Photo-induced release of active plasmid from crosslinked nanoparticles: O-nitrobenzyl/methacrylate functionalized polyethyleneimine

Article abstract of DOI:10.1039/b922613c

To facilitate DNA packaging and photolytic release, o-nitrobenzyl and methacrylate functionalized PEI (P10A) was synthesized for condensing DNA into nanoparticles as small as 160 nm after radical polymerization with initiator. The gene expression followin

Full text of DOI:10.1039/b922613c

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PAPER
www.rsc.org/materials | Journal of Materials Chemistry
Photo-induced release of active plasmid from crosslinked nanoparticles:
o-nitrobenzyl/methacrylate functionalized polyethyleneimine†
ab
a
a
a
*
*
Jeffrey Gruneich, Huiyan Jing and Scott L. Diamond
Moon Suk Kim,
Received 29th October 2009, Accepted 3rd February 2010
First published as an Advance Article on the web 4th March 2010
DOI: 10.1039/b922613c
To facilitate DNA packaging and photolytic release, o-nitrobenzyl and methacrylate functionalized
PEI (P10A) was synthesized for condensing DNA into nanoparticles as small as 160 nm after radical
polymerization with initiator. The gene expression following delivery of uncross-linked P10A/DNA
was unchanged by photoirradiation of cells. However, exposure to photo-irradiation caused a 3-fold
increase in gene expression in cells transfected with cross-linked P10A/DNA. These polyplexes were
designed to mimic viral particles that condense and protect DNA while allowing subsequent
triggered release of DNA.
use have been synthesized, including, but not limited to, ATP and
1. Introduction
analogues, alanine, nitric oxide, nitric oxide inhibitors, receptor
ligands, DNA, and inositol trisphosphate (IP3); many are
commercially available.18,19
Non-viral vectors for gene delivery offer several potential
advantages over viral vectors such as ease of scalable manufac-
ture and safety.1,2 Since its first use as a non-viral vector in 1995
by Boussif, Lezoualc’h et al., polyethylenimine (PEI) with
densely positively charged cationic amine has remained
a commonly used cationic polymer for polyplex formation.3
Early studies focused on the physical properties of the PEI/
plasmid DNA (DNA) polyplexes such as size and zeta potential
and transfection efficiency in different cell lines as a function of
polymer nitrogen to DNA phosphate ratio (N/P ratio).4,5
Chemical modifications of PEI are directed at enhancing
transfection efficiency and reducing aggregation, cytotoxicity,
and the lack of cell selectivity.6–10 There have been several
attempts at engineering temperature-sensitive, pH-sensitive, or
biodegradable PEI for controlled DNA release.11–14 A polyplex
should remain intact in the endosomes and lysosmes to protect
the DNA from cellular nucleases, but should degrade or disas-
semble to facilitate DNA release once the polyplex has reached
the cytoplasm. Thus, a fine-tuned balance needs to be achieved
between a polyplex that is packaged too loosely, allowing for
DNA degradation by cellular nucleases and a polyplex that is
packaged too tightly, not allowing for dissociation and eventual
transcription of the released plasmid.
The potential to use this technology for gene delivery,
however, has not been fully realized. Monroe et al. synthesized
a caged DNA for photolytically activated gene delivery via direct
chemical modification of the DNA backbone.19 They found
a significant decrease in the amount of DNA that could be
transcribed for the caged DNA compared to both uncaged DNA
where the caging group had been photocleaved and native DNA.
Additionally, the photolysis of the caged DNA had the potential
to cause DNA nicking. However, the method was the first report
of successful light activation of DNA and transgene expression
within a living cell.
Viral vectors such as adenovirus provide functionality for
DNA packaging and protection, cellular uptake and endosome
escape, and triggered release of DNA at the nuclear pore. The
packaging of DNA and the release of DNA by a single polymeric
material presents competing material design goals. We report the
synthesis of a crosslinkable PEI that is amenable to photolytic
degradation for the controlled intracellular release of plasmid
DNA. In this work, we attempted to design a novel photolabile
gene carrier. The designed photolabile carrier for gene delivery
consist of three functional domains: a cationic domain to elec-
trostatically interact with and condense the DNA to nano-
particle, a cross-linking domain to entrap the DNA within the
polyplex, and a photolabile domain to release the DNA with the
addition of light of an appropriate wavelength. A photoactivated
cross-linked DNA nanoparticle could release DNA nanoparticle
and transgene expression for spatial, temporal, and metered
dosing.
