C and stirred for 12
h. After, it was cooled down to room temperature, triethylamine was
added to neutralize the solution. Post-neutralization, the solution was
concentrated under reduced pressure, and the crude mixture was dried
under vacuum. The product was purified via column chromatography
by eluting the product in a 20:1 CHCl3/MeOH mobile phase.
Semipure oxazoline (3, 4.00 g) was isolated from the column and
taken directly to the next step.
Polyplex Formulation and DNA Binding Studies by Gel
Electrophoresis. All polyplex solutions were prepared in DNase/
RNase-free water, unless otherwise specified. Stock solutions of
polymer were prepared in water at a concentration of 15 mM ionizable
amines. The stock solutions were diluted in water to appropriate
concentrations necessary for the desired N/P ratios. Polyplexes were
formulated by adding an equal volume of polymer solution to pDNA
in water. For DNA-binding studies by gel electrophoresis, aqueous
polymer solutions (10 μL) at appropriate concentrations were added
to 10 μL of pDNA solution (50 ng/μL) to make polyplexes at various
N/P values. For example, to achieve N/P = 5, the stock polymer
solution was diluted to 0.75 mM of ionizable amines before adding 10
μL of it to pDNA solution at 50 ng/μL (0.15 mM phosphates).
Polyplex formulations were kept at room temperature to equilibrate
for 1 h before running them on a 0.6% agarose gel containing 0.3 μg/
mL ethidium bromide. Gel electrophoresis was carried out at 80 V for
45 min. The binding of pDNA to cationic polymers resulted in its
retardation on the gel which was visualized and imaged (Figure S10)
with a Fotodyne FOTO/Analyst Luminary/FX workstation from
Fotodyne (Hartland,WI).
Particle Size and Zeta Potential Measurements. The particle
size and zeta potential measurements were carried out by dynamic
light scattering using Zetasizer Nano-ZS from Malvern Instruments
Ltd. (Malvern, U.K.) For particle size measurements, polypexes were
prepared by adding 25 μL of aqueous polymer solutions to 25 μL of
pDNA (20 ng/μL in water). The polymer concentrations were
adjusted to achieve N/P ratios of 2.5, 5, and 7.5, as described earlier.
The mixture was incubated for an hour before adding 100 μL of Opti-
MEM. The sample was analyzed immediately to determine the particle
size at 0 h. Additional measurements were performed after 2 and 4 h to
study the stability of polyplexes over time. Z-average diameters
calculated by the instrument at 0, 2, and 4 h time points are reported.
For zeta potential measurements, 150 μL of aqueous polymer solution
was added to 150 μL pDNA (20 ng/μL in water) to achieve N/P
values of 2.5 and 5. After equilibration at room temperature for an
Synthesis of Acetylated MAGalNAc (5). To a solution of 3 (4.00 g)
in 1,2-dichloroethane (72 mL) under a nitrogen atmosphere was
added 2-hydroxyethylmethacrylamide (4; 5.55 g, 43 mmol) and
camphor sulfonic acid (CSA, 0.333 g, 1.43 mmol) and refluxed for 12
h. After, the solution was cooled to room temperature and neutralized
with triethylamine. The crude was then concentrated under reduced
pressure and dried under high vacuum. The product was then purified
via column chromatography where it was eluted with a 20:1 CHCl3/
MeOH mobile phase. IR (film) 3293, 3078, 2935, 1744, 1658, 1619,
1533, 1433, 1369, 1304, 1221, 1166, 1135, 1074, 1044, 929, 733, 589
1
cm−1; H NMR (500 MHz) δ 6.44 (m, 1H), 5.89 (m, 1H), 5.76 (s,
1H), 5.35 (s, 1H), 5.35 (m, 1H), 5.11 (dd, J = 15, 5 Hz, 1H), 4.62 (d, J
= 5 Hz, 1H), 4.14 (m, 3H), 3.91 (m, 2H), 3.71 (m, 2H), 3.39 (m, 1H),
2.15 (s, 3H), 2.05−1.94 (m, 12H); 13C NMR (125 MHz) δ 171.11,
170.65, 170.46, 170.21, 168.81, 139.65, 120.2, 100.97, 70.83, 70.30,
68.04, 66.57, 61.48, 51.05, 39.12, 23.26, 20.70 (3C), 18.65; HRMS
(ESI) Calcd for C20H30N2O10 + Na, 481.1798; found, 481.1806.
Synthesis of MAGalNAc (6). To a solution of 5 (3.60 g, 8.10 mmol)
in MeOH (300 mL), sodium methoxide (2.72 g, 33.2 mmol) was
added to obtain a solution pH of ∼9. The reaction was stirred for 12 h,
and then the solution was neutralized with Dowex 50W×2 hydrogen
form resin. The resin was filtered and the solution was concentrated
under reduced pressure and dried under vacuum. The product was
then redissolved in water and lyophilized to dryness. No further
purification was required. IR (film) 3306, 2926, 1651, 1613, 1529,
1
1433, 1372, 1310, 1206, 1156, 1115, 1051, 930, 584 cm−1; H NMR
(500 MHz) δ 5.62 (s, 1H), 5.38 (s, 1H), 4.36 (d, J = 10 Hz, 1H), 3.9−
3.5 (m, 8H), 3.45−3.27 (m, 2H), 1.9 (s, 3H), 1.84 (s, 3H); 13C NMR
(125 MHz) δ 174.66, 171.80, 138.89, 121.23, 101.48, 75.05, 70.96,
67.85, 67.72, 60.93, 52.34, 39.36, 22.15, 17.61; HRMS (ESI) Calcd for
C14H24N2O7 + Na, 355.1481; found, 355.1484.
