Engineering buffering and hydrolytic or photolabile charge shifting in a polycarboxybetaine ester gene delivery platform

Article abstract of DOI:10.1021/bm400221r

Polycarboxybetaine esters (PCB-esters) can condense plasmid DNA into nanosized polyplexes for highly effective gene delivery with low toxicity. The design and characterization of tertiary CB-ester monomers and PCB-ester polymers are presented here to study the effects of molecular variation on functions important to nonviral gene transfer. Both buffering capacity and charge-shifting behavior can be tuned by modifying the distance between the charged groups and the ester size or type. A carbon spacer length (CSL) of one was found to bring the pK_a of the tertiary amine into the optimal range for proton buffering. Ester hydrolytic degradation switches this polymer from cationic (DNA binding) to zwitterionic (DNA releasing) form while conferring nontoxicity. To allow rapid and externally controlled degradation, the effect of this charge-switching behavior on DNA release from polyplexes was directly studied with a novel photolabile PCB-nitrobenzyl ester (PCB-NBE). Photoinitiated ester degradation precipitated the rapid release of 72 ± 5% of complexed DNA from PCB-NBE polyplexes. These insights reveal the key parameters important for the PCB-ester platform and the significance of charge switching to an effective and nontoxic nonviral gene delivery platform.

Full text of DOI:10.1021/bm400221r

Article
pubs.acs.org/Biomac
Engineering Buffering and Hydrolytic or Photolabile Charge Shifting
in a Polycarboxybetaine Ester Gene Delivery Platform
Andrew Sinclair, Tao Bai, Louisa R. Carr, Jean-Rene Ella-Menye, Lei Zhang, and Shaoyi Jiang*
Department of Chemical Engineering, University of Washington, Seattle, Washington 98195, United States
S
* Supporting Information
ABSTRACT: Polycarboxybetaine esters (PCB-esters) can
condense plasmid DNA into nanosized polyplexes for highly
effective gene delivery with low toxicity. The design and
characterization of tertiary CB-ester monomers and PCB-ester
polymers are presented here to study the effects of molecular
variation on functions important to nonviral gene transfer.
Both buffering capacity and charge-shifting behavior can be
tuned by modifying the distance between the charged groups
and the ester size or type. A carbon spacer length (CSL) of
one was found to bring the pKa of the tertiary amine into the
optimal range for proton buffering. Ester hydrolytic degradation switches this polymer from cationic (DNA binding) to
zwitterionic (DNA releasing) form while conferring nontoxicity. To allow rapid and externally controlled degradation, the effect
of this charge-switching behavior on DNA release from polyplexes was directly studied with a novel photolabile PCB-nitrobenzyl
ester (PCB-NBE). Photoinitiated ester degradation precipitated the rapid release of 72 5% of complexed DNA from PCB-NBE
polyplexes. These insights reveal the key parameters important for the PCB-ester platform and the significance of charge
switching to an effective and nontoxic nonviral gene delivery platform.
Most of these systems necessarily operate on a principle of
compromiseone function of the vector (e.g., toxicity or DNA
release) is improved at the expense of another (e.g.,
transfection rate or polyplex stability) until a balance is
found. We have developed a gene delivery platform based on
polycarboxybetaines (PCBs) modified with degradable esters,
with the goal of optimizing each function without detriment to
the others. PCB is a zwitterionic polymer that has recently
shown many important biomedical uses.13,14 When the anionic
carboxylate groups in zwitterionic PCB side chains are
esterified, the polymer is rendered cationic and thus able to
bind and condense nucleic acids.15 A tertiary amine can be
introduced in place of the quaternary ammonium to make the
polymer pH-responsive, buffering pH changes during polyplex
trafficking and providing a mechanism via the “proton sponge”
for endosomal escape.16,17 Another key function of this
platform is the hydrolytic conversion of cationic PCB esters
to zwitterionic PCB. The rate of this charge shifting can be
easily modified by the incorporation of different ester leaving
groups and different distances between the ester and the
tertiary amine groups. This appends a “smart” characteristic to
this polymer; DNA is first strongly complexed by cationic PCB-
esters, but is efficiently released when ester degradation reveals
the anionic carboxylate groups, rendering PCB zwitterionic and
nontoxic. Thus, the PCB-ester platform is of great potential
1. INTRODUCTION
The safe and controlled delivery of genes to targeted cells
promises exceptional advancements in clinical disease treat-
ment, next-generation vaccines, and tissue engineering.1−3
Nonviral transfection vectors have gained increasing attention
as capable platforms that mitigate the safety, immunogenicity,
manufacturing, and scalability concerns of viruses.4,5 In
particular, DNA-complexing polymers are highly customizable
and can be engineered with tunable parameters by which to
optimize each of a vector’s duties. For over a decade,
polyethyleneimine (PEI) has been the benchmark in this
class, mediating effective complexation and protection of
anionic nucleic acids by virtue of its plentiful amines.