Photolabile protecting groups have been used extensively for
controlled release, spatial, temporal, concentration dependent,
and kinetic studies.15–17 The advantage of using photolabile
protecting groups is that the ‘‘caged’’ compounds are rendered
inert until photolysis which should liberate the compound in msec
to msec time frames. Numerous caged compounds for biological
aInstitute for Medicine and Engineering, Departments of Chemical and
Biomolecular Engineering, 1024 Vagelos Research Laboratory,
University of Pennsylvania Philadelphia, PA, 19104, USA. E-mail: sld@
seas.upenn.edu; Fax: +1-215-573-7227; Tel: +1-215-573-5702
bDepartment of Molecular Science and Technology, Ajou University,
Suwon, 443-749, Korea. E-mail: moonskim@ajou.ac.kr; Fax: +82-31-
219-3931; Tel: +82-31-219-2608
2. Experimental
2.1 Materials
Commercially available extra pure o-nitroacetophenone
(Aldrich), benzoyl chloride (Aldrich), acetovanillone (Aldrich),
N-hydroxysuccinimide (Aldrich), N,N-dicyclohexylcarbodiimide
† This paper is part of a Journal of Materials Chemistry themed issue on
Actively Moving Polymers. Guest editor: Andreas Lendlein.
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(Aldrich), trimellitic anhydride chloride (Aldrich) poly-
ethyleneimine MW 600 (Aldrich), MW 10000 (Polyscience), and
N-(3-aminopropyl)methacrylamide hydrochloride (Polyscience)
were used as received without further purification.
2.5 Synthesis of N-(3-(4-acetyl-2-methoxy-5-
nitrophenoxy)acetamide)propyl methacry-lamide (MCO)
A slurry of (4-acetyl-2-methoxy-5-nitrophenoxy) acetic acid (B2)
(0.6 g, 2.25 mmol), N-hydroxysuccinimide (0.6 g, 5.2 mmol), and
N,N-dicyclohexylcarbodiimide (0.8 g, 3.9 mmol) in CH2Cl2
(15 mL) was stirred at room temperature for 1 h under nitrogen
to make the activated carboxylic group. N-(3-amino-
propyl)methacrylamide hydrochloride (0.6 g, 3.4 mmol) was
poured into the activated carboxylic solution. Triethylamine
(TEA) (0.4 ml, 2.9 mmol) added after 30 min. The solution was
stirred for 48 h at room temperature. The solution was filtered
and poured into water. The solution was extracted with ethyl
acetate and saturated NaHCO3. The combined organic solution
dried with anhydrous MgSO4 and concentrated by evaporation.
The obtained mixture was dissolved in acetonitrile, and the
resulting solution was filtrated. The combined organic solution
concentrated by evaporation to give the MCO (0.72 g, 1.8 mmol,
80%) as viscous liquid of slight yellow, which was used at next
step without further purification. 1H NMR (CDCl3): 7.68 (s, 1H,
C6H2), 7.39 (s, 1H, C6H2), 7.30 (m, 1H, NH), 6.81(s, 1H, C6H2),
6.75 (m, 1H, NH), 5.77 (s, 1H, ¼CH2), 5.35 (s, 1H, ¼CH2), 4.63
(s, 2H, –CH2–), 4.01 (s, 3H, OCH3), 3.44 ꢁ 3.34 (m, 4H,
NHCH2CH2CH2NH), 2.51 (s, 3H, CH3), 1.99 (s, 3H, CH3C¼),
1.74 (m, 2H, NHCH2CH2CH2NH).
2.2 Measurements
1H NMR and 13C NMR were recorded with Bruker AC-250
spectrometer, using tetramethylsilane (TMS) as an internal
standard. HPLC was performed on HP-series 1050 HPLC
system consisting of HAMILTON PRP-1 column and photo-
diode array detectors. Elution was performed using a linear
gradient (30–70%) of acetonitrile in 1% TFA aqueous with a flow
rate of 1.0 ꢀ 4.0 mL minꢁ1. Photoirradiation was carried out with
Model B-100A Hi Intensity UV Lamp (UVP Inc) at a distance of
10 cm (emission peak is at 365 nm, 3.5 mW intensity at 30 cm).
