Structure-function assessment of mannosylated poly(β-amino esters) upon targeted antigen presenting cell gene delivery

Article abstract of DOI:10.1021/acs.biomac.5b00062

Antigen presenting cell (APC) gene delivery is a promising avenue for modulating immunological outcomes toward a desired state. Recently, our group developed a delivery methodology to elicit targeted and elevated levels of APC-mediated gene delivery. During these initial studies, we observed APC-specific structure-function relationships with the vectors used during gene delivery that differ from current non-APC cell lines, thus, emphasizing a need to re-evaluate vector-associated parameters in the context of APC gene transfer. Thus, we describe the synthesis and characterization of a second-generation mannosylated poly(β-amino ester) library stratified by molecular weight. To better understand the APC-specific structure-function relationships governing polymeric gene delivery, the library was systematically characterized by (1) polymer molecular weight, (2) relative mannose content, (3) polyplex biophysical properties, and (4) gene delivery efficacy. In this library, polymers with the lowest molecular weight and highest relative mannose content possessed gene delivery transfection efficiencies as good as or better than commercial controls. Among this group, the most effective polymers formed the smallest polymer-plasmid DNA complexes (～300 nm) with moderate charge densities (<10 mV). This convergence in polymer structure and polyplex biophysical properties suggests a unique mode of action and provides a framework within which future APC-targeting polymers can be designed.

Full text of DOI:10.1021/acs.biomac.5b00062

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Article
Structure-function assessment of mannosylated poly(beta-
amino esters) upon targeted antigen presenting cell gene delivery
Charles H. Jones, Mingfu Chen, Akhila Gollakota, Anitha Ravikrishnan, Guojian
Zhang, Sharon Lin, Myles Tan, Chong Cheng, Haiqing Lin, and Blaine A. Pfeifer
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00062 • Publication Date (Web): 07 Apr 2015
Downloaded from http://pubs.acs.org on April 11, 2015
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Structureꢀfunction assessment of mannosylated
poly(betaꢀamino esters) upon targeted antigen
presenting cell gene delivery
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Charles H. Jones‡, Mingfu Chen‡, Akhila Gollakota, Anitha Ravikrishnan, Guojian Zhang,
Sharon Lin, Myles Tan, Chong Cheng, Haiqing Lin, Blaine A. Pfeifer*
Department of Chemical and Biological Engineering, University at Buffalo, The State University
of New York, Buffalo, New York, USA
Keywords: Gene therapy, nonviral vector, cationic polymers, antigen presenting cells, poly(betaꢀ
amino esters)
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ABSTRACT
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Antigen presenting cell (APC) gene delivery is a promising avenue for modulating
immunological outcomes towards a desired state. Recently, our group developed a delivery
methodology to elicit targeted and elevated levels of APCꢀmediated gene delivery. During these
initial studies, we observed APCꢀspecific structureꢀfunction relationships with the vectors used
during gene delivery that differ from current nonꢀAPC cell lines, thus emphasizing a need to reꢀ
evaluate vectorꢀassociated parameters in the context of APC gene transfer. Thus, we describe
the synthesis and characterization of a secondꢀgeneration mannosylated poly(betaꢀamino ester)
library stratified by molecular weight. To better understand the APCꢀspecific structureꢀfunction
relationships governing polymeric gene delivery, the library was systematically characterized by
(1) polymer molecular weight, (2) relative mannose content, (3) polyplex biophysical properties,
and (4) gene delivery efficacy. In this library, polymers with the lowest molecular weight and
highest relative mannose content possessed gene delivery transfection efficiencies as good as or
better than commercial controls. Among this group, the most effective polymers formed the
smallest polymerꢀplasmid DNA complexes (~300 nm) with moderate charge densities (<10 mV).
This convergence in polymer structure and polyplex biophysical properties suggests a unique
mode of action and provides a framework within which future APCꢀtargeting polymers can be
designed.
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INTRODUCTION
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Gene delivery is a therapeutic treatment that is defined as the transport of recombinant
genetic material (DNA and/or RNA) into target cells for the eventual modulation of host cell
expression patterns. This form of treatment has been commercially applied to combat cancer or
restore deficient enzyme activity and has been documented as a strategy for addressing notable
elusive diseases (e.g., HIV).1, 2 Driving the approach is the choice of delivery vector or
technique, including biomaterials (polymers and lipids), biological vehicles (bacteria, viruses,
and other alternatives), and physicallyꢀderived mechanisms (e.g., electroporation). Of particular
interest, cationic polymers (CP) represent a viable gene delivery option due to their notable
design plasticity, low cytotoxicity, and effective delivery outcomes.3ꢀ6 Within this set of vectors,
poly(betaꢀamino esters) (PBAEs) have been used extensively in the successful delivery of
various genetic cargo in both in vitro and in vivo applications.7ꢀ10 Current gene delivery research
has predominately focused on engineering criteria towards optimal uptake and processing that is
applicable for all cell types.1 Designing CPs for universal cellular uptake directs polyplexes
(polymer complexed to plasmid DNA [pDNA]) through generalized mechanisms that result in
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ubiquitous (nonꢀspecific) delivery outcomes when applied in vivo.
