1,4-Butanediol diglycidyl ether (BDE)-crosslinked PEI-g-imidazole nanoparticles as nucleic acid-carriers in vitro and in vivo

Article abstract of DOI:10.1039/c1mb05049d

Previously reported 1,4-butanediol diglycidyl ether (BDE) crosslinked PEI (branched polyethylenimine, 25 k) nanoparticles (A. Swami, R. Kurupati, A. Pathak, Y. Singh, P. Kumar and K. C. Gupta, A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles, Biochem. Biophys. Res. Commun., 2007, 362, 835-841) (PN NPs) were reacted with varying proportions of a novel linker, 2-(N-1-tritylimidazol-4-yl)-N-(6- glycidyloxyhexyl)-acetamide (IGA linker, 3), to yield PN-g-imidazolyl nanoparticles (PNIm) with improved transfection efficiency. Here, the IGA linker (3) reacted through an epoxy ring to partially convert the residual 1° and 2° amines present in PN NPs to 2° and 3°, respectively, without altering the total number of amines and additionally incorporating the delocalized positive charge of the imidazolyl moiety. The resulting particles were characterized for their size, zeta potential and DNA complexing ability. PNIm/DNA nanoplexes, in the size range of 120-400 nm, were evaluated for transfection efficiency in HeLa, HEK293 and CHO cell lines, which was found to be ～11, ～2-3 and ～2-17 folds higher than PEI, PN-2 (the best working sample of the PN series) (A. Swami, R. Kurupati, A. Pathak, Y. Singh, P. Kumar and K. C. Gupta, A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles, Biochem. Biophys. Res. Commun., 2007, 362, 835-841) and commercial transfection reagents tested in this study, respectively. Also, flow cytometric analysis showed ～78% (ca. ～43% in PN-2) cells transfected with the PNIm 10(6)/DNA complex (the best working sample of the PNIm series) in HEK293 cells. Transfection of GFP specific siRNA in HEK293 cells suppressed the gene expression by ～90% (ca. ～70% in PN-2). All the cell lines treated with PNIm/DNA nanoplexes showed >90% viability. In vivo gene expression of luciferase enzyme in Balb/c mice showed highest expression in spleen after seven days.

Full text of DOI:10.1039/c1mb05049d

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PAPER
1,4-Butanediol diglycidyl ether (BDE)-crosslinked PEI-g-imidazole
nanoparticles as nucleic acid-carriers in vitro and in vivow
Ritu Goyal,a Ruby Bansal,a Shilpa Tyagi,b Yogeshwer Shukla,b Pradeep Kumara
and Kailash Chand Gupta*ab
Received 7th February 2011, Accepted 23rd March 2011
DOI: 10.1039/c1mb05049d
Previously reported 1,4-butanediol diglycidyl ether (BDE) crosslinked PEI
(branched polyethylenimine, 25 k) nanoparticles (A. Swami, R. Kurupati, A. Pathak, Y. Singh,
P. Kumar and K. C. Gupta, A unique and highly efficient non-viral DNA/siRNA delivery system
based on PEI-bisepoxide nanoparticles, Biochem. Biophys. Res. Commun., 2007, 362, 835–841)
(PN NPs) were reacted with varying proportions of a novel linker, 2-(N-1-tritylimidazol-4-yl)-N-
(6-glycidyloxyhexyl)-acetamide (IGA linker, 3), to yield PN-g-imidazolyl nanoparticles (PNIm)
with improved transfection efficiency. Here, the IGA linker (3) reacted through an epoxy ring to
partially convert the residual 11 and 21 amines present in PN NPs to 21 and 31, respectively,
without altering the total number of amines and additionally incorporating the delocalized
positive charge of the imidazolyl moiety. The resulting particles were characterized for their size,
zeta potential and DNA complexing ability. PNIm/DNA nanoplexes, in the size range of
120–400 nm, were evaluated for transfection efficiency in HeLa, HEK293 and CHO cell lines,
which was found to be B11, B2–3 and B2–17 folds higher than PEI, PN-2 (the best working
sample of the PN series) (A. Swami, R. Kurupati, A. Pathak, Y. Singh, P. Kumar and
K. C. Gupta, A unique and highly efficient non-viral DNA/siRNA delivery system based on
PEI-bisepoxide nanoparticles, Biochem. Biophys. Res. Commun., 2007, 362, 835–841) and
commercial transfection reagents tested in this study, respectively. Also, flow cytometric analysis
showed B78% (ca. B43% in PN-2) cells transfected with the PNIm 10(6)/DNA complex
(the best working sample of the PNIm series) in HEK293 cells. Transfection of GFP specific
siRNA in HEK293 cells suppressed the gene expression by B90% (ca. B70% in PN-2). All the
cell lines treated with PNIm/DNA nanoplexes showed >90% viability. In vivo gene expression
of luciferase enzyme in Balb/c mice showed highest expression in spleen after seven days.