Particle size was measured at 25 ꢂC with Dynapro Dynamic
Light Scattering of Protein solution. Fluorescence was measured
with SLM-AMINCO Model 8100 spectrofluorometer.
2.3 Synthesis of tert-butyl (4-acetyl-2-
methoxyphenoxy)acetate (B1)
Acetovanillone (2 g, 12 mmol) was placed in a flask and dissolved
in MeOH (10 mL). NaOH pellets (0.626 g, 14.2 mmol) were
added to the solution. The color of the solution turned deep
yellow as the NaOH gradually dissolved. The solution was kept
at room temperature for 15 h. Methyl bromoacetate (2.59 g, 16.9
mmol) was added and the solution was refluxed for 15 h. After
cooling, the MeOH was evaporated and the solid dissolved in
EaOAc. The resulting solution was washed with 5% HCl aqueous
solution and water several times. The organic phase was dried
over anhydrous magnesium sulfate and concentrated by evapo-
2.6 Synthesis of N-(3-(4-(1-hydroxylethyl)-5-nitrophenoxy)
acetamide) propyl methacryl-amide (MOH)
To a solution of MCO (0.71 g, 1.8 mmol) in EtOH (10 mL) was
added NaBH4 (0.132 g, 3.5 mmol) at 0 ꢂC. The reaction mixture
was stirred at room temperature for overnight. After the removal
of solvent, the residue was dissolved in ethyl acetate, and the
resulting solution was washed with NH4Cl solution several times.
The organic phase was dried over anhydrous MgSO4 and
concentrated by evaporation to give yellow oil, which was
purified by silica gel column chromatography using a solution of
CHCl3 and acetonitrile (v/v ¼ 60/40, Rf 0.45) as an eluent to
1
ration to give to afford quantitative B1 of light yellow solid. H
NMR (CDCl3): 7.55–7.51 (m, 2H, C6H2), 6.78 (d, J ¼ 8.8 Hz,
1H, C6H2), 4.66 (s, 2H, –CH2–), 3.94 (s, 3H, OCH3), 2.57 (s, 3H,
CH3), 1.48 (s, 9H, 3CH3). 13C NMR (CDCl3): 196.6, 167.1, 149.2,
131.3, 122.7, 111.6, 110.8, 82.7, 66.0, 56.0, 28.0, 26.2. Anal. Calcd
for C15H20O5: C; 64.27, H; 7.19. Found: C; 64.65, H; 7.40.
1
afford 0.52 g (1.32 mmol, 73.3%) of light yellow oil. H NMR
(CDCl3): 7.61 (s, 1H, C6H2), 7.41 (s, 1H, C6H2), 7.23 (m, 1H,
NH), 6.67 (m, 1H, NH), 5.76 (s, 1H, ¼CH2), 5.55 (m,
1H, HOC(H)-), 5.35 (s, 1H, ¼CH2), 4.57 (s, 2H, –CH2–), 4.02
(s, 3H, OCH3), 3.53 ꢁ 3.24 (m, 4H, NHCH2CH2CH2NH), 1.98
(s, 3H, CH3C¼), 1.73 (m, 2H, NHCH2CH2CH2NH), 1.54 (d,
J ¼ 6.2 Hz, 3H, CH3). 13C NMR (CDCl3): 168.66, 168.28, 154.21,
145.17, 139.59, 139.48, 139.24, 119.84, 111.31, 109.44, 68.89,
65.43, 56.35, 35.87, 35.76, 29.42, 24.47, 18.47. HRMS Anal.
Calcd for C18H25N3O7 (M + Na)+: 418.1590. Found: 418.1566.
2.4 Synthesis of (4-acetyl-2-methoxy-5-nitrophenoxy)acetic
acid (B2)
A solution of B1 (5.8 g, 20.7 mmol) in 15 mL of acetic anhydride
was added to a solution 15 mL of 70% HNO3 and 10 mL of acetic
anhydride at 0 ꢂC. The solution was stirred for 2 h and then room
temperature for 4 h. The solution was poured into water and
chilled to 4 ꢂC overnight. The product was isolated by filtration,
and the precipitate was washed with water to give the B2 (4.73 g,
17.6 mmol, 85%) as yellow solid. 1H NMR (CD3OD): 7.62 (s, 1H,
C6H2), 7.05 (s, 1H, C6H2), 4.83 (s, 2H, –CH2–), 3.98 (s, 3H,
OCH3), 2.47 (s, 3H, CH3). 13C NMR (CD3OD): 202.0, 171.6,
168.5, 155.9, 134.6, 110.8, 66.9, 57.3, 30.4. Anal. Calcd for
C11H11NO7: C; 49.08, H; 4.12, N; 5.20. Found: C; 48.72, H; 4.02,
N; 5.24.
