Low molecular weight PEI-based fluorinated polymers for efficient gene delivery

Article abstract of DOI:10.1016/j.ejmech.2018.11.041

Fluorinated biomaterials have been reported to have promising features as non-viral gene carriers. In this study, a series of fluorinated polymeric gene carriers were synthesized via Michael addition from low molecular weight polyethyleneimine (PEI) and f

Full text of DOI:10.1016/j.ejmech.2018.11.041

Accepted Manuscript
Low molecular weight PEI-based fluorinated polymers for efficient gene delivery
Ya-Ping Xiao, Ji Zhang, Yan-Hong Liu, Ju-Hui Zhang, Qing-Ying Yu, Zheng Huang,
Xiao-Qi Yu
PII:
S0223-5234(18)31000-6
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.041
EJMECH 10899
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 September 2018
Revised Date: 16 November 2018
Accepted Date: 16 November 2018
Please cite this article as: Y.-P. Xiao, J. Zhang, Y.-H. Liu, J.-H. Zhang, Q.-Y. Yu, Z. Huang, X.-Q. Yu,
Low molecular weight PEI-based fluorinated polymers for efficient gene delivery, European Journal of
Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.ejmech.2018.11.041.
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ACCEPTED MANUSCRIPT
Graphical Abstract:

ACCEPTED MANUSCRIPT
Low molecular weight PEI-based fluorinated polymers for efficient
gene delivery
*
Ya-Ping Xiao, Ji Zhang , Yan-Hong Liu, Ju-Hui, Zhang, Qing-Ying Yu, Zheng Huang, and
*
Xiao-Qi Yu
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan
University, Chengdu 610064, P. R. China
*
Corresponding authors: jzhang@scu.edu.cn (J. Zhang); xqyu@scu.edu.cn (X.-Q. Yu); Fax: + 86 28
5415886
8
Abstract: Fluorinated biomaterials have been reported to have promising features as non-viral
gene carriers. In this study, a series of fluorinated polymeric gene carriers were synthesized via
Michael addition from low molecular weight polyethyleneimine (PEI) and fluorobenzoic acids
(FBAs)-based linking compounds with different numbers of fluorine atoms. The
structure-activity relationship (SAR) of these materials was systematically investigated. SAR
studies showed that fluorine could screen the positive charge of these polymers. However, this
shielding effect of fluorine would endow fluorinated polymers with good balance between
DNA condensation and release. In vitro transfection results suggested that these fluorinated
polymers could mediate efficient gene delivery. Flow cytometry and confocal microscopy
studies demonstrated that more efficient cell uptake could be achieved by fluorinated materials
with more fluorine atoms. Cytotoxicity assays showed that these fluorinated materials
exhibited very low cytotoxicity even at high mass ratios. This study demonstrates that
FBA-based fluorinated biopolymers have the potential for practical application.
Keywords: non-viral gene vector, FBA-based fluorinated biopolymers, structure-activity
relationship, gene delivery,
1
. Introduction
Cationic polymers are widely used as promising gene vectors to introduce exogenous
nucleic acid into cells for activating normal function or correcting abnormal function of the
cells [1]. These materials can be prepared reproducibly, designable, inexpensively, and have
superior safety compared with viral gene vectors [1j,2]. Such polymers can compact nucleic

ACCEPTED MANUSCRIPT
acids through electrostatic interaction and the condensation may facilitate cellular uptake
through various endocytic pathways, inhibiting intra- or extracellular enzymatic degradation
[
3]. Among different varieties of cationic polymers, poly(ethyleneimine) (PEI) is the most
widely studied [4], and its high condensation capability for DNA and highly efficient delivery
of DNA made 25 kDa PEI the “gold standard” for newly designed polymeric vectors [5]. It is
also well known that PEI with a low molecular weight (LMW) shows a relatively lower
toxicity and transfection efficiency, while those with high molecular weight (HMW) always
have a higher transfection efficiency but higher toxicity [6].
Over the past few decades, varieties of PEI derivatives have been developed to enhance
transfection efficiency and reduce cytotoxicity. These PEI derivatives might be synthesized by
many strategies such as covalent grafting [7], cross linking [8] and electrostatic coating [9].
Among such polymers, crosslinked LMW PEIs have been examined and various types of
degradable PEI polymers and networks have been designed for in vitro transfection [10]. Our
group also studied a type of biodegradable polymer prepared via Michael addition of LMW
PEI and a linking compound, and these materials showed distinctly higher transfection
efficiency and serum tolerance than 25 kDa PEI [11].
Recently, fluorinated cationic polymers have shown great promise as alternatives to
traditional polymers in DNA and siRNA delivery, since fluorination increases the efficacy of
cationic polymers while reducing their cytotoxicity, representing a promising tool in the design
of safe and efficient non-viral gene vectors [10c, 12]. Such excellent performance is attributed
to the “fluorophilic effect” [13], which endows fluorinated polymers with manipulable
capacities such as serum resistance ability, efficient cellular uptake and successful endosomal
escape [14]. More recently, it was also found that the integration of rigid elements might
benefit the gene vectors with enhanced biocompatibility and efficiency [10c,15]. Such
encouraging results inspired us to develop fluorinated polymers with the characteristic of
rigidity to further improve the performance of cationic gene carriers.
Fluorobenzoic acids (FBAs) are a class of synthetic intermediates in medicinal chemistry,
and about 20% - 25% of the therapeutic drugs on the market contain fluorine [16]. Fluorine on
the aromatic ring of FBA can improve the stability and/or therapeutic activity of these drugs.
Therefore, we synthesized a series of fluorinated biopolymers via Michael addition from LMW

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PEI 600 Da and FBA-based linking compounds with different numbers of fluorine atoms, and
the polymer synthesized from PEI 600 Da and benzoic acid (BA)-based linking compound was
used for comparison. The structure-activity relationship (SAR) of such series of polymers was
systematically investigated. The transfection efficacy and cytotoxicity were compared with PEI
2
5 kDa. Parameters such as the number of the fluorine atoms were discussed. The aim of this
study is to provide a new strategy to improve the transfection efficacy and biosafety of
fluorinated polymers with the ability of rigidity and to understand the SAR of these
biopolymers in gene delivery.
2
. Experimental section
2
.1 Materials. Unless specially noted, all chemicals and reagents were obtained commercially
and used without further purification. Dichloromethane (CH Cl ) and Methanol (CH OH) were
2
2
3
dried and purified according to standard methods and were distilled immediately before use.
Column chromatography was performed using 200-300 mesh silica gel. All aqueous solutions
were prepared from deionized or distilled water. Fluorobenzoic acids (FBA) or benzoic acid
(BA) were purchased from Energy Chemical. LMW-PEI (branched, average molecular weight
6
00 Da) was purchased from Aladdin (Shanghai, China). HMW-PEI (branched, average
molecular weight 25 kDa) and MTS (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide) were purchased from Sigma-Aldrich (St Louis, MO, USA). pGL-3 (Promega,
Madison, WI, USA) coding for luciferase DNA as the plasmids and pEGFP-N1 (Clontech, Palo
Alto, CA, USA) coding for EGFP DNA were used in the study. Cy5™ was obtained from
Molecular Probe (Mirus, Madison, WI, USA). The Dulbecco's Modied Eagle's Medium
(DMEM), 1640 Medium and fetal bovine serum (FBS) were purchased from Invitrogen
Corporation. HeLa cells, 7702 cells, HepG2 cells and A549 cells were purchased from
Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences.
MicroBCA protein assay kit was obtained from Pierce Biotechnology (Rockford, IL, USA).
The luciferase assay kit was purchased from Promega (Madison, WI, USA). The
endotoxin-free (EndoFree) plasmid purification kit was purchased from TIANGEN
Biotechnology (Beijing, China).
1
13
19
H NMR, C NMR and F NMR spectra were obtained on a Bruker AV400 spectrometer.