Earlier, we had crosslinked PEI with BDE to address the
Introduction
concern of cytotoxicity with concomitant improvement in the
transfection efficiency.1 The resulting nanoparticles (PN NPs)
Non-viral vectors have emerged as promising candidates for the
treatment of genetic diseases.2–4 However, a challenge still
exhibited 2.5–5 folds higher transfection efficiency than native
remains where these vectors can be employed for in vivo
therapeutic applications.5 Over the past few decades, PEI based
delivery systems have shown potential as transfection agents.6–8
PEI and showed minimal cytotoxicity. In the present study,
we have tried to further improve the transfection properties
of PN-2 using a novel IGA linker (3), one end of which
However, polymer associated cytotoxicity limits its applications
in vivo.9,10 Reports indicate that higher charge density of PEI
consists of an epoxy ring that would react partially with
residual 10 and 20 amino groups in PN NPs and convert them
into 20 and 30, respectively, thereby maintaining the overall
contributes to its cytotoxicity, which has been circumvented by
acylation,11 alkylation,12 PEGylation,13 coating with sugar
number of amines intact. The other end of the linker bears an
moieties14–16 and incorporation of imidazolyl groups.17–19
imidazole moiety, which would incorporate a delocalized
charge (i.e. an endosomolytic agent) that would also help in
improving the transfection efficiency of the resulting particles.
a Institute of Genomics and Integrative Biology (CSIR), Mall Road,
As the beneficial role of imidazole in transfection is well
documented,17–19 its accumulation inside the endosomes
causes an increase in buffering capacity and thereby results
in the increase of transfection efficiency by several orders of
magnitude.17
Delhi University Campus, Delhi-110 007, India
b Indian Institute of Toxicology Research (CSIR),
Mahatma Gandhi Marg, Lucknow-226 001, Uttar Pradesh, India.
E-mail: kcgupta@iitr.res.in; Tel: +91-522-2621856
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c1mb05049d
c
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The synthesized PNIm NPs were evaluated in terms of their
size, zeta potential, buffering capacity, cell viability, trans-
fection efficiency, DNA release and in vivo gene expression.
One of the formulations, PNIm 10(6), scored B2–3 and B11
folds higher transfection efficiency than PN-2 and PEI,
respectively. In vivo gene expression studies revealed highest
expression in spleen followed by heart and lungs. The PNIm
NPs showed marked improvement over PN-2 NPs in terms of
transfection and therefore, hold great potential as transfection
agents.
the PN series) comparable to PN-3/PN-4 without compromising
on the transfection efficiency, we have now designed a unique
linker, which contains an epoxy group at one end, that could
partially convert the residual 11 and 21 amines to 21 and 31
(i.e. bad charge of 11 amines to good charge of 21 amines),
thereby improving the cell viability (comparable to PN-3 or
PN-4). The selection of a group for the other end was done
carefully, keeping the following points in mind, viz., (i) it
should help in improving the transfection efficiency of the
modified NPs, (ii) it should not adversely affect the cytotoxi-
city of the system, (iii) it should not involve time consuming
synthesis, and (iv) the required reagents should be commonly
available. Having gone through the literature and keeping
these points into consideration, we decided to have an
imidazole moiety, which is known for its beneficiary properties
in enhancing the transfection efficiency of the cationic polymers,19
on the other end of the linker (Scheme 1). This linker (3),
prepared from imidazole-4-acetic acid hydrochloride, was
reacted with the preformed PN NPs (Scheme 1) in increasing
percentage and a series (PNIm) was synthesized, which was
evaluated in terms of transfection efficiency and cyto-
toxicity. The best working sample of the series, PNIm 10(6),
dramatically enhanced the transfection efficiency over PEI
(B10–11 folds), PN-2 (B2–3 folds) and commercial trans-
fection reagents (B2–17 folds), which indicated that the
Results and discussion
Preparation of PNIm particles
In a previous publication from our laboratory, PEI was
converted into nanoparticles by crosslinking with increasing
amounts of BDE to synthesize a series of NPs (PN-1 to PN-4)
and evaluated them in terms of transfection efficiency and cell
viability properties.1 It was observed that cell viability
increased on increasing the percentage of crosslinking following
the trend PN-1oPN-2oPN-3oPN-4, and transfection
efficiency increased upto PN-2 and then started decreasing
on deviating from this percentage. Here, in order to further
improve the cell viability of PN-2 (the best working system of
Scheme 1 Schematic representation of preparation of IGA linker (3) and PNIm nanoparticles.
c
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projected linker not only improved the cell viability (comparable
to PN-3/PN-4) of the grafted NPs but also enhanced
the transfection efficiency by improving the endosomolytic
properties of the resulting polymeric NPs.
increase with increase in percentage grafting of (3), confirming
the conjugation of the same with PN NPs. On complexation
with pDNA (at a w/w ratio of 1), the zeta potential decreased,
which might be due to neutralization of some positive charge
with the negatively charged phosphate backbone of DNA.
However, in 10% FBS, the zeta potential became negative the
reason for which is well documented in the literature.20
Characterization of PNIm particles
The synthesized PNIm series was characterized by various
1
techniques, viz., H-NMR, IR, DLS and Zeta potential. The
DNA retardation assay
percentage grafting of linker (3) was analyzed on the basis of
1H-NMR (Table 1). The peaks at d 6.8 and 7.4 confirmed the
presence of an imidazole moiety in the synthesized PNIm
series. In IR, the peaks at 1620 cmꢀ1 (amide stretching) and
1130 cmꢀ1 (ether twisting) also confirmed the presence of
linker (3) in the nanoparticles.
The amines present in nanoparticles interact with the negatively
charged phosphate backbone of DNA electrostatically. This
property is used to see the effect of nanoparticles in retarding
the mobility of a fixed amount of pDNA on agarose gels. To
determine the amount of nanoparticles needed to retard the
mobility of a fixed amount (0.3 mg) of pDNA, the complexes
were prepared at increasing w/w ratios of nanoparticle : DNA
and loaded onto 1% agarose gel. PNIm particles retarded at
w/w ratios of 0.33 or 0.66, while PEI retarded the mobility of
the same amount of pDNA (0.3 mg) at a w/w ratio of 1. PN
NPs retarded the mobility of DNA (0.3 mg) at a higher w/w
ratio than PEI (Fig. 1).1 These observations show that a lesser
amount of PNIm particles and more amount of PN NPs are
required in comparison to PEI in retarding the fixed amount
of DNA. The reason may be attributed to the presence of extra
amines available for binding with DNA in the imidazole ring
in PNIm while conversely, in the case of PN NPs, some
amount of charge is embedded inside the NPs, so a higher
amount is required for retardation.