2.7 Synthesis of N-(3-(4-(1-(2,3-anhydridehydroxylethyl)-5-
nitrophenoxy)acetamide)propyl methacryl amide (AMCO)
To a solution of MOH (0.25 g, 0.63 mmol) and pyridine (0.1 g,
1.3 mmol) in CH2Cl2 (10 mL) was added a solution of trimellitic
anhydride chloride (0.18 g, 0.85 mmol) in CH2Cl2 (5 mL) at 0 ꢂC
under an atmosphere of nitrogen. The mixture was stirred at
room temperature overnight. The precipitate formed in the
reaction mixture was filtered off and washed with CH2Cl2. After
the removal of CH2Cl2, the residue was dissolved in chloroform,
and the resulting solution was washed with water several times
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and a dilute NaHCO3 aqueous solution. The organic phase was
dried over anhydrous MgSO4 and concentrated by evaporation
to give light yellow solid (0.32 g, 0.56 mmol, 89%). It was further
purified by HPLC using acetonitrile as an eluent. 1H NMR
(CDCl3): 8.66 (s, 1H, C6H3), 8.56 (d, J ¼ 7.7 Hz, 1 H, C6H3) 8.13
(d, J ¼ 8.0 Hz, 1 H, C6H3) 7.67 (s, 1H, C6H2), 7.23 (m, 1H, NH),
7.09 (s, 1H, C6H2), 6.77 (m, 1H, –OC(H)–), 6.56 (m, 1H, NH),
5.77 (s, 1H, ¼CH2), 5.35 (s, 1H, ¼CH2), 4.60 (s, 2H, –CH2–),
3.96 (s, 3H, OCH3), 3.49 ꢁ 3.30 (m, 4H, NHCH2CH2CH2NH),
1.98 (s, 3H, CH3C¼), 1.84 (d, J ¼ 6.6 Hz, 3H, CH3), 1.74 (m, 2H,
NHCH2CH2CH2NH). HRMS Anal. Calcd for C27H27N3O11
(M + Na)+: 592.1543. Found: 592.1449.
2.12 DNase protection
The naked DNA and P10A/DNA complexes were incubated
with DNase (Promega, Madison, WI) to confirm the protection
of complexes of DNA with P10A with experimental procedure
modified from a reported method.20 DNase (2 U) was added to
100 ml of the corresponding solution of DNA and P10A/DNA
complexes, containing 1 mg/100 ml of DNA. The mixture was
incubated at 37 ꢂC and aliquots of 20 ml were taken at 10, 20, 40,
60 min, to which 8 ml of 100 mM EDTA were immediately added
to inactivate DNase degradation and incubated for 15 min.
Then, 10 ml of 2 mg mlꢁ1 of heparin was added to the mixture and
incubated for 2 h at room temperature. To the time 0 min sample,
the same proportions of EDTA and DNase solutions were
added, but starting with the EDTA solution to prevent any
DNase degradation.
2.8 Synthesis of PEI with AMCO (P6A and P10A)
To a solution of PEI (MW ¼ 600) (75 mg, 0.125 mmol) in 5 ml
H2O–THF (v/v ¼ 40/60) was added a solution of AMCO (19 mg,
33.4 mmol) in THF (2 mL) at room temperature under an
atmosphere of nitrogen. The mixture was stirred at room
temperature for 30 min. After evaporation of THF, the residue
was dissolved in distillated water. P6A was purified by HPLC
using a solution of acetonitrile and 1% TFA as an eluent to
afford 58.9 mg (62.7%) of slight light yellow oil. According to
similar procedure, a 48.1 mg (82%) of P10A was obtained by
using of PEI (MW ¼ 10,000) (51 mg, 0.05 mmol) and AMCO
(7.6 mg, 13.4 mmol). The concentration of the photolabile group
can be calculated by integration of the 1H NMR spectrum that is
by calculating the ratio of the vinyl protons to the characteristic
methylene protons of PEI main chain. The concentration values
of approximately 20 mol% calculated in this manner are in good
agreement with the theoretical values.