Unfortunately, PEI remains cationic under physiological
conditions and is not biodegradable, resulting in its well-
known cytotoxicity through cell membrane destabilization.6 An
ideal polymeric vector would condense nucleic acids into stable
polyplexes that can successfully navigate the extracellular
environment and pass through the cell membrane, as well as
protect genes from degradation inside and outside the target
cells and assist with escape from the endosome or trafficking
vesicle. However, many current polymers including PEI fall
short when it comes to two final desired functions: controlled
DNA release and a lack of inherent toxicity.
Many researchers have modified PEI or other well-studied
cationic polymers to reduce their toxicity or boost efficiency.7−9
Others have focused on engineering new polymers with a
rational design or combinatorial chemistry approach, incorpo-
rating environmental responsiveness or biodegradability.10−12
Received: February 11, 2013
Revised: March 25, 2013
Published: March 26, 2013
© 2013 American Chemical Society
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Figure 1. Structures of tertiary (A−D) and quaternary (E) carboxybetaine methacrylate esters designed in this study. Key modifications studied are
color-coded: hydrolytic esters in blue, CSLs in red, and the photodegradable ester in purple. (A) CBMA1-ethyl ester (CB1-EE); (B) CBMA1-t-butyl
ester (CB1-Tbu); (C) CBMA2-ethyl ester (CB2-EE); (D) CBMA2-t-butyl ester (CB2-Tbu); (E) CBMA-o-nitrobenzyl ester (CB-NBE).
Scheme 1. Synthesis of CBMA-o-nitrobenzyl-ester
because its charge-shifting ability remedies the delicate binding-
release compromises of other systems.
degradation step to DNA release deserved further study.
Therefore, we developed a novel photolabile o-nitrobenzyl ester
of polycarboxybetaine (PCB-NBE) to give this platform a UV-
sensitive “switch” for active degradation control. The monomer
structure of CB-NBE is shown in Figure 1E. Similar
photoresponsive linkers have been utilized by others to develop
photodegradable hydrogels20,21 and release DNA from the
surface of gold nanoparticles.22 We have incorporated it into
the multifunctional PCB platform to study how the charge
neutralization caused by ester degradation directly catalyzes
DNA release from a polyplex. The library of tertiary PCB esters
and the photolabile PCB-NBE shows that the rational
adjustment of buffering ability and degradation character can
tune desired functions of PCB-esters and endow then with
“smart” abilities.
Previously, we have demonstrated the in vitro gene transfer
potential of the PCB-ester platform: an optimized tertiary/
quaternary PCB-ethyl-ester copolymer mediated luciferase gene
transfection approximately an order of magnitude higher than
PEI, but without toxicity.18 A similar strategy has also been
applied to DNA vaccine delivery.19 This motivated a
fundamental study of how altering specific characteristics of
the monomers and polymers influenced their functions.
Therefore, additional tertiary monomers were developed in
this study to arrive at a small library of four unique monomers.
Specifically, the side chain length was varied to include either
one or two carbon “spacers” denoted as a carbon spacer length
(CSL) of 1 or 2 between the amine and the carboxylate/ester
groups. Additionally, each chain length was synthesized with
both an ethyl ester and a tert-butyl ester hydrolytic group.
These molecular modifications result in monomers and
resulting polymers with varying proton buffering capacities
and hydrolytic profiles. The monomer structures are pictured in
Figure 1A−D.
2. EXPERIMENTAL SECTION
2.1. Materials. Chemicals used in the synthesis, purification, and
characterization of CB-ester monomers and polymers, and phosphate-
buffered saline (PBS, 138 mM NaCl, 2.7 mM KCl, pH = 7.4) were
purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s Modified
Eagle Medium, fetal bovine serum, nonessential amino acids,
penicillin-streptomycin, and PicoGreen kit were purchased from
While the degraded, zwitterionic form of PCB does not
condense DNA,15,18 the direct contribution of the ester
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Invitrogen Corp (Carlsbad, CA). COS-7 cells were purchased from the
American Tissue Culture Collection (Manassas, VA). All water used
had been purified to 18.2 mU with a Millipore Simplicity water
purification system.
