Side chain and backbone structure-dependent subcellular localization and toxicity of conjugated polymer nanoparticles

Article abstract of DOI:10.1039/c3cc43015d

The subcellular localization and toxicity of conjugated polymer nanoparticles (CPNs) are dependent on the chemical structure of the side chains and backbone. Primary amine-containing CPNs exhibit high Golgi localization with no toxicity. Incorporation of

Full text of DOI:10.1039/c3cc43015d

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Side chain and backbone structure-dependent
subcellular localization and toxicity of conjugated
polymer nanoparticles†
Cite this: Chem. Commun., 2013,
49, 6048
Received 23rd April 2013,
Accepted 16th May 2013
Eladio Mendez and Joong Ho Moon*
DOI: 10.1039/c3cc43015d
www.rsc.org/chemcomm
The subcellular localization and toxicity of conjugated polymer degradation routes because macropinosomes do not fuse with
nanoparticles (CPNs) are dependent on the chemical structure of the lysosomes, and the membranes of macropinosomes are
the side chains and backbone. Primary amine-containing CPNs highly leaky.10 Therefore, systematic investigation to understand
exhibit high Golgi localization with no toxicity. Incorporation of and modulate the cellular interaction and pathways will have a
short ethylene oxide and tertiary amine side chains contributes to significant impact on designing efficient labelling and delivery
decreased Golgi localization and increased toxicity, respectively.
vehicles.
Previously, we demonstrated that CPNs fabricated by treating a
Semiconducting conjugated polymer nanoparticles (CPNs) and CP containing both short ethylene oxide (EO) and primary amine
conjugated polyelectrolytes (CPEs) are emerging fluorescent (e.g., P1, Fig. 1) with organic acids followed by dialysis exhibit
biomaterials for cellular labelling,1 sensing,2 therapeutics,3 efficient cellular labelling and delivery of small interfering RNA
and delivery4 of biological substances. Excellent photophysical without toxic effects.4a,b Mechanistic studies further indicate that
properties of conjugated polymers (CPs) including high molar CPNs use both energy dependent and independent entry path-
absorptivity, quantum yield, and energy transfer efficiency ways. Among the energy dependent pathways, CPNs enter cancer
make them suitable for various biological applications.5 Well- cells via caveolae-mediated endocytosis as one of the entry path-
established synthetic methods also allow facile modifications ways.11 It is not clear why the CPNs use the specific entry pathway,
of both p-electron conjugated backbones and side chains with however, the positive charges and hydrophobicity of CPNs play
various sensing or targeting units. By treating non-aqueous important roles in interaction with various serum proteins and
soluble CPs under various particle formation conditions, non- the cell membranes, which will significantly influence the subse-
toxic soft nanoparticles have been fabricated and used for quent cellular uptake.12 It is also known that materials having
cellular labelling and nucleic acid delivery.6
Understanding cellular interactions and entry pathways of group-dependent cellular interactions and subsequent entry.13
CPNs is paramount to improving the overall labelling and delivery Based on the results and observations, we hypothesized that
high surface-to-volume ratios exhibit size, shape, and functional
efficiency. Depending on the entry pathways, the materials and chemical modifications in the side chains of CPs will change
their payloads (i.e., drugs or genes) will be trafficked into different
organelles, which will significantly influence the overall efficiency.7
For example, carriers entrapped in endosomes or lysosomes
trafficked via a certain type of endocytosis will experience recycling
of the contents back to the cell surface and degradation processes
in acidic lysosomes, lowering the overall labelling and delivery
efficiency.8 Meanwhile, exogenous materials trafficked by non-
destructive organelles such as caveosomes to the Golgi apparatus
(i.e., caveolae-mediated endocytosis) have high intracellular reten-
tion.9 Delivery via macropinocytosis can also avoid lysosomal
Department of Chemistry and Biochemistry, Florida International University,
Fig. 1 Chemical structures of CPs. Poly(p-phenyleneethynylenes) (PPEs) with different
side chains (P1–P3) and poly(p-phenylenebutadiynylene) (PPB) containing a small
amount of flexible unit in the backbone (P4) were synthesized and compared for
cellular behaviour.
11200 SW 8th St., Miami, FL, 33199, USA. E-mail: jmoon@fiu.edu;
Tel: +1-305-348-1368
† Electronic supplementary information (ESI) available: Detailed synthetic procedures,
characterization, and co-localization analyses. See DOI: 10.1039/c3cc43015d
c
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the subcellular localization of CPNs, because the modulated
surface properties will influence the cellular interactions of
CPNs and their subsequent entry into cells.
To test the hypothesis, we synthesized four CPNs with
different side chain (P1–P3) and backbone (P4) structures
(Fig. 1). Since the cellular interactions and entry processes of
nanomaterials are collectively influenced by the physicochemical
properties, it is important to keep other physicochemical proper-