2.13 Photo irradiation
Photolysis was performed at room temperature using Model
B-100A Hi Intensity UV Lamp (UVP Inc, Optical density is
3.5 mW cmꢁ2 at 30 cm) with distance of 10 cm.
2.14 Cell culture and transfection
The plasmid pEGFP-N3 was obtained from Clontech, amplified
in high copy E. Coli, and column purified by the standard
procedure of the manufacturer (Qiagen, Germany). The
concentration was determined by UV absorbance at 260 nm.
COS cells were cultured in Dulbeccos’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal calf serum (Hyclone),
0.3 mg mlꢁ1 L-glutamine (Gibco BRL), and 150 U/ml penicillin
(Gibco BRL), and 0.15 mg mlꢁ1 streptomycin (Gibco BRL) and
maintained at 37 ꢂC in 5% CO2 humidified atmosphere. Cells
were seeded in 24-well plates at 1 ꢃ 105 cells/well 24 h prior to
transfection. At the time of transfection, cells were at about
60%ꢁ70% confluence. P10A/DNA complexes were prepared by
the addition of P10A solution to pEGFP plasmid DNA solution
to make the required N/P ratios for 1 mg of plasmid DNA in
200 ml of Opti-MEM medium, and mixed by vortexing, and
incubated for 20 min at room temperature. Culture medium of
cells removed. Then the complexes were added to COS cells and
then incubate for 3 h at 37 ꢂC (5% CO2). In case of photo-
irradiation, the well plates exposed to 365 nm at distance of
10 cm for 10 min. The medium was replaced with growth medium
to remove the complexes after 4 h and incubated for 24 h. Gene
transfection efficiency was measured by analyzing the expression
of the EGFP by flow cytometry. All samples were measured in
triplicate.
2.9 Monitoring complexes formation by gel electrophoresis
P6A/DNA or P10A/DNA complexes, containing 1 mg DNA,
with N/P charge ration from 1 to 24 were visualized on 1%
agarose gel stained with 0.125 mg mlꢁ1 ethidium bromide (EtBr).
2.10 By fluorescence
Complex formation of P6A/DNA or P10A/DNA was measured
with a loss of fluorescence intensity by intercalating of EtBr. A 20
ml of EtBr solution (0.625 mg mlꢁ1) was added to 50 ml solution of
P6A/DNA or P10A/DNA complexes with N/P charge ratio from
1 to 24. After incubation of 10 min, the solution was measured
for the excitation of 510 nm and 590 nm emission with a fluo-
rescence spectrofluorometer.
2.11 Polymerization of P10A/DNA nanoparticles
3. Results and discussion
The complexes were formed by the addition of P10A solution to
DNA solution to achieve the 16 or 24 N/P charge ratio in 40 ml of
Opti-MEM medium. The ammonium persulfate initiator solu-
tion (1 mM) was added to the complexes and then purged with
dry nitrogen gas for 10 min. The final concentration of P10A,
DNA, and initiator in the polymerization was 2.53 nmol, 3 nmol,
and 1 nmol, respectively. The mixture was polymerized at 37 ꢂC
for 2 h, and then 160 ml of additional Opti-MEM was added to
use for transfection.
o-Nitrobenzyl group is one of the most useful photolabile
compounds available for light triggered investigations of bio-
logical processes.21–23 Moreover, the acrylate group facilitates
radical or anion (co)polymerization with acrylate monomer of
other series as well as homopolymerization.24 Therefore,
o-nitrobenzyl and methacrylate groups were selected for the
synthesis of functionalized PEI.
The functional reaction of PEI was carried out according
to Scheme 1. The acid group of B2 was activated with
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Scheme 1
N-hydroxysuccinimide
(NHS)
and
N,N0-dicyclohexyl-
of P6A alone was carried out in distilled water and monitored for
the cleavage under the irradiation (365 nm) using HPLC as
shown in Fig. 2. The carboxylate functional group was cleaved
by internal reaction with the nitrobenzyl functional group,
thereby separating the polyamine ligand from the cross-linkable
moiety. The photodecomposition reaction of P6A was shown in
Scheme 2. The intensity of signal A decreased with an increase of
the irradiation time, while the signal B increased. Moreover,
signal A slightly shifted to the region of fast retention time,
indicating change of polarity of decomposed PEI by irradiation.
The signal B in UV detector showed high intensity and reached
maximum value after 4 min. The UV spectra of signal B showed
an absorbance around 300 nm due to nitrosoacetophenone.