Synthesis and Characterization of MAGalNAc Polymers.
Synthesis of PMAGalNAc62 by RAFT Polymerization. A solution of
CPP (0.00900 g, 0.0320 mmol), MAGalNAc (0.765 g, 2.3 mmol), and
V-501 (8.80 × 10−4 g, 0.00320 mmol) in 4.6 mL of 4:1 H2O/methanol
C
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hour, 600 μL of water was added to each sample. The zeta potential
was measured at a detection angle of 17°.
Luciferase Assay. For luciferase gene expression assays, HepG2 or
HeLa cells were transfected in 24-well plates at a density of 50000
cells/well in 1 mL of DMEM containing 10% FBS. Polyplexes were
prepared an hour before transfection by adding 175 μL of aqueous
polymer solutions at appropriate concentrations to 175 μL of pDNA
(20 ng/μL in water) to achieve desired N/P values (2.5 and 5), as
described earlier. The polymer−pDNA mixtures were vortexed and
kept at room temperature for an hour. Transfections were carried out
24 h after plating. DMEM was aspirated off from each well and cells
were washed with 1× PBS. Polyplexes were diluted two times with
either reduced-serum Opti-MEM or DMEM containing 10% FBS. The
diluted solution (300 μL) was then added to each well. Four hours
after transfection, 1 mL of DMEM containing 10% FBS was added to
each well. A total of 24 h after transfection, culture medium was
replaced with fresh DMEM containing 10% FBS. A total of 48 h after
transfection, the culture medium was aspirated off and the cells were
washed with 1× PBS. The luciferase assay system was purchased from
Promega (Madison, WI). An aliquot (100 μL) of 1× lysis buffer was
added to each well and kept at room temperature for a minimum of 10
min to allow cell lysis to occur. Cell lysate (5 μL) was then pipetted
into an opaque white 96-well plate. After addition of 100 μL of
luciferase substrate, the luminescence was measured using a TECAN
Cell Culture. HepG2 and HeLa cells were cultured in DMEM
containing 10% FBS in 75 cm2 flasks at 37 °C under 5% CO2
atmosphere to maintain physiological pH. The culture medium was
supplemented with Antibiotic−Antimycotic solution from Life
Technologies (Grand Island, NY) at a final concentration of 10 μg/
mL penicillin, 10 μg/mL streptomycin, and 25 ng/mL Fungizone.
Cells were monitored for confluency regularly and passaged as
necessary. For plating, cells were trypsinized and suspended in DMEM
containing 10% FBS. A hemocytometer was used to count cells prior
to plating. Trypan blue was used to distinguish between viable and
dead cells. For MTT and luciferase assays, 50000 viable cells in 1 mL
of DMEM containing 10% FBS were plated per well in 24-well plates.
Cell Viability Assay. Polyplexes were prepared an hour before
transfection by adding 175 μL of aqueous polymer solutions at
appropriate concentrations to 175 μL of pDNA (20 ng/μL in water)
to achieve desired N/P values (2.5, 5, and 7.5), as described earlier.
Prior to transfection, the polymer−pDNA mixtures were vortexed and
incubated at room temperature for an hour. Transfections were carried
out 24 h after plating. The medium was aspirated from each well and
then cells were washed once with 1× PBS. Polyplexes were diluted 2-
fold with Opti-MEM. The diluted solution (300 μL) was then added
to each well. Four hours after incubation at 37 °C and 5% CO2, 1 mL
of DMEM containing 10% FBS was added to each well. At 24 h post-
transfection, the media was replaced with 1 mL of DMEM containing
10% FBS and 0.5 mg/mL MTT. Cells were incubated at 37 °C and 5%
CO2 for an hour and then the medium was aspirated from each well.
The cells were then washed once with 1× PBS, and 600 μL of DMSO
was added to each well to lyse the cells. The plates were placed on an
orbital shaker for 15 min to allow complete cell lysis. An aliquot (200
μL) was removed from each well and transferred to a clear 96-well
plate. Absorbance was measured at 570 nm using a TECAN GENios
GENios Pro microplate reader (Tecan, Mannedorf, Switzerland).
̈
Protein concentration in each sample was measured using Quick Start
Protein Assay Kit from Bio-Rad Laboratories (Hercules, CA).
