Beyond Gene Transfection with Methacrylate-Based Polyplexes - The Influence of the Amino Substitution Pattern

Article abstract of DOI:10.1021/acs.bioconjchem.8b00074

Methacrylate-based polymers represent promising nonviral gene delivery vectors, since they offer a large variety of polymer architectures and functionalities, which are beneficial for specific demands in gene delivery. In combination with controlled radical polymerization techniques, such as the reversible addition-fragmentation chain transfer polymerization, the synthesis of well-defined polymers is possible. In this study we prepared a library of defined linear polymers based on (2-aminoethyl)-methacrylate (AEMA), N-methyl-(2-aminoethyl)-methacrylate (MAEMA), and N,N-dimethyl-(2-aminoethyl)-methacrylate (DMAEMA) monomers, bearing pendant primary, secondary, and tertiary amino groups, and investigated the influence of the substitution pattern on their gene delivery capability. The polymers and the corresponding plasmid DNA complexes were investigated regarding their physicochemical characteristics, cytocompatibility, and transfection performance. The nonviral transfection by methacrylate-based polyplexes differs significantly from poly(ethylene imine)-based polyplexes, as a successful transfection is not affected by the buffer capacity. We observed that polyplexes containing a high content of primary amino groups (AEMA) offered the highest transfection efficiency, whereas polyplexes bearing tertiary amino groups (DMAEMA) exhibited the lowest transfection efficiency. Further insights into the uptake and release mechanisms could be identified by fluorescence and transmission electron microscopy, emphasizing the theory of membrane-pore formation for the time-efficient endosomal release of methacrylate-based vectors.

Full text of DOI:10.1021/acs.bioconjchem.8b00074

Subscriber access provided by Kaohsiung Medical University
Article
Beyond gene transfection with methacrylate-based
polyplexes – the influence of the amino substitution pattern
Ulrich S. Schubert, Tanja Bus, Martin Reifarth, Johannes C. Brendel,
Stephanie Hoeppener, Anja Traeger, and Anne-Kristin Trützschler
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00074 • Publication Date (Web): 01 May 2018
Downloaded from http://pubs.acs.org on May 2, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
Beyond Gene Transfection with Methacrylateꢀbased
Polyplexes – The Influence of the Amino
Substitution Pattern
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
‡
,§,†
‡,§,†
‡,§,#,$
‡,§
AnneꢀKristin Trützschler,
Tanja Bus,
Martin Reifarth,
Johannes C. Brendel,
‡,§
‡,§,
‡,§,
Stephanie Hoeppener, Anja Traeger, * Ulrich S. Schubert, *
‡
Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich Schiller University Jena,
Humboldtstrasse 10, 07743 Jena, Germany
§
Jena Center for Soft Matter (JCSM), Friedrich Schiller University Jena, Philosophenweg 7, 07743 Jena,
Germany
#
Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich Schiller University Jena,
Helmholtzweg 4, 07743 Jena, Germany
$
Leibniz Institute of Photonic Technology, AlbertꢀEinsteinꢀStrasse 9, 07745 Jena, Germany
†
The authors contributed equally to this work
Email: ulrich.schubert@uniꢀjena.de, anja.traeger@uniꢀjena.de
1
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 2 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ABSTRACT
Methacrylateꢀbased polymers represent promising nonꢀviral gene delivery vectors, since they offer a large
variety of polymer architectures and functionalities, which are beneficial for specific demands in gene
delivery. In combination with controlled radical polymerization techniques, such as the reversible
additionꢀfragmentation chain transfer polymerization, the synthesis of wellꢀdefined polymers is possible.
In this study we prepared a library of defined linear polymers based on (2ꢀaminoethyl)ꢀmethacrylate
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
AEMA), Nꢀmethylꢀ(2ꢀaminoethyl)ꢀmethacrylate (MAEMA) and N,Nꢀdimethylꢀ(2ꢀaminoethyl)ꢀ
methacrylate (DMAEMA) monomers, bearing pendant primary, secondary and tertiary amino groups, and
investigated the influence of the substitution pattern on their gene delivery ability. The polymers and the
corresponding plasmid DNA complexes were investigated regarding their physicochemical characteristics,
cytocompatibility and transfection performances. The nonꢀviral transfection by methacrylateꢀbased
polyplexes differs significantly from poly(ethylene imine)ꢀbased polyplexes, as a successful transfection is
not affected by the buffer capacity. We observed that polyplexes containing a high content of primary
amino groups (AEMA) offered the highest transfection efficiency, whereas polyplexes bearing tertiary
amino groups (DMAEMA) exhibited the lowest transfection efficiency. Further insights into the uptake
and release mechanisms could be identified by fluorescence and transmission electron microscopy,
emphasizing the theory of membraneꢀpore formation for the timeꢀefficient endosomal release of
methacrylateꢀbased vectors.
2
ACS Paragon Plus Environment

Page 3 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
In the last century the intensive research on major human diseases has shown that a wide variety of
diseases originate from dysfunction in protein expression. With the increasing knowledge about gene
regulation and the role of RNA, personalized therapies on the basis of nucleic acids became feasible. Due
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
to the instability of naked nucleic acids against nucleases and the limited direct cellular uptake, the use of
delivery systems, called vectors, is essential for efficient therapeutic approaches. Besides viral vectors,
synthetic systems gain increasing attention in the field of gene delivery. Despite the high transfection
2
efficiency of viral gene delivery vectors, the potential immune response on the natural viral proteins, the
limitations in the size of the DNA and the difficulties in the scaleꢀup of these systems make nonꢀviral
3
systems more favorable. In accordance, the structures and efficiencies of polymeric delivery systems
4
have constantly been improved, since the first polycationꢀmediated transfection in 1965 and the first
5, 6
polymerꢀtargeted gene delivery in the late 1980s have been introduced. Different bottlenecks during the
7
transfection processes have been described so far. Thereby, a high molar mass, the presence of cationic
charges as well as a buffering capacity at low pH values were discussed to be beneficial for (i) the
formation of the polymer/nucleic acid complex, (ii) the attachment of this complex to cellular membranes
and the associated uptake by endocytosis, (iii) as well as the endosomal escape and dissociation of the
cargo from the polymer. To date linear poly(ethylene imine) (lPEI) represents the most frequently applied
and efficient polymer for gene delivery. The secondary amino groups along the backbone are partially
protonated at physiological conditions, which enable the binding of nucleic acids and the buffering of
8, 9
acidifying endosomes as postulated by the popular “proton sponge” hypothesis. This theory describes a
potential escape strategy from endoꢀlysosomes by the increase of the charge density due to further
protonation leading to an enhanced influx of chloride ions and water. Consequently, the increasing
osmotic pressure causes the rupture of the endosomal membrane and hence, the release of the vector
within the cytsoplasm. Besides lPEI, which comprises mainly secondary amino groups, other materials
and amino functionalities have been studied regarding their transfection performance including polymers
like polyꢀ
L
ꢀlysine (PLL, primary amino groups), poly(N,Nꢀdimethyl(aminoethyl) methacrylate)
3
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 4 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
PDMAEMA, tertiary amino groups) and branched PEI (bPEI, primary, secondary and tertiary amino
groups). To gain deeper insights into the impact of the different amino functionalities it is important to
investigate the transfection performance with defined structures. As a consequence, the reversible
additionꢀfragmentation chain transfer polymerization technique (RAFT), which tolerates a wide range of
functional groups and enables the controlled synthesis of wellꢀdefined structures, represents a promising
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0, 11
tool in polymerꢀbased gene delivery.
In particular, the structural versatility of methacrylateꢀbased
polymers allows the introduction of all mentioned different amino moieties into a similar polymer
structure and, thus, a direct comparison of the impact of primary, secondary and tertiary amino groups. So
far, only a few studies investigated the influence of this degree of substitution on the final transfection
efficiency. Primary, secondary as well as tertiary amino groups were studied separately on polymer
12ꢀ14
structure and functionality use by groups like Reineke and coworkers as well as by Zhu et al.
.
Palermo
et al. revealed that primary and tertiary amino groups in methacrylateꢀbased copolymers complex the
phosphate head groups of an artificial lipid bilayer, showing higher membrane internalization of the
1
5
primary amino groups. This theory is supported by earlier findings of disruptive peptides, which evolve
16ꢀ18
into the membrane to create pathways for other molecules to escape into the cytosol.
Nevertheless, a
thorough comparison of structurally similar polymers comprising primary, secondary and tertiary amino
groups is still missing. Up to our knowledge, previous reports included either additional functional
domains or monomers, or lack at least one of the structural amino varieties.
Within this study we focused on the controlled RAFT synthesis of 2ꢀ(N,Nꢀdimethylamino)ethyl
methacrylate, NꢀmethylꢀNꢀtertꢀbutyloxycarbonylꢀ(2ꢀaminoethyl)ꢀmethacrylate (BocMAEMA) and Nꢀtertꢀ
butyloxycarbonylꢀ(2ꢀaminoethyl)ꢀmethacrylate (BocAEMA) to create a library of linear statistical homoꢀ
and copolymers with varying amino functionalities. Based on this library, we were able to investigate the
influence of different compositions of primary, secondary and tertiary amino groups in linear polymers on
the transfection performance and to elucidate an optimal polymer composition. The polymers were
characterized concerning their polyplex formation, cytocompatibility, their extraꢀ and intracellular fate
focusing on uptake and endosomal release.
4
ACS Paragon Plus Environment