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HRMS spectral data was recorded on a Bruker Daltonics Bio TOF mass spectrometer. The
molecular weights of polymers were determined by gel permeation chromatography (GPC),
which consisted of a Waters 515 isocratic HPLC pump, a Linear 7.8×300 mm column (Waters
Corp, Milford, MA, USA), an 18-angle laser scattering instrument (Wyatt Technology
Corporation, USA), and an OPTILAB DSP interferometric refractometer (Wyatt Technology
-1
Corporation, USA). A filtered mixture of 0.5 mol L HAc/NaAc buffer was used as the mobile
-1
phase, with a flow rate of 1 mL min . Molecular weights were calculated against polyethylene
glycol standards, with average molecular weights ranging from 601 to 106000.
Some of the experiments, including the agarose gel retardation assay, ethidium bromide (EB)
exclusion assay, dynamic light scattering (DLS) assay for the measurement of particle size and
zeta potential, transmission electron microscopy (TEM), amplification and purification of
plasmid DNA, cell culture, in vitro transfection experiments, flow cytometry assay and
cytotoxicity assay, were carried out following previously reported procedures except using
various cell lines [10c.] MTS, instead of MTT, was used in the cytotoxicity assay.
2
.2 Synthesis and characterization of target polymers
2.2.1 Synthesis and characterization of 2.
Diethanolamine (10 g, 0.095 mol) was dissolved in 50 ml CH OH. After addition of
3
ditertbutylpyrocarbonate ((Boc) O) (24.9 g, 0.114 mol), the solution was stirred at room
2
temperature for at least 30 min. The obtained mixture was evaporated under reduced pressure
to give a colorless oil product. The crude product was then used without further purifying.
Acryloyl chloride (9.42 g, 0.104 mol) in anhydrous dichloromethane (80 mL) was added
dropwise to a stirred solution of compound 1 (10 g, 0.048 mol) and triethylamine (10.52 g,
0
.104 mol) in anhydrous dichloromethane (100 mL) in an ice bath. The mixture was then
stirred overnight at room temperature. After the reaction, the solution was washed with
saturated NaHCO (2 × 50 ml) and brine (50 ml). The organic layer was dried over anhydrous
3
Na SO and concentrated under reduced pressure. The residue was purified by silica gel
2
4
column chromatography (PE : EA = 3:1, v/v) to give the product 2 as a colorless liquid.
1
2
: 85.7% yield. H NMR (400 MHz, CDCl , TMS) δ: 1.43 (s, 9H, -Boc), 3.49-3.56 (m, 4H,
3
-NCH CH O-), 4.23-4.29 (m, 4H, -NCH CH O-), 5.83 (t, J = 8 Hz, 2H, -OCOCHCH ), 6.10
2 2 2 2 2
13
(
dd, J = 12 Hz, J = 20 Hz, 2H, -OCOCHCH ), 6.39 (d, J = 20 Hz, 2H, -OCOCHCH ). C
2
2

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NMR (CDCl , 100 MHz) δ: 28.44, 47.06, 62.78, 80.47, 128.24, 131.33, 155.27, 166.05.
3
HR-MS (ESI): Calcd for: C H NO : 336.1418 ([M + Na]+), Found: 336.1427 ([M + Na]+).
15
23
6
2
.2.2 Synthesis and characterization of compound 3a-g.
Compound 2 (3 g, 9.58 mmol) was dissolved in anhydrous dichloromethane (30 mL), and
then trifluoroacetic acid (5 mL) was added dropwise under an ice bath. The mixture was
stirred overnight at room temperature. After the solvent and trifluoroacetic acid were
removed, dissolve the obtained mixture in 50 mL dichloromethane and adjust the pH of the
solution greater than 7 by dilute ammonia. The organic layer was dried over anhydrous
Na SO for 6 h, and then concentrated under reduced pressure. The crude product was used
2
4
without further purifying. Subsequently, dissolve the crude product (1.2 equivalent),
Fluorobenzoic acids (FBAs) or benzoic acid (BA) (1 equivalent), 1-hydroxybenzotriazole
(HOBt) (1 equivalent) and triethylamine (1 equivalent) in 50 mL dichloromethane, and then
1
-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (1.2 eq.) dissolved in
0 mL dichloromethane was added dropwise under an ice bath. The mixture was stirred
3
overnight at room temperature. After the reaction, the solution was washed with saturated
NaHCO (2 × 50 ml) and brine (50 ml). Then the organic layer was dried over anhydrous
3
Na SO and concentrated under reduced pressure. The residue was purified by silica gel
2
4
column chromatography (PE : EA = 1:1, v/v) to give compound 3a-g.
1
3
a: 70.2% yield. H NMR (400 MHz, DMSO, TMS) δ: 3.53 (s, 2H, -NCH CH O-), 3.76
2
2
(
s, 2H, -NCH CH O-), 4.16 (s, 2H, -NCH CH O-), 4.35 (s, 2H, -NCH CH O-), 5.95 (d, J = 8
2 2 2 2 2 2
Hz, 2H, -OCOCHCH ), 6.14-6.16 (m, 2H, -OCOCHCH ), 6.24-6.36 (m, 2H, -OCOCHCH ),
2
2
2
1
3
7
.12 (d, J = 8 Hz, 2H, PhH), 7.26 (t, J = 4 Hz, 1H, PhH), 7.46 (q, J= 12 Hz, 1H, PhH). C
NMR (DMSO, 100 MHz) δ: 43.45, 47.74, 61.34, 113.70, 116.22, 122.70, 128.16, 130.80,
19
1
31.91, 138.54, 160.53, 162.97, 169.84. F NMR (DMSO) δ: -112.27. HR-MS (ESI): Calcd
+
+
for: C H FNO : 358.1061 ([M + Na] ), Found: 358.1073 ([M + Na] ).
1
7
18
5
1
3
b: 63.4% yield. H NMR (400 MHz, DMSO, TMS) δ: 3.54 (s, 2H, -NCH CH O-), 3.77
2 2
(
s, 2H, -NCH CH O-), 4.19 (s, 2H, -NCH CH O-), 4.37 (s, 2H, -NCH CH O-), 5.96-5.98 (m,
2 2 2 2 2 2
2
7
4
H, -OCOCHCH ), 6.15-6.18 (m, 2H, -OCOCHCH ), 6.26-6.33 (m, 2H, -OCOCHCH ),
2 2 2
1
3
.06-7.07 (m, 2H, PhH), 7.34-7.36 (m, 1H, PhH). C NMR (DMSO, 100 MHz) δ: 43.60,
7.74, 61.30, 104.88, 110.07, 110.15, 110.26, 110.34, 128.01, 131.07, 139.62, 160.94, 163.42,

