An in vivo evaluation of amphiphilic, biodegradable peptide copolymers as siRNA delivery agents

Article abstract of DOI:10.1016/j.ijpharm.2014.03.011

A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA (short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer was controlled by reaction stoichiometry for the 11.5, 17.2, and 24.6 kDa polypeptides, and the highest molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in increasing potency was found with increasing molecular weight of the polymers examined (at a constant polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery vectors.

Full text of DOI:10.1016/j.ijpharm.2014.03.011

International Journal of Pharmaceutics 466 (2014) 58–67
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
An in vivo evaluation of amphiphilic, biodegradable peptide
copolymers as siRNA delivery agents
a,
b
c
c
d
Stephanie E. Barrett *, Marc T. Abrams , Rob Burke , Brian A. Carr , Louis S. Crocker ,
a
b
d
a
Robert M. Garbaccio , Bonnie J. Howell , Eric A. Kemp , Robert A. Kowtoniuk ,
d
b
d
d
Andrew H. Latham , Karen R. Leander , Anthony M. Leone , Mihir Patel ,
Sergey Pechenov , Nicole T. Pudvah , Sean Riley , Laura Sepp-Lorenzino ,
Eileen S. Walsh , J. Michael Williams , Steven L. Colletti
Department of RNA Medicinal Chemistry, Merck Research Laboratories, Merck & Co., Inc., West Point, PA 19486, USA
Department of RNA Biology, Merck Research Laboratories, Merck & Co., Inc., West Point, PA 19486, USA
d
c
d
b
b
a
a
a
b
c
Department of Pharmacokinetics & Drug Metabolism RNA, Merck Research Laboratories, Merck & Co., Inc., West Point, PA 19486, USA
Department of RNAi Analytical Sciences, Merck Research Laboratories, Merck & Co., Inc., West Point, PA 19486, USA
d
A R T I C L E I N F O
A B S T R A C T
Article history:
A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA
Received 8 December 2013
Received in revised form 28 February 2014
Accepted 2 March 2014
(short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer
was controlled by reaction stoichiometry for the 11.5, 17.2, and 24.6 kDa polypeptides, and the highest
molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide
having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems
designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate
the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in
increasing potency was found with increasing molecular weight of the polymers examined (at a constant
polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these
polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery
vectors.
Available online 05 March 2014
Keywords:
siRNA
RNA delivery
Polymer conjugates
Poly(peptides)
ã 2014 Elsevier B.V. All rights reserved.
1. Introduction
control over the structure of the conjugate (Convertine et al.,
2
009; Rozema et al., 2007; Matsumoto et al., 2008; Gaspar and
Since the discovery of RNA interference (RNAi) by Fire and
Mellow in 1998 (Fire et al., 1998), both academic and industrial
scientists worldwide have been working towards the realization
of siRNA as a therapeutic option for patients. The key challenge
that all scientists face is delivery of the siRNA. Successful systemic
delivery of siRNA into cells depends on the ability of the delivery
vehicle to protect siRNA from degradation while in circulation,
deliver the genetic material to the target cell of interest, and assist
the siRNA with endosomal escape (Fig. 1) (Whitehead et al., 2009;
Stanton and Colletti, 2010; Zimmermann et al., 2006; Convertine
et al., 2009; Itaka et al., 2004; Wang et al., 2008, 2007). Polymer
conjugates have proven to be an effective targeted delivery
strategy, offer small sizes for improved tissue penetration
Duncan, 2009). Various polymer-based carriers have been
developed for this purpose, but most suffer from toxicity derived
from the carrier components, limiting the practical application of
such systems (Gaspar and Duncan, 2009; Fischer et al., 2003;
Morille et al., 2008; Kichler, 2004; Kircheis et al., 2001).
A recent report describing a dynamic polyconjugate delivery
system for siRNA that demonstrated in vivo messenger RNA
(mRNA) knockdown in mouse liver was described by Rozema et al.
(Rozema et al., 2007). This design employed non-degradable
cationic amphiphilic endosomolytic polymers as their backbones
with a reversible disulfide bond to siRNA and pH-sensitive masking
groups to mitigate polycation-associated toxicity. The disulfide
link to the siRNA is thought to be stable in circulation, but to release
siRNA in cells where glutathione concentrations are increased. The
pH-sensitive masking groups (carboxydimethyl maleic anhydride
or CDM) possessed a targeting ligand (N-acetylgalactosamine,
GalNAc) to promote uptake into parenchymal liver cells using the
asialoglycoprotein receptor (ASGPr) or a stealth agent (poly
(
diameters <50 nm), and provide a high degree of chemical
*
Corresponding author. Tel.: +1 215 652 7348; fax: +1 215 652 7310.
E-mail address: stephanie_barrett@merck.com (S. E. Barrett).
http://dx.doi.org/10.1016/j.ijpharm.2014.03.011
378-5173/ã 2014 Elsevier B.V. All rights reserved.
0

S.E. Barrett et al. / International Journal of Pharmaceutics 466 (2014) 58–67
59
Fig. 1. The process of in vivo siRNA delivery using targeted siRNA-polymer conjugates. Components highlighted in red are thought to be the most significant constituent for
that particular step.
(
ethylene glycol), or PEG) to avoid unfavorable interactions in
inevitable polymer degradation that will occur once the system is
in vivo. In this study, siRNA polymer conjugates were prepared
using biodegradable polymers at various molecular weights or
polymer chain lengths to determine the effect of molecular weight
on siRNA delivery. These polymers were conjugated to siRNA, and
masked with CDM-based targeting ligands and PEG that are labile
and triggered by the chemical environment of the endosome and
cytosol. Once released in the acidified endosomal component, the
endosomolytic polymer aids in the endosomal escape of the siRNA
only then to be degraded in vivo.
circulation. Successful delivery of the siRNA cargo depends on
delivery to the target cell of interest, receptor mediated
endocytosis and then endosomal acidification that results in
demasking and endosomal escape. Following disulfide cleavage,
the siRNA in the cytosol is able to load into the RNA-induced
silencing complex (RISC) and engage in RNA interference. In this
delivery system, the polymer plays a crucial role as the vehicle for
the drug (i.e., siRNA), the targeting ligand (GalNAc), and the stealth
agent (PEG) and must be synthesized from biodegradable
components that can be excreted from the body once the delivery
of siRNA to the target cells is complete.