Page 5 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
RESULTS AND DISCUSSION
Polymer preparation and characterization
To systematically investigate the influence of secondary amino groups in combination with primary and
tertiary amino groups on the transfection efficiency, a library of linear statistical polymers was synthesized
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10, 19
via RAFT polymerization.
The direct polymerization of AEMA with MAEMA and/or DMAEMA led
to hydrogels during the polymerization process. This side reaction is probably caused by the basic effect
of the amino groups containing methacrylate monomers, which resulted in the aminolysis of the esters or
the RAFT group. The cycling or branching reaction induced by the aminolysis of the methacrylate ester
20
unit was previously described and is commonly used for crosslinking of polymers. Another possible
reason for the gel formation might be the aminolysis of the RAFT end group causing intermolecular
2
1ꢀ23
disulfide bridges.
Therefore, BocAEMA and BocMAEMA were synthesized starting with the Bocꢀ
24ꢀ27
protection of the corresponding ethanolamine according to literature reports.
Scheme 1. Schematic representation of the RAFTꢀpolymerization and deprotection reaction of the terpolymer
containing Bocꢀprotected primary (red), secondary (blue) and tertiary (green) amino groups containing monomers.
Due to the Boc–protection of the primary and secondary amino groups during the synthesis the hydrogel
formation can be avoided during the polymerization process. The polymerization was performed in DMF
at 70 °C for 40 h with a M/CTA feed of 240, with a CTA/I ratio of 4 and monomer ratios of the
copolymers or terpolymers of 50:50 or 1/3, respectively (Scheme 1). The kinetics of the coꢀ and
terpolymerization revealed a controlled polymerization with narrow dispersity and a statistical distribution
of the monomers (see SI Figure S1 and S2). For the copolymerization of BocAEMA and DMAEMA
1
2
similar reactivity rations were already described by Zhu et al. The aim for the final molar mass of the
5
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 6 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
synthesized polymers was at least 20 kDa, which represents a compromise considering the enhancement
of the transfection efficiency versus an increasing toxicity with increasing molar mass and the opportunity
28, 29
for controlled polymer synthesis.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
An overview of the synthesized Bocꢀprotected polymer library (PBM to PBMA) is provided in Table 1.
In the following, the polymers are named using “P” for polymer, “B” for the presence of Bocꢀprotected
AEMA and/or MAEMA units and the combination of letters “D”, “M” and “A” for the presence of
primary (A), secondary (M) or tertiary amino group containing monomers (D), respectively. All polymers
1
featured molar masses in the range of 33 to 54 kDa and narrow distributions (see Figure 1A). In the Hꢀ
NMR spectra of the homoꢀ and copolymers characteristic methylene signals of the protection group can be
1
seen at 1.43 ppm (see SI Figure S3). The resulting compositions were calculated from HꢀNMR using the
ratio of the specific signals of the secondary (δ 2.93 ppm) and tertiary amine (δ 2.25 ppm) containing
units, which are assigned to the methyl groups at the amino function. The ratio of those methyl signals to
the methylene signal (δ 4.02 ppm) of all units neighboring the ester function in the backbone provides the
mole fraction of each monomer.
Table 1. Selected characterization data of the Bocꢀprotected polymers.
b
b
b
n [mol%]
n [mol%]
n [mol%]
a
ꢀ1
Polymer
M [g mol ]
Ɖ
n
DMAEMA
BocMAEMA
BocAEMA
PBM
PBA
35,600
33,800
34,400
49,800
54,600
46,200
1.08
1.10
1.16
1.19
1.14
1.25
ꢀ
ꢀ
100
ꢀ
ꢀ
100
PBDM
PBDA
PBMA
PBDMA
60
57
ꢀ
40
ꢀ
ꢀ
43
50
33
50
33
33
a
Molar mass and dispersity were determined by size exclusion chromatography (DmAc, 0.21% LiCl, PMMA
b
1
calibration). The molar percentage was determined by HꢀNMR.
6
ACS Paragon Plus Environment