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1
9
1
65.53, 168.60. F NMR (DMSO) δ: -108.50. HR-MS (ESI): Calcd for: C H F NO :
17 17 2 5
+
+
3
76.0967 ([M + Na] ), Found: 376.0948 ([M + Na] ).
1
3
c: 67.8% yield. H NMR (400 MHz, DMSO, TMS) δ: 3.55 (s, 2H, -NCH CH O-), 3.76
2
2
(
s, 2H, -NCH CH O-), 4.18 (s, 2H, -NCH CH O-), 4.35 (s, 2H, -NCH CH O-), 5.95-5.98 (m,
2 2 2 2 2 2
2
H, -OCOCHCH ), 6.14-6.20 (m, 2H, -OCOCHCH ), 6.26-6.32 (m, 2H, -OCOCHCH ), 7.34
2 2 2
1
3
(
t, J = 8 Hz, 2H, PhH). C NMR (DMSO, 100 MHz) δ: 43.80, 47.82, 61.41, 111.95, 112.11,
19
1
12.17, 128.15, 131.88, 132.72, 148.98, 151.36, 168.02. F NMR (DMSO) δ: -160.15, -134.02.
+
+
HR-MS (ESI): Calcd for: C H F NO : 394.0873 ([M + Na] ), Found: 394.0873 ([M + Na] ).
17
16
3
5
1
3
d: 74.1% yield. H NMR (400 MHz, DMSO, TMS) δ: 3.57 (t, J = 4 Hz, 2H,
-
NCH CH O-), 3.83 (t, J = 4 Hz, 2H, -NCH CH O-), 4.14 (t, J = 4 Hz, 2H, -NCH CH O-),
2
2
2
2
2
2
4
.32 (t, J = 4 Hz, 2H, -NCH CH O-), 5.91-5.94 (m, 2H, -OCOCHCH ), 5.98-6.13 (m, 2H,
2 2 2
1
3
-
OCOCHCH ), 6.21-6.27 (m, 2H, -OCOCHCH ), 7.95-8.01 (m, 1H, PhH). C NMR (DMSO,
2
2
19
1
00 MHz) δ: 43.24, 47.13, 60.86, 108.04, 127.49, 128.01, 131.98, 159.25, 165.28. F NMR
+
(
DMSO) δ: -142.30, -137.65. HR-MS (ESI): Calcd for: C H F NO : 412.2918 ([M + Na] ),
17
15
4
5
+
Found: 412.2250 ([M + Na] ).
1
3
e: 60.3% yield. H NMR (400 MHz, DMSO, TMS) δ: 3.61 (t, J = 4 Hz, 2H,
-
NCH CH O-), 3.86 (t, J = 4 Hz, 2H, -NCH CH O-), 4.17 (t, J = 4 Hz, 2H, -NCH CH O-),
2 2 2 2 2 2
4
.35 (t, J = 4 Hz, 2H, -NCH CH O-), 5.94-5.98 (m, 2H, -OCOCHCH ), 6.03-6.17 (m, 2H,
2 2 2
1
3
-
OCOCHCH ), 6.26-6.34 (m, 2H, -OCOCHCH ). C NMR (DMSO, 100 MHz) δ: 43.32,
2
2
19
4
7.06, 60.82, 127.76, 132, 158.50, 165.26. F NMR (DMSO) δ: -160.53, -153.34, -142.01,
+
-
141.98. HR-MS (ESI): Calcd for: C H F NO : 430.0684 ([M + Na] ), Found: 430.0692 ([M
1
7
14
5
5
+
+
Na] ).
f: 80.2% yield. H NMR (400 MHz, DMSO, TMS) δ: 3.54 (s, 2H, -NCH CH O-), 3.81 (s,
1
3
2
2
2
H, -NCH CH O-), 4.19 (s, 2H, -NCH CH O-), 4.41 (s, 2H, -NCH CH O-), 5.93-5.95 (m, 2H,
2 2 2 2 2 2
-
OCOCHCH ), 6.10-6.36 (m, 4H, -OCOCHCH and -OCOCHCH ), 8.02 (s, 2H, PhH), 8.20 (s,
2 2 2
1
3
2
H, PhH). C NMR (DMSO, 100 MHz) δ: 43.61, 47.92, 61.23, 121.64, 123.11, 124.36,
1
9
1
27.63, 130.58, 131.85, 138.85, 165.12, 165.61, 168.31. F NMR (DMSO) δ: -61.52. HR-MS
+
+
(
ESI): Calcd for: C H F6 NO : 476.0903 ([M + Na] ), Found: 476.0895 ([M + Na] ).
1
9
17
5
5
1
3
g: 83.6% yield. H NMR (400 MHz, DMSO, TMS) δ: 3.53 (s, 2H, -NCH CH O-), 3.75
2
2
(
s, 2H, -NCH CH O-), 4.15 (s, 2H, -NCH CH O-), 4.35 (s, 2H, -NCH CH O-), 5.95 (d, J = 8
2
2
2
2
2
2

ACCEPTED MANUSCRIPT
Hz, 2H, -OCOCHCH ), 6.16-6.31 (m, 4H, -OCOCHCH and -OCOCHCH ), 7.27-7.29 (m, 2H,
2
2
2
1
3
PhH), 7.39-7.41 (m, 2H, PhH). C NMR (DMSO, 100 MHz) δ: 44.57, 47.77, 61.32, 126.54,
28.43, 129.28, 132, 136.41, 171.36. HR-MS (ESI): Calcd for: C H NO : 340.1155([M +
1
1
7
19
5
+
+
Na] ), Found: 340.1156 ([M + Na] ).
.2.3 Synthesis and characterization of target polymers.
Polymers were successfully synthesized by Michael addition reaction as reported previously.
Briefly, PEI 600 (1.26 mmol) and compound 3a-g (1.26 mmol) were separately dissolved in
2
1
.5 mL of anhydrous methanol and 1.5 mL anhydrous dichloromethane. The reaction mixtures
o
were heated at 45 C with constant stirring for 72 h. After the reaction, the mixtures were
diluted with 3 mL of anhydrous methanol, and the crude product was precipitated by the
addition of diethyl ether.
1
PF1: 45.8% yield. H NMR (400 MHz, D O, TMS) δ: 2.70-3.07 (m, 45H, -NHCH CH N-,
2
2
2
and -OCOCH CH -), 3.53 (t, J = 4 Hz, 2H, -NCH CH O-), 3.65 (t, J = 4 Hz, 2H,
2
2
2
2
-
NCH CH O-), 3.73 (t, J = 4 Hz, 2H, -NCH CH O-), 3.88 (t, J = 4 Hz, 2H, -NCH CH O-),
2 2 2 2 2 2
1
9
7
.21-7.29 (m, 3H, PhH), 7.49-7.53 (m, 1H, PhH). F NMR (D O) δ: -112.37. GPC: Mw = 5.7
2
kDa, PDI = 1.52.
PF2: 56.3% yield. H NMR (400 MHz, D O, TMS) δ: 2.70-3.15 (m, 58H, -NHCH CH N-,
1
2
2
2
and -OCOCH CH -), 3.52 (t, J = 4 Hz, 2H, -NCH CH O-), 3.66 (t, J = 4 Hz, 2H,
2
2
2
2
-
NCH CH O-), 3.72 (t, J = 4 Hz, 2H, -NCH CH O-), 3.87 (t, J = 4 Hz, 2H, -NCH CH O-),
2 2 2 2 2 2
1
9
7
.08-7.10 (m, 3H, PhH). F NMR (D O) δ: -108.58. GPC: Mw = 6.1 kDa, PDI = 1.69.
2
1
PF3: 69.7% yield. H NMR (400 MHz, D O, TMS) δ: 2.68-3.03 (m, 56H, -NHCH CH N-,
2
2
2
and -OCOCH CH -), 3.51-3.54 (m, 4H, -NCH CH O-), 3.79 (t, J = 4 Hz, 4H, -NCH CH O-),
2
2
2
2
2
2
1
9
7
.53-7.56 (m, 3H, PhH). F NMR (D O) δ: -133.54, -133.49. GPC: Mw = 4.7 kDa, PDI =
2
1
.41.
1
PF4: 65.4% yield. H NMR (400 MHz, D O, TMS) δ: 2.69-3.00 (m, 36H, -NHCH CH N-,
2
2
2
and -OCOCH CH -), 3.53 (t, J = 4 Hz, 2H, -NCH CH O-), 3.66 (t, J = 4 Hz, 2H,
2
2
2
2
-
NCH CH O-), 3.78 (t, J = 4 Hz, 2H, -NCH CH O-), 3.88 (t, J = 4 Hz, 2H, -NCH CH O-),
2 2 2 2 2 2
1
9
7
.49-7.51 (m, 1H, PhH). F NMR (D O) δ: -137.72, -137.71, -137.68, -137.65, -137.62. GPC:
2
Mw = 8.1 kDa, PDI = 1.90.
1
PF5: 45.7% yield. H NMR (400 MHz, D O, TMS) δ: 2.71-3.08 (m, 35H, -NHCH CH N-,
2
2
2