2. Materials and methods
A potential limitation of this design is the non-degradable nature
of the poly(vinyl ether) (PBAVE) endolytic polymers that have the
potential to induce acute and chronic toxicity once demasked. In
contrast to these PBAVE-based systems, the poly(peptides)
employed in this study bear several advantages including the
potential for biodegradability and excretion, as well as control over
monomer incorporation, polymer architecture and polydispersity
2.1. Materials
d
N -Boc-
L-ornithine was obtained from Chem-Impex Interna-
tional (product# 05367).
L
-phenylalanine, triphosgene, n-butyl-
amine, tetrahydrofuran (THF), cyclopentylmethyl ether (CPME),
hexanes, anhydrous N,N-dimethylacetamide (DMA), anhydrous
dimethylsulfoxide (DMSO), dichloromethane (DCM), trifluoro-
acetic acid (TFA), tris(hydroxymethyl) aminomethane (TRIS), 4-
(2-hydroxyethyl) piperazine-1-ethanesulfonic acid, (HEPES), glu-
cose, sucrose, sodium chloride (NaCl), N,N-dimethylformamide
(DMF), lithium chloride (LiCl), poly(styrene) gel permeation
chromatography (GPC) standards, acetonitrile (ACN), tyloxapol
(Triton WR1339), and Streptomyces griseus were purchased from
Sigma–Aldrich. 4-Succinimidyloxycarbonyl-alpha-methyl-alpha
(2-pyridyldithio) toluene (SMPT), the BCA (bicinchoninic acid)
assay kit, and DyLight 650 NHS ester was obtained from Thermo
Scientific. Female Sprague-Dawley rats were purchased from
Charles River. Carboxydimethylmaleic anhydride-N-acetylgalac-
tosamine (CDM-GalNAc) and carboxydimethylmaleic anhydride
poly(ethylene glycol) (CDM-PEG) were prepared according to
literature procedures (Rozema et al., 2007).
(Sunetal.,2011).Byvirtueofalivingpolymerization,thesynthesisof
poly(peptides) allows for tremendous diversity to be built into the
side chain functionality of the polymer (Deming, 2000; Habraken
et al., 2011; Vayaboury et al., 2004), along with improved
maneuverability in chemical space available for improving potency
and reducing toxicity. The poly(peptide) polymers presented herein
were synthesized from N-carboxyanhydrides via ring-opening
polymerization using high-vacuum techniques with a primary
amine initiator (Hadjichristidis et al., 2009; Pickel et al., 2009;
Aliferis et al., 2004; Habraken et al., 2011).
This study assesses the potential of poly(peptides) to serve as
biodegradable delivery vehicles for siRNA. In order to obtain efficient
endosomal escape, it was thought that the appropriate amphiphilicity
or balance of hydrophilic to hydrophobic monomers must be met and
the two monomer components chosen for this initial study were
ornithine and -phenylalanine. Both monomers represent natural
-amino acids, with the potential of being degraded enzymatically in
vivo. Additionally, -ornithine has the potential to undergo
L-
L
a
2.2. Preparation of Boc-
(Scheme 1)
L
-ornithine N-carboxyanhydride (NCA)
L
spontaneous lactamization once in vivo as an alternative degrada-
tionpathway(WeberandMiller,1981).Itwas hypothesizedthatthis
lactamization process could aid in the overall biodegradability of
polymer, which is likely to occur after the polyconjugate had
undergone endocytosis, demasked and affected endosomal escape.
Given the potential for endopeptidase and exopeptidase
degradation, we suspected that polymer molecular weight or
polymer chain length may be an important component of the
structure activity relationship for polymer based siRNA delivery to
balance the need for liver targeting and siRNA delivery with the
A 2 L round bottom was dried in an oven prior to use (oven
temp = 120 C). The glassware was cooled under an inert nitrogen
atmosphere. Boc-L-ornithine (35 g, 151 mmol) was added to the
ꢀ
flask, along with anhydrous THF (1.2 L, 0.13 M). To the slurry was
added a solution of triphosgene (16.6 g, 55.8 mmol, 0.37 equiv-
alents) in anhydrous THF (240 mL, 0.6 M). The reaction was heated
ꢀ
at 50–55 C for 1 h then cooled to ambient temperature. The
remaining solid was removed by filtration washing with 100 mL of
THF. The filtrate was concentrated by vacuum distillation to 350 mL

6
0
S.E. Barrett et al. / International Journal of Pharmaceutics 466 (2014) 58–67
O
Cl
O
Cl
O
Cl
Cl
Cl
Cl
O
NH2
HO
O
O
O
NH
5
0oC, THF
NH
NH
O
O
O
O
Scheme 1. Synthesis of boc-L-ornithine N-carboxyanhydride.
2
.4. General polymer synthesis
Cl
O
Cl
O
Cl
Cl
Cl
Cl
O
O
O
In a typical polymerization, L-Boc-ornithine-N-carboxyanhy-
dride (1 g, 3.87 mmol) and phenylalanine-N-carboxyanhydride
0.170 g, 0.891 mmol, 0.23 equivalents) were placed in an oven-
NH2
O
O
HO
NH
(
dried round-bottom flask and the flask was purged with an
atmosphere of nitrogen. 10 mL of anhydrous DMA (water
content = 68 ppm) was added and the solution was stirred until
Scheme 2. Synthesis of L-phenylalanine N-carboxyanhydride.
it became clear. N-butylamine (11
mL, 0.116 mmol, 0.03 equiv-
alents) was added to the roundbottom as a solution and the entire
ꢁ6
flask was put under vacuum (<10 mmHg). The solution bubbled,
suggesting the release of CO . The solution continued to stir at
2
and the solvent was switched to cyclopentylmethyl ether (CPME,
50 mL). The resulting slurry was cooled to ambient temperature
room temperature overnight. The next day, the solution was clear.