Page 7 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 1. (A) Size exclusion chromatography curves (DmAc, 0.21% LiCl, PMMA calibration) of the Bocꢀprotected
polymer library showing narrow dispersities. (B) SEC curves (water, 0.1% trifluoroacetic acid, 0.1 M NaCl, system
peak @ 20 mL) of deprotected polymer library showing monomodal distributions.
As no Bocꢀprotection is required, the polymerization of the PDMAEMA homopolymer (PD) was directly
performed in ethanol using 4,4′ꢀazobis(4ꢀcyanopentanoic acid) as initiator at 70 °C for 20 h. The other
Bocꢀprotected polymers of the library (PBM to PBDMA) were finally deprotected using 1 M
hydrochloric acid in methanol. A complete deprotection of the polymers was confirmed by HSQCꢀNMR
ꢀ1
1
at a polymer concentration of 200 mg mL , which did not show any residual Hꢀsignal in the typical range
13
at 1.44 ppm for the methyl groups of the Bocꢀprotection group and no corresponding C signal (see SI
Figure S4). The results of the characterization experiments are summarized in Table 2. The
characterization of the waterꢀsoluble deprotected library was performed by size exclusion chromatography
on an AppliChrom ABOA CatPhil system, which revealed only small interactions with the cationic
polymers (Figure 1B). The determination of the molar masses was conducted by asymmetric flow fieldꢀ
flow fractionation (AF4) coupled with multiꢀangle laser light scattering (MALLS) detection showing
narrow distributions and molar masses in the range of 21 to 37 kDa. With regard to the missing
appropriate size exclusion standards (dextrane used for aqueous SEC) for cationic methacrylate systems
the determination of the molar masses and the dispersities is more accurate by utilizing the MALLS
7
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 8 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
0
detection. However, the AF4 measurement will always tend to underestimate the values for dispersitiy in
comparison with other MALLS coupled techniques due to the architecture of the AF4 channel including a
31
membrane with a cutoff of 10 kDa.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Selected characterization data of the coꢀ and homopolymers.
a
a
b
b
d
Polymer
MnAF4
ƉAF4
MnSEC
ƉSEC
DP
pKa
c
c
7.45
PD
26,200
24,000
1.09
1.21
29,000
1.09
185
PM
13,400
11,700
2.04
1.98
167
163
8.40
8.19
PA
21,400
31,500
1.10
1.14
PDM
17,900
16,500
20,000
21,800
2.15
2.19
2.26
2.35
208
249
348
250
7.97
PDA
PMA
37,000
46,000
36,000
1.12
1.28
1.27
7.83
8.29
PDMA
8.03
a
b
and c
Molar masses and dispersities were determined by AF4 using the MALLS detector.
Molar mass was
b
c
determined by size exclusion chromatography ( 0.1 % TFA, 0.1M NaCl, dextran calibration/ DmAc, 0.21% LiCl,
d
1
PMMA calibration). The degree of polymerization was calculated from AF4 results and HꢀNMR determining the
ratio of monomers. pK value was calculated from the titration curves (see SI Figure S5).
a
In additional titration experiments, the pK values of the homoꢀ and copolymers PD to PDMA were
a
determined to be in the range from pH 7.45 to 8.40 (Table 2). The later used physiological ionic strength
was mimicked by the addition of 150 mM NaCl. In Figure S5, the titration curves are shown starting at
around pH 2 and plotted against the volume of 0.1 M NaOH solution added to the polymer solution of
ꢀ
1
1
0 mg mL . The calulation of the pK value was performed by fitting the obtained curves with a FTT fit
a
after smoothing the plot to avoid artefacts. The turning points were determined by the differentiation of
the fitted curves and the pK value was calculated by the HendersonꢀHasselbalch equation (2) (see
a
experimental section). For PDMAEMA the pK was previously described to be 7.5 by van de Wetering et
a
32
al., which is in accordance with our result for the PD polymer (pK = 7.45). These results confirm that at
a
8
ACS Paragon Plus Environment

Page 9 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
typical cell culture conditions at a pH value of 7.2 to 7.4, at least half of the amino functions of the
polymers are in a protonated state and can, therefore, bind and condense negatively charged nucleic acids,
thus forming polyplexes.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The calculation of the buffer capacity was performed for qualitative comparison from the titration data
according to equation (1) and was plotted as a function of the pH value (Figure S6). For the calculation of
ꢀ
β, d(pH) was set to 1 as it is defined from IUPAC. D(OH ) was calculated from the volume of sodium
33
hydroxide solution used for the change of 1 pH unit.
ꢁ
ꢂꢃ(ꢄꢅꢆ)
ꢀ =
ꢂꢂꢂꢂꢂꢂꢂꢂꢂꢂꢂꢂꢂꢂꢂꢂꢂ(1)
ꢁ
ꢂꢇꢅ
Tested polymers revealed buffer capacities in the range of pH 6.0 to 10.0. Homoꢀ or copolymers with a
high content of primary and secondary amino groups (PM, PA, PMA and PDMA) exhibited good buffer
capacities at more basic conditions of pH 8.0 and above, while polymers with increasing content of
tertiary amino groups (PD, PDM, PDA) tend to show also buffer capacities from neutral to acidic
conditions in agreement with the increase in DMAEMA content. In particular, for PD, increased buffer
capacities over the whole range from pH 6 to pH 8 could be observed. With regard to the “proton sponge”
theory, it can be assumed that the sample PD most probably possesses the highest potential of enhancing
endosomal release. Therefore, we examined the transfection potential of the methacrylateꢀbased polymer
library with a particular focus on the influence of different amino functionalities in the following studies,
which includes cytocompatibility, cellular uptake and polymerꢀmediated escape.
Cytocompatibility
The success of newly designed synthetic transfection vectors crucially depends on their interaction with
cellular membranes, the first biological barrier along the intracellular uptake process, which correlates
with the overall cell viability. To investigate the cytotoxic potential of the methacrylateꢀbased homoꢀ and
copolymers in vitro, we performed a resazurinꢀbased cell viability assay with L929 cells, as recommended
9
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 10 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
by ISO10993ꢀ5. Due to high cationic charge densities within the polymer structure, cytotoxic effects
34, 35
known from literature can be expected.
As depicted in Figure 2, a concentrationꢀdependent
cytotoxicity of all polymers was observed after 24 h. The lowest cell viability was observed for the homo
polymers PD, PM and PA as well as for the copolymer PDM revealing CC50 values (50% cytotoxic
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
ꢀ1
concentration) of 23 µg mL for PM and 19 µg mL for the remaining polymers. Whereas, the coꢀ and
terpolymers containing primary amino groups showed moderate cytotoxicities with CC50 values of
ꢀ1
ꢀ1
ꢀ1
6
2 µg mL (PDA), 38 µg mL (PMA) and 31 µg mL (PDMA), respectively. The CC values of the
50
polymers, except PDA, are within the range of linear polyethylene imine (lPEI 25 kDa, CC50
=
ꢀ1
25 µg mL ), which is the most prominent polymerꢀbased vector used for gene delivery. The molar massꢀ
dependent cytotoxicity of lPEI can be explained by its high cationic charge density. But it should be
considered that the polymerꢀmediated gene delivery mostly correlates with the cytotoxicity of polyamines,
which is the main issue in transfection.
Figure 2. Cytotoxicity evaluation of the methacrylateꢀbased homoꢀ and copolymers PA to PDMA including lPEI
25 kDa (as control) at indicated concentrations after 24 h. CC50 values represented in the table were calculated
applying the Boltzman fit (see Figure S7). Values represent the mean ± S.D. (n=3).
1
0
ACS Paragon Plus Environment