ACCEPTED MANUSCRIPT
and -OCOCH CH -), 3.51-3.54 (m, 2H, -NCH CH O-), 3.65-3.67 (m, 2H, -NCH CH O-),
2
2
2
2
2
2
1
9
3
.75-3.76 (m, 2H, -NCH CH O-), 3.85-3.88 (m, 2H, -NCH CH O-). F NMR (D O) δ:
2 2 2 2 2
-
160.44, -160.39, -160.34, -156.46, -151.90, -144.42, -143.39, -141.59. GPC: Mw = 5.4 kDa,
PDI = 1.73.
PF6: 76.9% yield. H NMR (400 MHz, D O, TMS) δ: 2.68-3.00 (m, 36H, -NHCH CH N-,
1
2
2
2
and -OCOCH CH -), 3.55 (t, J = 20 Hz, 4H, -NCH CH O-), 3.78 (t, J = 4 Hz, 4H,
2
2
2
2
1
9
-
NCH CH O-), 8.06-8.36 (m, 3H, PhH). F NMR (D O) δ: -63.01. GPC: Mw = 3.7 kDa, PDI
2
2
2
=
1.27.
1
PF0: 77.3% yield. H NMR (400 MHz, D O, TMS) δ: 2.69-3.01 (m, 40H, -NHCH CH N-,
2
2
2
and -OCOCH CH -), 3.53 (t, J = 4 Hz, 2H, -NCH CH O-), 3.63 (t, J = 4 Hz, 2H,
2
2
2
2
-
NCH CH O-), 3.73 (t, J = 4 Hz, 2H, -NCH CH O-), 3.88 (t, J = 4 Hz, 2H, -NCH CH O-),
2 2 2 2 2 2
7
.45 (m, 2H, PhH), 7.51-7.53 (m, 3H, PhH). GPC: Mw = 12.4 kDa, PDI = 2.66.
2
.3 Confocal laser scanning microscopy analysis (CLSM)
To study the intracellular delivery of the polyplexes after endocytosis, confocal laser scanning
microscopy (CLSM) was applied to visualize the distribution of Cy5-labeled DNA (red) at the
optimal weight ratios. The HeLa cells were incubated with the polyplexes for 4 h. The acidic
vesicles and the nuclei of HeLa cells were then stained with LysoTraker Green and Hoechest
3
3342 (blue), respectively. Co-localizations of the polyplexes with acidic vesicles in HeLa cells
were observed by the CLSM observation, which was performed using an Olympus
FV1000-IX81 (Leica, Germany) at excitation wavelengths of 405 nm for Hoechest 33342
(blue), 633 nm for Cy5 (red) and 504 nm for LysoTracker Green (green). The co-localization
ratio of the complexes containing Cy5-labeled pDNA with endosome/lysosomes was
calculated by Image Pro Plus 6.0.
3
. Results and discussion
3
.1 Polymer preparation

ACCEPTED MANUSCRIPT
Scheme 1. Schematic illustration for the preparation of target polymers.
The preparation of target polymers is shown in Scheme 1. Compound 2 was prepared by
Boc-protected diol and 2 equiv of acryloyl chloride. After removal of the Boc group by
trifluoroacetic acid, the compound was subsequently reacted with fluorobenzoic acids (FBAs)
or benzoic acid (BA) in the presence of 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide
hydrochloride (EDCI) and N-hydroxybenzotriazole (HOBt) to give compounds 3a-g, and
compound 3g without fluorine was used for comparison. Michael addition polymerization took
place between the linkers 3a-g and PEI 600 Da with the mole ratio of 1:1 in anhydrous
o
methanol and dichloromethane at 45 C for 72 h. The crude products were recrystallized by
methanol and diethyl ether for 3 times to ensure their polydispersity. Besides PEI 600 Da, we
also tried small polyamine molecules such as tri(β-aminoethyl)amine to construct polymers.
Unfortunately, the products exhibited poor DNA encapsulation ability and gave almost no

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transfection efficiency. After lyophilization, the target cationic polymers were obtained. The
target materials were named PFn, in which “n” represents the number of fluorine atoms in each
linking bridge. The molecular weights (M s) together with the poly-dispersity index (PDIs) of
w
PFn were measured using gel permeation chromatography (GPC), and the results revealed that
PFn have M in the range of 3.7 - 8.1 kDa except PF0, whose measured M was higher and
w
w
1
the PDI was also higher. H NMR spectrum revealed that the unit ratio of the bridge to PEI was
1
9
more than 1 : 1, indicating the formation of a reticular but not linear structure. Besides, F
NMR also confirmed the successful formation of such FBA-based biopolymers.
3
.2 Interaction with DNA and polyplex formation
To identify the appropriate material concentration required for efficient condensation, the
agarose gel electrophoresis assay, which has been widely used for the detection of the
interaction between cationic polymers and DNAs [17], was first performed. As shown in Fig. 1,
although the M s of the polymers are relatively low, all of the polycations except PF5
w
exhibited good DNA encapsulation ability and could completely retard DNA migration at the
weight ratio (polymer / DNA) of 0.8. Among these materials, PF6 with the most amount of
fluorine atoms that locate on the methyl group had stronger DNA compressibility. As for PF5,
the total DNA was retarded at the ratio of 1.6. This result could be explained by the fact that
fluorination would reduce the positive charge of cationic polymers [18] and lead to poor
interaction between the cationic materials and DNA. This was also proved by the quantitative
ethidium bromide (EB) exclusion assay (Fig. S1), in which the non-fluorinated polymer PF0
exhibited higher fluorescence quenching ability, indicating its higher DNA binding capability.
Among these fluorinated materials, PF1 and PF2 with less amount of fluorine atoms showed
weaker quenching ability. Different from the results of gel retardation assays, PF5 showed as
high DNA binding ability as PF6. This is reasonable since fluorinated chains might associate
with each other through the fluorophilic effect, facilitating the compression of PF5 to DNA
[
12c,20,21]. In addition, the stability of the formed DNA complexes (polyplexes) was
investigated by heparin competition experiment (Fig. S2). As the results revealed, when the
weight ratio (heparin/DNA) was 4 or 8, the original DNA bands clearly appeared. However, it
was found that at the ratio of 8, almost all of the DNA compressed by the fluorinated materials