Biodistribution Studies in Mice. All animal work was carried out
in accordance with IACUC guidelines. Animals were housed in the
Research Animal Resources facility at the University of Minnesota. For
in vivo biodistribution studies, a 40 μg dose of pDNA was delivered
with polymers P(MAGalNAc62-b-AEMA33), P(MAG46-b-AEMA13), or
in vivo-jetPEI via tail-vein injections in C57BL/6J mice. Three mice
were injected for each study group, unless otherwise stated. Polyplexes
were formulated in a 5% dextrose solution. At day 1, mice were given
intraperitoneal injections of 100 μL of 25 mg/mL D-luciferin and
imaged with an IVIS Spectrum Preclinical In Vivo Imaging System
from PerkinElmer Inc. (Waltham, MA). Animals were euthanized
immediately using carbon dioxide, and tissues (liver, left lung, heart,
left kidney, spleen) and blood were collected. All the samples were
flash-frozen in liquid nitrogen and stored at −80 °C until further
processing. DNA was purified from the tissues by phenol−chloroform
extraction. The amount of plasmid DNA in each tissue was determined
by quantitative polymerase chain reaction (qPCR), run by the
University of Minnesota Genomics Center using primers 5′-tgagtact-
tcgaaatgtccgttc-3′ and 5′-gtattcagcccatatcgtttcat-3′. The data are
presented as fold difference from background (tissues from wild-type
C57BL/6J mouse) of pDNA per genome equivalent of total DNA.
To quantify the amount of polymer in various tissues, P-
(MAGalNAc62-b-AEMA33) and P(MAG46-b-AEMA13) were labeled
with a Cy7 fluorophore using Cy7-NHS ester from Lumiprobe
(Hallandale Beach, FL) according to manufacturer’s protocol. The
extent of labeling was calculated based on the absorbance due to Cy7
at 747 nm. Approximately 2% of the amines in both the polymer
samples were found to carry the Cy7 label. Dynamic light scattering of
labeled polymer−DNA complexes showed no significant change from
unlabeled samples (data not shown). Three mice each were injected
with 40 μg of pT2/CAL complexed with the Cy7-labeled polymers at
desired N/P ratios. Mice were euthanized 30 min after injections, and
the tissues (liver, left lung, heart, left kidney, spleen) and blood were
harvested. All the samples were stored at −80 °C. Tissues were imaged
for Cy7 content with the IVIS Spectrum Preclinical In Vivo Imaging
System from PerkinElmer Inc. (Waltham, MA). The background
fluorescence was subtracted from each sample. The distribution data
for individual tissues are presented as a fraction of total fluorescence in
all the harvested samples.
Pro microplate reader from Tecan (Mannedorf, Switzerland). Data
̈
were normalized such that the negative control (cells that were not
transfected) had a cell viability of 100%.
Competitive Inhibition of Cy5-Labeled Asialofetuin. Asialo-
fetuin, a known ligand of ASGPRs, was purchased from Aldrich
(Milwaukee, WI). Cy5-NHS ester from Cyandye (Sunny Isles Beach,
FL) was dissolved in PBS containing 50% DMSO creating a stock
solution of 2.5 mM. The Cy5 labeling reaction contained 0.1 mM ASF
and 0.5 mM Cy5-NHS in a total volume of 1.1 mL. The mixture was
kept on ice for 10 h to allow the reaction to proceed. After 10 h,
protein was purified from the mixture using Amicon Ultra-0.5
centrifugal filters (30 kDa nominal molecular weight limit) by buffer
exchange with Imject purification buffer from Life Technologies
(Grand Island, NY). The buffer exchange was performed to achieve a
million-fold dilution of the original buffer. The final volume (1 mL) of
purified ASF-Cy5 was aliquoted into 100 μL fractions, flash frozen in
liquid nitrogen, and stored at −80 °C until further use. The protein
concentration and Cy5 labeling efficiency were determined by UV−vis
using a NanoDrop 2000c instrument from NanoDrop Products
(Wilmington, DE). The labeling reaction achieved approximately 1.7
Cy5 labels per molecule of ASF, as determined by UV−vis. For
competitive inhibition studies, HepG2 cells were cultured and
trypsinized, as described earlier. Cells were washed once with
DMEM containing 10% FBS to deactivate and remove trypsin/
EDTA. The inhibition experiments were carried out in a volume of 1
mL of DMEM containing 10% FBS, 6 nM ASF-Cy5, and various
concentrations of inhibitors prepared by serial dilutions. Each sample
contained 200000 cells, which were incubated at 37 °C for 2 h in 5%
CO2 environment prior to analysis. Cells were washed twice with PBS
before running them on a BD FACSVerse from BD Biosciences (San
Jose, CA). Measurements were performed in triplicate, and the median
Cy5 intensity of each cell population was used in data analysis. The
median intensity of samples containing no inhibitor was normalized to
100 and the uptake of ASF-Cy5 in all other samples was reported as a
percentage of that value in Figure 4. The IC50 values were estimated by
fitting data to the Hill equation.56
RESULTS AND DISCUSSION
■
Synthesis and Characterization of Diblock Glycopol-
ymers. To create the targeted delivery vehicles, two
methacrylamide-based monomers were selected for controlled
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a
Scheme 1. Synthesis of Methacrylamido N-Acetyl-D-galactosamine
a
Reagents and conditions: (Step 1) Acetic anhydride (Ac2O), 4-dimethylaminopyridine (DMAP), pyridine (yield = 85%). (Step 2) Trimethylsilyl
trifluoromethanesulfonate (TMSOTf), ethylene dichloride (EDC), 50 °C. (Step 3) N-Hydroxyethyl acrylamide (HEMAA; structure shown in the
red box), camphorsulfonic acid (CSA), EDC, 90 °C (overall yield = 54%). (Step 4) Sodium methoxide (NaOCH3), pH = 9.0, methanol (overall
yield = 35%).