Page 11 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
In addition to that, the membrane activity of the polymers was investigated using erythrocytes suspended
in PBS (Figure 3 A and B). All polymers revealed a strong interaction with cellular membranes indicated
by the increased hemolytic activity (Figure 3 A) and erythrocyte aggregation (Figure 3 B). The membrane
activity was higher for polymers containing primary amino groups. As a significantly higher erythrocyte
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
damage was observed at transfection relevant concentration (see values for 10 µg mL ). Membrane
13, 29
destabilization and rupture indicate a strong membrane activity resulting in a severe cytotoxicity.
Figure 3. Hemocompatibility studies of metharcylateꢀbased homoꢀ and copolymers PD to PDMA. (A) Erythrocyte
hemolysis assay of polymers at indicated concentrations. Triton Xꢀ100 served as positive control (99,8% hemolysis)
and PBS as negative control (0%). A value less than 2% hemoglobin release was classified as nonꢀhemolytic and
ꢀ
1
values > 5% as hemolytic. Tꢀtest of the hemolysis values at 10 µg mL in comparison to PMA; ** ≤ 0.005; # > 0.05.
(B) Aggregation of erythrocytes after polymer treatment at indicated concentrations. PBS was used as negative
control and bPEI (25 kDa) as postitive control. Values represent the mean ± S.D. (n=3).
Methacrylateꢀbased polyplexes
In order to examine the binding affinity of all polymers to nucleic acids, the ethidium bromide quenching
36, 37
assay (EBA) was performed using pDNA (4.8 kb) as genetic material to be transfected.
Different
nitrogen (polymer) to phosphate (DNA) ratios (N/P) were used for the polyplex preparation in order to
assess the optimal conditions for a stable polyplex formation. All tested polymers revealed the ability to
1
1
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 12 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
condense pDNA and to form polyplexes with increasing N/P ratios (Figure 4 A). This was indicated by
the decrease in fluorescence intensities, as a result of the exclusion of ethidium bromide from its binding
site within the pDNA. At higher N/P ratios (10 to 20) a plateau of constant fluorescence values is formed
in all cases, which indicates a stable polyplex formation. However, the overall fluorescence intensities
vary depending on the type of composition of the polymers. Homoꢀ and copolymers based on DMAEMA
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
PD, PDM, PDA) displayed higher relative fluorescence intensities between 60 to 45%. Those polymers
demonstrated only a reduced ability for pDNA condensation, which can be related to the sterically
hindered tertiary amino groups. The presence of primary and secondary amino groups (PM, PA, PMA
and PDMA) resulted in improved DNA condensation properties (fluorescence intensities below 40%).
These observations clearly confirmed the trend for an improved DNA complexation by primary and
1
3, 28, 38
secondary amino groups.
Figure 4. Polyplex stability: (A) Ethidium bromide quenching assay with polyplexes of PD to PDMA prepared at
different N/P ratios (pDNA). (B) Dissociation assay of polyplexes PD to PDMA (N/P 20) using heparin at different
concentrations. Values represent the mean ± S.D. (n=3).
Despite differences in the complexation behavior and the resulting relative fluorescence intensities, all
polyplexes featured comparable sizes below 200 nm at N/P 20 with a high cationic surface charge
39ꢀ41
between 35 to 39 mV (Table 3), which is typical for cationic methacrylates.
Nevertheless, the prepared
polyplexes exhibited a broad size distribution as indicated by the relatively high PDI values. This fact
indicates the presence of a disperse polyplex population and suggests the formation of aggregates, which
1
2
ACS Paragon Plus Environment

Page 13 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
is a consequence of the preparation method as well as the used complexation buffer. The high dispersity is
most likely caused by the preparation of polyplexes with higher N/P ratios than necessary for
neutralization of the pDNA charges. However, this method (vigorous mixing of pDNA and polymer) is
42
known to show improved polyplex stability and transfection performance.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Size and zeta potential of pDNAꢀpolyplexes prepared with PD to PDMA at N/P 20.
Polyplexes of
ZꢀAverage [d.nm]
PDI
ζ (Zeta potential) [mV]
PD
84
0.4
0.4
0.4
0.3
0.4
0.4
0.3
37
37
39
37
35
38
39
PM
97
PA
180
138
144
115
148
PDM
PDA
PMA
PDMA
Polyplexes were measured in 20 mM 4ꢀ(2ꢀhydroxethyl) piperazineꢀ1ꢀethanesulfonic acid (HEPES) and 5% (w/v)
glucose, at pH 7.2 by dynamic and electrophoretic light scattering.
The dissociation capability of the genetic material from polymer was investigated by the heparin
4
3
dissociation assay (Figure 4 B). The previously formed polyplexes were treated with different
concentrations of heparin to force the release of pDNA within the polymer complex as competitor to the
polymer. Hence, ethidium bromide will be able to intercalate into the displaced nucleic acid once more,
leading to an increase in fluorescence intensity. The tested methacrylateꢀbased polymers seem to force a
ꢀ1
strong condensation of pDNA, since very high concentrations of heparin (50 U mL ) were required to
reach a (almost) full release of the pDNA from the polyplexes. The swiftest release was achieved with PD,
PDM and PDA, although such copolymers comprising DMAEMA units only revealed reduced pDNA
dissociation rates (80 to 85%).
Polyplex performance
1
3
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 14 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Since various studies reported good transfection efficiencies (TE) of methacrylateꢀbased polymers or
12, 44, 45
particles,
the transfection capability of the polymer library was evaluated at various conditions. For
this purpose, HEKꢀ293 cells were transfected with pDNA encoding the enhanced green fluorescence
protein (EGFP). The transfection efficiency was determined by flow cytometry (Figure 5) analyzing all
viable cells (counterstaining with propidium iodide, red) successfully expressing EGFP (green). Despite
the fact that PD possessed the highest buffer capacity at slightly acidic conditions, which is thought to be
beneficial for endosomal release, it demonstrated the lowest transfection levels (< 20% TE) at different
N/P ratios independent of serumꢀreduced or serumꢀcontaining cell media. Similar observations were
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0, 46
reported previously.
The best transfection efficiencies were achieved with PA (66 %TE) and PMA
(
56% TE) revealing comparable gene expression to lPEI, the standard of transfection (25 kDa,
Polyscience) at a N/P ratio of 20. The terpolymer PDMA showed also good transfection levels (43% TE)
at N/P 20, while PM, PDM and PDA revealed moderate TEs between 20 and 30%. A concentrationꢀ
dependent impairment of the overall cell viability during transfection in serumꢀreduced medium was
observed (Figure S8) and showed similar tendencies compared to the cytotoxicity profile of polymers (see
Figure 2). It has to be mentioned that the assessment of the viability by flow cytometry (PI staining) is
based on membrane leakage and, therefore, not directly comparable to the alamarBlue assay.
Nevertheless, it is known that polyplexes revealed decreased cytotoxicity compared to free polymers, as
47
the cationic charge is partially masked upon complexation with nucleic acids. In the presence of serum
proteins, the intracellular level of EGFP decreases drastically, meaning maximum TEs below 20% for all
methacrylateꢀbased polymers as well as lPEI. Furthermore, improved cell viabilities for tested polyplexes
at these conditions were observed (Figure S8). Due to their strong cationic character, negatively charged
proteins may aggregate with the polyplexes, which causes an alteration in the surface charges and the sizes
of the complex, finally leading to the formation of larger structures and the masking of the positive net
48, 49
charge.
This effect in turn may lead to a reduced affinity to cell membranes (polymerꢀmembrane
interaction), diminished uptake of polyplexes and, consequently, to a reduction in transfection
50, 51
efficiencies.
1
4
ACS Paragon Plus Environment