Indeed, general
administration can prompt powerful systemic responses that are often critical towards alleviating
diseases that afflict multiple tissue/organ systems but are largely ineffective when targeting
diseases that require spatially acute responses.1
In an effort to provide cell or tissue targeting, numerous strategies have been employed
that include restriction of genetic cargo activity to a desired cell type via tissueꢀspecific
promoters.11 Specificity has also been achieved by engineering the PBAE component via
grafting of cellꢀhoming ligands,12 tuning of the polyplex physical characteristics,13, 14 or the
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inclusion of stimulusꢀdriven release.15 We previously demonstrated the ability to simultaneously
restrict and increase delivery to antigen presenting cells (APCs; macrophages and dendritic cells)
both in vitro and in vivo via novel chemical linkage of mannose to the terminal ends of amineꢀ
capped PBAEs to serve as polymeric genetic vaccine vectors.12, 16 Mannose was selected as the
grafting ligand due to the upregulation of the corresponding receptor (CD206) on the surface of
APCs.17 CD206 is a member of the calciumꢀdependent lectin family that selectively recognizes
polysaccharides (mannose, fucose, and Nꢀacetylglucoseamine) which are commonly present on
pathogens.18 In addition, previous studies have suggested that inclusion of mannose induces an
alternative processing mechanism of the polyplex that is more permissive towards gene delivery
and eventual production of antigenic peptides.12, 19
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During our initial studies using mannosylated PBAEs (PBAEꢀMan), a library of
structurallyꢀdiverse polymers was assessed for optimal gene delivery. Due to the limited number
of polymers assessed and an incomplete understanding of fundamental APCꢀassociated vector
processing steps, limited information was ascertained regarding the polymeric features governing
gene delivery results. However, initial trends suggested that, contrary to the existing paradigm
(as it relates to nonꢀAPCs),1, 3 gene delivery was inversely correlated with molecular weight.
Specifically, the lowest molecular weight polymers provided optimal gene delivery.12 However,
recent studies have highlighted the need for cellꢀspecific evaluation of gene delivery vectors as
structureꢀfunction relationships are not universal.20
Given the contradictory nature of these findings, we designed the present study to analyze
gene delivery outcomes by utilizing a library of stratified molecular weights of the previously
identified optimally performing polymer. In so doing, statistically significant structureꢀfunction
trends emerged that prompt the reconsideration of design parameters for CPꢀbased APC gene
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delivery vectors. In addition, out of necessity to improve the throughput of fundamental polymer
characterization techniques, we developed an NMRꢀbased approach to evaluate polymer
degradation profiles that significantly reduces experimental cost and instrumentation time.
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Scheme 1. Polymer synthesis methodology for mannosylated poly(betaꢀamino esters). (1) Allylꢀ
αꢀDꢀmannopyranoside (ADM) was synthesized as a precursory reaction. (2) Diacrylate and
amine monomers were coꢀpolymerized to synthesize acrylateꢀterminated polymers (D4A4) that
were then (3) amineꢀcapped using an excess of ethylenediamine. (4) Amineꢀcapped D4A4
(D4A4ꢀEDA) molecules were then reacted with ADM to yield mannoseꢀcapped polymers.
MATERIALS AND METHODS
1
Measurements. All HꢀNMR spectra were measured at 500 MHz in dꢀDMSO using a
Varian INOVAꢀ500 spectrometer maintained at 25ºC with tetramethylsilane (TMS) as an
internal reference standard. GPC data were acquired from a Viscotek system equipped with a
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VEꢀ3580 refractive index (RI) detector, a VE 1122 pump, and two mixedꢀbed organic columns
(PASꢀ103M and PASꢀ105M). Dimethylformamide (DMF; HPLC) containing 0.01 M LiBr was
used as a mobile phase with a flow rate of 0.5 mL/min at 55ºC. The GPC instrument was
calibrated through narrowlyꢀdispersed linear polystyrene standards purchased from Varian.