2.2. Synthesis of Tertiary (3°) Carboxybetaine Esters.
Detailed synthesis procedures, schematics, and characterization of
tertiary amine carboxybetaine ester monomers can be found in the
Supporting Information, sections S1−S2.
hydrophilic due to their zwitterionic character.25 Peak areas were
normalized and compared to show hydrolytic rate over 48 h. The
photodegredation rate of the UV-sensitive CBMA-nitrobenzyl-ester
monomer was determined with a similar method. A hand-held UV
lamp emitting 365 nm wavelength light was positioned directly above
an open-top sample of a 1 mg/mL solution of CB-NBE in pH 7.4
NaAc in a shallow 3 cm2 glass dish for a radiation rate of 10 mW cm−2.
A 10 μL sample was removed after each 5 min until a complete shift in
peak areas confirmed photodegredation was complete.
2.3. Synthesis of CB-Nitrobenzyl Ester Monomer (CB-NBE).
To design a photoresponsive ester of PCB able to fully switch from
cationic to zwitterionic upon irradiation, we decided to synthesize an
o-nitrobenzyl CBMA monomer as a starting point. Polymerization of a
photolabile ester monomer ensured each PCB side chain is
functionalized, for a more complete charge shift than postpolymeriza-
tion esterification. The o-nitrobenzyl photoresponsive group was
chosen based on its recent prevalence in photodegradable linker
chemistries for biomedical applications.23,24 The monomer was
synthesized in a two-step process, as shown in Scheme 1. First, 2-
nitrobenzyl-bromoacetate (2) was generated. Bromoacetic acid (11.2
g, 80.6 mmol), p-toluenesulfonic acid (2.78 g, 16.1 mmol), and 2-
nitrobenzyl alcohol (14.8 g, 96.6 mmol) were successively dissolved in
anhydrous toluene (200 mL). The solution was refluxed at 120 °C for
24 h and toluene was removed via evaporation on a rotovap. The
resulting residue was dissolved in ethyl acetate (200 mL), and the
organic solution was washed with H2O (4 × 50 mL). The organic
phase was dried over Na2SO4, after which the solvent was removed
under reduced pressure and the residue was purified by silica gel
chromatography using a gradient of pure hexane to hexane:ethyl
acetate 3:1. The pure product was obtained as a light yellow oil (19.6
2.6. PicoGreen DNA Binding and Release Quantification.
PicoGreen quantitative binding assays were adapted from Green et
al.12 PCBMA-NBE and quaternary PCBMA-EE solutions were
prepared at 1 mg/mL in 25 mM NaAc buffer, pH 5.2. Then, 50
μL/well of 30 μg/mL gWiz-luciferase DNA plasmid was added to each
well of a 96-well plate. The polymer solutions were diluted to
correspond to [polymer/DNA] weight ratios from 1:1 to 20:1 and
added to the DNA samples. Initial DNA concentration in each well
was set to 1 μg/mL and then condensed with PCB-NBE or PCB-EE in
NaAc buffer. Solutions were gently mixed with pipetting and allowed
to sit for 10 min for polyplex formation. Half of each sample was
transferred to another open-top plate and exposed to 365 nm UV light
for 1 h via a hand-held lamp. Then, 100 μL/well of PicoGreen solution
was added. PicoGreen working solution was prepared by diluting 80
μL of the purchased stock into 15.2 mL NaAc buffer. After 5 min, 30
μL/well of polymer−DNA−PicoGreen solution was added to 150 μL/
well of NaAC in black 96-well polystyrene plates. The plate
fluorescence was then measured on a Perkin-Elmer Victor 3 plate
reader using a FITC filter set (excitation 485 nm, emission 535 nm).
The relative fluorescence (RF) was calculated using the following
relationship:
1
g, 71.5 mmol). Yield: 89%. H NMR (300 MHz, CDCl3) δ (ppm):
7.99 (d, 1H, J = 8.0 Hz), 7.59 (m, 1H), 7.43 (m, 2H), 5.49 (s, 2H),
3.89 (s, 2H). 13C NMR (75 MHz, CDCl3) δ (ppm): 166.3, 146.8,
133.7, 130.8, 128.7, 128.5, 124.7, 63.9, 25.3. Following this, CBMA-
nitrobenzyl-bromoacetate (CB-NBE; 4) was synthesized. 2-Nitro-
benzyl-bromoacetate (2; 19.5 g, 71.1 mmol) was dissolved in
anhydrous acetonitrile (150 mL). Dimethylaminoethylmethacrylate
(DMAEMA; 10.8 mL, 63.3 mmol) and hydroquinone (150 mg, 1.36
mmol) were added to the mixture and the solution was stirred at 60
°C for 18 h. The solvent was removed under reduced pressure and
ether (400 mL) was added to the oily residue. The resulting
suspension was stirred for 4 h and the ether phase was decanted. The
process was repeated three times and the residue was concentrated on
the rotovap. The resulting oil was dried further under high vacuum to
afford the pure product as a yellow oil (24.6 g, 57.0 mmol). Material
was protected from light to avoid unintentional degradation. Yield:
90%. 1H NMR (300 MHz, CDCl3) δ (ppm): 8.05 (d, 1H, J = 8.3 Hz),
7.71 (m, 1H), 7.47 (m, 2H), 6.68 (s, 1H), 6.02 (s, 1H), 5.53 (s, 2H),
5.09 (s, 2H), 4.60 (m, 2H), 4.26 (m, 2H), 3.59 (s, 6H), 3.35 (s, 3H),
1.82 (s, 3H).