Page 15 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Transfection study of adherent HEKꢀ293 cells in serumꢀreduced (OptiMEM, light grey) and serumꢀ
containing media (RPMI + 10% FCS, black) with all polymers of the library and commercial 25 kDa lPEI as positive
control at different N/P ratios after 24 h. Values represent the mean ± S.D. (n=3).
Based on the results obtained from flow cytometry experiments, we conclude that polymers with tertiary
amino groups and an average molar mass in the range of 20 kDa are not beneficial for transfection,
whereas primary amino groups have a substantial impact on the successful delivery of nucleic acids, as the
highest transfection efficiency was observed for the polymer with the highest primary amine content
(
serumꢀreduced conditions). It should be mentioned, that the transfection efficiency of PDMAEMA was
52
previously shown to increase with the increase of the molar mass. The best polymer for transfection was
PA, which composes 100% AEMA units. Polymers including additional secondary amino groups (PMA
and PDMA) also demonstrated improved outcomes compared to polymers without any primary amino
groups. Homopolymers with 100 mol% tertiary or secondary amino groups as well as copolymers
consisting of varying ratios of the corresponding monomers lacked the ability to efficiently transfect
1
5
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 16 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
HEKꢀ293 cells. Despite the beneficial buffer capability based on the presence of tertiary amino groups
,
various studies have been questioning the endosomal escape mechanism of PDMAEMA by the proton
16, 53, 54
sponge effect
. Our results further undermine this theory, since the buffer capacities of the
55, 56
methacrylateꢀbased polymers do not correlate with their ability for gene expression.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To gain a deeper insight of the differences in the gene delivery performance of the methacrylates, we
investigated the uptake behavior of the polyplexes. It has been reported that cationic polyplexes are
5
7, 58
internalized into cells by endocytosis.
Since we obtained polyplexes with favorable sizes below
2
00 nm and cationic net charges, their cellular uptake was investigated by flow cytometry measurements
+
using YOYOꢀ1 labeled pDNA and bafilomycin, a vacuolar type H ꢀATPase inhibitor (see SI Figure S9 A
and B). Uptake studies at 4 °C confirmed an energyꢀdependent internalization pathway via endocytosis,
shown by the inhibition of the polyplex uptake into HEKꢀ293 cells (Figure S9 A). Furthermore, results
obtained by the treatment of cells with bafilomycin further support an uptake by endocytosis, since an
almost complete inhibition of EGFP transfection was observed (Figure S9 B). This effect is caused by the
inhibition of the acidification of endoꢀlysosomal organelles, which prevents the endosomal release of
polyplexes into the cytoplasm and thus a successful transfection. This experiment further supports the
requirement of polymer protonation through acidification to achieve sufficient endosomal escape.
In contrast, uptake studies at 37 °C revealed a timeꢀdependent uptake of YOYOꢀ1 labeled polyplexes,
where about 80% of HEKꢀ293 cells presented internalized polyplexes after 4 h (Figure 6 A). In particular,
the homopolymers PD (MFI = 188) and PA (MFI = 159) followed by PDM (MFI ~ 150) revealed the
highest amount of YOYOꢀ1 positive cells.
1
6
ACS Paragon Plus Environment

Page 17 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. (A) Polyplex uptake of adherent HEKꢀ293 cells in serumꢀreduced media using YOYOꢀ1 labeled pDNA.
The uptake efficiency (columns) and the mean fluorescence intensity of all viable cells (dots) measured by flow
cytometry after certain time points. Values represent the mean ± S.D. (n=3). (B) Quantification of endosomal release
by the calcein quenching assay. HEKꢀ293 cells were treated with PD, PM, PA, PMA and PDMA polyplexes and
simultaneous supplementation of calcein (25 µM). After 4 h and 24 h the endosomal release of polyplexes was
evaluated by the release of calcein from endosomes into the cytoplasm of the cells, leading to high fluorescence
signals. Ratio of green fluorescent calcein area in % over the number of detected Hoechst stained nuclei in HEKꢀ293
cells were detected by fluorescence microscopy and quantified using ImageJ.
59
Our results indicate that a high uptake is not the limiting step for high transfection levels, since the
29, 60
endosomal release represents a further critical hindrance in the gene delivery process.
Therefore, the
endosomal escape of the homopolymers PD, PM and PA as well as the best performing copolymer PMA
and terpolymer PDMA was further examined via a calcein quenching assay after 4 and 24 h (see Figure
6 B and SI Figure S10 and S11). Calcein is a membrane impermeable fluorophore, which is not able to
enter cells by diffusion, but by fluidꢀphase endocytosis. As calcein is, therefore, trapped within the endoꢀ
lysosomes, it can only escape by membrane disruption, which leads to a bright fluorescence in the cytosol
54, 61
by the distribution of calcein throughout the whole cell.
Since our polymers and polyplexes revealed
increased interaction with cellular membranes (see hemolysis and aggregation data), they may promote
the release of calcein from endoꢀlysosomes within the cytoplasm compared to untreated cells. This
experimental setup is designed to model the potential release mechanisms hypothesized by earlier studies
1
7
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 18 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
5
on methacrylates with artificial membranes. Representative images of HEKꢀ293 cells with calcein
remaining captured within the endoꢀlysosomes (control) as well as images of successfully released calcein
(
PMA) are provided within the Supporting Information (Figure S10 and S11). After endosomal release of
the dye into the cytosol, the fluorescent area of the calcein signal should increase while the relative
fluorescence intensity should decrease in comparison to the stained cell nuclei. The evaluation of the
endosomal release was done by image analysis of the captured fluorescence pictures using the ImageJ
software as described in the experimental section focusing on the increase in relative fluorescent area to
the number of stained nuclei. The differentiation between endosomal and cytosolic fluorescence was
limited using fluorescence microscopy regarding spatial information and resolution quality. Therefore the
overall fluorescent area was plotted against the number of Hoechst stained nuclei. The results revealed an
increased fluorescent area of HEKꢀ293 cells treated with methacrylateꢀbased polyplexes compared to cells
treated only with calcein (control). The methacrylateꢀbased polyplexes showed a up to eightꢀtimes higher
relative fluorescent area after 4 h in contrast to the control, which represents a hint for an early endosomal
escape by membrane disruptive activity. In particular, PA, PMA and PDMA showed the fastest
endosomal release, whereas PD revealed a lower ratio of escape after 4 h. This trend may relate to its poor
transfection performance. PD seems to be unable to escape the endosomes in a time efficient manner as
for instance PA, thus being kept entrapped within endoꢀlysosomes despite its high buffer capacity. As
mentioned earlier, various studies have been questioning the endosomal escape of PDMAEMA by the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
6, 53, 54
proton sponge effect
. For example, Jones et al. reported the lack of an endosome disruptive activity
54
of PDMAEMA as an explanation for its diminished transfection performance. After 24 h, the
fluorescence area of PA and PMA treated cells decreases compared to the other measured polyplexes,
which indicates a fast release within few hours. A decrease in calcein fluorescence intensity after 4 h was
also reported by others and explained by the activity of Pꢀglycoprotein pumps, which might transport
6
2
calcein out of the cell. A rapid endosomal release of polyplexes within the first hours during the
transfection process seems to correlate with high transfection efficiency, as the highest transfection levels
were observed for PA and PMA.
1
8
ACS Paragon Plus Environment