Average hydrodynamic diameters (Dh) and zeta potential of polyplexes were obtained using DLS
on a Zetasizer nanoꢀZS90 (Malvern, Inc.) at 25°C. All experiments were conducted using a 4
mW 633 nm HeNe laser as the light source at a fixed measuring angle of 90° to the incident laser
beam. The correlation decay functions were analyzed by cumulants method coupled with Mie
theory to obtain volume distribution. Microplate experiments were analyzed using a Synergy 4
MultiꢀMode Microplate Reader (BioTek Instruments, Inc.). FACS analysis was conducted using
a Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ).
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Materials. Monomers were purchased from SigmaꢀAldrich (St. Louis, MO). Acetone
(HPLC), DMF (HPLC), DMSO (≥99.7%), heat inactivated FBS, MEM sodium pyruvate,
HEPES buffer, penicillin/streptomycin solution,
D
ꢀ(+)ꢀglucose, and RPMIꢀ1640 were purchased
ꢀ(+)ꢀmannose (cell culture
from Fisher Scientific (Pittsburgh, PA). Allyl alcohol (≥99%),
D
grade) and 4ꢀtoluenesulfonyl chloride (pꢀTsCl) were purchased from SigmaꢀAldrich (St. Louis,
MO). Phosphate buffered saline (PBS) and 3 M sodium acetate (NaOAc) were purchased from
Life Technologies (Grand Island, NY).
Synthesis of Allylꢀαꢀ
Dꢀmannopyranoside (ADM). ADM was synthesized by dissolving
3 g of
Dꢀ(+)ꢀmannose and 18 mg pꢀTsCl in allyl alcohol (20 mL) at 90°C under reflux for 24 h.
The resulting reaction solution was concentrated by vacuum distillation at 35°C.
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Synthesis of Mannosylated Poly(betaꢀamino esters). MannosylatedꢀPBAEs (PBAEꢀ
Man) were generated as before12, 16 except acrylateꢀterminated PBAEs were synthesized using a
modified previous protocol20 in DMSO at fourteen different amine/diacrylate molar ratios (using
1,000 mg amine) for 5 days at 60°C (Schematic 1). Variance of molar ratios permitted tunable
control of the base polymer molecular weight (Table 1). Acrylateꢀterminated polymers were
then reacted with excess ethylenediamine to amineꢀcap the terminal ends. Specifically, acrylateꢀ
terminated polymers were dissolved in DMSO at 167 mg/mL and reacted with 5 M
ethylenediamine (in DMSO) at room temperature for 24 h. Amineꢀcapped polymers were
purified by dialysis followed by evaporation under vacuum. Dialysis procedures were conducted
against acetone using molecular porous membrane tubing (Spectra/Por Dialysis Membrane,
Spectrum Laboratories Inc.) with an approximate molecular weight cut off at 3,500 Da. Amineꢀ
capped PBAEs were then reacted with ADM at a 1:2 molar ratio in DMSO at 90°C for 24 h and
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1
then purified via dialysis as before. Structure and purity of polymers were confirmed using H
NMR spectroscopy.
Degradation Studies. Desiccated PBAE samples were resuspended to a concentration of
20 mg/mL. RPMIꢀ1640 (pH 7.4) and 25 mM NaOAc buffer (pH 5.15) with 50% FBS were used
as the suspension media. Alternatively, for some samples, water (pH 7.4) was used as the
suspension medium. Solutions were sealed in glass vials and incubated at 37ºC with mild
shaking. At predefined time intervals, aliquots of 0.5 mL of polymer solution were withdrawn
1
and fully dried under vacuum. The resulting polymer was dissolved in dꢀDMSO for HꢀNMR
analysis.
1
To perform degradation studies in various buffers, H NMR spectroscopy was utilized to
calculate the relative hydroxyl group generation based on the resonance intensities of the OH
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protons of the degradants at 4.2ꢀ4.5 ppm relative to the 12 CH2 protons at 3.95ꢀ4.05, 2.6ꢀ2.7, and
1.3ꢀ1.4 ppm of the undegraded polymer (Index). This value was then analyzed to yield a relative
degradation metric, which has been termed the Relative Hydroxyl Group Increase (RHGI).