2.4. Titration of Tertiary CB-Ester Monomers. To titrate the
monomers, 5 μL of each monomer was dissolved in 0.1 M NaCl to a
concentration of 1 mg/mL. The pH of the solution was lowered to 2
with 1 M HCl. Initial titration curves were obtained with sequential 0.1
mL additions of 0.1 M NaOH, with sufficient time between each
addition to allow for pH stabilization. After initial curves were obtained
and regions of buffering were identified, more thorough titration
curves were obtained with additions of 0.02 mL 0.1 M acid or base in
the buffering regions. Buffering capacity was calculated with molar
equivalency.
RF = (F
− Fblank)/(FDNA − Fblank)
sample
(1)
where Fsample is the fluorescence of the polymer−DNA−PicoGreen
sample, Fblank is the fluorescence of a sample with no polymer or DNA
(only PicoGreen), and FDNA is the fluorescence of DNA−PicoGreen
(no polymer), but an equivalent amount of DNA. The binding
percentage was determined by 1 − RF, because DNA bound to
polymer or entrapped in a polyplex does not contribute to dye
fluorescence.12 A standard curve of free DNA concentration was used
to ensure linear correlation between free DNA content and
fluorescence for all measurements and quantify the free DNA
concentration of the samples.
3. RESULTS AND DISCUSSION
To study the influence of molecular variations on the buffering
capacity and degradation behavior of tertiary PCB esters, four
monomers were synthesized for this study. These monomers
are referred to as CB1-EE, CB2-EE, CB1-Tbu, and CB2-Tbu, in
reference to their carbon atom spacing length (CSL) between
the charged groups (1 or 2) and identity of ester leaving group
(2-carbon ethyl ester or 4-carbon tert-butyl).
3.1. Buffering Capacity of Tertiary CB Esters.
Monomers were titrated to identify the pKa of their tertiary
amines, with the goal to evaluate how the chemical structure of
the monomers affects their potential buffering capacities. For
clinical applications, a vector encounters changes in pH as it is
trafficked to its target, which can degrade DNA if it is not
sufficiently protected. Endocytosed materials are exposed to an
increasingly acidic environment; the pH in the endosome is
lowered via proton pumps in the endosomal membrane, from
physiological pH of 7.4 to approximately 5. Therefore, materials
with a pKa and buffering capacity in this endosomally
appropriate range can absorb protons as they are pumped
into the endosome and help to create an osmotic pressure
gradient across the endosomal membrane that may ultimately
lead to membrane rupture and endosomal escape, though this
“proton sponge” mechanism is still being studied.16,17,26,27 One
2.5. Hydrolysis Rates of Tertiary CB-Ester Monomers.
Hydrolytic rates of the tertiary CBMA-ethyl-ester and CBMA-t-
butyl-ester monomers were determined using reverse-phase high
performance liquid chromatography (HPLC), with a C18 column
(Econosil, 250 × 4.6 mm, 5 μm, Alltech, Deerfield, IL, U.S.A.), and a
UV detector (wavelength of 227 nm). Monomers at a concentration of
1 mg/mL in 100 mM sodium phosphate (pH 7.4) or 100 mM sodium
citrate buffer (pH 5.1) were held at 37 °C. A chromatography buffer
solution of 0.50 vol/vol acetonitrile and aqueous sodium phosphate
(100 mM) was used. This buffer caused the monomer to elute based
on hydrophilicity; hydrolyzed monomers are significantly more
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reason for the high transfection efficiency of PEI may be due to
its buffering capacity in this range.28 Dimethylaminoethyl
methacrylate (DMAEMA) has been used as a gene transfer
vector, but mediates inferior transfection to PEI.29,30 The pKa
of its tertiary amine is around 8.4, which is not well-suited for
endosomal buffering.31 Both of these polymers remain
substantially protonated at physiological pH, leading to post-
transfection toxicity.
monomers will be protonated at physiological pH, and the
fraction available for endosomal buffering will greatly increase.