Page 19 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
In order to elucidate the location and membrane interaction of the polyplexes, highꢀangle annular darkꢀ
field scanning transmission electron microscopy (HAADFꢀSTEM) was applied. This method provides a
resolution down to a few nanometers and combines it with the capability to reveal the ultrastructure of the
cellular interior. Figure 7 shows representative images of HEKꢀ293 cells, embedded into an Eponꢀbased
resin, after exposure to polyplexes based on PMA for 4 h incubation time. In addition to the cellular
organelle structures (i.e. mitochondria, vesicles, nucleus), structures of high electron density (dark/black)
were observed. We attribute these structures to polymerꢀDNA complexes, since the functional groups of
the polyplex (amines and phosphates) exhibit a strong chemical affinity to the standard staining reagents
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(OsO and uranyl acetate) resulting in an efficient staining of polyplexes. This staining ability of
4
63
mentioned reagents has been observed for a variety of different polyplexes so far. In Figure 7 A and B,
PMA polyplexes are located within endosomal structures and partially a high number of polyplexes is
observed within these structures. In the periphery of the cells the formation of aggregates is evident (red
arrow), however, also smaller aggregates are observed to approach the cells. Next to completely filled
endosomal structures some intracellular vesicles are filled with a low number of densely packed
polyplexes alongside a large amount of intracellular fluids. In Figure 7 B an increased interaction of the
polyplexes with the endosomal membrane is observed in some cases (yellow arrows), which can be
explained by the electrostatic interaction between the positively charged polyplexes and the negatively
charged membranes. A close vicinity of the polyplexes to the endosomal membrane was also observed for
6
4
PEIꢀbased polyplexes. Moreover, in some cases alterations of the endosomal membrane structures
become evident. Here, the polyplexes are partially observed in close vicinity to the endosomal structure,
which are associated with deformed membranes (green arrows). These structural changes can be attributed
to endosomal escape events. An alternative escape strategy to the proton sponge effect might be induced
by the direct interaction of methacrylateꢀbased polyplexes with the endoꢀlysosomal membrane causing a
membrane destabilization and a subsequent release due to pore formation without complete rupture. This
59, 60, 65ꢀ67
effect was previously reported for cationic polymers.
A disturbed membrane integrity of
polyplexꢀcontaining vesicles was observed, as parts of the electron dense structures were detected outside
1
9
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 20 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of the vesicular membrane and reaching into the cytoplasm of HEKꢀ293 cells (indicated by red arrow
heads). This observation, as well as our previous results (hemolysis and calcein assay), support an
endosomal escape by holeꢀformation due to an increased tension on the membrane rather than a complete
osmotic rupture of the endosome.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. HAADFꢀSTEM images of polyplex treated HEKꢀ293 cells for 4 h in OptiMEM. Polyplexes can be
observed as electron dense structures (black). (AꢀB) PMA polyplex treated cells: Red arrows indicate polyplex
aggregation in the extracellular environment. Yellow arrows indicate interaction of polyplexes with the membrane of
endoꢀlysosomal vesicles. Green arrows indicate deformed membrane structures of endoꢀlysosomes caused by
polylpex interaction. Red arrowheads indicated disturbed membrane integrity of polyplexꢀcontaining vesicles and
potential membrane disruption or pore formation. Polyplex structures (black) seems to pass from endosomes to
cytoplasm.
2
0
ACS Paragon Plus Environment

Page 21 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
CONCLUSION
In an effort to understand the influence of different amino functionalities in methacrylateꢀbased polymers
on their transfection efficiency, we have synthesized a library of defined linear homoꢀ and copolymers
bearing primary, secondary and tertiary amino groups. The polymers were prepared using the RAFT
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
process and revealed similar molar masses with a DP of 163 to 250 and narrow dispersities (Ɖ
= 1.09 to
1
.28) as well as varying compositions of DMAEMA, MAEMA and AEMA monomers. pK values
a
between 7.45 and 8.40 were observed, leading to a partially protonation of all polymers at physiological
conditions. The buffer capacities, in particular for PD, are in the pH range of endosomal environments,
which is supposed to be beneficial for transfection. All synthesized polymers were able to bind and
condense pDNA resulting in the formation of stable, nanosized polyplexes (< 200 nm). Furthermore, the
performance of these polymers as gene delivery vehicles was examined at different conditions by
microscopic techniques (fluorescence and scanning transmission electron microscopy) as well as flow
cytometry measurements. It was found that several parameters (Figure 8) show a relevant impact on the
successful transfection: Most importantly (i) the type and content of the amino group, (ii) the interaction
of the polymer with the membrane and (iii) a rapid endosomal release. However, the size of polyplexes,
their uptake rate and interestingly, the buffer capacity revealed no direct correlation with the observed
transfection levels. Based on our results, the transfection efficiency of such polymers can be summarized
regarding their amino functionalities as the following: Primary amino groups > Secondary amino groups >
Tertiary amino groups. High contents of primary amino groups in homoꢀ (PA) and copolymers (PMA)
resulted in an improved transfection efficiency, whereas higher amounts of tertiary amino groups within
the polymer structure seem to hamper the transfection process (PD and PDM). A primary amino content
of at least ~ 40 to 50 mol% was found to be necessary for efficient transfection, since already the
copolymer PDA (AEMA30%ꢀDMAEMA70%) comprising ~70 mol% of tertiary amino groups showed
reduced results. With the addition of 30 mol% secondary amino groups, the transfection performance of
the terpolymer PDMA (AEMA30%ꢀMAEMA30%ꢀDMAEM40%) in contrast, significantly increased
compared to PDA. This effect was only observed for copolymers of MAEMA, since the homopolymer
2
1
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 22 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
PM also demonstrated reduced transfection levels. The good performance of the presented homoꢀ and
copolymers could furthermore be explained by a strong polymerꢀmembrane interaction rather than by a
rupture mediated by osmotic swelling, as it is postulated for the proton sponge effect. However, it is
demonstrated that the endosomal acidification is still required for further protonation of the polymers and
therefore for their sufficient interaction with the endoꢀlysosomal membrane (see bafilomycin treatment).
In fact, all polymers exhibited high hemoglobin release and erythrocyte aggregation, indicating their
ability to destabilize membranes and to cause a partial rupture. Despite the fact that such a high membrane
interaction leads to an increased cytotoxicity, this property seemed to be beneficial for a fast endosomal
release. This assumption is in accordance to the results of our calcein quenching assay, which revealed a
fast endosomal escape, in particular for PA and PMA. Based on this investigations, we conclude that the
mechanisms for endosomal escape during transfection of methacrylate based polymers differ significantly
from PEI, as they are unaffected by the respective buffer capacity and support the theory of the disruptive
formation of pores.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
ACS Paragon Plus Environment