RHGI was calculated by:
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OH
− H
Index
RHGI =
H
Hydroxyl group from the degradation resultant:
OH = I(ꢀ.ꢁꢂꢀ.ꢃ)
Methylene groups (as a reference of hydrogen amount) from the degradation resultant:
H = (I(
) + I(
ꢀ.ꢄꢁꢂꢅ.ꢆꢁ
) + I(
ꢃ.ꢇꢂꢃ.ꢈ
))/12
ꢉ.ꢀꢂꢉ.ꢅ
Index from the original unꢀdegraded polymer:
I(ꢀ.ꢁꢂꢀ.ꢃ)
Index =
(I(
) + I( ) + I(
ꢃ.ꢇꢂꢃ.ꢈ
))/12
ꢉ.ꢀꢂꢉ.ꢅ
ꢀ.ꢄꢁꢂꢅ.ꢆꢁ
Degradation for each polymer was determined by linear regression using the RHGI value of the
7th day divided by two as the as respective halfꢀlife value. For example, P14 in RPMI with serum
has a 7th day RGHI value of 2.3 (SI NMR Degradation Data Set), which when halved is 1.15.
Using regression analysis and other RGHI values, the halfꢀlife is approximately 8 h. For
comparison to established techniques, degradation samples prepared and processed as described
above were withdrawn and fully dried under vacuum, and each resulting polymer sample was
dissolved in DMF for GPC analysis (Figure S1).
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Preparation of Polymer:pDNA Polyplexes. Polyplexes were prepared by electrostatic
interaction between respective PBAEs and pDNA by mixing three different weight ratios (50:1,
100:1, and 200:1) of polymer to 500 ng of pDNA. First, working dilutions of PBAEs were
prepared in 25 mM NaOAc (pH 5.15) to a final concentration of 4 mg/mL. Depending on the
desired weight ratio, differing amounts of PBAE stock solution were mixed with pDNA in 25
mM NaOAc (pH 5.15), gently vortexed, and incubated for 30 min. Taking a 200:1 polyplex as
an example, 125 ꢁL of PBAE stock solution was mixed with 2,500 ng pDNA and 25 mM
NaOAc (pH 5.15), as needed, to provide a final volume of 150 ꢁL, which was then distributed as
500 ng pDNA/well.
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Polyplex Characterization. Using polyplexes prepared as described above, zeta
potential and effective diameter were measured in either PBS (pH 7.4) or 25 mM NaOAc (pH
5.15). To assess polyplex serum stability via monitoring particle diameter changes over time,
200:1 polyplexes were incubated in either PBS (pH 7.4) or 25 mM NaOAc (pH 5.15) that each
contained 50% v/v FBS. Polyplexes were analyzed for effective diameters monitored every 15
minutes and these values were then standardized by their respective serumꢀfree diameter. All
data points result from three independently formulated polyplex samples measured five times per
sample.
Gene Delivery Assessments. For gene delivery experiments, a RAW264.7 cell line
(kindly provided by Dr. Terry Connell, Department of Microbiology and Immunology,
University at Buffalo) was cultured in growth medium (50 mL of FBS (heat inactivated), 5 mL
of 100 mM MEM sodium pyruvate, 5 mL of 1 M HEPES buffer, 5 mL of penicillin/streptomycin
solution, and 1.25 g of Dꢀ(+)ꢀglucose added to 500 mL RPMIꢀ1640 and filter sterilized) in T75
flasks at 37°C/5% CO2 prior to seeding at a density of 3 × 104 cells/well in clear, tissue cultureꢀ
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treated, sterile, polystyrene 96ꢀwell plates in 100 ꢁL growth medium/well and allowed 24 h for
attachment. Following the 24 h incubation, the original medium was removed and replaced with
100 ꢁL fresh growth medium plus 30 ꢁL of PBAE:pDNA polyplexes, prepared as described
above (total volume of 130 ꢁL), and incubated for an additional 4 h. Polyplexꢀcontaining media
was then removed using a 12ꢀchannel aspirating wand and replaced with 100 µL fresh,
antibioticꢀfree growth medium preheated to 36°C. After 48 h (72 h after initial seeding), plates
were analyzed for transfection efficiency using flow cytometry. To conduct flow cytometry
assessment, cells were first washed with iceꢀcold PBS and detached from the well surface using
cell scrapers. The fraction of EGFPꢀexpressing cells was quantified using gating based upon
transfections using pDNA not encoding EGFP (negative control). Commercial controls,
FuGENE HD, Xfect, and JETꢀPEI were utilized according to different manufacturer’s
instructions. Results derive from four independent experiments and twelve replicates per
experiment.
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To determine if transfection was affected by the presence of free mannose and
physiological levels of serum, polyplexes were prepared at a 200:1 PBAEꢀtoꢀpDNA weight ratio.
Vectors were then transfected as before with the following alterations. Prior to transfection, cell
media was replaced with growth medium containing 1 mM mannose or with 50% v/v FBS,
incubated 30 minutes, and transfected as before. After the 4 h transfection incubation, the altered
media was replaced with regular growth media and assessed for gene delivery as described
above.