The size of PN NPs after grafting with (3) increased,
although remained in the nano range (data not shown), as
determined using DLS. This increasing trend in size also
confirmed the grafting of the IGA linker on the pre-formed
nanoparticles, which might be acting as a hanging pendant.
The pDNA complexes of PNIm particles exhibited size in the
range of 120–400 nm (Table 2). Further, in the presence of
10% FBS, the sizes decreased (49–212 nm), which might be
due to adsorption of water molecules from the cationic
surfaces by the serum proteins leading to dehydration of the
particles.20 The zeta potential of nanoparticles was found to
Table 1 Percent grafting of imidazole on PN NPs as estimated by
1H-NMR spectroscopy
Attempted
substitution of
the IGA linker (%)
Found substitution
of the IGA linker
by H-NMR (%)
Cell viability assay
1
PNIm
The synthesized PNIm series was evaluated in terms of cell
viability using the MTT assay in three different cell lines, viz.,
HEK293, HeLa and CHO. The PNIm series was found to be
almost non-toxic (cell viability ranged between 90–98%) in all
the cell lines tested. However, PEI and LipofectamineTM
showed a significantly reduced cell viability (ranging between
45–60% in all the cell lines tested; P o 0.01) (Fig. S1, ESIw).
For the commercial reagents, the cell viability was found
to be in the range of 80–82% (SuperfectTM) and 87–92%
(GenePORTER 2TM). PN-2 showed the cell viability ranging
between 84–91% in the cell lines tested in this study.
PN-1
PN-2
PN-3
PNIm 5(2)
PNIm 5(4)
PNIm 5(6)
PNIm 5(8)
PNIm 10(2)
PNIm 10(4)
PNIm 10(6)
PNIm 10(8)
PNIm 15(2)
PNIm 15(4)
PNIm 15(6)
PNIm 15(8)
2
4
6
8
2
4
6
8
2
4
6
8
0.65
1.25
1.74
2.11
0.62
1.19
1.69
2.07
0.66
1.29
1.79
2.12
Table 2 Particle size and zeta potential measurements of PNIm particles and their corresponding DNA complexes in water and serum at a w/w
ratio of 1 : 1 for PNIm particles and PEI
Size ꢁ SD (PDI)/nm
With DNA
Zeta potential ꢁ SD/mV
Native particle
With serum
With DNA
With serum
PNIm 5(2)
PNIm 5(4)
PNIm 5(6)
PNIm 5(8)
PNIm 10(2)
PNIm 10(4)
PNIm 10(6)
PNIm 10(8)
PNIm 15(2)
PNIm 15(4)
PNIm 15(6)
PNIm 15(8)
PEI
396.0 ꢁ 3.78 (0.165)
386.7 ꢁ 4.29 (0.276)
330.0 ꢁ 3.10 (0.292)
321.1 ꢁ 5.92 (0.189)
280.0 ꢁ 2.05 (0.156)
248.6 ꢁ 7.29 (0.208)
226.8 ꢁ 6.20 (0.143)
202.1 ꢁ 4.95 (0.187)
169.4 ꢁ 6.20 (0.387)
168.0 ꢁ 3.87 (0.170)
148.2 ꢁ 4.76 (0.186)
123.5 ꢁ 6.38 (0.241)
287.8 ꢁ 2.97 (0.675)
211.1 ꢁ 4.61 (0.271)
105.2 ꢁ 2.91 (0.153)
100.4 ꢁ 3.79 (0.189)
98.4 ꢁ 4.76 (0.297)
95.5 ꢁ 5.39 (0.129)
76.5 ꢁ 2.07 (0.275)
68.4 ꢁ 3.87 (0.128)
59.4 ꢁ 3.92 (0.274)
54.0 ꢁ 4.73 (0.186)
53.5 ꢁ 4.93 (0.252)
50.1 ꢁ 4.27 (0.127)
49.1 ꢁ 3.68 (0.138)
125.6 ꢁ 2.95 (0.489)
30.6 ꢁ 2.17
33.9 ꢁ 1.98
36.4 ꢁ 1.76
40.6 ꢁ 3.48
29.2 ꢁ 2.63
30.2 ꢁ 3.73
32.4 ꢁ 3.79
36.4 ꢁ 3.92
26.4 ꢁ 3.72
28.3 ꢁ 2.61
29.6 ꢁ 2.02
32.8 ꢁ 2.81
31.8 ꢁ 2.90
15.9 ꢁ 1.83
17.8 ꢁ 1.12
17.8 ꢁ 1.95
17.9 ꢁ 2.63
16.6 ꢁ 1.98
16.7 ꢁ 2.76
19.6 ꢁ 3.65
20.6 ꢁ 2.93
17.2 ꢁ 1.98
19.5 ꢁ 2.75
20.5 ꢁ 3.81
21.5 ꢁ 2.72
21.4 ꢁ 2.81
ꢀ23.0 ꢁ 1.07
ꢀ19.3 ꢁ 1.67
ꢀ19.2 ꢁ 1.92
ꢀ17.5 ꢁ 2.72
ꢀ20.3 ꢁ 2.02
ꢀ17.4 ꢁ 2.83
ꢀ16.9 ꢁ 2.67
ꢀ16.4 ꢁ 1.29
ꢀ20.9 ꢁ 2.90
ꢀ14.2 ꢁ 1.83
ꢀ14.2 ꢁ 1.28
ꢀ14.0 ꢁ 1.29
ꢀ13.6 ꢁ 1.98
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and PN-2 NPs. The enhanced buffering capacity of the PNIm
series may be due to the presence of an imidazole moiety as
this moiety is well documented in the literature to be a good
buffering provider in the pH range of endosomes. We, there-
fore, analyzed the transfection efficiency of these particles.