The polymer solution was precipitated in water (20ꢃ reaction
volume) and then filtered. The collected precipitate was frozen and
placed on the lyophilizer for 48 h to dry the product. Table 1 shows
the exact amounts added of each component for each polymeri-
zation. The last line in the table shows the amount added for the
polymer having the highest molecular weight, where a second
3
and stirred under nitrogen overnight. The solid was isolated by
filtration washing with 70 mL of CPME and vacuum dried to give a
white crystalline product (35 g, 92%). Solid was collected and
ꢀ
stored at ꢁ20 C in a sealed bottle. GC/MS was used to confirm the
1
product. H NMR (500 MHz, DMSO-d
6
):
d
9.08 (s,1 H); 6.86 (s,1 H);
4
.44 (t, J = 6.15 Hz,1 H); 2.92 (t, J = 6.41 Hz, 2 H); 1.75 ꢁ 1.67 (m,1 H);
0
aliquot of monomers (denoted as 4 ) was added the following
1.65 ꢁ 1.57 (m, 1 H); 1.51 ꢁ 1.30 (m, 2 H); 1.38 (s, 9 H) .
morning along with an additional 5 mL of DMA and allowed to
polymerize for an additional 8 h at room temperature, under
vacuum. This sequential addition was required to achieve the
highest molecular weight and was prepared according to literature
procedures (Kowtoniuk et al., 2014).
2.3. Preparation of
L
-phenylalanine N-carboxyanhydride (NCA)
(
Scheme 2)
A 1 L round bottom was dried in an oven prior to use (oven
The protected polymer was added to a round bottom and
dissolved in dichloromethane (35 mg/mL polymer). The solid
dissolved readily to give a hazy solution. The solution was stirred at
room temperature under nitrogen. Trifluoroacetic acid was added
to the solution (1:1 dichloromethane:trifluoroacetic acid by
volume). The solution became clear immediately and was allowed
to react for 20 min. The deprotected polymer was obtained as a
trifluoroacetic acid salt after the solvent and volatile byproducts
were removed by vacuum (Scheme 3).
ꢀ
temp = 120 C). The glassware was cooled under an inert nitrogen
atmosphere. Phenylalanine (50.0 g, 303 mmol) was added to the
flask. Anhydrous THF (600 mL, 0.5 M) was charged to give a
ꢀ
suspension of white solid. The mixture was heated to 50 C and
triphosgene (35.9 g, 121 mmol, 0.4 equivalents) was added as a
solid. The suspension was stirred until the reaction was clear
(
ꢂ30 min). The reaction mixture was cooled to ambient temper-
ature then concentrated to an oil. The oil was then slowly poured
into 3 L of hexanes with rapid stirring to yield a white precipitate.
The resulting suspension was capped using aluminum foil and
placed in the freezer for 3 h. The white precipitate was then
filtered via vacuum filtration while maintaining an inert
environment. The white solid was rinsed with hexanes
Table 1
Material requirements for polymerization.
Sample
L
-ornithine NCA
L
-phenylalanine
DMA
(mL)
n-butyl amine
(mL, mmol)
(
3 ꢃ 20 mL) to give the product. The white solid was collected
and dried overnight under vacuum. GC/MS was used to confirm
the product (54.0 g, 94% yield). 1H NMR (500 MHz, CHCl
-d):
(g, mmol)
NCA (g, mmol)
1
2
3
4
1, 3.87
1, 3.87
1, 3.87
1, 3.87
0.170, 0.891
0.170, 0.891
0.170, 0.891
0.170, 0.891
0.131, 0.686
10
10
10
10
5
11, 0.116
3.83, 0.039
1.91, 0.019
1.91, 0.019
0, 0
3
d
7
.37 ꢁ 7.30 (3 H, m); 7.18 (2 H, d, J = 7.21 Hz); 6.23 (1 H, s); 4.53 (1 H,
dd, J = 8.18, 4.21 Hz); 3.27 (1 H, dd, J = 14.14, 4.20 Hz); 3.00 (1 H, dd,
J = 14.13, 8.17 Hz) .
0
4
0.770, 2.98
Scheme 3. Synthesis of linear amphiphilic statistical copolymers by living ring-opening polymerization.

S.E. Barrett et al. / International Journal of Pharmaceutics 466 (2014) 58–67
61
2.5. GPC analysis
buffer for the retained polymer conjugate was exchanged. After
TFF, the final product was concentrated to 0.4–2.0 mg/mL of siRNA
Molecular weight and molecular weight distributions were
and sterile filtered using a 0.2
ꢁ20 C until use (Scheme 4).
m
m PES syringe filter and stored at
ꢀ
estimated using a gel-permeation chromatography (GPC) (Waters
Alliance 2695 Separations Module) system equipped with a TOSOH
TSKgel Alpha 3000 column and a Waters 2414 refractive index
detector. The columns were eluted with dimethylformamide (DMF)
2
.7. siRNA conjugation efficiency
ꢀ
containing lithium chloride (10 mM) (0.5 mL/min) at 40 C. The
Free RNA duplex as well as free RNA duplex-dimer was
determined by aqueous SEC using a GE Healthsciences Superdex
5HR 10/300 column. The mobile phase was composed of 100 mM
molecular weights and molecular weight distributions of poly
amide) polymers were compared to poly(styrene) standards
Sigma–Aldrich).