ties constant when a specific parameter is tested. Because of the
particle formation mechanism (i.e., molecular weight indepen-
dent phase inverse precipitation driven by aqueous insolubility
of CPs),14 the shapes and hydrodynamic radii of CPNs are
relatively constant.15 CPN-2 was designed and synthesized to
check the EO side chain effects on the toxicity and localization by
removing the EO unit from the repeating unit of CPN-1. CPN-3
was synthesized to increase amine density using branched
Fig. 2 Cellular toxicity of CPNs measured by cell viability inhibition at various
concentrations.
amine side chains containing tertiary amines. CPN-4 was synthe- buffering capacity of tertiary amines. Membrane disruption
sized to compare the backbone flexibility effects while amine properties of synthetic carriers containing tertiary amines have
density was maintained close to that of CPN-2. Synthesis of P4 been used to increase payload escape from the endosomes or
was reported in our recent publication.16 All polymers were treated lysosomes, however, these classes of materials often cause
with a series of organic acids followed by dialysis, affording CPNs toxicity issues.17
that are physically stable in water. Physicochemical properties of
Subcellular localization of CPNs was monitored by fluores-
CPNs are listed in Table 1. Since aggregation behaviour is concen- cent microscopic imaging. HeLa cells incubated with CPNs
tration dependent, the concentrations of all CPN solutions were (green) overnight were co-stained with pHRhodo Dextran (10 kDa)
adjusted to be 0.5 mM. Non-EO containing CPN-2 and CPN-4 (red) and BODIPY-TR C5-ceramide–BSA complex (red) for labelling
exhibited slightly larger hydrodynamic radii than those of CPN-1 acidic organelles (i.e., endosomes and lysosomes) and Golgi appa-
and CPN-3 fabricated with EO containing polymers. The difference ratus, respectively (Fig. 3a and ESI†). CPNs were mainly found at
in hydrodynamic radius among the CPNs is expected to have the perinuclear regions (punctuated green dots) and exhibited
minimal effects on the cellular interaction and subcellular locali- overlaps with both pHRhodo and BODIPY. Co-localization patterns
zation due to the polydisperse nature of CPNs. Zeta potentials of with the Golgi were clearly distinguishable among CPNs having
CPNs were determined to be B+42–46 mV, except for CPN-1 different side chain or backbone structures (Fig. 3a), while over-
exhibiting B+20 mV (Table 1).
lapping patterns with pHRhodo were relatively uniform (ESI†).
To test how the side chain structure influences cellular CPN-2 and CPN-4, which only contain primary amine side chains,
toxicity, CPNs were incubated with human cervical carcinoma exhibit high Golgi localization (Fig. 3a), while CPN-1 and CPN-3,
cells (HeLa) overnight at various concentrations. Zeta potentials which contain both EO and amine side chains, exhibit a relatively
of CPNs had no direct correlation with the toxicity, but the low Golgi overlap.
chemical structure (i.e., type and density of amine) of the side
To obtain quantitative co-localization information, all
chains was related to the cellular toxicity. As shown in Fig. 2, images were further analysed using Pearson’s Correlation
CPN-3 containing the highest amine density, including tertiary Coefficient (PCC) method. The PCC method gauges the level
amines, exhibited substantial toxicity starting from 10 mM, of overlap by measuring the pixel-by-pixel covariance in the
while no cell viability inhibition was observed up to 40 mM signals of two images. Because the PCC method uses normalized
for the primary amine containing CPNs, whether they contain signals by subtracting the mean intensity from each pixel’s intensity
EO side chains or flexible backbones (i.e., CPN-1, -2, and -4). value, PCC is independent of signal levels (probe brightness) and
Compared to CPN-2, which contains the same amount of signal offset (background).18 PCC values of 0 and 1 correspond to
primary amines per repeating unit as CPN-3, toxicity of CPN-3 uncorrelated and perfectly linear correlated images, respectively.
can be attributed to both increased amine density and the high Instead of picking small, subjective regions of interest within an
Table 1 Physicochemical properties of CPNs
Hydrodynamic
radiuse (nm)
Zeta potential f
(mV)
CPN
Type
Mna (kDa)
PDIb
lmax,absc (nm)
lmax,emd (nm)
PDIe
1
2
3
4
P1 (PPE)
P2 (PPE)
P3 (PPE)
P4 (PPB)
16.4
11.8
10.7
22.3
1.49
1.43
1.64
2.28
433
427
420
444
496
492
496
500
61 Æ 6.7
71 Æ 7.9
58 Æ 3.4
87 Æ 6.1
0.27 Æ 0.02
0.29 Æ 0.02
0.33 Æ 0.06
0.51 Æ 0.03
+20 Æ 0.4
+42 Æ 5.1
+44 Æ 1.1
+46 Æ 2.3
a
b
c
Determined by gel permeation chromatography in THF relative to polystyrene standard. Polydispersity index (PDI) = Mw/Mn. Measured in
d
e
f
water. Measured in water, excitation wavelength 400 nm. Measured by DLS at 500 mM in water. Mean Æ standard deviation. Electrophoretic
measurement at pH 7.0. Mean Æ standard deviation.
c
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and biodegradability of CPN-4, we are currently investigating the
delivery of small RNA molecules to target cells.
This work was supported by National Institute of Health/
National Institute of General Medical Sciences Grant No.
SC1GM092778 and R25GM61347.
Notes and references
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6050 Chem. Commun., 2013, 49, 6048--6050