Interestingly, the transfection efficiency initially increased
with increase in buffering capacity but showed a decreasing
trend after attaining a maximum efficiency. This increase in
buffering may be responsible for the enhanced transfection
efficiency of the proposed nanoparticles.
In vitro transfection studies
Transfection efficiency of PNIm and PN-2 nanoplexes,
PEI polyplexes and DNA complexes of Superfectt,
GenePORTER 2t and Lipofectaminet was evaluated in
HEK293, CHO and HeLa cells using EGFP (Enhanced Green
Fluorescent Protein) as a reporter gene in the presence and
absence of serum. The study was performed at various w : w
ratios (0.33, 0.66, 1.0, 1.66, 2.33). It was found that PNIm
10(6) performed the best in terms of transfection efficiency and
scored B10.4, B11.6 and B12.7 folds higher in HEK293,
CHO and HeLa cells, respectively, as compared to PEI. Also,
the transfection efficiency of PNIm 10(6) was B2.3, B3.0, and
B2.0 folds higher in HEK293, CHO and HeLa cells, respec-
tively, than PN-2 (Fig. 2a). Moreover, in the presence of 10%
serum, the transfection efficiency did not reduce significantly
(P > 0.05) (Fig. 2b), which further implicates the potential of
modified nanoparticles for in vivo gene delivery applications.
Flow cytometry was employed to evaluate the reporter gene
expression at the individual cell level. PNIm 10(6) showed
maximum transfection efficiency, i.e. 78 ꢁ 4.8 and 72 ꢁ 3.9%
in HEK293 and CHO cells, respectively. In contrast, PEI
showed only 23 ꢁ 2.3% and 20 ꢁ 3.4% GFP positive cells
in HEK293 and CHO cells, respectively (Fig. 3). Also, PN-2
showed 48 ꢁ 5.3% in HEK293 and 45 ꢁ 2.7% GFP positive
cells in CHO cells. PEI and PNIm particles worked best at a
w : w ratio of 1 : 1 (NP : DNA) while PN-2 worked best at
1.66 : 1. This might be due to the presence of an imidazole
moiety, which provided an extra delocalized charge to bind
pDNA. One of the plausible explanations for the enhanced
transfection efficiency may be attributed to the enhanced
buffering of PNIm nanoparticles in the pH range 3–10 due
to the imidazole group. Secondly, PNIm particles are almost
non-toxic (see Cell viability assay), which may further account
for the enhanced transfection efficiency.
Fig.
1 DNA retardation assay of PNIm/DNA and PEI/DNA
complexes. pDNA (0.3 mg) was incubated with increasing amounts
of nanoparticles in 5% dextrose for 20 min. Samples were electro-
phoresed in a 1% agarose gel at 100 V for 45 min.
Therefore, it is evident from the figure that after grafting with
the IGA linker (3), the cell viability improved in comparison to
crosslinked nanoparticles (PN-2). The possible reasons for the
increased cell viability of PNIm particles over PN-2 may be (i)
further reduction in the number of toxic primary amino
groups, and (ii) the presence of an imidazole moiety, as it is
reported that a protonated imidazole ring is less cytotoxic than
protonated amines.17
Buffering capacity
PEI exhibits a considerable buffering capacity in the pH range
of endosomes and lysosomes,21,22 however, displays consider-
able toxicity due to the presence of primary amines.10 There-
fore, PEI was crosslinked using BDE to result in well defined
nanoparticles with reduced toxicity.1 This process also
enhanced the transfection efficiency of the PEI by several folds
maintaining the buffering capacity of the resulting nano-
particles to the level of PEI. In this study, we have tried to
enhance the buffering capacity of PN NPs by grafting the
imidazole moiety on these nanoparticles through an epoxy
linker (3).
DNA release assay
The imidazole ring helps in the proper binding with DNA
and also in the escape of pDNA from endosomes.17 Due to the
protonation tendency of the imidazole ring at pH 6.0, it is a
weak base and, therefore, has been found to be suitable to
modify the surface of cationic polymers.17 To see the effect of
imidazole grafting on the buffering capacity of PNIm NPs, an
acid–base titration was performed in the pH range 3–10.
Fig. S2 (ESIw) shows the buffering capacity of PNIm particles,
PN-2 NPs and PEI under different pH conditions. It was
found that the proton capturing tendency of the PNIm
particles was slightly enhanced as compared to native PEI
The DNA carrier after carrying the desired therapeutic
inside the cell must be able to release it to facilitate efficient
transfection.23 The extent of pDNA binding with the series of
PNIm 10 nanoparticles was evaluated and compared with
the release pattern of PEI. A fixed amount of PNIm 10
nanoparticle/DNA and PEI/pDNA complexes were incubated
with the increasing units of heparin, a competitive anionic
moiety, and the samples run on 1.0% agarose gel. On quanti-
fication by densitometry, it was observed that on descending
the series (PNIm 10), the binding with DNA became stronger.