(
(
7
TRIS with 2 M NaCl, pH 8.4. Total RNA (both free and bound) was
determined by using inductively coupled plasma (ICP) spectrosco-
py. Since the RNA is the only phosphorus containing species in the
formulations, determining the total phosphorus content can be
used to directly determine the total RNA concentration. Once the
free RNA (duplex and duplex-dimer) and total RNA is determined,
the amount of RNA conjugated to the polymer can be calculated (i.
e., conjugation efficiency, see Supporting Information Figures S1
and S2).
2
.6. General polyconjugate synthesis
Step 1: Activation of polymer. Polymer (122 mg, 41 wt% purity
with the major component being TFA) was dissolved in 1.67 mL of
anhydrous DMSO. The solution was mixed until the polymer was
completely dissolved and 150
loxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)
SMPT) in DMSO (1 mg/100 L) was added (corresponding to
.5 wt% with respect to the polymer weight).
Step 2: Activation of oligonucleotide. Amine terminated Sci10
mL of a solution of 4-succinimidy-
toluene
(
1
m
2
.8. Masking efficiency
ApoB oligonucleotide (1 g, 0.0714 mmol) was dissolved in 0.1 M
sodium bicarbonate buffer (20 ml, 50 mg/mL) in a vial with
magnetic stir bar and cooled to 0–5 C in an ice water bath. In a
Total concentrations of CDM-GalNAc and CDM-PEG were
determined using reverse-phase HPLC with mobile phases of
.1% TFA in water and 0.1% TFA in acetonitrile with UV detection.
Rapid demasking of the polymer after injection onto the column
allowed quantitation of CDMs with the polymer removed using a
C18 guard column to prevent chromatographic interference. Free
ꢀ
0
separate vial, N-succinimidyl S-acetylthioacetate (SATA) (83 mg,
0
.357 mmol, 5 equivalents) was dissolved in 0.78 mL of DMSO. The
SATA solution was added over 1 min and the clear, colorless
ꢀ
reaction mixture was stirred at 0–5 C. After 2 h, the reaction
(
i.e., unbound) CDM-GalNAc and CDM-PEG was analyzed by first
mixture was sampled and analyzed by UPLC or HPLC (Dionex
DNApac) for completion of the reaction. Additional SATA can be
added to effect complete conversion of the oligonucleotide (<5%
remaining unreacted). The reaction mixture was purified by
tangential flow filtration (TFF) using water (ꢂ2 L). The retentate
was lyophilized to give a white solid. The recovery was ꢂ95% and
the purity was greater than 70% by UPLC.
filtering through a 10 kDa centrifuge filter followed by analysis
using the same reverse-phase HPLC method. Masking efficiency
was calculated by first calculating the bound RNA, CDM-GalNAc
and CDM-PEG. The polymer molecular weight in combination with
the total amines available for conjugation was then used with the
bound ligands to calculate masking efficiency (see Supporting
Information Fig. S3).
Step 3: Polymer-oligonucleotide conjugation. The entire sample
of activated polymer from Step 1 was diluted with 19.7 mL of
2
.9. Polymer assay
100 mM TRIS, 5% glucose buffer at pH 9 resulting in a final polymer
concentration of ꢂ2.5 mg/mL. 10 mg of oligonucleotide was added
to the activated polymer solution and allowed to react at room
temperature for one hour until the final masking step. In situ, the
primary amine on the polymer is assumed to deprotect the SATA
modified siRNA to produce the free thiol siRNA, which can then
react with the SMPT-modified polymer.
Polymer purity was determined by using a LECO TruSpec N
nitrogen combustion analyzer. Approximately 50 mg of polymer
was combusted in an atmospher using the LECO TruSpec N
software (v1.26). Quantitation of nitrogen was achieved by using
urea as an external standard. Instrument parameters are summa-
rized in the supporting information section (Table S1). Polymer
purity (free base) was calculated as the ratio between the
measured nitrogen content and the stoichiometric amount of
nitrogen.
Step 4: Masking of the polymer conjugate. In a separate vial,
1
03 mg (0.218 mmol, 50 equivalents) of carboxydimethylmaleic
anhydride-N-acetylgalactosamine (CDM-GalNAc) and 444 mg
0.653 mmol, 150 equivalents) of carboxydimethylmaleic anhy-
(
dride poly(ethylene glycol (CDM-PEG) were weighed out. The
siRNA-polymer conjugate solution was then transferred into the
vial containing CDM-GalNAc and CDM-PEG and the resulting
solution was stirred at room temperature for 10 min. The final pH
of the polyconjugate solution was 8.3–8.5.
Step 5: Purification of the polymer conjugate. Tangential flow
filtration (TFF) was used to purify polymer conjugate formulations
of unincorporated components and to exchange buffer to a
pharmaceutically acceptable formulation vehicle using a TFF
system. The TFF filter material was made of regenerated cellulose.
The selection of molecular weight cutoff for these membranes was
chosen with efficiency of purification and retention of polymer
conjugate in mind. The processing parameters included feed
pressure, retentate pressure, crossflow rate and filtrate flux and
were set to allow reproducibility from batch to batch and linear
scaling of the process. Using the difiltration mode of TFF, the
reaction impurities were filtered out into the permeate and the
With polymer purity established, an accurate HPLC standard
was prepared by dissolving the polymer in water. Masked polymer-
conjugated siRNA test articles were treated with dithiothreitol
(
DTT), followed by a two-step dilution with 0.1 N NaOH and 4 M
NaCl dissolved in 10 mM HCl. The final method concentration for
the polymer assay was 0.04 mg/mL (free base). Samples and
standards were analyzed using an Agilent 1100 with an online
NQAD 500 (Quant Technologies) universal detector. All instrument
control and data analyses were performed using the Thermo Atlas
Chromatography Data System (v8.2). Quantitation of nitrogen was
achieved by using an external standard. Method conditions are
summarized in the Supporting Information section (Table S2).