Mannose and serum sensitivities of gene delivery (as described in the main text) were
calculated using the following formula:
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GD − IGD
Sensitivity =
GD
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Where, GD = Uninhibited Gene Delivery and IGD = Inhibited Gene Delivery
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Statistical Analysis. Unless otherwise indicated, data presented were generated from
three independent experiments. Error bars represent standard deviation values. All statistical
significance comparisons between groups were performed using a oneꢀway ANOVA with
Dunnett (to compare within groups) postꢀtests.
RESULTS AND DISCUSSION
Polymer Synthesis and Characterization. Polymers were synthesized using our
recently developed mannose endꢀcapping strategy (Scheme 1).12 However, unlike before, the
ratio of diacrylate to amine monomers (D:A ratio) used in the synthesis of the base polymer
(Scheme 1, Step 2) was systematically varied. Variation of the D:A ratio was used to generate
stratified molecular weight polymers with the same chemical background. Each respective
1
PBAE structure and purity was confirmed using H NMR (Table 1). Gel permeation
chromatography (GPC) and dynamic light scattering (DLS) were used to measure molecular
weight, polydispersity index (PDI), and zeta potential (in two buffers) of the polymers (Table 1;
Figure 1A). The generated polymer library spanned a molecular weight (weighted) range from
6.8 to 33.7 kDa with PDI values (~1.4ꢀ2.5) characteristic of PBAEs. Furthermore, zeta potential
values of the polymer library increased with increasing molecular weight regardless of buffer
(Figure 1B and C).
Table 1. Polymer Synthesis and Characterization.
D:A
Ratio
Zeta Potential (mV) Zeta Potential (mV)
PBS (pH 7.4) NaOAc (pH 5.15)
PDIGPC
Polymer
MnGPC (Da)
MwGPC (Da)
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P1
P2
1.025
1.0375
1.05
2.477
1.641
1.536
1.370
1.629
1.642
1.833
1.468
1.699
1.549
1.590
1.600
1.638
1.615
13,623
13,425
11,961
11,848
10,474
10,130
7,902
7,312
6,589
5,966
5,510
5,112
4,673
4,479
33,745
22,031
18,372
16,232
17,063
16,633
14,484
10,734
11,195
9,241
-8.6
-5.3
32.6
34.0
33.6
32.4
32.7
34.6
31.9
27.5
28.6
24.9
26.4
24.0
20.9
21.4
4
5
6
7
8
9
P3
-6.2
P4
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1.075
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P5
-7.7
P6
-8.8
P7
1.125
1.15
-8.0
P8
-11.1
-12.1
-11.2
-11.4
-12.7
-12.9
-11.2
P9
1.175
1.2
P10
P11
P12
P13
P14
1.225
1.25
8,761
8,180
1.275
1.3
7,654
7,234
Polymer Degradation Studies. A prerequisite for the translational usage of biomaterialꢀ
based delivery vectors is the successful demonstration of a balanced degradation profile. In
particular, the degradation profiles of CPs play a critical role in predicting potential circulation
residency time, genetic cargo unpackaging, gene delivery efficiency, and vectorꢀassociated
cytotoxicity.1 Premature degradation profiles, for example, indicate potential fragility of
polyplexes; whereas, extended stability may result in the inability to unpackage cargo in addition
to increased cytotoxicity.1, 3, 21 Previously, we developed methodology that more accurately
assessed degradation profiles in media that mimicked physiologicallyꢀrelevant settings.12
Specifically, polymers were incubated for various time points in water (pH 7.4; traditional
degradation buffer) or either RPMI (pH 7.4; mimics the circulation/extracellular environment) or
25 mM NaOAc (pH 5.15; mimics the lysosome) with and without serum. However, a limitation
of the previous technique was the reliance on GPC, which introduced time and cost constraints.
For example, use of GPC for degradation studies requires regimented sample preparation, an
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hour instrumentation time per sample, and is not able to distinguish false positives or failures
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Table 2. Polymer Degradation in Various Buffers.
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Half-life (h)
RPMI w/ Serum
(pH 7.4)
25 mM NaOAc
w/ Serum (pH 5.15)
Polymer
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Taking into account the limitations of the previous methodology, we developed an NMRꢀ
based alternative that significantly reduces timeꢀ and costꢀdemands of degradation studies. In
doing so, a new variable (Relative Hydroxyl Group Increase [RHGI]) was developed and used in
the context of physiologicalꢀrelevant buffers to calculate the relative halfꢀlife of each polymer
(Table 2). The results indicate that degradation was independent of molecular weight and
primarily influenced by buffer pH.