With 5 U of heparin, the amount of pDNA released from
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Fig. 2 GFP fluorescence intensity in HEK293, CHO and HeLa cells in the (a) absence of serum, and (b) presence of serum, transfected with
PNIm/DNA, PEI/DNA, SuperfectTM/DNA, GenePORTER 2TM/DNA and LipofectamineTM/DNA complexes. The transfection profiles show
fluorescence intensity expressed in terms of arbitrary units per mg of total cellular protein obtained at a w/w ratio of 1 for PNIm/DNA and
PEI/DNA polyplexes. Cells were incubated with the complexes for 4 h and the expression of GFP was monitored after 36 h. The fluorescent
intensity of GFP fluorophore in the cell lysate was measured on a spectrofluorometer. The results represent the mean of three independent
experiments performed in triplicates. *P o 0.05 vs. PEI and Lipofectamine.
complexes decreased from 60 to 38% on increasing the percent
of IGA linker (3), while under the same conditions, PEI
released only 19% of bound DNA. Further, on increasing
the heparin units (10 U), PNIm 10(2) and PNIm 10(6) released
upto 98% and 75% of pDNA, respectively, while PEI released
only 55% of it (Fig. 4). These results suggest that PEI binds
very strongly to DNA, which may be due to the presence of a
high amount of cationic charge (11 amines). In PNIm particles,
some amount of charge is probably embedded inside the
particles, which is not available to bind with DNA, and hence
a comparatively loose complex is formed. Moreover, in a
recent report, it has been demonstrated that transfection
efficiency depends on the pDNA binding ability of NPs.24
The synthesized nanoparticles not only carried the bound
DNA efficiently to inside of the cell but also released it in
sufficient amount in the cellular milieu to result in enhanced
gene expression.
by confocal laser scanning microscopy. DAPI (blue) was used
to stain the nucleus. Within 30 min of the incubation, red
and green particles were seen in the cells near the plasma
membrane, and in the overlaid images yellow fluorescence was
observed. Red and green fluorescence was also observed inside
the cytoplasm and nucleus, which indicated the dissociation of
the complex within the cytoplasm or nucleus. PNIm 10(6) was
found to carry pDNA inside the cytoplasm within 1 h and to
the nucleus within 2 h of the addition of complexes to the cells
(Fig. 5). The nanoplexes were localized not only in the
cytoplasm but also in the nucleus. This observation is in
agreement with a previously reported study where PEI delivers
nucleic acids to the nucleus.25 The finding clearly demonstrates
efficient intracellular delivery of DNA using PNIm 10(6).
Delivery of siRNA
RNA interference is a technology that allows fully or partially
suppressing the expression of a specific gene, allowing targeted
gene knockout and gene knockdown.26,27 As PNIm 10(6)
efficiently transfected pGFPDNA into the cell, its ability to
knockdown this gene was evaluated using GFP specific
siRNA in HEK293 cells and the results were compared
with GenePORTER 2TM. The cells were transfected first
with the PNIm 10(6)/GFPDNA complex and after 3 h
(the optimum time for cellular entry; see Confocal study)
Intracellular localization of nanoplexes
The intracellular localization of DNA is an important factor
for successful gene therapy. To determine the intracellular
localization of PNIm 10(6)/pDNA nanoplexes after cellular
uptake, HeLa cells were incubated with tetramethylrhodamine-
labeled PNIm 10(6) nanoparticles (red fluorescence) complexed
with YOYO-1 labeled DNA (green fluorescence) and observed
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present in the cellular environment from degradation.28 The
PNIm 10(6)/pDNA formulation and pDNA alone were
incubated with DNase I for different time intervals (0.25,
0.50, 1 and 2 h) and run in 1% agarose gel. Quantitative
analysis of the gel showed that in contrast to the free DNA
(0.3 mg), which was degraded completely by DNase I within
15 min, PNIm 10(6) effectively protected bound DNA (Fig. 7)
and only 15% of it was found to be degraded even after 2 h of
treatment. Thus, the projected nanoparticles are suitable for
in vivo administration of pDNA.
In vivo gene expression studies
In vivo transfection efficiency of the complexed PNIm 10(6)
was examined in Balb/c male mice by luciferase activity in all
the vital organs of the organism 7 d post intravenous injection.
A significant (P o 0.05) increase in luciferase activity was
observed in spleen and heart in PNIm 10(6) polyplexes
compared to unmodified PEI (Fig. 8). Naked DNA performed
least out of all the three tested. PEI also showed highest gene
expression in the spleen for which the mechanism is still
not clear.
Fig. 3 Percent transfection efficiency of PNIm 10/DNA complexes
Methods
determined using FACS in (a) CHO and (b) HEK293 at various w/w
ratios compared with PN-2, PEI, SuperfectTM and LipofectamineTM
Cell culture and materials
.
*P o 0.05 vs. PEI and Lipofectamine.