Page 23 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
Figure 8. Overview of factors (main polyplex properties) with impact on the transfection efficiency using the
Pearson correlation coefficient. Highest proportional correlation is represented by 1.0, whereas
̶ 1.0 means the
highest invers proportional correlation. Values around zero indicate no correlation. For membrane disruption, the
ꢀ1
hemoglobin release at 10 µg mL polymer concentration was used. For cytotoxicity the half maximum (50%) critical
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
concentration (CC ) was used. The binding affinity represents the measured relative fluorescence of ethidium
50
bromide at N/P 20. To compare the mean fluorescence intensity (MFI) of polyplex uptake, the time (min) that was
needed to reach 100 relative MFI (where the control is 1) was determined. The calcein release represents the ratio of
green fluorescent calcein area in % over the number of detected Hoechst stained nuclei of the polymers after 4 h.
2
3
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 24 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EXPERIMENTAL
Materials
Unless otherwise stated, the chemical were used without further purification. 2ꢀ(N,NꢀDimethylamino)ethyl
methacrylate (DMAEMA), methacryloyl chloride, anhydrous triethylamine, 4ꢀcyanoꢀ4ꢀ
phenylcarbonothioylthio) pentanoic acid (CPDBꢀCOOH), neutral aluminium oxide and 2ꢀ
methylamino)ethanol, propidium iodide and calcein were purchased from Sigma Aldrich (Merck). The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
(
inhibitor removal for DMAEMA was performed using the inhibitor remover from Sigma Aldrich. Linear
and branched poly(ethylene imine) (lPEI/bPEI, 25 kDa) was obtained from Polyscience. Diꢀtertꢀ
butyldicarbonate for the protection was from Alfa Aesar and 2ꢀaminoethanol was purchased by TCI. 2ꢀ
Bisazobutyronitile was received from Acros and recrystallized from methanol prior to use. Hydrochloric
acid, dimethylformamide and tetrahydrofurane were purchased from VWR Chemicals, all other solvents
ꢀ1
used were obtained from standard suppliers. Ethidium bromide solution (1%, 10 mg mL ) was obtained
from Carl Roth. AlamarBlue, YOYOꢀ1 iodide and Hoechst 33342 (10 mg mLꢀ1 solution) were obtained
from Life Technologies (Thermo Fisher). If not stated otherwise, cell culture consumables, cell culture
media and supplements (LꢀGlutamin, antibiotics) were obtained from Greiner BioꢀOne, Lonza and
Biochrom (Merck Millipore), respectively. Plasmid pEGFPꢀN1 (4.7 kb, Clontech, USA) encoding green
fluorescent protein (EGFP) was isolated with the Giga Plasmid Kit provided by Qiagen. Doubleꢀstranded
DNA (dsDNA, 23 nucleotides) was purchased from Jena Bioscience.
Synthesis of Nꢀtertꢀbutyloxycarbonylꢀ(2ꢀaminoethyl)ꢀmethacrylate (BocAEMA)
NꢀtertꢀButyloxycarbonylꢀ(2ꢀaminoethyl)ꢀmethacrylate was synthesized according to a procedure of
2
4
Kuroda et al.. 10 g of 2ꢀAminoethanol were dissolved in 120 mL THF and 200 mL of a 1 M aqueous
sodium hydroxide solution were added. 35.28 g of diꢀtertꢀbutyldicarbonate in 80 mL THF were added
dropwise while cooling the reaction mixture in an ice bath and stirred overnight. The mixture was diluted
with ethyl acetate and washed with water, NaHCO and brine and the aqueous phase was reextracted with
3
ethyl acetate, dried over sodium sulfate and the solvent was removed under reduced pressure. Without
2
4
ACS Paragon Plus Environment

Page 25 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
further purification 18.5 g of Nꢀtertꢀbutyloxycarbonylꢀ2ꢀaminoethanole was diluted with 30 mL dry
dichloromethane under schlenk conditions and 22.6 mL of dry triethylamine was added while cooling the
mixture in an ice bath. 15 mL Methacryloylchloride were added dropwise and the reaction was stirred
overnight. The mixture was washed with water, brine and sodium hydrogen carbonate and dried over
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
sodium sulfate. The crude off white product was finally recrystallized from nꢀhexane. H NMR (300 MHz,
CDCl ): δ [ppm] = 1.42 (s, 9H), 1.93 (s, 3H), 3.43 (m, 2H), 4.18 (t, 2H), 4.8 (br. s, 1H), 5.57 (s, 1H), 6.10
3
13
(
s, 1H). C NMR (75 MHz, CDCl ): δ [ppm] = 18.2 (CH3), 28.3 (CH3), 39.6 (CH2), 63.9 (CH2),
3
79.5(CH2), 125.8 (Cquart), 136.0 (Cquart), 155.7 (Cquart), 167.2 (Cquart).
Synthesis of NꢀmethylꢀN-tertꢀbutyloxycarbonylꢀ(2ꢀaminoethyl)ꢀmethacrylate (BocMAEMA)
NꢀMethylꢀNꢀtertꢀbutyloxycarbonylꢀ(2ꢀaminoethyl)ꢀmethacrylate was synthesized according to a procedure
2
5
of Sinclair et al. . 10 g NꢀMethylaminoethanol was dissolved in 80 mL chloroform was cooled in an ice
bath and 29 g diꢀtertꢀbutylcarbonate in 80 mL was added dropwise and stirred at room temperature for 1 h.
The solvent was removed under reduced pressure and the mixture was purified by distillation (30 mbar,
1
80 °C). Under schlenk conditions 21.4 g of NꢀmethylꢀNꢀtertꢀbutyloxycarbonylꢀ2ꢀaminoethanol were
diluted with 100 mL dry dichloromethane, 49.4 mL triethylamine was added and the reaction mixture
cooled in an ice bath. 17.7 mL methacryloyl chloride in 100 mL dichloromethane were added dropwise
and the reaction was stirred at room temperature overnight. The mixture was washed with water and brine
and dried over sodium sulfate. Further purification was done by column chromatography using a mixture
1
of cyclohexane an ethyl acetate (9:1ꢀ3:1). H NMR (300 MHz, CDCl ): δ [ppm] = 1.96 (s, 9H), 2.76 (m,
3
13
3H), 3.30 (m, 2H), 4.56 (m, 2H), 5.65 (s, 1H), 6.30 (s, 1H). C NMR (75 MHz, CDCl ): δ [ppm] = 18.2
3
(CH ), 28.3 (CH ), 35.2 (CH ), 47.9 (CH ), 62.7 (CH ), 79.7(CH ), 126.0 (Cquart), 136.1 (Cquart), 155.8
3
3
2
3
2
2
(Cquart), 167.1 (Cquart).
Synthesis of homoꢀ and copolymers
Homoꢀ and copolymers of BocAEMA, BocMAEMA and DMAEMA were prepared by the reversible
10
additionꢀfragmentation chain transfer (RAFT) polymerization method.
In a typical RAFT
2
5
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 26 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
ꢀ
3
copolymerization experiment, 0.735 g of BocAEMA (3.18 10 mol), 0,773 g of BocMAEMA (3.18
ꢀ3
ꢀ5
ꢀ5
1
0 mol), 0.98 mg of AIBN initiator (5.96 10 mol), 5.68 mg of CPDBꢀCOOH RAFT agent (20.33 10
mol) and 5.03 mL DMF were mixed together with anisole as internal standard (0.347 mL) in a 25 mL
ꢀ1
reaction vial. The monomer concentration was kept at 1 mol L . The reaction mixture was degassed with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
argon for at least 30 min and subsequently, the reaction solution was placed in a preheated oil bath at
7
0 °C for 38 h. The copolymer was purified by two times precipitation from THF into a minimum of 10ꢀ
fold amount large volume of nꢀhexane and dried under reduced pressure. Conversion was measured by
1
HꢀNMR spectroscopy using anisole as internal standard.
The RAFT polymerization of the DMAEMA homopolymers was carried out in ethanol in 1.73 molar
solution. 1.572 g DMAEMA (0.01 mol), 2.8 mg 4,4′ꢀazobis(4ꢀcyanovaleric acid) (0.001 mmol) and
11.1 mg 4ꢀcyanoꢀ4(pehnylcarbonthiothioylthio) pentanoic acid (0.004 mmol) were dissolved in 3.3 mL of
ethanol and 0.77 mL DMF were added as internal standard. The reaction mixture was degassed with argon
for 10 min and heated to 70 °C for 10 h. The resulting polymer was purified via precipitation from THF in
1
nꢀhexane twice and dried at high vacuum. Conversion was measured by HꢀNMR
Deprotection of Bocꢀprotected polymers
Bocꢀprotected homoꢀ and copolymers were deprotected using diluted hydrochloric acid in methanol. In a
typical procedure, 300 mg polymer was dissolved in 10 mL methanol and 1 mL of concentrated
hydrochloric acid was added dropwise and stirred at room temperature overnight. The solvent was
removed under reduced pressure, dissolved in water and freeze dried.
Asymmetric flow fieldꢀflow fractionation (AF4)
Asymmetric flow fieldꢀflow fractionation (AF4) was performed on an AF2000 MT system (Postnova
Analytics, Landberg, Germany) coupled to an UV (PN3211, 260 nm), RI (PN3150), MALLS (PN3070,
6
33 nm) detector. The eluent is delivered by two different pumps (tip and focusꢀflow) and the sample is
injected by an autosampler (PN5300) into the channel. The channel has a trapezoidal geometry and an
2
6
ACS Paragon Plus Environment