The formation of P6A/DNA and P10A/DNA nanoparticles
was performed by the adding of the P6A and P10A solution to
the DNA solution to make desired N/P charge ratios. Gel elec-
trophoresis showed the charge ratio where DNA completely
binds with P6A and P10A. DNA forms the nanoparticle complex
at N/P ratio of 16 with P6A and 8 with P10A (Fig. 3).
carbodiimide (DCC) at room temperature for 1 h. The addition
of N-(3-aminopropyl)methacrylamide hydrochloride gave the
MCO and dicyclohexylurea which was easily separated by
filtration. MOH was obtained by the reduction of acetophenone
site of MCO with NaBH4. In subsequent step, the hydroxyl
group was reacted with trimellitic anhydride chloride. Finally,
reaction with PEI (MW: 600 and 10,000) was carried out to give
the functionalized PEI (referred to as: P6A and P10A).
The 1H NMR spectra of the P6A and P10A showed a peak at
6.7 ppm and two separated peaks at 5.5–5.3 ppm, which were
attributed to methylidyne proton of o-nitrobenzyl group and
vinyl protons of methacrylate group, respectively, as well as
peaks assignable to PEI (Fig. 1). From NMR, the conjugation of
o-nitrobenzyl and methacrylate groups on PEI showed almost
similar molar ratio with targeted ratio. P6A and P10A showed
UV signal around at 270–400 nm due to nitro and aromatic
group.
Our photo labile compound contained a carboxylate and
a nitrobenzyl group cleavable upon exposure to light in the range
of 300 to 400 nm, especially 365 nm. Firstly, the decomposition
Fig. 4 shows the fluorescence change of P6A/DNA and P10A/
DNA nanoparticles for various N/P charge ratios. The
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Fig. 1 1H NMR spectra of B1, B2, MCO, MOH, AMCO, and P6A.
fluorescence reached on minimum at N/P charge ratio of 24 for
P6A and 8 for P10A, indicating that EtBr could not intercalate
with DNA further because the DNA was completely condensed
with P6A and P10A. The radius of the nanoparticles was
dependent upon the N/P ratio used. Moreover, particle size
showed diameters of about 40–80 nm in case of P10A. In
contrast, P6A of low molecular weight PEI showed slightly larger
diameters around 100–160 nm. These results indicated that DNA
condensed more sufficiently with P10A of high molecular weight.
To evaluate the protective effect of the complex, naked DNA
and DNA/P10A nanoparticles were incubated with DNase
(Fig. 5). Degradation was monitored by agarose gel electro-
phoresis. The DNA was competed off the polymer using the
anionic displacer, heparin. The condensation of DNA with P10A
protected DNA from DNase even after 60 min, whereas free
DNA was rapidly degraded by added DNase.
Fig. 2 HPLC charts were measured by (a) RI and (b) UV detector of
functionalized PEI (P6A) according to photo irradiation at 365 nm for
0–8 min. (a1) and (a2) represent chart at enlarge scale of peak A and B. S
is due to the solvent peak.
The polymerization of P10A alone was carried out with
ꢂ
ammonium persulfate (AS) as an initiator at 37 C for 2 h. In
monitoring by HPLC, the signal slightly shifted to a region of
slow retention time, indicating polarity change by polymeriza-
tion. Polymerization of the P10A/DNA nanoparticles with N/P
charge ratio of 16 and 24 was then carried out. Particle size of
P10A/DNA nanoparticles increased slightly from 40–80 nm to
60–120 nm after polymerization (Fig. 6). This might be due
to the inter-polymerization between methacrylate groups on
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Fig. 5 Gel electrophoresis exposed to DNase for free DNA and
P10A/DNA for 60 min.
P10A/DNA nanoparticles. P10A/DNA nanoparticles after
polymerization had limited mobility during gel electrophoresis
(Fig. 7). This suggested that the P10A/DNA nanoparticles
formed was coated by the polymerization of methacrylate group.
DNA cross-linked within the nanoparticle was also protected
against DNase.
Scheme 2
To examine transfection efficiency, several samples were
prepared and transfected into dividing COS cells. AS initiator
was used to form cross-linked P10A/DNA nanoparticle with N/P
charge ratio of 16. Post-transfection photo-irradiation was then
tested for release of DNA intracellularly. Cell viability and
growth were unaffected by the 10 min light exposure at 365 nm.