We expect CB esters with this shorter CSL to mediate optimal
DNA protection. Their inclusion in copolymers could extend
the polymer buffering range to a lower pH without resulting in
leftover charge that contributes to toxicity. The pKa of a
posthydrolysis CSL 1 monomer (tertiary CB1) is 8.3, very
similar to that of DMAEMA. Importantly, this implies that after
DNA delivery and transfection, the polymeric residue of the
vector will be almost entirely protonated at the amine and
unprotonated at the carboxylic acid, rendering it zwitterionic
and biocompatible. The size of ester leaving group (ethyl vs
tert-butyl) had no significant effect on the pKa, showing that the
ester size can be changed without sacrificing the desired
buffering range.
3.2. Hydrolytic Degradation Profiles of Tertiary CB
Esters. While others32,33 have incorporated hydrolytically
degradable bonds in polymers, the wide variation in possible
hydrolysis profiles necessitate a more discriminating selection
of ester leaving group. For example, it could be rationalized that
a bond stable at physiological pH (7.4) but labile at the lower
endosomal pH (5.1) would ensure that the DNA stays
protected for cellular uptake. However, such a strategy may
result in DNA release and degradation within the acidic
environment of the endosomal pathway.34 An ester more stable
in acidic conditions may remain intact in the endosome, but if it
is overly labile at a higher pH, it may experience premature
degradation. Therefore, an ester relatively stable at both pH
conditions may be desirable, and it is important to study their
hydrolytic profiles to help identify unsuitable leaving groups
and provide design insight.
The monomers in this study have a tertiary amine like
DMAEMA, but an electron-withdrawing carboxylate ester
connected to each amine. The proximity of this ester group
decreases the pH required to protonate the amine, shifting the
pKa and buffering range down into the pH environment of the
endosome, as compared to DMAEMA. Changing the
separation distance between the amine and ester changes the
extent of this shift. All of these monomers featured either a one-
or two-carbon distance (CSL = 1 or 2) between the ester and
amine and exhibited pKas that were significantly lower than the
pKa of DMAEMA. The four monomers showed pKas in two
distinct subsets: CSL 1 monomers had pKas of 5.5−6 and CSL
2 monomers had pKas of approximately 7. Titration curves and
the molar buffering capacities of the monomers are shown in
Figure 2. These data show that the pKa is sensitive to the
proximity of the electron-withdrawing group down to a one-
carbon difference in distance. The pKa values imply that half of
the amines on the CSL 2 monomers will be protonated
(cationic) at physiological pH, which may aid in DNA
packaging. Conversely, few of the amines in the CSL 1
The hydrolysis of each ester was monitored over a period of
40 h with reverse-phase HPLC. As seen in Figure 3, CB2-EE
and CB1-Tbu monomers exhibited pH-dependent hydrolysis
behavior. At physiological conditions, the CB2-EE hydrolyzed
much more rapidly, reaching more than 80% hydrolysis after 40
h, but only degrading 20% over the same time period at
endosomal pH. Conversely, CB1-Tbu was more stable at
physiological conditions than endosomal ones. The CB1-EE
and CB2-Tbu esters were relatively stable at both physiological
(pH 7.4) and endosomal (pH 5.1) conditions. These data show
that the parameters of the CB-ester platform can be modified to
confer ester stability while maintaining other desirable
functions, such as an optimal buffering range. It is apparent
that both types of molecular design variations, the ester size and
amine/ester CSL, contribute to the stability of these esters. The
protonation state of the amines (dependent on CSL) may thus
play a role. While this could explain the ester stability difference
at physiological pH between CB1-EE (deprotonated amine)
and CB2-EE (protonated amine), a larger library of monomers
is needed to support this hypothesis.
Overly rapid hydrolysis at any relevant pH may sacrifice
polyplex stability. Of the two most stable monomers, CB1-EE
features a more suitable pKa for endosomal buffering. The
hydrolytic product of ethyl esters, ethanol, also is of lowest
toxicity.35 In further platform development, we will use these
insights to design additional multifunctional and nontoxic
polymers and study the potential role played by enzymes in the
cell.