a
Scheme 2. Synthesis of GalNAc-Based Diblock Polymers
a
Reagents and conditions: (Step 1) 4-Cyano-4-(propylsulfanylthiocarbonyl) sulfanylpentanoic acid (CPP), 4,4′-azobis(4-cyanopentanoic acid) (also
known as V-501), 4:1 H2O/methanol mixture, 70 °C. (Step 2) V-501, 2-aminoethylmethacrylamide hydrochloride (AEMA·HCl) (structure shown
in the red box), 1 M acetate buffer (pH = 5.2), 70 °C.
polymerization by the Reversible Addition−Fragmentation
Chain Transfer (RAFT) mechanism.57 The first monomer, 2-
aminoethylmethacrylamide (AEMA), was selected to create the
polycation block to facilitate binding to pDNA, and a second
monomer based on GalNAc was designed to create the
ASGPR-targeting functionality. The GalNAc-derived monomer,
methacrylamido N-acetyl-D-galactosamine (denoted as MAG-
alNAc, 6), was synthesized in four steps, as shown in Scheme 1.
The hydroxyl groups of N-acetyl-D-galactosamine (GalNAc, 1)
were first acetylated to create O-acetyl GalNAc (2). The
acetylated product 2 was then treated with trimethylsilyl
trifluoromethanesulfonate (TMSOTf) in dichloroethane at 50
°C to yield 1,2-oxazoline (3).58 The oxazoline was ring-opened
with hydroxyethylmethacrylamide (HEMAA, 4) to synthesize
O-acetyl MAGalNAc (5). Lastly, the O-acetyl groups of 5 were
cleaved by methanolysis at pH = 9 to yield the final
methacrylamido-monomer structure, MAGalNAc (6).
A trithiocarbonate chain transfer agent (CTA), 4-cyano-4-
(propylsulfanylthiocarbonyl) sulfanylpentanoic acid (CPP) was
used to achieve control over radical polymerization. The initial
RAFT studies on MAGalNAc (6) were performed on a 100−
200 mg scale, which lead to approximately 60% conversion in 6
h with low dispersity values (Đ < 1.1). To produce a large batch
of polymer to carry through for numerous analytical and
biological characterizations, 1 g of MAGalNAc (6) was
polymerized in the presence of CPP and a free radical initiator
4,4′-azobis(4-cyanopentanoic acid) in a 4:1 H2O/MeOH
mixture at 70 °C for 6 h. As shown in Scheme 2, the
macroCTA poly(methacrylamido N-acetyl-D-galactosamine) or
PMAGalNAc62 was synthesized in high yield (92%) and low
dispersity (Đ = 1.17) at this larger scale. This macroCTA was
isolated by dialysis against water using a 3.5 kDa MWCO
dialysis bag. The PMAGalNAc62 macroCTA was subsequently
chain-extended with AEMA to produce three diblock polymers
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with AEMA repeat units of 19, 33, and 80 (Table 1). AEMA
polymerizations were performed in 1 M aqueous acetate buffer
(pH = 5.2) to minimize hydrolysis and maintain the
trithiocarbonate chain end groups.
Polyplex Formulation and Size Measurements. To
examine the interactions of pDNA with each of the block
copolycations, polymer solutions were added to pDNA
solutions (50 ng/μL), vortexed, and kept at room temperature
for 1 h. The N/P ratio of each polyplex formulation was
calculated as the molar ratio of ionizable amines on the polymer
(“cationic block”) to phosphates on the pDNA. In the
polymerized form, the average pKa of the AEMA amine groups
is 8.5.44 As a result, 93% of the amines are expected to be in the
protonated form when the polyplexes are formulated in water
and added to cell culture media (pH = 7.4). Polyplex formation
was confirmed by the lack of pDNA migration through agarose
gels (Figure S10). P(MAGalNAc62-b-AEMA19) and P(EG45-b-
AEMA32) significantly retarded the migration of pDNA at N/P
values of 2 and above. P(MAGalNAc62-b-AEMA33) and
P(MAGalNAc62-b-AEMA80) achieved the same effect at N/P
values above 2.5. Based on these results, N/P values of 2.5, 5,
and 7.5 were selected to evaluate polyplex stability and
cytotoxicity.