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was released, yet less DNA was released from PF0/DNA polyplexes, indicating its higher DNA
affinity. Nevertheless, excessively high DNA binding ability (e.g. PEI) may hinder the release
of DNA in the cells.
Fig. 1. DNA condensation by polycations at various weight ratios evaluated by the agarose gel retardation
assay.
The properties such as particle size and zeta-potential of the polyplexes were subsequently
investigated by DLS. Results in Fig. 2A showed that the particle sizes of these complexes was
irregular unless the weight ratio was up to 2, and became stable at similar size of ~250 nm. The
large particles formed at the w/w ratio of 1-2 might be attributed to their neutral zeta-potential,
which led to the minimized repulsion and resulting aggregation. The zeta potentials shown in
Fig. 2B also indicated that the charge of the complex reversed to positive from the w/w ratio of
2
. With the increase of w/w ratio, the positive charge finally reached a +40~50 mV plateau.
Since the cell membrane is negatively charged, the positive surface charge may facilitate their
interaction through electrostatic interaction and lead to better cellular uptake. In addition, a
visual image of PF3/DNA complexes in the absence of serum at w/w ratio of 6 by TEM (Fig.
S3) revealed regular spherical morphology features with diameter of ~50 nm, which was
smaller than that measured by DLS. This might be due to the different measuring conditions of
the two assays. Moreover, the polyplex size of PF3/DNA seemed to slightly decrease in the
presence of serum. We suppose that the components in the serum, such as protein and inorganic
ions, may increase the ionic strength of the solution, leading to stronger hydrophobic
interaction among the fluorinated chains (also known as the fluorophilic effect). As a result, the
polyplexes may be more compact with the addition of serum, and appear as smaller particles.

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Fig. 2. Particle sizes (A) and zeta-potentials (B) of the polyplexes. Data represent mean ± SD (n = 3).
3
.3 In vitro transfection studies
One of the most important characteristics of fluorinated materials as gene carriers is that
fluorination achieved increased transfection efficiency at very low w/w ratios, and the
transfection efficacy of fluorinated polymers increased [12].The enhanced green fluorescent
protein (EGFP) gene was first used to assess the in vitro transfection efficiency of the newly
synthesized polymers in HeLa cells (Fig. 3), and PEI 25 kDa was used as control at its optimal
w/w ratio of 1.4 (N/P ratio = 10) [5]. As the results shown, the fluorinated polymers PF3 and
PF4 induced considerable EGFP expression, and the fluorescent densities at optimized w/w
ratio were higher than those induced by the non-fluorinated polymer PF0, which gave similar
amount of green fluorescence compared with PEI 25 kDa. As the material with the least
amount of fluorine atoms, PF1 mediated modest EGFP transfection, which is comparable with
PF6 that had the greatest amount of fluorine atoms. Meanwhile, PF2 and PF5 induced almost
no EGFP expression. In addition, results also revealed some factors that are not consistent with
literature reports. First, fluorinated polymers achieved excellent gene transfection efficiency at
very low vector dosage, yet in this study, the optimal w/w ratio was as high as 6-8. Second, it
was accepted that the transfection efficacy of cationic polymers with fluorinated groups
increased with the increase of the number of fluorine. Nevertheless, the results showed that
there was no significant relationship between the transfection efficiency and the number of
fluorine atoms in this paper. Moreover, the same assay was also carried out in other cell lines
such as HepG2 and 7702 cells. As shown in Fig. S4 and S5, among the target materials, PF3

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always gave significantly better transfection efficiency. Besides, the results suggested that the
EGFP expression induced by PF0 was significantly suppressed by 10% serum, however, such
inhibition for fluorinated materials was weak, especially for PF3 (Fig. S5).
Fig. 3. Fluorescence microscopy images of EGFP expression in HeLa cells at various w/w ratios. PEI 25
kDa was used as control
To quantitatively study the in vitro transfection, pGL-3 reporter gene was also applied for the
transfection experiment. As shown in Fig. 4, the fluorinated polymers gave diverse efficiency,
and in all cases, PF3 and PF4 exhibited better transfection than PEI as well as PF0. Slightly
different from the results of the EGFP transfection experiment, PF1 gave the highest
transfection efficiency. Besides, the pGL-3 reporter gene expression induced by PF6 was lower
than that mediated by PF1. Such result is reasonable since the efficacy of a polymeric gene

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vector depends considerably on the type of plasmids transfected, which was also observed in
other studies [12]. Among these fluorinated materials, PF2 and PF5 gave the lowest
transfection efficiency. As the results in Fig. S2 revealed, DNA compressed by PF2 and PF5
was easy to release in the presence of heparin, so we suppose that the weak interaction might
lead to the release of DNA from the polyplexes early, and the DNA will be rapidly degraded,
leading to lower transfection level. Similarly, the transfection efficiency mediated by all target
polymers was inhibited by 10% serum. However, with the additional serum, the transfection
efficiency of PF0 and PEI dropped severely, and the efficiency decreased 30 and 70 times
respectively. On the contrary, the gene expression efficiency of fluorinated polymers gave
slight decreases, e.g. the efficiency of P3 only decreased 4 times. Additionally, compared with
PEI and PF0, P3 gave 87 and 36 times higher transfection efficiencies. These results showed
that P3 not only has better gene transfection ability than other materials, but also exhibits better
serum tolerance.
Fig. 4. Luciferase gene expression transfected by polyplexes at different weight ratios in comparison with 25

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kDa bPEI. Data represent mean ± SD (n = 3).
Besides high transfection efficiency, fluorinated cationic polymers are believed to cause low
cytotoxicity [22]. So the cytotoxicity of all fluorinated materials was then estimated using the
MTS assay toward two cell lines. As shown in Fig. 5, the viability of cells treated with newly
synthesized FBA or BA-based materials was distinctly higher than those treated with PEI, and
the cells treated with all polyplexes gave cell viability above 80% even at a relatively higher
w/w ratio of 8. This might be contributed to the integration of the aromatic ring, which was
found to benefit the gene vectors with enhanced biocompatibility [10c,15]. Besides, results
showed that the number of fluorine atoms seldom affected the cytotoxicity. Such results
suggested that these materials were suitable for potential gene delivery in vivo.
Fig. 5. Relative cell viabilities of polyplexes at different mass ratios. PEI/DNA polyplex was used as control.
Data shows a representative mean ± SD (n = 3).
3
.4 Mechanism studies
Apart from high transfection efficiency and low cytotoxicity, fluorinated cationic gene
carriers are also deemed to facilitate efficient cellar uptake and successful endosomal escape
13b,23]. Therefore, the effective cellular uptake of the polyplexes in the presence of 10%
[
serum at optimal w/w was investigated by fluorescence-activated cell sorting (FACS) analysis
in HeLa cells. After 4 h incubation of polyplexes with the cells, the percentage of positive cells
for Cy5-labeled DNA was calculated. Results shown in Fig.6 revealed that polyplexes formed
from the target polymers except PF2 exhibited excellent cellular uptake, and the percentage of
transfected cells was found to be close to 100%. Meanwhile, the fluorine atoms seldom