3.3. Transfection and Toxicity of Tertiary PCB Esters.
Tertiary PCB-ester copolymers were used to mediate luciferase
expression in COS-7 cells, and the polymer toxicity was
evaluated. As our previous work found 3:1 to be the optimal
Figure 2. (Top) Titration of monomers, showing their pH buffering
ability, tested in 100 mM NaCl. Region of interest is between pH 5.1
and 7.4. (Bottom) Molar buffering capacity of the tertiary amine in
each monomer. Vertical dashed lines show the pH range present in the
endosome. Curve peaks correspond to the pKa of each monomer.
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Figure 3. Hydrolysis rates of tertiary CB-ester monomers. (Top)
Hydrolytic degradation of PCB-esters at pH 7.4, representative of
physiological pH. (Bottom) Degradation at pH 5.1 under the same
conditions, representative of lowest endosomal pH.
Figure 4. Transfection efficacy and toxicity of tertiary-dominated
PCB-ester copolymers. (Top) Luciferase expression in COS-7 cells
mediated by each tertiary copolymer variation (RLU/mg lysate
protein). (Bottom) Relative toxicity, determined by BCA assay of μg
protein content in mL cell lysate, normalized to medium only.
tertiary to quaternary amine ratio in PCB side chains, all
polymers were generated with this monomer ratio in the
reaction mixture. RAFT polymerization resulted in the
generation of well-controlled molecular weights for each
polymer, but increased MW beyond 10 kDa (up to 40 kDa)
did not result in a statistically significant transfection increase.
Therefore, 10 kDa polymers were used to control for this
parameter. A nitrogen/phosphorus (N/P) ratio of 20 was
found to result in optimal expression for all PCB-based
polymers, while N/P ratios of 5 and 10 were used for the 25
kDa bPEI positive control. Copolymers based on the four
tertiary CB-esters were all able to mediate transfection without
any apparent cytotoxicity. Notably, the two copolymers
featuring ethyl esters (PCB1-EE and PCB2-EE) resulted in
expression about an order of magnitude higher than both those
featuring tert-butyl esters (PCB1-Tbu and PCB2-Tbu) and
bPEI. Transfection and toxicity data are shown in Figure 4. The
higher stability of ethyl esters at the lower endosomal pH
(PCB2-EE) or both pH conditions (PCB1-EE) might play a
role in their improved transfection, potentially by delaying any
DNA release until the polyplexes escape from endosomes.
While more stable esters may complex DNA more efficiently
and protect it for a longer time, substantial hydrolysis delay may
bottleneck gene release. As the charge-switching behavior of
PCB-esters is their key feature, the direct contribution of this
charge switch to DNA release from a polyplex deserved further
study. This motivated the design of a PCB-ester material
capable of rapid degradation upon external stimulus.
nitrobenzyl ester of PCB, was developed to quantitatively
study the charge-switching contribution to DNA release.
Photoconversion of CB-NBE was verified with reverse-phase
HPLC; after 1 h, degradation was complete.
We quantitatively measured DNA binding and release in
PCB-NBE polyplexes with a PicoGreen assay, in which the
fluorescence signal corresponds to the concentration of free
(unbound) DNA over a wide range of magnitudes.12,36 An
initial screening (Figure S2) determined the minimum weight
ratio of PCB-NBE and PCB1-EE required to condense nearly
all DNA (over 95%) was 5:1. As shown in Figure 5, both
PCB1-EE and PCB-NBE at this ratio complexed nearly all the
DNA in each sample, quenching fluorescence. A 365 nm
wavelength UV lamp was held directly above each polyplex
sample for 1 h. This wavelength was chosen because it is least
harmful to cells21 but still catalyzes degradation of the
nitrobenzyl ester. The released DNA in these irradiated
samples bound to the fluorescent dye, restoring fluorescence.
Photoinitiated ester degradation released 72 5% of the DNA
from the PCB-NBE polyplexes as separation of the ester
converted them to zwitterionic PCB. The nonphotosensitive
PCB1-EE released a statistically insignificant amount under the
same conditions, as expected based on its hydrolytic profile.
Previously, the postdegradation form of PCB esters have been
simply shown not to condense DNA15,18 due to their
zwitterionic nature. Here, this photolabile polymer shows that
the charge-switching step plays the key role in releasing DNA
from an entangled polyplex, which is vital for transfection. This
3.4. Controlled Degradation of PCB-NBE and DNA
Release from Polyplexes. PCB-NBE, a photolabile o-
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ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed synthesis procedures and schemes of tertiary and
quaternary carboxybetaine ester monomers, polymerization,
and characterization details, and the in vitro transfection
protocol. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: sjiang@u.washington.edu. Fax: (+01) 206-543-3778.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the National Science Foundation
(DMR 1005699).