Polyplex sizes were measured by dynamic light scattering to
study the colloidal stability in biological media over time
(Figure 2). The hydrodynamic diameters of polyplexes at N/P
ratios of 2.5, 5, and 7.5 ranged from 90 to 160 nm when
measured immediately after dilution with reduced-serum Opti-
MEM. The polyplexes formed with P(MAGalNAc62-b-
AEMA19) and P(MAGalNAc62-b-AEMA33) were found to be
more colloidally stable in reduced-serum Opti-MEM compared
to P(EG45-b-AEMA32) polyplexes. After 4 h at room temper-
ature, P(MAGalNAc62-b-AEMA19) and P(MAGalNAc62-b-
AEMA33) polyplexes formulated at N/P of 2.5 and 5 showed
no statistically significant increase in diameter. Similarly,
P(MAG46-AEMA13) polyplexes at N/P of 5 were previously
shown to maintain their size at approximately 100 nm over 4
h.45 In contrast, P(EG45-b-AEMA32) polyplexes exhibited an
increase of 140 and 56 nm during the same time period at N/P
ratios 2.5 and 5, respectively, which could be attributed to the
less bulky nature of the PEG45 block conferring less effective
steric stabilization. P(MAGalNAc62-b-AEMA80) polyplexes
were found to aggregate to a higher extent than polyplexes
formed with shorter cationic blocks. This difference is likely the
result of fewer polymer chains (and, thus, fewer hydrophilic
sugar blocks) present at the same N/P values with this polymer
compared to the shorter cationic block polymers. The presence
of fewer MAGalNAc blocks coating the polyplexes imparts
lower steric hindrance, and thus less protection from
aggregation. This highlights the role of the neutral/hydrophilic
sugar blocks in promoting polyplex colloidal stability.
Table 1. Molecular Weight, Dispersity, and Degree of
Polymerization (DP) of Polymers
a
a
a
a
polymer
PMAGalNAc62
Mn
Đ
MAGalNAc DP AEMA DP
20
23
1.17
1.29
62
62
N/A
19
P(MAGalNAc62-
b-AEMA19)
P(MAGalNAc62-
25
31
6.5
1.32
1.32
62
62
33
80
b-AEMA33)
P(MAGalNAc62-
b-AEMA80)
P(EG45-b-AEMA32)
1.11
1.02
N/A
N/A
32
13
b
P(MAG46-b-AEMA13)
13.1
a
Number-average molecular weight (Mn) in kilodaltons (kDa) and
dispersity (Đ), as determined by SEC using an aqueous eluent of 0.1
M Na2SO4 in 1 wt % acetic acid at a flow rate of 0.3 mL/min on
Eprogen columns [CATSEC1000 (7 μm, 50 × 4.6), CATSEC100 (5
μm, 250 × 4.6), CATSEC300 (5 μm, 250 × 4.6), and CATSEC1000
(7 μm, 250 × 4.6)] with a Wyatt HELEOS II static light scattering
detector (λ = 662 nm) and an Optilab rEX refractometer (λ = 658
b
nm). The synthesis and characterization of P(MAG46-b-AEMA13) has
been reported previously.45
As a control, a PEG-based block polycation was also
prepared by extending a PEG macroCTA (Mn = 2400, 45
repeat units of ethylene glycol) with AEMA (Scheme 3) to
create P(EG45-b-AEMA32). A previously published glucose-
derived block polycation, P(MAG46-b-AEMA13), containing
poly(methacrylamido glucopyranose) neutral block was used as
an additional control.45 These controls were studied to examine
the specificity of sugar blocks in hepatocyte-targeted delivery.
MAGalNAc and PEG block polycations were analyzed by
size-exclusion chromatography (SEC) to measure dispersities
and number-average molecular weights (Table 1). SEC
chromatograms of PMAGalNAc62 and the diblock polymers
were unimodal with moderate dispersities (Đ < 1.35). The
moderate dispersities of the diblock polymers may be attributed
to the monomer-starved conditions accompanying the high
percent conversion (92%) during the synthesis of PMAGal-
NAc62 macroCTA. The number-average molecular weights
were used to calculate degree of polymerizations. Block
polymer compositions were confirmed by comparing 1H
NMR resonances of the MAGalNAc block with those
associated with the AEMA block.
a
Scheme 3. Synthesis of PEG-Based Cationic Polymer P(EG45-b-AEMA32)
a
Reagents and conditions: 4,4′-azobis(4-cyanopentanoic acid) (also known as V-501), 2-aminoethylmethacrylamide hydrochloride (AEMA·HCl), 1
M acetate buffer (pH = 5.2), 70 °C.
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PMAGalNAc and PEG-based block polymers showed similar
cell viabilities compared to untransfected controls; however, a
statistically significant decrease in viability was observed at a N/
P ratio of 7.5. In contrast, earlier work has shown no
cytotoxicity up to a N/P ratio of 10 with P(MAG46-b-
AEMA13) polyplexes. Based on these results and the size
measurements discussed earlier, subsequent gene delivery
studies were carried out at N/P ratios of 2.5 and 5.
Competitive Inhibition of ASGPR-Mediated Uptake.