2.10 In vivo experiments
Sci10 ApoB siRNA was utilized in the experiments.
Sci10 ApoB siRNA

6
2
S.E. Barrett et al. / International Journal of Pharmaceutics 466 (2014) 58–67
Scheme 4. Synthesis of siRNA-polymer conjugates.

S.E. Barrett et al. / International Journal of Pharmaceutics 466 (2014) 58–67
63
0
0
5
-amino-iB-CUUUAACAAUUCCUGAAAUTsT-iB-3
(SEQ
ID
flushed with saline via hepatic portal vein, rinsed in saline, and
minced into 8 g pieces. Livers were homogenized in 0.25 M sucrose
with 20 mM HEPES (pH 7.4) at a 1:3 (w/v) ratio using a glass/Teflon
Potter-Elvehjem tissue grinder. The liver homogenates were
centrifuged at 1000 ꢃ g for 10 min using an Avanti J25I rotor
(Beckman) and the post-nuclear supernatant was collected. This
supernatant was then centrifuged at 34,000 ꢃ g for 10 min using
the same rotor, the fat layer and supernatant were discarded, and
the organelle pellet was resuspended in 45% sucrose. Ultracentri-
fuge tubes (Beckman #344058) were prepared with a discontinu-
ous gradient of 14.3% sucrose and 34.5% sucrose, the organelles in
45% sucrose were underlayed beneath the gradient, and then the
NO.:1)
0
0
3
-UsUGAAAUUGUUAAGGACUsUsUsA-5 (SEQ ID NO.:2)
Bases: A – Adenine; U – Uracil; G – Guanine; C – Cytosine; T –
Thymine. Modifications to the sugars: iB – Inverted deoxy abasic;
AGU – 2 Fluoro; T – 2 Deoxy; CU – 2 OCH ; s – phophorothioate
3
0
0
0
linkage between nucleotides.
Doses were administered to rats based on the weight of siRNA.
Five days post intravenous tail vein injection, the Sprague-Dawley
rats were sacrificed and liver tissue samples were immediately
preserved in RNALater (Ambion). Preserved liver tissue was
homogenized and total RNA isolated using a Qiagen bead mill
and the Qiagen miRNA-easy RNA isolation kit following the
manufacturer’s instructions. Liver ApoB mRNA levels were
determined by quantitative reverse transcription polymerase
chain reaction (RT-PCR). Message was amplified from purified
RNA utilizing primers against the mouse ApoB mRNA (Applied
Biosystems). The PCR reaction was run on an ABI 7500 instrument
with a 96-well Fast Block. The ApoB mRNA level was normalized to
the housekeeping genes peptidyl isomerase B (PPIB) and glyceral-
dehyde 3-phosphate dehydrogenase (GAPDH). PPIB and GAPDH
mRNA levels were determined by RT-PCR using a commercial
probe set (Applied Biosytems). Results are expressed as a ratio of
ApoB mRNA/ PPIB / GAPDH mRNA. All mRNA data is expressed
relative to the vehicle control.
ꢀ
tubes were centrifuged at 77,000 ꢃ g for 2 h at 4 C in a SW-28 rotor
(Beckman). Tritosomes were collected from the interface between
the 14.3% and 34.5% sucrose layers, diluted 1:2.5 in 0.25 M sucrose
with 20 mM HEPES, and centrifuged at 28,000 ꢃ g for 30 min at
ꢀ
4 C. The pellet containing the tritosomes was then resuspended in
0.25 M sucrose with 20 mM HEPES, the tritosomes were lysed
using a sonic dismembrator (Fisher) followed by one freeze/thaw
cycle, and the protein concentration was determined using a BCA
assay kit (Pierce #23227) according to the manufacturer’s
instructions. To ensure that the tritosome preparations were
representative of lysosomes, the activity of citrate synthase (a
mitochondrial enzyme) and acid phosphatase (a lysosomal
enzyme) were measured by enzyme assay kits (Sigma #CS0720
and #CS0740, respectively) according to the manufacturer’s
instructions.
Alanine aminotransferase (ALT) was measured using the ADVIA
Chemistry Systems Alanine Aminotransferase (ALT) method,
0
3815151, Rev. A (Berte et al., 2006). All animal studies were
During normal differential centrifugation to isolate specific
organelles, it can often be very difficult to separate lysosomes
from other organelles (especially mitochondria) due to the
organelles having very similar densities. The methods used in
this work are based upon the uptake and trafficking of Tyloxapol
conducted according to approved Institutional Animal Care and
Use Committee (IACUC) protocols.
2.11. Preparation of fluorophore-labeled polymers
(
Triton WR1339) to the lysosomes of hepatocytes, which alter the
density of the lysosomes and enable a very simple isolation of
liver lysosomes using a discontinuous sucrose gradient. Testing
the tritosome preparations for enzyme activity, it was determined
that the tritosomes had no measurable citrate synthase activity
and ꢂ1 unit/mg protein of acid phosphatase activity (average of
multiple batches), thus confirming that the tritosome prepara-
tions are highly representative of lysosomes and not of
mitochondria.
A statistical copolymer of -ornithine and -phenylalanine was
L
L
prepared for these labeling experiments. Here, poly(ethylene
glycol)-amine (2 kDa) was used to initiate the polymerization, and
the polymerization and characterization proceeded according to
the procedure described above. The final polymer showed a
number-average molecular weight of 11,400 g/mol and a PDI
(
polydispersity index) of 1.12 (DP = degree of polymerization,
DPORN = 74, DPPHE = 20).