HEK293, HeLa and CHO cells were procured from NCCS,
Pune, India. Cell cultures were maintained (37 1C, 5% CO2)
in Dulbecco’s Modified Eagle’s Culture Medium (DMEM)
(Sigma, USA) supplemented with 10% heat-inactivated fetal
bovine serum (GIBCO, Life Technologies, UK) and 1%
antibiotic cocktail of streptomycin and penicillin (Sigma,
USA). Branched PEI (Mw 25k), tetramethylrhodamine
isothiocyanate (TRITC), Bradford reagent, 40,6-diamidino-2-
phenylindole (DAPI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT), high retention dialysis tubing
(cut off 12k), N-hydroxysuccinimide (NHS) and N,N0-dicyclo-
hexylcarbodiimide (DCC) were procured from Sigma-Aldrich
Chemical Co., USA. Standard transfection agents, Super-
Fig. 4 DNA release assay of the PNIm 10 series and PEI. To a 20 ml
of the PNIm/DNA nanoplex, heparin, in increasing concentration,
was added and incubated for 20 min at 25 ꢁ 1 1C. The samples were
run on a 1% agarose gel at 100 V for 45 min. Error bars represent +/ꢀ
standard deviation from the mean.
fectTM
,
LipofectamineTM and GenePORTER 2TM were
procured from Qiagen, France; Invitrogen, USA; and
Genlantis, USA, respectively. YOYO-1 iodide was purchased
from Invitrogen, USA, and plasmid purification kit and the
plasmid pEGFPN3 were obtained from Qiagen, France, and
Clontech, USA, respectively. Infrared spectra of nanoparticles
were recorded on a single beam Perkin Elmer (Spectrum BX
Series), USA,ꢀw1 ith the following scan parameters: scan range,
PNIm 10(6)/GFPsiRNA was added. After 36 h of treatment
with the siRNA, the observed knockdown of GFP expression
by PNIm 10(6)/siRNA formulation was B90.2% (ca. 70% for
PN-2)1 (Fig. 6). In contrast, GenePORTER 2TM/siRNA
formulations knocked down GFP expression by B53.2% only
(Fig. 6). Hence, the delivery of GFP-specific siRNA oligo-
nucleotides by selected nanoparticle formulation clearly
down-regulated the GFP gene more efficiently than
GenePORTER 2TM. Therefore, the projected nanoparticles
may serve as an effective carrier for the delivery of siRNA as well.
4400–400 cm ; number of scans, 16; resolution, 4.0 cmꢀ1
;
interval, 1.0 cmꢀ1; unit, %T. 1H-NMR was recorded on a
Bruker Avance 400 MHz instrument. Size and zeta potential
measurements of the projected nanoparticles were carried
out on Zetasizer Nano-ZS (Malvern Instruments, UK).
Green fluorescent protein (GFP) in transfected cells was
assayed (lex: 488 nm; lem: 509 nm) on a NanoDropTM
ND-3300 spectrofluorometer (USA). The uptake and intra-
cellular passage of nanoparticles/DNA complexes were
monitored by confocal microscopy (LSM 510 Meta Zeiss,
Germany). Fluorescence activated cell sorting (FACS) analysis
was performed by Flow Cytometry (Guavas EasyCytet
DNase I protection assay
An efficient gene delivery system is the one which provides
sufficient protection to the gene of interest against nucleases
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Fig. 5 Confocal microscopic images of HeLa cells treated with the tetramethylrhodamine–PNIm 10(6)/YOYO-1-pDNA nanoplex at different
time points.
Animals
Six to seven week old male Balb/c mice (25 ꢁ 3 g), from the
animal breeding colony of Indian Institute of Toxicology
Research (IITR), Lucknow, were acclimatized under standard
laboratory conditions and given
a commercial pellet
diet (Ashirwad Industries, Chandigarh, India) and water
ad libitum. Animals were housed in plastic cages with rice
husk bedding and maintained at 22 ꢁ 2 1C with 12 h dark/light
and 50–60% humidity as per rules laid down by the Animal
Welfare Committee of IITR.
Synthesis of siRNA
Fig. 6 siRNA delivery in HEK293. PNIm 10(6) mediated delivery of
siRNA suppressed the expression of GFP in cells by more than 90%
in comparison to GenePORTER 2TM/pGFP DNA/siRNA, which
suppressed GFP expression by only 53.2%, as monitored by
measuring fluorescence on a spectrofluorometer. All the experiments
were performed at least thrice and error bars represent the standard
deviation.
The following oligonucleotide sequences were synthesized
using phosphoramidite chemistry and purified on RP-HPLC:
T7 primer: d (TAA TAC GAC TCA CTA TAG)
GFP sense: d (ATG AAC TTC AGG GTC AGC TTG
CTA TAG TGA GTC GTA TTA)
GFP antisense: d (CGG CAA GCT GAC CCT GAA GTT
CTA TAG TGA GTC GTA TTA)
Plus System, USA). Unless otherwise stated, milliQ water
filtered through 0.22 mm sterile filters was used in the present
study.
After annealing the T7 primer sequence to GFP sense or
antisense oligonucleotides, complementary strands were
synthesized by T7 RNA polymerase.29 The sense and antisense
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amount). Trityl chloride (5.5 mmol), dissolved in dry pyridine
(20 ml), was added to the reaction mixture and stirred for
B8 h at room temperature. Then, the reaction mass was
concentrated on a rotary evaporator, dried completely to
remove all traces of solvents and triturated with diethylether.
The residual mass was dissolved in ethyl acetate (20 ml) and
washed with a 5% aqueous solution of citric acid (2 ꢂ 10 ml).
The organic phase was separated, dried over sodium sulfate,
filtered and concentrated to obtain (N-1-tritylimidazol-4-yl)
acetic acid in almost quantitative yield and characterized using
LC-MS (m/z: 367, M ꢀ H+).
To
a solution of (N-1-tritylimidazol-4-yl) acetic acid
(4 mmol) in dry THF (10 ml), DCC (4.4 mmol) and NHS
(4.4 mmol) were added and the reaction mixture was stirred
for 4 h at room temperature. 6-Aminohexan-1-ol (4.5 mmol)
was then added to the above reaction mixture and stirring was
continued for 4 h at room temperature. Subsequently, the
reaction mixture was cooled to 4 1C and solid dicyclohexylurea
was filtered, which was washed with THF (2 ꢂ 5 ml) and the
combined filtrate was concentrated on a rotary evaporator.