■
Figure 5. DNA release from polyplexes by photodegredation of esters.
PCB-NBE polyplexes released 72 5% of their entrapped DNA when
polyplexes were exposed to 365 nm UV light to rapidly degrade side
chain esters. The limited release by the nonphotolabile PCB1-EE
control shows that the polyplex dissolution was due primarily to ester
degradation.
REFERENCES
■
(1) Sheridan, C. Nat. Biotechnol. 2011, 29, 121−128.
(2) Ferraro, B.; Morrow, M. P.; Hutnick, N. A.; Shin, T. H.; Lucke, C.
E.; Weiner, D. B. Clin. Infect. Dis. 2011, 53, 296−302.
(3) Vallet-Regi, M.; Ruiz-Hernandez, E.; Gonzalez, B.; Baeza, A. J.
Biomater. Tissue Eng. 2011, 1, 6−29.
result supports one of the key benefits of the PCB-ester system:
since tunable hydrolysis or photoresponsive “smart” ester
degradation is the primary driver of DNA release, strong initial
binding need not bottleneck eventual polyplex dissociation.
Thus, unlike many other platforms, PCB-esters do not need to
establish a fragile balance between DNA binding and release,
but these parameters can be engineered individually.
(4) Guo, X.; Huang, L. Acc. Chem. Res. 2012, 45, 971−979.
(5) Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2009, 109, 259−302.
(6) Teramura, Y.; Kaneda, Y.; Totani, T.; Iwata, H. Biomaterials 2008,
29, 1345−1355.
(7) Kichler, A. J. Gene Med. 2004, 6, S3−S10.
(8) Zhong, Z.; Feijen, J.; Lok, M. C.; Hennink, W. E.; Christensen, L.
V.; Yockman, J. W.; Kim, Y.-H.; Kim, S. W. Biomacromolecules 2005, 6,
3440−3448.
(9) Arote, R.; Kim, T.-H.; Kim, Y.-K.; Hwang, S.-K.; Jiang, H.-L.;
Song, H.-H.; Nah, J.-W.; Cho, M.-H.; Cho, C.-S. Biomaterials 2007, 28,
735−744.
4. CONCLUSIONS
This study demonstrates how rational molecular design can
tune the functionality of PCB-esters and add “smart”
responsiveness to environmental stimuli. A small library of
CB-esters was designed, synthesized, polymerized and charac-
terized to study the role of small modifications on the
platform’s buffering and hydrolytic characteristics. A carbon
spacer length (CSL) of 1 between the charged groups was
found to shift the pKa of the tertiary amine into the optimal
endosomal buffering range. Ester hydrolysis converts this
polymer from cationic (DNA binding) to zwitterionic (DNA
releasing) form, while conferring nontoxicity. CB1-EE was most
stable at both physiological and endosomal pH, while
possessing the highest buffering capacity. A copolymer
dominated by the CB1-EE monomer mediated transfection
an order of magnitude better than bPEI, with no toxicity. To
further study how the charge-switching feature affects polymer
binding with DNA in polyplexes, a photolabile o-nitrobenzyl
ester of PCB (PCB-NBE) was developed to allow rapid and
externally controlled hydrolysis. Quantitative PicoGreen DNA
binding assays demonstrated that photoinitiated ester degrada-
tion precipitates rapid release of approximately 72% of
complexed DNA from PCB-NBE polyplexes. Further work
will expand the library of CSL-1 nontoxic PCB-esters, and
study the role of enzyme-catalyzed hydrolysis. These
monomers provide crucial insight to the design of buffering
character and charge switching in an effective and nontoxic
nonviral gene delivery platform.
(10) Zhou, J.; Liu, J.; Cheng, C. J.; Patel, T. R.; Weller, C. E.;
Piepmeier, J. M.; Jiang, Z.; Saltzman, W. M. Nat. Mater. 2011, 11, 82−
90.
(11) Miyata, K.; Nishiyama, N.; Kataoka, K. Chem. Soc. Rev. 2012, 41,
2562−2574.
(12) Green, J. J.; Zugates, G. T.; Tedford, N. C.; Huang, Y.-H.;
Griffith, L. G.; Lauffenburger, D. A.; Sawicki, J. A.; Langer, R. S.;
Anderson, D. G. Adv. Mater. 2007, 19, 2836−2842.
(13) Jiang, S.; Cao, Z. Adv. Mater. 2009, 22, 920−932.
(14) Keefe, A. J.; Jiang, S. Nat. Chem. 2012, 4, 59−63.