Page 27 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
overall area of 31.6 cm². The nominal height of the spacer was 500 µm and a regenerated cellulose
ꢀ1
membrane with a molar mass cutꢀoff of 10,000 g mol was used as the accumulation wall. All
experiments were carried out at 25 °C and the eluent was 20 mM NaCl in 25 mM sodium acetate buffer at
ꢀ1
pH 3.5. The detector flow rate was set to 0.5 mL min for all samples and a sample volume of 50 µL
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ꢀ1
ꢀ1
(
10 mg mL ) were injected with an injection flow rate of 0.2 mL min for 7 min. For all samples the
ꢀ1
crossꢀflow was set to 2 mL min . After the focusing period and a transition time of 1 min, the cross flow
was kept constant for 1 min and then decreased under a power function gradient 0.40 to zero within 15
min. Afterwards the crossꢀflow was kept constant at zero for 20 min to ensure complete elution. For
calculation of the molar mass a Zimm plot was used. The refractive index increment (dn/dc) of all samples
was measured by manual injection of a known concentration directly into the channel without any
focusing or crossꢀflow. The dn/dc was calculated as the average of at least three injections from the area
under the RI curve.
NMR spectrometry
NMR spectra were recorded on a Bruker AC 300 MHz or on a Bruker AC 250 MHz spectrometer.
Titration
The titration for the detection of the pK value of the polymers was performed with the automated titrator
a
7
65 Dosimat (Metrohm, Herisau, Swiss) and the pH detector GMH3530 (GHM Messtechnik GmbH
Standort Greisinger, Regenstauf, Germany). For a typical measurement the polymer in ultrapure water
ꢀ1
(
10 mg mL ) was acidified by 10 µL hydrochloric acid and stirred while titrated against a 0.1 M sodium
ꢀ1
hydroxide solution (0.1 mL min ) to a pH value of 12. The pK value was using the Hendersonꢀ
a
Hasselbalch equation (2) by determining the equivalence point from the titration diagram.
ꢑ
ꢏꢐ ꢒ
ꢏꢉꢐꢒ
ꢂꢂꢂꢂꢂꢂꢂꢈꢉ = ꢈꢊ + ꢌꢍꢎ
ꢂꢂꢂꢂꢂꢂꢂꢂꢂꢂ(2)
ꢋ
Size exclusion chromatography (SEC)
2
7
ACS Paragon Plus Environment

Bioconjugate Chemistry
Page 28 of 42
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Size exclusion chromatography (SEC) was measured on a Agilent 1200 series system equipped with a
PSS degasser, a G1310A pump, a G1362A refractive index detector and a PSS GRAM guard column
running with dimethylacetamide (DmAc) with 0.21 % of lithium chloride. The Techlab oven was set to
5
0 °C and the molar masses were calculated using a poly(methyl methacrylate) (PMMA) standard. For the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
water soluble polymers a SEC system of Jasco was used with a DGꢀ980ꢀ50 degasser, a PUꢀ980 pump,
equipped with a RIꢀ930 RI detector and Jasco oven set to 50 °C. The column used was AppliChrom
ABOA CatPhil, which is operated with a water solution with 0.1 % trifluoric acid and 0.1 M NaCl.
Polyplex preparation
Polyplexes of plasmid DNA (pDNA) and polymers were prepared by mixing stock solutions of
ꢀ1
ꢀ1
1
5 µg mL pDNA and different amounts of polymers (1 mg mL ) to obtain various N/P ratios (nitrogen
of polymer to phosphate of pDNA) in HBG buffer (20 mM 4ꢀ(2ꢀhydroxethyl) piperazineꢀ1ꢀethanesulfonic
acid (HEPES) and 5% (w/v) glucose, pH 7.2). The solutions were vortexed for 10 sec at maximal speed
and incubated at room temperature for 15 min to ensure complex formation.
Ethidium bromide quenching assay
The formation of polyplexes with pDNA was examined by quenching of the ethidium bromide
ꢀ
1
fluorescence. Briefly, 15 µg mL pDNA in a total volume of 100 µL HBG buffer were incubated with
ꢀ1
ethidium bromide (0.4 µg mL ) for 10 min at room temperature. Subsequently, polyplexes with different
amounts of polymer (various N/P ratios) were prepared in black 96ꢀwell plates (Nunc Thermo Fisher) and
incubated at room temperature for 15 min before the fluorescence measurements. The fluorescence of the
samples was measured at an excitation wavelength of 525 nm and an emission wavelength of 605 nm
using a Tecan microplate reader. A sample containing only pDNA and ethidium bromide was used to
ꢀ1
calibrate the device to 100% fluorescence against a background of 0.4 µg mL of ethidium bromide in
HBG solution. The percentage of dye displaced upon polyplex formation was calculated using equation
(3):
2
8
ACS Paragon Plus Environment

Page 29 of 42
Bioconjugate Chemistry
1
2
3
4
5
6
7
8
9
ꢓꢔꢕꢖꢗꢘꢙꢑꢓꢚ
RFUꢂꢏ%ꢒ = ꢓꢗꢛꢜꢝꢑꢓꢚ * 100
(3)
Here, RFU is the relative fluorescence and Fsample, F , and F
represents the fluorescence intensities of a
pDNA
0
given sample, the ethidium bromide in HBG alone, and the ethidium bromide intercalated into pDNA
alone.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Heparin dissociation assay
Polyplexes with an N/P ratio of 20 were prepared as described above in a total volume of 100 µL HBG
ꢀ1
buffer containing ethidium bromide (0.4 µg mL ). After incubation in the dark at room temperature for
15 min, polyplexes were transferred into a black 96ꢀwell plate, and heparin of indicated concentrations
were added. The solution was mixed and incubated for further 30 min at 37 °C in the dark. The
fluorescence of ethidium bromide was measured at Ex 525 nm/Em 605 nm with a Tecan microplate
reader. The percentage of intercalated ethidium bromide was calculated as described before.
Dynamic and electrophoretic light scattering
Dynamic light scattering (DLS) was performed on a Zetasizer Nano ZS (Malvern Insturments,
Herrenberg) with a HeꢀNe laser operating at a wavelength of λ = 633 nm. All measurements (30 runs,
triplicate) were carried out at 25 °C after an equilibration time of 120 sec. The counts were detected at an
angle of 173°. The mean particle size was approximated as the effective (zꢀaverage) diameter and the
width of the distribution as the polydispersity index of the particles (PDI) obtained by the cumulants
method assuming a spherical shape. Electrophoretic light scattering (ELS) was used to measure the zeta
potential (ζ). The measurement was performed on a Zetasizer Nano ZS (Malvern Instruments, Herrenberg,
Germany) by applying laser Doppler velocimetry. For each measurement, 20 runs were carried out using
the slowꢀfield reversal and the fastꢀfield reversal mode at 150 V. Each experiment was performed in
2
9
ACS Paragon Plus Environment