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affected cellular uptake. On the other hand, the fluorescence intensities of the internalized
Cy5-DNA were also measured. As shown in Fig. 6, PF6 gave the highest fluorescent intensity.
However, although PF6 has better cellular uptake efficiency, its transfection efficiency was
lower, indicating that PF6 has much lower intracellular gene delivery efficiency, which might
largely be due to the difficulty of DNA release (Fig. S2). Although PF4, PF0 and PEI gave
similar cellular uptake and fluorescence intensities, the former mediated higher gene
transfection (Fig. 5), indicting its higher intracellular gene delivery efficiency.
Fig. 6. Cellular uptake (columns) and fluorescence intensity (dots) of polyplexes at optimal weight ratios in
comparison with 25 kDa bPEI (w/w = 1.4) in 10% concentrations of serum in HeLa cells quantified by flow
cytometry analysis. Data shows the representative mean ± SD (n = 3).
The intracellular gene delivery and distribution of internalized polyplexes after endocytosis
were further investigated by confocal laser scanning microscopy (CLSM). Beside the
Cy5-labelled DNA, the nuclei and endosomes/lysosomes were stained with Hoechest 33342
(blue) and LysoTracker Green (green), respectively. Fig. 7A shows the fluorescent images
obtained after 4 h incubation of HeLa cells with polymer/DNA polyplexes at optimal
transfection mass ratio in the presence of 10% serum. It was shown that in the case of PF6,
considerable amount of DNA cargos was still bound in the endosome, which was illustrated by
the yellow dots in the merged image. This result indicated that most of the internalized DNA
located in the late endosome, which has similar environment to the lysosome. For other
polymers, the yellow dots were fewer. Meanwhile, co-localization ratios (Pearson's correlation,
Pr) of DNA (red) with endosome/lysosomes (green) in the pictures were also calculated by
Image Pro Plus 6.0. As shown in Fig. 7B, PF6 polyplexes gave the highest co-localization ratio.
This result further confirmed that PF6 polyplexes might mediate efficient cell uptake, however,
its strong DNA binding ability might hinder the dissociation of the polyplex in the cells, which

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might be one of the reasons for the decreased transfection. Meanwhile, among these fluorinated
polymers, PF1 showed the lowest ratio, suggesting better endosome escape ability. Besides,
PF3 and PF0 gave similar ratios. However, PF3 gave higher transfection efficiencies than PF0
(Fig. 5). This might be due to the fact that PF3 would mediate more effective cellular uptake
(Fig. 6). Such results further indicated that fluorinated materials could facilitate efficient cellar
uptake and successful endosomal escape.

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Fig. 7 (A) Intracellular distribution of Cy5-labeled pDNA (red) complexes at optimal transfection weight
ratio for 4 h in the presence of 10% serum. The nuclei were stained with Hoechest 33342 (blue), and the
endosome/lysosomes were stained with LysoTracker Green (green). (B) The co-localization ratio of the
complexes containing Cy5-labeled pDNA with endosome/lysosomes was calculated by software.
4
. Conclusion
In summary, we synthesized a series of FBA-based cationic vectors via Michael addition.
These polymers were used for DNA delivery and their structure-activity relationship (SAR)
was studied. Results revealed that fluorine would screen the positive charge of cationic
polymers. Meanwhile, the fluorine atoms might strengthen the interaction between fluorinated
materials and nucleic acid through the fluorophilic effect and lead to more stable polyplexes.
Several assays confirmed that fluorination improved the uptake efficiency of these polymeric
vectors, and more fluorine atoms might lead to higher uptake efficiency. Transfection
experiments showed that these fluorinated polymers could mediate efficient gene transfection,
and exhibited very low cytotoxicity even at high weight ratios. To sum up, this work
demonstrates that fluorobenzoic acid-based linkage might distinctly improve the cellular
uptake and transfection abilities. Further studies including modification of current efficient
fluorinated vectors and cytosolic protein delivery are now in progress.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China
(Nos: 21472131, 21672155). We also thank the Comprehensive Training Platform of
Specialized Laboratory, College of Chemistry, Sichuan University for sample analysis.

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References
[1]. (a) C. Ornelas-Megiatto, P. R. Wich, J. M. J. Fréchet , Polyphosphonium Polymers for siRNA Delivery:
An Efficient and Nontoxic Alternative to Polyammonium Carriers. Journal of the American Chemical
Society 134(4) (2012) 1902-1905; (b) L. Cui, J. L. Cohen, C. K. Chu, P. R. Wich, P. H. Kierstead, J. M. J.
Fréchet, Conjugation Chemistry through Acetals toward a Dextran-Based Delivery System for Controlled
Release of siRNA. Journal of the American Chemical Society 134 (38) (2012) 15840-15848; (c) T. Niidome,
L. Huang, Gene Therapy Progress and Prospects: Nonviral vectors. Gene Therapy 9 (2002) 1647; (d) M. A.
Kay, J. C. Glorioso, L. Naldini, Viral vectors for gene therapy: the art of turning infectious agents into
vehicles of therapeutics. Nature Medicine 7 (2001) 33; (e) M. G. Sebestyén, J. O. Hegge, M. A. Noble, D. L.
Lewis, H. Herweijer, J. A. Wolff, Progress Toward A Nonviral Gene Therapy Protocol for The Treatment of
Anemia. Human Gene Therapy 18(3) (2007) 269-285; (f) J. L. Cohen, S. Schubert, P. R. Wich, L. Cui, J. A.
Cohen, J. L. Mynar, J. M. J. Fréchet, Acid-Degradable Cationic Dextran Particles for the Delivery of siRNA
Therapeutics. Bioconjugate Chemistry 22(6) (2011) 1056-1065; (g) J. A. Cohen, T. T. Beaudette, J. L. Cohen,
K. E. Broaders, E. M. Bachelder, J. M. J. Fréchet, Acetal-Modified Dextran Microparticles with Controlled
Degradation Kinetics and Surface Functionality for Gene Delivery in Phagocytic and Non-Phagocytic Cells.
Advanced Materials 22 (32) (2010) 3593-3597; (h) E. Mastrobattista, W. E. Hennink, Charged for success.
Nature Materials 11 (2011) 10; (i) D. G. Anderson, W. Peng, A. Akinc, N. Hossain, A. Kohn, R. Padera, R.
Langer, J. A. Sawicki, A polymer library approach to suicide gene therapy for cancer. Proceedings of the
National Academy of Sciences of the United States of America (2004) 101 (45) 16028-16033; (j) D. J.
Siegwart, K. A. Whitehead, L. Nuhn, G. Sahay, H. Cheng, S. Jiang, M. Ma, A. Lytton-Jean, A. Vegas, P.
Fenton, C. G. Levins, K. T. Love, H. Lee, C. Cortez, S. P. Collins, Y. F. Li, J. Jang, W. Querbes, C. Zurenko,
T. Novobrantseva, R. Langer, D. G. Anderson, Combinatorial synthesis of chemically diverse core-shell
nanoparticles for intracellular delivery. Proceedings of the National Academy of Sciences 108 (32) (2011)
1
2996-13001; (k) M. Gujrati, A. Malamas, T. Shin, E. Jin, Y. Sun, Z.-R. Lu, Multifunctional Cationic
Lipid-Based Nanoparticles Facilitate Endosomal Escape and Reduction-Triggered Cytosolic siRNA Release.
Molecular Pharmaceutics 11(8) (2014) 2734-2744; (l) Z. Chen, B. Krishnamachary, M.-F. Penet, Z. M.
Bhujwalla, Acid-degradable Dextran as an Image Guided siRNA Carrier for COX-2 Downregulation.
Theranostics 8 (1) (2018) 1-12; (m) C. Ruan, L. Liu, Q. Wang, X. Chen, Q. Chen, Y. Lu, Y. Zhang, X. He, Y.;
Guo, Q. Zhang, T. Sun, C. Jiang, ROS-Biodegradable Gene Carrier for Targeting Therapy of Breast Cancer.