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Figure 1. Polymer characterization data. (A) Numbered polymer molecular weight as a function
of diacrylate (D4) to amine (A4) monomer feed ratios. Data points have been labeled with the
associated polymer name. Zeta potential measurements in either PBS (B) or 25 mM NaOAc (C)
relative to numbered polymer molecular weight.
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Utilization of the RGHI methodology provides numerous advantages and opportunities
for timeꢀ and costꢀreductions (Figure S1). Specifically, using our 140 polymer samples (14
polymers × 5 time points × 2 buffers = 140) as an example, the traditional GPC method requires
140 h (1 h/sample) and $2,800 ($20/sample [University at Buffalo Instrumentation quote]). In
comparison, using the RGHI method reduces these requirements to 12 h and $700 and provides
time and cost reductions that range from 12ꢀ18× and 2ꢀ4×, respectively.
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Polyplex Preparation and Characterization.
To form polyplexes, three
polymer:pDNA weight ratios were selected based upon our previous report.12 Specifically, 50:1,
100:1, and 200:1 were used in initial gene delivery experiments; whereas, only 200:1 was used
during inhibition and structureꢀfunction studies (to be described later).
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Figure 2. Characterization summary of D4A4ꢀMan polyplexes. Polyplex diameter (A & B) and
zeta potential (C & D) in 25 mM NaOAc (left panels) or PBS (right panels). All panel B values
were significantly lower (95% confidence) than corresponding values in panel A.
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To assess the biophysical properties associated with polyplexes, DLS was used to
measure the particle effective diameter and zeta potential in both 25 mM NaOAc and PBS
(Figure 2). Polyplexes ranged in size from 300ꢀ1,000 nm, increasing with higher molecular
weights and (in most cases) polymer:pDNA weight ratios. Neutral pH conditions (PBS)
prompted statistically significant tighter condensation of polyplexes (Figure 2B), presumably due
to the lack of excessive protonation (which may promote aggregation1, 3) and neutralization of all
remaining charges by bound pDNA. Furthermore, polymer molecular weight was linearly
correlated with increasing polyplex zeta potential and polyplex size (Figure S2A). In addition,
for each unit molecular weight required to complex pDNA, there is a linear increase in mannose
content (each individual polymer chain is capped with two mannose molecules) that may further
influence condensation and zeta potential of polyplexes. When relative mannose content is taken
into account, higher mannose content (associated with smaller polymer chains) also leads to
reduced zeta potential and polyplex diameter values (Figure S2B).
As a last form of assessment, polyplexes were prepared as described above, incubated in
physiologicallyꢀrelevant media, and monitored for changes in relative diameter over time to
determine polyplex stability (Figure S3). Upon incubation in 25 mM NaOAc containing 50%
v/v serum (Figure S3A), relative polyplex diameters increased with respect to time for most
polymers. The upperꢀhalf molecular weight polymers (P1ꢀP7) displayed the largest relative
changes in polyplex diameter. This observation presumably is the result of excessive protonation
which may mediate nonꢀspecific serum deposition or polyplex aggregation. Conversely, the
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lowerꢀhalf molecular weight polymers (with exception to P8 and P10) did not display any
appreciable changes in diameter over time. Upon incubation in PBS containing 50% v/v serum
(Figure S3B), only P1 and P7 demonstrated changes in polyplex diameter. All other polymers
mediated the formation of smaller polyplexes. This may be the result of a shielding effect
conferred by the attachment of mannose combined with increased colloidal pressure (derived
from increased negativelyꢀcharged serum) driving the compaction of polyplexes.