The syrupy residue, so obtained, was dissolved in ethyl acetate
(20 ml) and washed successively with a 5% aqueous solution
of citric acid (2 ꢂ 10 ml) and saturated brine (1 ꢂ 10 ml). The
organic phase was separated, dried over sodium sulfate,
filtered and concentrated to obtain 2-(N-1-tritylimidazol-4-yl)-
N-(6-hydroxyhexyl) acetamide (1) in B85% yield, which was
characterized using LC-MS (m/z: 466, M ꢀ H+).
Fig. 7 DNase I protection assay. The PNIm 10(6)/DNA nanoplex
(w/w ratio 1) was treated with DNase I for different time intervals. The
complexed DNA was released by treating the samples with heparin.
The amount of DNA protected (%) after DNase treatment
was calculated as the relative integrated densitometry values (IDV)
quantified and normalized by that of pDNA values (untreated with
DNase I) using a Gel Documentation system (Syngene, UK).
Compound (1) (3 mmol) was suspended in epichlorohydrin
(10 ml) and N,N0-diisopropylethylamine (DIPEA) (100 ml) was
added. The resulting reaction mixture was stirred at 65 1C for
overnight followed by removal of unreacted epichlorohydrin
on a rotary evaporator to obtain a syrupy residue, which was
dissolved in ethyl acetate (20 ml) and washed successively with
a 5% aqueous solution of citric acid (2 ꢂ 10 ml) and saturated
brine (2 ꢂ 5 ml). The organic phase was separated, dried
over sodium sulfate, filtered and concentrated to obtain crude
2-(N-1-tritylimidazol-4-yl)-N-[6-(3-chloro-2-hydroxypropoxy)
hexyl] acetamide (2), which was purified by silica gel
column chromatography using the solvent system, ethylene
dichloride : methanol (8 : 2, v/v), as an eluant. The fractions
containing the desired compound were pooled together and
concentrated to obtain (2) in B80% yield, which was
characterized using LC-MS (m/z: 558.5, M ꢀ H+).
To a solution of compound (2) (2 mmol), dissolved in THF
(10 ml), was added an aqueous solution of 2 N NaOH (1.5 ml)
and the reaction mixture was stirred at 45 1C. After 1 h, the
reaction mixture was diluted with water (10 ml) and THF was
removed using a rotary evaporator. The desired compound
was extracted in dichloromethane (5 ꢂ 10 ml), dried over
sodium sulfate, filtered and concentrated to obtain IGA linker
(3), which was characterized by its 1H-NMR (CDCl3):
d 1.2–1.5 (8H, 4 ꢂ –CH2), 2.9–3.2 (5H, –CH–CH2, –NH–CH2),
3.75–3.85 (4H, 2 ꢂ –OCH2), 3.4 (2H, –CH2CO), 6.75 (1H,
–CH–N–), 7–7.2 (15 H, Ar–H), 7.38 (1H, –N–CH–N–).
Fig. 8 In vivo gene expression analysis in Balb/c mice 7 d post
intravenous injection using the pGL3 control vector as a reporter
gene. The mice were sacrificed post 7 d injection and all the vital
organs were dissected out. The organs were homogenized in a 1 ꢂ lysis
buffer and the lysate was read on a luminometer to quantify the
luciferase gene expression. *P o 0.05 vs. PEI and naked DNA in the
respective organs.
RNA strands were annealed and double stranded siRNA was
used for transfection.
Synthesis of IGA linker (3)
4-Imidazole acetic acid hydrochloride (5 mmol) was dissolved
in water, NaHCO3 (10 mmol) was added and the solution was
stirred for B1 h. The resulting sodium salt was dried in a
vacuum desiccator and dissolved in dry THF (minimum
Synthesis of BDE crosslinked PEI-g-imidazole nanoparticles (PNIm)
PEI was crosslinked with varying amounts of BDE to generate
PN-1 (5% crosslinking), PN-2 (10% crosslinking) and PN-3
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(15% crosslinking) using a previously reported procedure
from our laboratory.1 The synthesized nanoparticles were
grafted with 2, 4, 6 and 8% of IGA linker (3). Briefly,
for 2% grafting, PN-1 (25 mg, 10 ml H2O) was mixed with
(3) (4.2 mg, 1 ml THF). The reaction mixture was stirred
overnight at 65 1C and concentrated to obtain a syrupy
residue, which was suspended in 30% aqueous trifluoroacetic
acid (TFA, 10 ml). After stirring for 30 min at room tempera-
ture, the reaction volume was reduced to half on a rotary
evaporator and the aqueous phase was washed with diethyl
ether (2 ꢂ 5 ml). Subsequently, a saturated solution of sodium
bicarbonate was added (B2 ml) to bring the pH of the
solution to B7.5 and subjected to dialysis against water with
intermittent change of water for 2 d. The dialyzed solution was
lyophilized to obtain PNIm 5(2), which was characterized by
biophysical techniques. Similarly, the whole series was pre-
pared and characterized using DLS, zeta potential, 1H-NMR,
IR and gel retardation assays.