We hypothesized that incorporation of GalNAc ligands into a
polymeric chain would facilitate multivalent interactions with
the carbohydrate recognition domains of ASGPRs leading to
enhanced binding affinity and internalization in hepatocytes. To
test this hypothesis, the ability of GalNAc-derived glycopol-
ymers to inhibit internalization of asialofetuin (ASF) by a
hepatocyte cell line (HepG2) was examined. Asialofetuin is
known to contain bi- and triantennary ligands that facilitate
cellular binding and ASGPR-mediated uptake.59−62 Compet-
itive inhibition experiments were performed in suspended
HepG2 cells to estimate relative binding affinities of various
ligands including PMAGalNAc62, ASF, GalNAc, galactose,
PMAG62, and glucose as inhibitors. Cells were incubated with
6 nM Cy5-labeled ASF (ASF-Cy5) in DMEM containing 10%
FBS for 2 h at 37 °C in the presence of various concentrations
of the inhibitors. Figure 4 shows the uptake profile of ASF-Cy5
into HepG2 cells, as measured by flow cytometry. PMAGal-
NAc62 effectively inhibited the uptake of Cy5-labeled
asialofetuin at considerably lower concentrations (half-maximal
Figure 2. Hydrodynamic diameters of polyplexes as measured by
dynamic light scattering. Polyplexes were formed by mixing equal
volumes of pDNA and polymer solutions at N/P ratios of 2.5, 5, and
7.5. The mixtures were incubated at room temperature for 1 h before
adding twice the volume of reduced-serum Opti-MEM. The time of
Opti-MEM addition is indicated as 0 h, with additional measurements
reported at 2 and 4 h as mean standard deviation (n = 3). Data were
analyzed by paired t test. The asterisks (*) indicate that the
measurements were statistically different (p < 0.05).
Cell Viability Studies. The viability of HepG2 cells (Figure
3), derived from human hepatocellular carcinoma, and HeLa
Figure 3. HepG2 cell viability as measured by MTT assay. Cells were
transfected with polyplexes formed at N/P ratios of 2.5, 5, and 7.5 in
Opti-MEM. At 24 h post-transfection, cells were incubated with
DMEM containing 10% FBS and 0.5 mg/mL MTT for 15 min,
washed with PBS, and lysed by the addition of DMSO. Data were
normalized to indicate 100% cell viability for the untransfected control
(cells only) and reported as mean standard deviation (n = 3). A
number sign (#) is used to mark measurements that were statistically
different (p < 0.05) from the cells only control.
Figure 4. (A) Competitive inhibition of Cy5-labeled asialofetuin
(ASF-Cy5) uptake by HepG2 cells in the presence of various
compounds: PMAGalNAc62 (blue diamonds), ASF (green triangles),
62*PMAGalNAc62 (PMAGalNAc62 concentration expressed in
GalNAc equivalents; purple crosses), GalNAc (blue dashes), galactose
(orange circles), and glucose (light blue triangles), and PMAG62 (pink
dashes). Data are reported as mean standard deviation (n = 3). (B)
IC50 values estimated by fitting data to the Hill equation. N.D. = not
determined.
cells (Figure S12), derived from human cervical adenocarcino-
ma, following treatment with the polyplex formulations in Opti-
MEM was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay. In general, treatment with
PMAGalNAc and PEG polyplexes resulted in lower HepG2 cell
viability at higher N/P ratios. At N/P ratios of 2.5 and 5, the
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inhibitory concentration, IC50 = 20 nM) than monomeric
GalNAc (IC50 = 1 mM) and asialofetuin (IC50 = 1 μM).
Consistent with earlier reports, IC50 values followed the order
ASF < GalNAc < galactose < glucose.60,63,64 To further
highlight the strong affinity of PMAGalNAc62, we plotted these
data using a “GalNAc equivalent” concentration (i.e.,
concentration of PMAGalNAc62 polymer chains multiplied by
62, denoted as 62*PMAGalNAc62 in Figure 4). The inhibition
by one GalNAc unit in the polymer was comparable to ASF
and much greater than free GalNAc when compared with the
GalNAc equivalent concentration of PMAGalNAc62 chains.
These data suggest that multivalent binding imparts higher
receptor affinity, in accordance with previous findings on the
effect of ligand valency on binding affinity to ASGPRs.50,51,65,66
Previous studies have used ligand-functionalized, radiolabeled
proteins at 4 °C for measuring IC50 values to show that
biantennary and triantennary structures can have 3−4 orders of
magnitude higher binding constants compared to monovalent
structures.50,54 Uptake studies of fluorescently labeled com-
pounds at 37 °C (similar to our current study) have also shown
a similar trend51,66 with biantennary and triantennary ligands,
even though the IC50 values differ between these two
approaches due to differences in experimental design. These
data suggest that a serial arrangement of GalNAc in a block of
62 repeat units also provides high binding affinity due to the
cluster glycoside effect (yielding 5 orders of magnitude higher
affinity than free GalNAc).67 This work is the first report of
using polymerized GalNAc ligands to achieve high ligand−
receptor affinity and provides a facile alternative to the labor-
intensive synthesis of bi- or triantennary structures currently
used for ASGPR targeting.
Transgene Expression in Transfected Cultured Cells.