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Gene Delivery Studies. Previously, we identified that mannosylation (either by
chemical grafting or free addition) positively affected gene delivery in direct response to binding
and processing through a CD206ꢀdependent mechanism.12 However, despite successful
transfection data, clear molecular weight trends were not observed due to the narrow range of
molecular weights evaluated. Thus, the purpose of the present study is the systematic analysis of
molecular weight upon APCꢀmediated gene delivery. Furthermore, polyplexes formed using
three polymer:pDNA ratios were compared to select commercial controls (representing distinct
categories of gene delivery vectors – lipidꢀbased [FuGENE HD], unmodified CP [Xfect],
mannosylated CP [JETꢀPEI]) during gene delivery to a murine macrophage cell line known to
express CD206 (Figure 3).22ꢀ24 Transfection efficiency of polyplexes was found to correlate
positively with increasing polymer:pDNA ratios (Figure 3A). Unexpectedly, for the highest
polymer:pDNA ratio, gene delivery increased linearly with decreasing polymer molecular
weight; the lowest molecular weight polymer (P14) mediated the highest gene delivery at a 200:1
ratio (Figure 3A and B). This finding is interesting because it is commonly accepted that higher
molecular weight polymers mediate the highest gene delivery.1 However, previous studies were
not applied to APCs, which are noted for their unique processing of external cargo.12
Accordingly, it is possible that the mannoseꢀmediated uptake directs polyplexes toward
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endocytic mechanisms that are more permissive to gene delivery by lower molecular weight
polymers. Alternatively, gene delivery improvements may be explained by a sizeꢀexclusion
effect. Specifically, polyplexes larger than 500 nm are biased for phagocytosisꢀmediated uptake,
which is known to be more degradative and less prone to phagosomal escape.4, 5 At the lowest
polymer:pDNA ratio (50:1), gene delivery is negatively correlated with decreasing molecular
weight (Figure 3A). Presumably, this occurs because lower molecular weight polymers do not
effectively condense pDNA at lower weight ratios, which results in increased fragility and
degradation of the associated polyplexes. Thus, taking into account the optimal levels of gene
delivery, 200:1 polyplexes were selected for subsequent gene delivery experiments.
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Figure 3. Gene delivery summary. (A) Transfection efficiency (% EGFP Positive Cells) of
commercial controls and D4A4ꢀMan polyplexes at three different polymerꢀtoꢀpDNA weight
ratios. (B) Standardization of 200:1 transfection efficiency by top performing D4A4ꢀMan (P14)
variant. (C) Evaluation of mannose (1,000 µM) and serum (50% v/v) inhibition of 200:1
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polyplexes gene delivery efficiency. (D) Standardization of inhibited gene delivery with
uninhibited values from (C). Colored arrows were added to help visualize the connection of the
data to expected vector properties.
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For PBAEꢀMan polyplexes to be successful in translational applications, gene delivery
vectors must overcome the barriers present during circulation. Key gene delivery limitations
include target specificity and the documented lossꢀofꢀactivity in the presence of heightened
levels of extracellular protein.1 These specific limitations result in significant offꢀtarget effects
and premature aggregation, degradation, and clearance.3, 25 As such, transfection was conducted
using 200:1 polyplexes in the presence a CD206ꢀinhibiting concentration of free mannose and a
physiologicallyꢀrelevant level of serum (Figure 3C). All polyplexes exhibited significant (95%
confidence) drops to both mannose and serum inhibitions. However, decreasing molecular
weight resulted in the formation of polyplexes that were increasingly sensitive to mannose
inhibition and decreasingly sensitive to serum inhibition. To better assess the capabilities of the
polyplexes, inhibition values were standardized by their respective uninhibited gene delivery
capabilities (Figure 3D). These findings demonstrate that the lower molecular weight polyplexes
possess the highest specificity and stability. Moreover, these results support previous studies
that demonstrated increased PBAE molecular weight does not always improve transfection.26, 27
Collectively, these results suggest translational APCꢀtargeting gene delivery polymers should be
designed towards moderately charged and lower molecular weight alternatives.
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Figure 4. Structureꢀfunction relationships of 200:1 polyplexes. (A) Mannoseꢀinhibited
transfection efficiencies for polymers P1ꢀP14 standardized by P1 transfection values. Total %
gene delivery sensitivity (B) as a function of polyplex zeta potential (C) and diameter (D) in 25
mM NaOAc (Ph 5.15). *Indicates statistical decrease (95% confidence) between samples
compared to P1.
Structureꢀfunction Relationships. During the initial steps of gene delivery, vector
design and polyplex biophysical characteristics significantly influence the eventual outcome.
First, mannoseꢀgrafting is a potent strategy toward improving gene delivery outcomes in APCs.
Due to the complexity of polymer design, however, it is often difficult to identify the influence
of targeting agents (i.e., mannosylation) from various polymer chemical properties. Our current
experimental plan introduces the same chemical background to each polymer and, therefore,
allows direct comparison of parameters such as polymer molecular weight, polyplex zeta
potential and diameter, and mannosylation. Furthermore, given the size range of the polyplexes
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prepared in the current study (i.e., >~400 µm), internalization and processing is primarily
restricted to phagocytic mechanisms.4, 5, 28, 29
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Polyplex zeta potential and size (diameter) are two critical properties mediating uptake,
endosomal release, and subsequent gene delivery (Figure S4). Specifically, uninhibited gene
delivery was negatively correlated with increasing polyplex zeta potential and diameter. In
addition, there was not a statistical dependence of gene delivery upon charge or size in the
presence of inhibiting concentrations of mannose; whereas, in physiological levels of serum,
gene delivery was negatively correlated with increasing charge and size.