A solution of polymer (200 mg, 0.0018 mmol) was prepared in
anhydrous DMSO (5 mL, 3.51 mM). Separately, a solution of
DyLight 650 (Thermo Pierce) (1.29 mg,1.28 mmol, 0.07 equivalents)
2
.13. Gel electrophoresis-based polymer degradation assay
was prepared in anhydrous DMSO (0.2 mL, 6.40 mM). The entire
solution of fluorophore was then added to the solution of polymer
and allowed to react at room temperature overnight. The
A cocktail of bacterial proteases from Streptomyces griseus
Sigma #P5147, also called Pronase E) was used as a degradation
(
matrix in addition to the lysosomes prepared above. Fluorescently
labeled free polymer or polyconjugate was spiked into these
matrices at a final polymer concentration of 0.02 mg/mL, with the
matrices diluted to a protein concentration of 1 mg/mL and
buffered to pH 6.0 with 20 mM TRIS. One parent reaction vial was
fluorophore-labeled conjugate was then dialyzed using
a
10,000 g/mol molecular weight cut-off centrifuge dialysis mem-
brane (centrifuged at 3800 ꢃ g = 4600 rpm) against 100 mM TRIS,
pH 9, with 1.5 N NaCl (four times). The labeled polymer was then
dialyzed four additional times using distilled water. Using water,
the polymer was transferred and filtered through a 0.22 um filter
into a pre-weighed scintillation vial, and lyophilized to dryness
yielding a blue powder (85 mg, 43%).
ꢀ
incubated at 37 C and a 10 uL aliquot was removed at each desired
time point (0, 2, 4, 6, and 24 h) and flash frozen on dry ice. After
completion of the incubations, the samples were thawed, each
aliquot was mixed 1:1 (v/v) with dye-free gel loading buffer
(
450 mM TRIS HCl, 12% Glycerol, and 4% sodium dodecyl sulfate
ꢀ
2.12. Lysosomal lysate generation
(SDS) at pH 8.45), and these mixtures were heated at 85 C for
2
min. After cooling, 10 uL of each sample was loaded into the wells
Tritosomes, which are highly representative of lysosomes, were
of a Novex 16% Tricine gel (Invitrogen #EC66955BOX) and run at
125 V for 60 min using a 1X Novex Tricine SDS running buffer
(Invitrogen # LC1675). After running, the gel was quickly rinsed
isolated using a modified protocol based on published methods of
Bagshaw et al. (Bagshaw et al., 2003) Briefly, female Sprague-
Dawley rats (Charles River) were dosed at 450 mg/kg body weight
with tyloxapol (Triton WR1339; Sigma #T0307) prepared in
normal saline via intraperitoneal injection. Five days post-dose,
with distilled H O and then imaged with a FluorChem Q
2
MultiImage III (Alpha Innotech) with the excitation/emission
channel set to Cy5 and using the autoexposure settings within the
AlphaView Q software (Alpha Innotech).
2
animals were sacrificed using CO and livers were harvested,

6
4
S.E. Barrett et al. / International Journal of Pharmaceutics 466 (2014) 58–67
Table 2
Characterization of the (
L
-ornithine:
L-phenylalanine) statistical copolymers.
Sample
Mn of the protected
PDI of the protected polymer Projected Mn after Absolute MW from
DPORN DPPHE
L
-orn:
L
-phe
Wt% purity by
polymer (g/mol)
(from DMF GPC)
deprotection
g/mol)
aqueous GPC (g/mol)
(NMR ratio)
nitrogen analysis
(
1
2
3
19,200
28,300
41,100
64,300
1.11
1.11
1.19
1.09
11,500
17,200
24,600
38,600
7400
11,700
22,400
32,300
77
112
165
258
18
30
40
63
4.17
3.70
4.16
4.12
41
40
nd
43
4
‘
Mn = number ꢁ average molar mass; MW = absolute molecular weight; PDI = polydispersity index; GPC = Gel permeation chromatography; DP = degree of polymerization as
1
determined by H NMR (see Supporting Information for calculations); nd = not determined).
3
. Results and discussion
siRNA cargo. Once the siRNA was added to the polyconjugate, the
remaining available amines were masked with CDM-GalNAc and
CDM-PEG in a 1:3 w/w GalNAc:PEG ratio. These masking groups
were utilized as pH-sensitive moieties that remain on the
polyconjugate throughout in vivo circulation (pH ꢂ7.4), but
demask from the polymer conjugates once they have reached
the acidic endosomal compartment (pH #16 12#6). It was
important that the polymer conjugates were kept at a slightly
basic pH in order to ensure that the pendant amine groups
remained masked with the CDM groups before dosing. The
polymer conjugates were characterized for pH, particle size, siRNA
conjugation efficiencies, masking efficiencies, and measured
polymer:siRNA ratio (Table 3). In order to leverage the chemical
control of this living polymerization to create clear structure-
activity relationships, we chose to adopt rigorous analytical
characterization and purification for each conjugate. The availabil-
ity of measured conjugation efficiency, masking efficiency, and
polymer:siRNA ratio enables clear conclusions to be drawn from
the in vivo data.
3.1. Polymer design and synthesis
To investigate the effect of molecular weight or polymer chain
length of linear amphiphilic copolymer structures on their ability
to knockdown mRNA, we prepared a series of amphiphilic poly(
ornithine: -phenylalanine) statistical copolymers from the ring-
opening polymerization of N-carboxyanhydrides (NCA). This
polymerization method has been previously used to prepare
living polymers under vacuum conditions (Habraken et al., 2011;
Hadjichristidis et al., 2009). In this study, the polymers were
prepared from the addition of a boc-protected
along with -phenylalanine NCA to yield cationic amphiphilic
statistical copolymers in a 4:1 ratio of -ornithine to -phenylala-
nine (see Supporting Information Figs. S4, S5 and Table S3). This
ratio was chosen to mimic the hydrophilic to hydrophobic ratio
used in the synthesis of poly(vinyl ether) polymers in the
preparation of dynamic polyconjugates by Rozema et al. to
enhance the polymer’s ability for endosomal escape (Rozema
et al., 2007). Organic GPC analysis showed that the protected
polymers had a narrow molecular weight distribution (Table 2,
L
-
L
L-ornithine NCA,
L
L
L
Further, the polymer conjugates were dosed into rats to
investigate their potential for siRNA delivery and mRNA knock-
down in liver. From this work, it is clear that an increase in
molecular weight leads to increased potency from polymer
1
PDI < 1.2) and H NMR analysis allowed for the determination of
the final monomer composition. Aqueous GPC of the deprotected
polymers confirmed the molecular weights of the polymer series,
as well as the narrow PDI (Fig. S6). The polymers were also
analyzed for weight percent purity as a function of TFA content. In
these experiments, the purity was determined as a function of
measured nitrogen content and then back calculated to determine
the mass of polymer in a sample. Any additional mass was
attributed to the trifluoroacetic salt component of the polymer. The
obtained percent purity was used as a correction factor in any
further experiments.