Transfection efficiency was studied by quantifying expression of
a luciferase reporter gene from each polyplex formulation in cell
culture. HepG2 and HeLa cells were transfected with
polyplexes containing gWiz-luc plasmid (sourced from
Aldevron, Fargo, ND) at N/P ratios of 2.5 and 5. We
hypothesized that ASGPR receptors on HepG2 cells will
facilitate uptake of plasmids complexed with the cationic block
polymers containing polymerized GalNAc ligands. In agree-
ment with our hypothesis, HepG2 cells exhibited higher
luciferase activity than HeLa cells when gWiz-luc was delivered
with GalNAc-derived block polymers (Figure 5). HepG2 cells
display approximately 76000 ASGPRs per cell that can bind
with the ligands in MAGalNAc polyplex formulations.68 The
lack of these receptors on HeLa cells leads to lower gene
uptake, and therefore a lower transgene expression compared to
HepG2 cells. In contrast to MAGalNAc-containing formula-
tions, the P(MAG46-b-AEMA13), P(EG45-b-AEMA32), and
JetPEI controls showed similar or higher gene expression in
HeLa compared to HepG2 cells. Interestingly, efficient gene
delivery is achieved at low N/P ratios (2.5 and 5) compared to
previously reported nontargeted glycopolycations that required
N/P ratios up to 20.21,43,45,69 These data suggest that the
positive charge or excess free polymer may play a more
important role in nonspecific cell surface interactions and
uptake of nontargeted systems compared to the receptor-
mediated uptake mechanism promoted by targeting li-
gands.70,71 When polyplexes were formulated with a mixture
of P(MAGalNAc62-b-AEMA33) and P(EG45-b-AEMA32), the
selectivity toward HepG2 was dose-dependent on P-
(MAGalNAc62-b-AEMA33), which further validates the role of
Figure 5. Comparison of luciferase activity in HepG2 (blue, solid fill)
and HeLa cells (red, diagonal fill). The luciferase assay was performed
48 h after transfection. Relative light units per mg of protein (RLU/mg
protein) are reported as mean
standard deviation (n = 3). The
asterisks indicate statistically different measurements (p < 0.05). The
GalNAc-derived polymers show higher gene expression in HepG2
than HeLa, whereas the nontargeted jetPEI, P(MAG46-b-AEMA13),
and P(EG45-b-AEMA32) vehicles do not show this preference.
GalNAc ligands (Figure S13) in promoting higher efficacy for
gene delivery.
Biodistribution Studies in Mice. Distribution of pDNA
and MAGalNAc block copolycations in mice was studied by
qPCR and fluorescence imaging, respectively. For qPCR
studies, a dose of 40 μg pT2/CAL pDNA was formulated
with P(MAGalNAc62-b-AEMA33) at N/P = 2.5 and adminis-
tered to C57BL/6 mice via tail-vein injection.29 In vivo-jetPEI
was formulated according to manufacturer’s recommendation
(N/P = 8) and used as a control. The nontargeted P(MAG46-b-
AEMA13) polymer was formulated at an N/P of 5 as it showed
negligible cellular uptake at lower N/P ratios in previous
experiments.45 At day 1 after systemic administration of
polyplexes, mice were euthanized to extract DNA from each
tissue. The amount of pDNA in each tissue was determined by
quantitative PCR (qPCR) against the luciferase gene on the
pT2/CAL plasmid (Figure 6). P(MAGalNAc62-b-AEMA33)
formulation at N/P = 2.5 delivered 70-fold higher pDNA to
liver as compared to lungs. In contrast, in vivo-jetPEI showed
similar levels of pDNA in both of these organs, possibly due to
aggregation of nanoparticles following systemic injection
leading to the entrapment in lungs after first-pass circula-
tion.28,29 As a nontargeted control, P(MAG46-b-AEMA13)
showed 4 orders of magnitude lower uptake of pDNA in
liver compared to P(MAGalNAc62-b-AEMA33) or in vivo-
jetPEI. The low amount of pDNA in all tissues is consistent
with the low polyplex uptake at an N/P of 5 seen in a previous
study,45 suggesting that this vehicle type circulates well in vivo
and discourages nonspecific tissue interactions.
To further understand biodistribution of the polyplexes,
P(MAGalNAc62-b-AEMA33) and P(MAG46-b-AEMA13) were
labeled with a Cy7 fluorophore, complexed with 40 μg pDNA,
and administered to mice via tail-vein injections. The time-
course images of fluorescence in whole animal (Figure 7A)
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showed the smallest area over which the fluorescence was
spread. The decreased fluorescence at 3 h and 1 day post-
injection suggests that the polyplexes localize to the liver
quickly and are cleared from the body over time. To better
understand the biodistribution of polyplexes, individual tissues
were harvested 30 min post-injection and imaged to quantify
fluorescence intensity (Figure 7). The results for each tissue
type are presented in Figure 7B as the fraction of the total
measured fluorescence in all harvested tissues. The liver
samples were responsible for approximately 80% of the total
measured fluorescence from P(MAGalNAc62-b-AEMA33),
whereas the P(MAG46-b-AEMA13) formulation resulted in a
lower amount of polymer in the liver (64% of total
fluorescence). The lower amount of P(MAG46-b-AEMA13) in
liver was accounted for by the higher amount present in
kidneys (20% of total fluorescence) compared with P-
(MAGalNAc62-b-AEMA33) (p < 0.05). Because the MAG
block does not interact as strongly with ASGPRs on the liver,
P(MAG46-b-AEMA13) is not sequestered within liver and is
excreted through kidney. The higher localization of P-
(MAGalNAc62-b-AEMA33) in liver tissue compared to P-
(MAG46-b-AEMA13) is in agreement with our hypothesis that
GalNAc ligands on the MAGalNAc block facilitate interactions
with the ASGPRs on liver cells and therefore provides a novel
material for GalNAc-based ASGPR targeting in vivo.