ACCEPTED MANUSCRIPT
ACS Applied Materials & Interfaces (2018); (n) J.-H. Gong, Y. Wang, L. Xing, P.-F. Cui, J.-B. Qiao, Y.-J. He,
H.-L. Jiang, Biocompatible fluorinated poly(β-amino ester)s for safe and efficient gene therapy. International
Journal of Pharmaceutics 535(1) (2018) 180-193.
[2]. (a) C. Boyer, J. Teo, P. Phillips, R. B. Erlich, S. Sagnella, G. Sharbeen, T. Dwarte, H. T. T. Duong, D.
Goldstein, T. P. Davis, M. Kavallaris, J. McCarroll, Effective Delivery of siRNA into Cancer Cells and
Tumors Using Well-Defined Biodegradable Cationic Star Polymers. Molecular Pharmaceutics 10(6) (2013)
2
435-2444; (b) M. S. Shim, Y. J. Kwon, Ketalized poly(amino ester) for stimuli-responsive and
biocompatible gene delivery. Polymer Chemistry 3(9) (2012) 2570-2577.
3]. (a) H.-L. Jiang, Y.-K. Kim, R. Arote, J.-W. Nah, M.-H. Cho, Y.-J. Choi, T. Akaike, C.-S. Cho,
[
Chitosan-graft-polyethylenimine as a gene carrier. Journal of Controlled Release 117(2) (2007) 273-280; (b)
M. H. Allen, M. D. Green, H. K. Getaneh, K. M. Miller, T. E. Long, Tailoring Charge Density and Hydrogen
Bonding of Imidazolium Copolymers for Efficient Gene Delivery. Biomacromolecules 12(6) (2011)
2
243-2250.
[4]. (a) Y. Nakayama, Hyperbranched Polymeric “Star Vectors” for Effective DNA or siRNA Delivery.
Accounts of Chemical Research 45 (7) (2012) 994-1004; (b) Y.-H. Zhang, Y. Chen, Y.-M. Zhang, Y. Yang,
J.-T. Chen, Y. Liu, Recycling Gene Carrier with High Efficiency and Low Toxicity Mediated by
L-Cystine-Bridged Bis(β-cyclodextrin)s. Scientific Reports 4 (2014) 7471; (c) Y. Tao, Z. Li, E. Ju, J. Ren, X.
Qu, Polycations-functionalized water-soluble gold nanoclusters: a potential platform for simultaneous
enhanced gene delivery and cell imaging. Nanoscale 5(13) (2013) 6154-6160.
[5]. (a) L. Bonetta, The inside scoop—evaluating gene delivery methods. Nature Methods 2 (2005) 875; (b)
U. Lungwitz, M. Breunig, T. Blunk, A. Göpferich, Polyethylenimine-based non-viral gene delivery systems.
European Journal of Pharmaceutics and Biopharmaceutics 60(2) (2005) 247-266.
[6].(a) K. Kunath, A. von Harpe, D. Fischer, H. Petersen, U. Bickel, K. Voigt, T. Kissel,
Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of
physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight
polyethylenimine. Journal of Controlled Release (2003) 89(1) 113-125; (b) D. Fischer, Y. Li, B. Ahlemeyer, J.
Krieglstein, T. Kissel, In vitro cytotoxicity testing of polycations: influence of polymer structure on cell
viability and hemolysis. Biomaterials (2003) 24(7) 1121-1131.
[7]. H. Petersen, P. M. Fechner, A. L. Martin, K. Kunath, S. Stolnik, C. J. Roberts, D. Fischer, M. C. Davies,
T. Kissel, Polyethylenimine-graft-Poly(ethylene glycol) Copolymers:ꢀ Influence of Copolymer Block

ACCEPTED MANUSCRIPT
Structure on DNA Complexation and Biological Activities as Gene Delivery System. Bioconjugate
Chemistry (2002) 13(4) 845-854.
[
8]. J.-Z. Deng, Y.-X. Sun, H.-Y. Wang, C. Li, F.-W. Huang, S.-X. Cheng, R.-X. Zhuo, X.-Z. Zhang,
Poly(β-amino amine) cross-linked PEIs as highly efficient gene vectors. Acta Biomaterialia (2011) 7(5)
200-2208.
9]. T. Ito, C. Yoshihara, K. Hamada, Y. Koyama, DNA/polyethyleneimine/hyaluronic acid small complex
particles and tumor suppression in mice. Biomaterials (2010) 31 (10) 2912-2918.
10]. (a) F. Alexis, E. Pridgen, L. K. Molnar, O. C. Farokhzad, Factors Affecting the Clearance and
2
[
[
Biodistribution of Polymeric Nanoparticles. Molecular Pharmaceutics (2008) 5(4) 505-515; (b) H. Koo, G.-w.
Jin, H. Kang, Y. Lee, K. Nam, C. Zhe Bai, J.-S. Park, Biodegradable branched poly(ethylenimine sulfide) for
gene delivery. Biomaterials (2010) 31(5) 988-997; (c) Y.-P. Xiao, J. Zhang, Y.-H. Liu, Z. Huang, B. Wang,
Y.-M. Zhang, X.-Q. Yu, Cross-linked polymers with fluorinated bridges for efficient gene delivery. Journal
of Materials Chemistry B (2017) 5(43) 8542-8553; (d) Q. Guo, Y.-H. Liu, M.-M. Xun, J. Zhang, Z. Huang,
X.-D. Zhou, X.-Q. Yu, Diol glycidyl ether-bridged low molecular weight PEI as potential gene delivery
vehicles. Journal of Materials Chemistry B (2015) 3(13) 2660-2670.
[11]. M.-M. Xun, Y.-P. Xiao, J. Zhang, Y.-H. Liu, Q. Peng, Q. Guo, W.-X. Wu, Y. Xu, X.-Q. Yu, Low
molecular weight PEI-based polycationic gene vectors via Michael addition polymerization with improved
serum-tolerance. Polymer (2015) 65 45-54.
[12]. (a) M. Wang, H. Liu, L. Li, Y. Cheng, A fluorinated dendrimer achieves excellent gene transfection
efficacy at extremely low nitrogen to phosphorus ratios. Nature Communications (2014) 5 3053; (b) H.
Wang, Y. Wang, Y. Wang, J. Hu, T. Li, H. Liu, Q. Zhang, Y. Cheng, Self-Assembled Fluorodendrimers
Combine the Features of Lipid and Polymeric Vectors in Gene Delivery. Angewandte Chemie International
Edition (2015) 54 (40) 11647-11651; (c) M. Wang, Y. Cheng, The effect of fluorination on the transfection
efficacy of surface-engineered dendrimers. Biomaterials (2014) 35(24) 6603-6613; (d) M. Wang, Y. Cheng,
Structure-activity relationships of fluorinated dendrimers in DNA and siRNA delivery. Acta Biomaterialia
(2016) 46 204-210; (e) M. E. Johnson, J. Shon, B. M. Guan, J. P. Patterson, N. J. Oldenhuis, A. C. Eldredge,
N. C. Gianneschi, Z. Guan, Fluorocarbon Modified Low-Molecular-Weight Polyethylenimine for siRNA
Delivery. Bioconjugate Chemistry (2016) 27 (8) 1784-1788.
[13]. (a) J. M. Criscione, B. L. Le, E. Stern, M. Brennan, C. Rahner, X. Papademetris, T. M. Fahmy,
Self-assembly of pH-responsive fluorinated dendrimer-based particulates for drug delivery and noninvasive