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By normalizing mannoseꢀinhibited gene delivery values with the gene delivery of the
highest molecular weight polymer (P1), the resulting data allow an assessment of gene delivery
as a function of polymer molecular weight without the influence of mannosylation (Figure 4A).
Moreover, without mannoseꢀmediated improvements, the established higher molecular weight
paradigm is restored. Namely, gene delivery is correlated with higher molecular weight
polymers (which also possessed the largest polyplex diameters and zeta potential values). These
observations highlight the importance of biasing polyplexes towards mannoseꢀmediated
processing that in turn prompts the redirection into the recycling endocytic mechanism that is
more permissive to gene delivery outcomes.12
Interestingly, the relative degree of inhibition (both mannoseꢀ and serumꢀdependent)
demonstrated by each polyplex varied in a linear fashion with polymer molecular weight
(Figures 3C and D), thus, prompting the development of a new comparative “sensitivity” metric
(Figure 4B). Here, the percent change between uninhibited and inhibited gene delivery values
was utilized to represent the likelihood of a particular parameter to inhibit gene delivery (i.e.,
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higher sensitivity values indicate the higher likelihood of inhibited gene delivery in the presence
of that particular parameter). Accordingly, mannose sensitivity was positively correlated with
decreasing molecular weight; whereas, serum sensitivity was negatively correlated with
decreasing molecular weight. This phenomenon is best explained by the interplay of polyplex
relative mannose content, zeta potential, and diameter (Figure 4C and D). Polymers possessing
the lowest molecular weight contain the highest relative mannose content, driving the formation
of increasingly smaller sized and less cationic polyplexes (Figures S2 and S5). Consequently,
the resulting polyplexes have smaller surface areas, which in turn enrich the relative mannose
surface density. Collectively, these results suggest that increased serum surface deposition
(attributed to electrostaticallyꢀdriven interactions between the residual cationic charges of the
polyplex and the anionic serum proteins coupled with increasing surface area) promotes the
physical coverage of mannose molecules and, thus, inhibits mannoseꢀmediated uptake
mechanisms. However, at the smallest polyplex sizes, the increased relative mannose surface
density may serve a dual function in providing a shielding layer (similar to PEGylation) that
simultaneously prevents serum deposition while also providing targeting capabilities.
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In our initial studies using mannosylated PBAEs, the best performing polymer
background, D4A4 (used in the current study), was further analyzed in a preliminary in vivo
DNA vaccination study.12 Although both nonmannosylated and mannosylated D4A4 polymers
elicited strong immune responses, only D4A4ꢀMan was able to evoke a response statistically
higher than the commercial controls. Furthermore, the structureꢀfunction analysis presented in
the previous work was limited but provided a polymer chemical background basis for
engineering polyplex gene delivery specific for APCs. Given the success of the initial study, the
results in this work provide new in vitro findings that support further in vivo translation.
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CONCLUSIONS
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The library of polymers developed in this study highlights the unique ability of small,
moderately charged, mannosylated PBAEs to elicit statistically significant levels of gene
transfections in APCs. Taken together, the study provides fundamental insight into underlying
structureꢀfunction relationships for APCꢀtargeted gene delivery while also providing a costꢀ
effective alternative to an established polymer degradation methodology.
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ASSOCIATED CONTENT
Supporting Information. Additional gene delivery and structure functions are presented. In
addition, NMR data is presented including an example of the polymer degradation analysis. This
material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Corresponding authors. Department of Chemical and Biological Engineering, University at
Buffalo, State University of New York, Buffalo, NY 14260ꢀ4200, USA, Phone: 716ꢀ645ꢀ1198,
Fax: 716ꢀ645ꢀ3822. Email: blainepf@buffalo.edu.
Author Contributions
CHJ conceptualized and designed the overall study, conducted all biological experiments, and
analyzed all data. MC, AG, AR, GZ, SL, and MT conducted and analyzed data for all materialꢀ
related studies. HL and CC assisted with NMR spectra analysis. The manuscript was written
through contributions of all authors. All authors have given approval to the final version of the
manuscript. ‡These authors contributed equally.
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Funding Sources
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The authors recognize support from NIH award AI088485 (BAP), a SUNYꢀBuffalo Schomburg
fellowship (CHJ), and two Mark Diamond Research Fund Grants (AR and MC).
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Notes
The authors declare no competing financial interest.
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