Enhanced green fluorescence protein (EGFP) was monitored by
flow cytometry (Fig. 8). The histograms in flow cytometry show
the count of cells in each sample that showed expression of the
transfected DNA. The control sample shows a control DNA,
transfected into dividing COS cells, which produced little fluo-
rescence as shown in Table 1. Delivery of EGFP plasmid with
uncross-linked P10A, which was not regulated by light, resulted
in 6.3% transfection. Delivery of EGFP plasmid with uncross-
linked P10A, which was exposed to 365 nm light showed 5.7%
transfection. This demonstrates that the uncross-linked P10A
had no effect even though the P10A can be photocleaved.
Delivery of EGFP plasmid with cross-linked P10A without
photoactivation resulted in 5.7% transfection. In contrast, the
Fig. 3 Gel electrophoresis of (a) P6A/DNA and (b) P10A/DNA
complexes with different N/P charge ratios. P6A/DNA or P10A/DNA
complexes, containing 1 mg DNA, with N/P charge ration from 1 to 24 are
visualized on 1% agarose gel stained with 0.125 mg mlꢁ1 ethidium bromide
(EtBr).
Fig. 4 Fluorescence change of (a) DNA/P10A and (b) DNA/P6A with
different N/P charge ratios. Complex formation of DNA is measured
with a loss of fluorescence intensity by intercalating of EtBr. EtBr solu-
tion alone showed fluorescence intensity of 400–600.
Fig. 6 Particle size before and after polymerization, and after photo
irradiation for P10A/DNA complexes with N/P charge ratios (a) 16 and
(b) 24.
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Table 1 Gene expression efficiency
Sample
% FI > 100a
Mean FI
Control
0.01%
6.3%
5.7%
5.7%
18.4%
55
Uncross-linked P10A/DNA
Uncross-linked P10A/DNA + light
Cross-linked P10A/DNA
Cross-linked P10A/DNA + light
3266
3206
3187
3274
a
The percent transfection is defined as fluorescence intensity (FI) of
greater than 100 (background cellular fluorescence < 10). FI incubated
with P10A only showed <0.01%.
prior studies,2 the resulting transfection efficiency after photo-
lytic release is comparable, but not exceeding, that obtained with
lipofectamine. Future improvements of this approach will seek to
increase DNA caging prior to light exposure and to increase the
functionally of DNA after light exposure.
4. Conclusions
The present work provides a newly designed photolabile gene
carrier that is cationic, cross-linkable, and photolytic, permitting
the light-triggered, controlled release of a plasmid. In addition,
this methodology does not seek to directly alter the DNA
backbone with a photolabile moiety. The photolabile gene carrier
allowed a 3-fold increase in transgene expression following light
exposure. Moreover, the nanoparticles yielded a small packing
size, DNA protection, and reduced DNA leakage until photo-
triggering. Accordingly, these materials are directed at providing
timed delivery of DNA without endosomal degradation, poten-
tially bypassing the deleterious endosomal toll-like receptor
response. Consequently, such photo-regulated, cross-linkable
reagents offer new options in the useful generation of stable
DNA nanoparticles for temporally-controlled, spatial-addressed,
Fig. 7 Gel electrophoresis of (a) naked DNA, (b) before polymerization
of P10A/DNA (N/P charge ratio 16), and (c) without and (d) with pho-
toirradiation (365 nm) for 10 min after polymerization with ammonium
persulfate.
amount of transfection was dramatically increased from 5.7% to
18.4% when cells transfected with cross-linked P10A/DNA were
exposed to 365 nm light. This experiment demonstrated that
DNA was caged by the cross-linked P10A and that gene
expression increased markedly when the o-nitrobenzyl in P10A
was broken down with light.
The present study sought to demonstrate the phototriggered
release of DNA from cationic polymers. In comparison with our
Fig. 8 Histograms for the expression of enhance green fluorescence protein transfected to dividing COS cells.
3402 | J. Mater. Chem., 2010, 20, 3396–3403
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and metered dosing of DNA for gene transfer. Even though the
material in this work acts as a photolabile gene carrier, a special
photo-device for in vivo working of this material due to the
penetration depth of 365 nm light (<0.1mm into the tissue) and
an additional procedure for residue initiators might be necessary
for the future work.
Acknowledgements
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This work was supported in part by NIH, KMOHW (grant no
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