Figure 6. Fold difference from background measured by quantitative
polymerase chain reaction against the pT2/CAL plasmid in various
tissue samples 1 day following systemic delivery: liver (blue circles),
lung (red squares), heart (green triangles), kidney (black diamonds),
and spleen (blue horizontal lines). Data are reported as mean
standard error (n = 3).
CONCLUSION
■
We reported the synthesis and polymerization of a novel N-
acetylgalactosamine-derived monomer (MAGalNAc) that
yielded a series of diblock glycopolycations to improve delivery
specificity through binding ASGPRs on liver hepatocytes. The
MAGalNAc block polycations readily formed polyplexes with
pDNA that exhibited higher colloidal stability in cell culture
media than polyplexes formed with similar polycations
copolymerized from a PEG block. The presence of GalNAc
moieties on the polyplexes promoted cell type-dependent
delivery, with higher transgene expression in HepG2 (a
hepatocyte-derived cell line that displays ASGPRs) as
compared to HeLa cells (which lack ASGPRs). Competitive
inhibition experiments with HepG2 cells revealed relative
binding affinities as follows: PMAGalNAc62 ≫ ASF ≫ GalNAc
> galactose ≫ glucose. PMAGalNAc62 exhibited 2 orders of
magnitude higher binding affinity than ASF and five orders
higher than monomeric GalNAc, which suggests enhanced
binding due to the cluster glycoside effect. Biodistribution
experiments in mice revealed a higher amount of pDNA in liver
as compared to lungs and other organs when delivered with
P(MAGalNAc62-b-AEMA33) at N/P = 2.5. Nontargeted in vivo-
jetPEI showed higher lung uptake in comparison and
P(MAG46-b-AEMA13) showed low internalization by all tissues.
In addition, Cy7-labeled P(MAGalNAc62-b-AEMA33) revealed a
higher fraction of polymer localization to the liver as compared
to the nontargeted P(MAG46-b-AEMA13) control. This study
offers a facile design motif for functionalizing nanomedicines to
enhance delivery specificity to the liver, an organ of wide
interest for drug, siRNA, and gene therapy/genome editing
applications. Further investigation of the effect of blood
components on these polyplexes, kinetics of their systemic
distribution, localization at the cellular level within the liver, and
immunogenicity are in progress and are expected to provide
useful fundamental insights to guide the future design of
efficacious targeted delivery vehicles.
Figure 7. (A) Time-course imaging of fluorescence in live mice after
injection of 40 μg pDNA complexed with Cy7-labled P(MAGalNAc62-
b-AEMA33) at N/P = 2.5. (B) Biodistribution of Cy7-labeled
P(MAGalNAc62-b-AEMA33) at N/P = 2.5 (blue, solid fill) and Cy7-
labeled P(MAG46-b-AEMA13) at N/P = 5 (red, diagonal fill) in various
tissues, as determined by fluorescence imaging 30 min post-
administration. Data are reported as mean
standard deviation (n
= 3). The asterisks (*) indicate that the measurements are statistically
different (p < 0.05).
revealed localization of P(MAGalNAc62-b-AEMA33) to the liver
within 30 min post-injection. The highest total fluorescence
was seen at 1 h post-injection, whereas the image at 30 min
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.5b01555.
■
S
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Biomacromolecules 2014, 15 (5), 1716−1726.
NMR spectra and SEC traces of polymers synthesized in
this study, gel electrophoresis of polyplexes, and
additional cell culture experiments (PDF).
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(3), 237−247.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: treineke@umn.edu.
■
(25) Ingle, N. P.; Xue, L.; Reineke, T. M. Mol. Pharmaceutics 2013,
10 (11), 4120−4135.
Notes
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Drug Discovery 2005, 4 (7), 581−593.
The authors declare no competing financial interest.
(27) Smith, A. E.; Sizovs, A.; Grandinetti, G.; Xue, L.; Reineke, T. M.
Biomacromolecules 2011, 12 (8), 3015−3022.
ACKNOWLEDGMENTS
■
(28) Sharma, V. K.; Thomas, M.; Klibanov, A. M. Biotechnol. Bioeng.
2005, 90 (5), 614−620.
We acknowledge the University of Minnesota and the National
Institutes of Health (NIH 1DP2 OD00666901, NIH/NIDDK
5R01-DK082516, NIH/NICHD 5P01-HD032652) for funding
this work and the University of Minnesota Genomics Center
for running quantitative polymerase chain reactions.
(29) Podetz-Pedersen, K. M.; Bell, J. B.; Steele, T. W.; Wilber, A.;
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