ACCEPTED MANUSCRIPT
imaging. Biomaterials (2009) 30(23) 3946-3955; (b) M. J. Patrick, J. M. Janjic, H. Teng, M. R. O’Hear, C. W.
Brown, J. A. Stokum, B. F. Schmidt, E. T. Ahrens, A. S. Waggoner, Intracellular pH Measurements Using
Perfluorocarbon Nanoemulsions. Journal of the American Chemical Society (2013) 135(49), 18445-18457;
(c) V. Percec, M. R. Imam, M. Peterca, P. Leowanawat, Self-Organizable Vesicular Columns Assembled
from Polymers Dendronized with Semifluorinated Janus Dendrimers Act As Reverse Thermal Actuators.
Journal of the American Chemical Society (2012) 134(9) 4408-4420.
[14]. (a) M. C. Z. Kasuya, S. Nakano, R. Katayama, K. Hatanaka, Evaluation of the hydrophobicity of
perfluoroalkyl chains in amphiphilic compounds that are incorporated into cell membrane. Journal of
Fluorine Chemistry (2011) 132(3) 202-206; (b) E. Neil, G. Marsh, Towards the nonstick egg: designing
fluorous proteins. Cell Chemical Biology 7(7) R153-R157.
[15]. (a) A. Tschiche, A. M. Staedtler, S. Malhotra, H. Bauer, C. Bottcher, S. Sharbati, M. Calderon, M. Koch,
T. M. Zollner, A. Barnard, D. K. Smith, R. Einspanier, N. Schmidt, R. Haag, Polyglycerol-based amphiphilic
dendrons as potential siRNA carriers for in vivo applications. Journal of Materials Chemistry B (2014) 2(15)
2
153-2167; (b) V. Bagnacani, V. Franceschi, M. Bassi, M. Lomazzi, G. Donofrio, F. Sansone, A. Casnati, R.
Ungaro, Arginine clustering on calix[4]arene macrocycles for improved cell penetration and DNA delivery.
Nature Communications (2013) 4 1721; (c) D. Joester, M. Losson, R. Pugin, H. Heinzelmann, E. Walter, H.
P. Merkle, F. Diederich, Amphiphilic Dendrimers: Novel Self-Assembling Vectors for Efficient Gene
Delivery. Angewandte Chemie International Edition (2003) 42(13) 1486-1490; (d) J. Liu, M. Feng, D. Liang,
J. Yang, X. Tang, Vitamin E-Labeled Polyethylenimine for in vitro and in vivo Gene Delivery.
Biomacromolecules (2016) 17(10) 3153-3161.
[
(
[
16]. K. Müller, C. Faeh, F. Diederich, Fluorine in Pharmaceuticals: Looking Beyond Intuition. Science
2007) 317(5846) 1881-1886.
17]. I. Yudovin-Farber, A. J. Domb, Cationic polysaccharides for gene delivery. Materials Science and
Engineering: C (2007) 27(3) 595-598.
18].(a) V. Percec, M. Glodde, T. K. Bera, Y. Miura, I. Shiyanovskaya, K. D. Singer, V. S. K. Balagurusamy,
P. A. Heiney, I. Schnell, A. Rapp, H. W. Spiess, S. D. Hudson, H. Duan, Nature, 2002, 417 (6905), 384-387;
[
(b) S. M. Brittain, S. B. Ficarro, A. Brock, E. C. Peters, Nat. Biotech., 2005, 23 (4), 463-468.
[19]. (a) M. Ahmed, R. Narain, The effect of molecular weight, compositions and lectin type on the
properties of hyperbranched glycopolymers as non-viral gene delivery systems. Biomaterials (2012) 33(15)
990-4001; (b) K. Morimoto, M. Nishikawa, S. Kawakami, T. Nakano, Y. Hattori, S. Fumoto, F. Yamashita,
3

ACCEPTED MANUSCRIPT
M. Hashida, Molecular weight-dependent gene transfection activity of unmodified and galactosylated
polyethyleneimine on hepatoma cells and mouse liver. Molecular Therapy (2003) 7(2), 254-261; (c) J. Zhu,
A. Tang, L. P. Law, M. Feng, K. M. Ho, D. K. L. Lee, F. W. Harris, P. Li, Amphiphilic Core−Shell
Nanoparticles with Poly(ethylenimine) Shells as Potential Gene Delivery Carriers. Bioconjugate Chemistry
(2005) 16(1) 139-146.
[20]. S. M. Brittain, S. B. Ficarro, A. Brock, E. C. Peters, Enrichment and analysis of peptide subsets using
fluorous affinity tags and mass spectrometry. Nature Biotechnology (2005) 23 463.
21]. V. Percec, M. Glodde, T. K. Bera, Y. Miura, I. Shiyanovskaya, K. D. Singer, V. S. K. Balagurusamy, P.
[
A. Heiney, I. Schnell, A. Rapp, H. W. Spiess, S. D. Hudson, H. Duan, Self-organization of supramolecular
helical dendrimers into complex electronic materials. Nature (2002) 419 384.
[22]. H. M. Courrier, M. P. Krafft, N. Butz, C. Porté, N. Frossard, A. Rémy-Kristensen, Y. Mély, F. Pons, T. F.
Vandamme, Evaluation of cytotoxicity of new semi-fluorinated amphiphiles derived from
dimorpholinophosphate. Biomaterials (2003) 24(4) 689-696.
[
23]. A.-M. Caminade, C.-O. Turrin, P. Sutra, J.-P. Majoral, Fluorinated dendrimers. Current Opinion in
Colloid & Interface Science (2003) 8(3) 282-295.
24]. C.-R. Luan, Y.-H. Liu, J. Zhang, Q.-Y. Yu, Z. Huang, B. Wang, X.-Q. Yu, Low Molecular Weight
Oligomers with Aromatic Backbone as Efficient Nonviral Gene Vectors. ACS Applied Materials & Interfaces
2016) 8(17) 10743-10751.
[
(

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Highlights
Effect of fluorine atom number and location on gene delivery ability was investigated
Fluorine atoms may facilitate DNA condensation and stabilize the polymer/DNA
complex
Fluorination of the polymeric gene vectors enhanced their transfection efficiency
Fluorination improved uptake efficiency, and more fluorine atoms led to higher
uptake
All fluorinated polymeric vectors exhibited very low cytotoxicity even at high dosage

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Electronic supplementary information
Low molecular weight PEI-based fluorinated polymers for efficient
gene delivery
*
Ya-Ping Xiao, Ji Zhang , Yan-Hong Liu, Ju-Hui, Zhang, Qing-Ying Yu, Zheng Huang, and
*
Xiao-Qi Yu
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan
University, Chengdu 610064, P. R. China
*
Corresponding authors: jzhang@scu.edu.cn (J. Zhang); xqyu@scu.edu.cn (X.-Q. Yu); Fax: + 86 28
5415886
8

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Fig. S1. Fluorescence quenching assay by the addition of polymers.
Fig. S2. Release of DNA with the addition of heparin at various concentrations via electrophoretic gel
retardation assay. Polymer/DNA: w/w = 6.
Fig. S3. TEM images of PF3/DNA polyplexes at w/w of 6 in deionized water in the absence or presence of
1
0% serum. Scale bar = 100 nm.

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Fig. S4. EGFP gene transfection efficacy in HepG2 cells in the absence of serum.

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Fig. S5. EGFP gene transfection efficacy in 7702 cells.