́
(15) Zhang, Z.; Cheng, G.; Carr, L. R.; Vaisocherova, H.; Chen, S.;
Jiang, S. Biomaterials 2008, 29, 4719−4725.
(16) Sonawane, N. D.; Szoka, F. C.; Verkman, A. S. J. Biol. Chem.
2003, 278, 44826−44831.
(17) Benjaminsen, R. V.; Mattebjerg, M. A.; Henriksen, J. R.;
Moghimi, S. M.; Andresen, T. L. Mol. Ther. 2012, 21, 149−157.
(18) Carr, L. R.; Jiang, S. Biomaterials 2010, 31, 4186−4193.
(19) Zhang, L.; Sinclair, A.; Cao, Z.; Ella-Menye, J.-R.; Xu, X.; Carr,
L. R.; Pun, S. H.; Jiang, S. Hydrolytic Cationic Ester Microparticles for
Highly Efficient DNA Vaccine Delivery. Small 2013, accepted for
publication.
(20) Kloxin, A. M.; Kasko, A. M.; Salinas, C. N.; Anseth, K. S. Science
2009, 324, 59−63.
(21) Adzima, B. J.; Tao, Y.; Kloxin, C. J.; DeForest, C. A.; Anseth, K.
S.; Bowman, C. N. Nat. Chem. 2011, 3, 258−261.
(22) Han, G.; You, C.-C.; Kim, B.-J.; Turingan, R. S.; Forbes, N. S.;
Martin, C. T.; Rotello, V. M. Angew. Chem. 2006, 118, 3237−3241.
(23) Agasti, S. S.; Chompoosor, A.; You, C.-C.; Ghosh, P.; Kim, C.
K.; Rotello, V. M. J. Am. Chem. Soc. 2009, 131, 5728−5729.
(24) Pasparakis, G.; Manouras, T.; Argitis, P.; Vamvakaki, M.
Macromol. Rapid Commun. 2011, 33, 183−198.
1592
dx.doi.org/10.1021/bm400221r | Biomacromolecules 2013, 14, 1587−1593

Biomacromolecules
Article
(25) Cao, Z.; Yu, Q.; Xue, H.; Cheng, G.; Jiang, S. Angew. Chem., Int.
Ed. 2010, 49, 3771−3776.
(26) Wong, S. Y.; Pelet, J. M.; Putnam, D. Prog. Polym. Sci. 2007, 32,
799−837.
(27) Behr, J. P. Chimia 1997, 51 (1), 34−36.
(28) Wang, D.-A.; Narang, A. S.; Kotb, M.; Gaber, A. O.; Miller, D.
D.; Kim, S. W.; Mahato, R. I. Biomacromolecules 2002, 3, 1197−1207.
(29) Chu, D. S. H.; Schellinger, J. G.; Shi, J.; Convertine, A. J.;
Stayton, P. S.; Pun, S. H. Acc. Chem. Res. 2012, 45, 1089−1099.
(30) Mao, B.; Gan, L.-H.; Gan, Y.-Y.; Li, X.; Ravi, P.; Tam, K.-C. J.
Polym. Sci., Part A: Polym. Chem. 2004, 42, 5161−5169.
(31) Van Tomme, S. R.; van Steenbergen, M. J.; De Smedt, S. C.; van
Nostrum, C. F.; Hennink, W. E. Biomaterials 2005, 26, 2129−2135.
(32) Sun, B.; Lynn, D. M. J. Controlled Release 2010, 148, 91−100.
(33) Lynn, D. M.; Langer, R. S. J. Am. Chem. Soc. 2000, 122, 10761−
10768.
(34) Aa, M. A. E. M.; Huth, U. S.; Hafele, S. Y.; Schubert, R.;
̈
Oosting, R. S.; Mastrobattista, E.; Hennink, W. E.; Peschka-Suss, R.;
̈
Koning, G. A.; Crommelin, D. J. A. Pharm. Res. 2007, 24, 1590−1598.
(35) Fortman, J. L.; Chhabra, S.; Mukhopadhyay, A.; Chou, H.; Lee,
T. S.; Steen, E.; Keasling, J. D. Trends Biotechnol. 2008, 26, 375−381.
(36) Sun, S.; Wang, M.; Knupp, S. A.; Soto-Feliciano, Y.; Hu, X.;
Kaplan, D. L.; Langer, R. S.; Anderson, D. G.; Xu, Q. Bioconjugate
Chem. 2012, 23, 135−140.
1593
dx.doi.org/10.1021/bm400221r | Biomacromolecules 2013, 14, 1587−1593