conjugates prepared from statistical copolymers of
L-ornithine
and -phenylalanine (in a 4:1 ratio) (Fig. 2, Table 4). Also, the
L
efficacy of mRNA knockdown shows a dose-dependent potency
gain with increasing molecular weight as the siRNA dose of 18 mg/
kg showed greater mRNA knockdown than 3 mg/kg, which in turn
showed greater mRNA knockdown than 1 mg/kg. While small
differences are noted in the measured polymer:siRNA ratios
between conjugates, the range of doses and dose response clearly
support a relationship between molecular weight and in vivo
mRNA knockdown. From Fig. 2, it can be seen that at a dose of 3 mg/
kg polymer conjugates with a number-average molecular weight of
24.6 kDa (polymer:siRNA = 7.9) have an mRNA knockdown greater
than that of a polyconjugate comprised of polymers having a
number-average molecular weight of 11.5 kDa (polymer:siRNA of
5.9) at 18 mg/kg.
3.2. siRNA delivery
In this study, the effect of molecular weight or polymer chain
length of linear statistical copolymers of
L-ornithine and L-
phenylalanine was examined. Based on previous work by Rozema
et al., all polymer conjugates were prepared at a 5:1 w/w polymer:
siRNA where the siRNA was covalently bound to the polymer
backbone via a reversible disulfide bond (Rozema et al., 2007). It is
assumed that once these polymer conjugates reach the target cell
of interest, the disulfide is cleaved intracellularly, releasing the
Furthermore, it should be noted that none of the polymer
conjugates tested showed any signs adverse physical effects at
doses of 1 or 3 mg/kg, nor did they show any signs of liver damage,
relative to a buffer control (Fig. 3). Clinical biochemistry was
performed on the blood of the rats tested, and alanine transami-
nase (ALT) was the marker used to assess toxicity since it is known
Table 3
Characterization of polymer conjugates prepared from (
L
-ornithine:L-phenylalanine) statistical copolymers; nd = not determined.
Sample
DPORN
DPPHE
Projected M
deprotection
g/mol)
n
after
pH
Particle size
(radius, nm)
Measured polymer:siRNA
Ratio (w/w)
siRNA conjugation
efficiency (%)
Masking efficiency (%)
(
1
2
3
4
77
112
165
258
18
30
40
63
11,500
17,200
24,600
38,600
8.3 ꢄ 0.1
8.5 ꢄ 0.1
8.3 ꢄ 0.1
8.3 ꢄ 0.1
23 ꢄ 2
nd
5.9 ꢄ 0.6
nd
98 ꢄ 1
82 ꢄ 0.8
98 ꢄ 1
97 ꢄ 1
50 ꢄ 3
nd
23 ꢄ 2
7.9 ꢄ 0.8
47 ꢄ 2
25 ꢄ 3
7.8 ꢄ 0.8
48 ꢄ 3

S.E. Barrett et al. / International Journal of Pharmaceutics 466 (2014) 58–67
65
100
100
Rat Data (1 mg/kg)
Rat Data (3 mg/kg)
Rat Data (18 mg/kg)
80
50
60
40
0
20
0
Fig. 2. mRNA knockdown data of poly(L-ornithine:
L
-phenylalanine) statistical
copolymers as
a
function of molecular weight. Data are expressed as the
mean ꢄ SEM (each point represents the ApoB mRNA levels of an individual animal).
to rise dramatically in the case of acute liver damage. Since all
groups tested showed no statistical difference in ALT levels at 1,
and 3 mg/kg it was assumed that the polymer conjugates tested
were safe at these doses. The high dose (18 mg/kg) was used as a
means for assessing toxicity of all the polymer conjugates tested.
At this high dose, the lowest molecular weight polyconjugate
showed no rat deaths, with no observable adverse physical effects
in all of the rats dosed with test article. The higher molecular
weight polymer conjugates (24.6 kDa or 38.6 kDa) were found to be
lethal when tested at 18 mg/kg within 24 h of dosing (with the
exception of the 17.2 kDa polyconjugate which was not tested in
this toxicity screen), indicating that although the higher molecular
weight polymer conjugates were more potent, they were also more
toxic. Based on this work, the potential for a defined therapeutic
window has emerged for the polymer conjugates having a
molecular weight of 24,600 g/mol or 38,600 g/mol, which yield
ꢂ70–90% mRNA knockdown data at doses of 1–3 mg/kg with no
toxicity observed at those doses.
Fig. 3. ALT levels of poly(
function of molecular weight and dose, 48 h post-dose. Data are expressed as the
mean ꢄ SEM (each point represents the U/L levels of the individual animals).
L
-ornithine:
L
-phenylalanine) statistical copolymers as a
bacterial protease cocktail mixture was used as a positive control,
since it is a non-specific enzymatic matrix ideal when complete
degradation of protein is desired (Burrell, 1993). This cocktail is
composed of at least ten proteases, including five serine-type
proteases, two zinc endopeptidases, two zinc leucine amino-
peptidases and one zinc carboxypeptidase (Burrell, 1993). In the
degradation experiments involving the protease cocktail matrix,
near complete degradation of the fluorophore-labeled polymer
was observed after 2 h (Fig. 5). It was hypothesized that our
polyconjugates were endocytosed and trafficked to lysosomes
during the normal course of siRNA delivery, so the lysosomal
The increase in mRNA knockdown correlated well with higher
levels of siRNA delivered to the livers of the dosed rats as the
molecular weight of the polymer increased (Fig. 4). In this assay,
whole rat livers were homogenized and examined for siRNA
content. From this study, it was clear that polymer conjugates with
polymers of higher molecular weight delivered more siRNA to the
liver of the rats despite having similar polymer:siRNA (w/w) ratios.
The higher liver levels obtained from polymer conjugates of higher
molecular weight correlate with the ability of these polymer
conjugates to be more potent, as more siRNA was delivered to the
hepatocyte target cells.
0.15
0.10
0.05
3.3. Polymer degradability
0.00
Since the structure of these polyamide-based polymers is
chemically similar to that of a peptide, the potential for these
polymers to be biodegradable was assessed using gel electropho-
resis in both protease cocktail and lysosomal matrices. In these
experiments, a single representative statistical copolymer was
fluorophore-labeled with Dylight 650 for detection purposes. The
Fig. 4. siRNA liver levels of poly(L-ornithine:L-phenylalanine) statistical copolymers
as a function of molecular weight, 48 h post-dose all dosed at 3 mg/kg. Data are
expressed as the mean ꢄ SEM (each point represents the siRNA levels in the liver of
the individual animals).
Table 4
mRNA knockdown data of poly(L-ornithine:L-phenylalanine) statistical copolymers as a function of molecular weight; nd = not determined; n/a = not applicable.
Sample
Projected M
deprotection
n
after
mRNA knockdown
at 1 mg/kg
mRNA knockdown
at 3 mg/kg
mRNA knockdown
at 18 mg/kg
(
g/mol)
1
2
3
4
11,500
17,200
24,600
38,600
7.3 ꢄ 5.1
25 ꢄ 13
65 ꢄ 6
33 ꢄ 7
72 ꢄ 4
88 ꢄ 3
89 ꢄ 1
73 ꢄ 7
nd
n/a
65 ꢄ 9
n/a

6
6
S.E. Barrett et al. / International Journal of Pharmaceutics 466 (2014) 58–67
Fig. 5. A time course degradation study of a fluorescent poly(L-ornithine:L-phenylalanine) statistical copolymer using gel electrophoresis.
Fig. 6. Cartoon depiction of the effect of molecular weight on siRNA delivery.
lysates were used as a matrix with more physiological relevance to
determine what kind of degradation may occur in vivo. The
fluorophore-labeled polymer did show bands consistent with
degradation products after 2 h in the lysosomal matrix. However,
we found that the polymer degradation appeared to be less
efficient in the lysosomal matrix when compared to the protease
cocktail matrix. This finding is consistent with the fact that at a
constant concentration of protein in the matrices (1 mg/mL as
measured by BCA assay), the protease cocktail contains 100%
proteases whereas only a fraction of the protein within the
lysosomal lysates is actually proteases. Taken together, these data
show there is a high probability that these copolymers are
degraded during the process of delivering siRNA in vivo.
4. Conclusion
In summary, polymer conjugates were prepared of biodegrad-
able statistical poly(peptides) composed of a 4:1 ratio of
ornithine and -phenylalanine at a range of molecular weights, in
L-
L
an attempt to decipher the effect of molecular weight or polymer
chain length on siRNA delivery. Using polymers prepared via a
living polymerization enabled the synthesis molecular weight
controlled polymers with a low polydispersity index to facilitate
this comparison. A robust suite of analytical assays and purification
of the final conjugates further strengthens the clarity of these
results. It was found that polymer conjugates show an increase in
efficacy in vivo with an increase in molecular weight when held at
the same polymer:siRNA ratio. In accordance with increasing
potency, these polymer conjugates also show an increase in their
ability to deliver siRNA to the liver of the animals with increasing
molecular weight. In addition, the toxicity of the resulting polymer
conjugates appears to increase as the molecular weight of the
polymer increases, but no toxicity was observed at 1–3 mg/kg
when >90% mRNA knockdown was achieved suggesting the
potential for a defined therapeutic index with these polymer
conjugates. The biodegradability potential of these polymer
conjugates was shown with both protease cocktail and lysosomal
matrices. This work proves the utility of biodegradable polymer
conjugates as delivery vehicles for siRNA. It is hypothesized that
this type of system could be broadly utilized for siRNA delivery to
target liver-specific clinically relevant indications. Future studies
will continue to leverage the incorporation of poly(amides) into
siRNA delivery systems.
The findings of this study show a clear advantage in mRNA
knockdown with polyconjugates composed of increased molecular
weight, even when holding the polymer:siRNA (w/w) ratio constant
(Fig. 6). We have shown that the statistical polymers used herein
have the potential for enzymatic degradation. We hypothesize that
this potential for degradation may provide an explanation for our
observation that in vivo efficacy is dependent on polymer molecular
weight or polymer chain length. This hypothesis assumes that
polymers of increasing molecular weight are more efficient at
promoting endosomal escape of siRNA and that as the polymer
degrades (shortens) it is less competent for delivery. We view the
biodegradability of the polymer backbone as an advantage for this
delivery system and expect that it contributes to the defined
therapeutic window observed in rodents. It is clear that for these
polymer-based siRNA systems, a balance must be met between
molecular weight based efficacy, toxicity and degradation.

S.E. Barrett et al. / International Journal of Pharmaceutics 466 (2014) 58–67
67
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Supplementary data associated with this article can be found, in
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molecular weight, and monomer purity. Journal of Polymer Science, Part A:
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