Cell viability
The cell viability of PEI and PNIm/pDNA nanoplexes as well
as SuperfectTM, GenePORTER 2TM and LipofectamineTM
/
pDNA complexes was evaluated by the MTT colorimetric
assay. HEK293, CHO and HeLa cells were seeded in 96-well
plates one day prior to performing the assay. Nanoparticle/
pDNA nanoplexes were prepared by adding an aqueous
solution of PNIm nanoparticles (1 mg mlꢀ1) to 1 ml of pDNA
(0.3 mg mlꢀ1) at various w/w ratios (0.33, 0.66, 1.0, 1.66 and
2.33) and 5 ml dextrose (20%) was added before making up the
final volume to 20 ml with water. The complexes were gently
vortexed, incubated for 30 min at 25 ꢁ 1 1C, diluted with
serum-free DMEM (60 ml) and gently added to HEK293,
CHO and HeLa cells. After 4 h, the transfection medium
was replaced by 200 ml fresh DMEM containing 10% FBS
and cells were incubated for 36 h. After 36 h, MTT (100 ml,
0.5 mg mlꢀ1 in DMEM) was added to the cells and incubated
for 2 h at 37 1C. Then the supernatant was aspirated, the cells
were rinsed with PBS and the formazan crystals, so formed,
were suspended in 100 ml of isopropanol containing 0.06 M
HCl and 0.5% SDS. Absorbance of the resulting solution was
measured spectrophotometrically in an ELISA plate reader
(MRX, Dynatech Laboratories) at 540 nm. Untreated cells
were taken as the control with 100% viability and cells without
addition of MTT were used as blank to calibrate the spectro-
photometer to zero absorbance. The relative cell viability (%)
compared to control cells was calculated by the formula
[Abs]sample/[Abs]controlꢂ100.
DNA retardation assay
An aqueous solution of PEI/PNIm (1 mg mlꢀ1) was added to
1 ml of pDNA (0.3 mg mlꢀ1) at w/w ratios 0.16, 0.33, 0.66 and
1.0 to form PEI/DNA and PNIm/pDNA polyplexes and the
final volume was made up to 20 ml with water. The resulting
samples were gently vortexed and incubated at room tempera-
ture for 30 min. All the polyplexes and nanoplexes (20 ml) were
mixed with 2 ml xylene cyanol (in 20% glycerol), electro-
phoresed (100 V, 1 h) in 1% agarose, stained with ethidium
bromide and visualized on a UV transilluminator using a
Gel Documentation system (Syngene, UK). From the gel,
the amount of sample required for complete retardation of
0.3 mg of pDNA was obtained.
In vitro transfection
PEI/DNA, PNIm/DNA complexes were prepared and
transfected to HEK293, CHO and HeLa cells, as described
above. Similarly, transfection was performed in a 10% serum
containing medium. DNA complexes of SuperfectTM
,
GenePORTER 2TM and LipofectamineTM were also prepared
following manufacturers’ protocols and transfected onto the
cells for comparison studies.
Physical characterization of nanoparticles
The hydrodynamic diameters of the PNIm series (1 mg mlꢀ1
)
and nanoplexes, suspended separately in water and 10%
serum, were measured by DLS in triplicate. The data analysis
was performed in automatic mode and measured sizes were
presented as the average value of 20 runs.
For delivery of GFP specific siRNA, pGFP DNA (1 ml, 1.4 nM)
was transfected with PNIm 10(6) as above, and after 3 h, the
transfection medium was replaced by GFP specific siRNA
(2 ml, 2.5 mM) complexed with PNIm 10(6) in a 80 ml reaction
mixture containing 60 ml of DMEM. Post 3 h, the medium was
again replaced with fresh DMEM containing 10% serum and
the cells were incubated for 36 h. The PNIm 10(6)/DNA
Zeta potential measurements of nanoparticles and nano-
plexes in water and 10% serum were carried out on a Zetasizer
Nano ZS, carried out 30 runs in triplicate and the average
values were estimated by the Smoluchowski approximation
from the electrophoretic mobility.
nanoplex alone was used as the control. LipofectamineTM
/
DNA/siRNA and LipofectamineTM/DNA complexes were
also prepared and all of these formulations were added on
to seeded HEK293 cells. The experiment was carried out as
described above. The expression levels were monitored by
quantifying GFP. All experiments were performed at least in
triplicate.
Buffering capacity
The ability of PEI and PNIm (5, 10 and 15) series to resist
change in pH was experimentally demonstrated by an acid
titration assay following a method reported by Tseng et al.30
A suspension of PNIm 5(2) (6 mg per 30 ml) in 0.1 N NaCl
was adjusted to pH 10 with 0.1 N NaOH and then the pH was
brought to 3.0 with 50 ml aliquots of 0.1 N HCl. pH values
were recorded after each addition. The slope in the plot
between pH and the amount of HC1 consumed indicates
the intrinsic buffering capacity of the system. Similarly, the
projected assay was repeated with the whole series.
Quantification of EGFP expression
The quantification of EGFP expression was made using a
NanoDrop ND-3300 spectrofluorometer. After 36 h of trans-
fection, cells in each well were washed with PBS (2 ꢂ 100 ml)
and incubated with cell lysis buffer (10 mM Tris, 1 mM EDTA
and 0.5% SDS, pH 7.4, 100 ml) for 45 min at 37 1C. The cell
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lysates were centrifuged and EGFP was estimated spectro-
fluorometrically by taking 2 ml of lysate. The fluorescence
intensity values for background and auto-fluorescence were
determined using mock-treated cells. The total protein content
in cell lysate from each well was estimated using the Bradford
reagent (Bangalore Genei, India), taking BSA as the standard.
The level of fluorescence intensity of GFP was calculated by
subtracting the background values and normalized against
protein concentration in the cell extract. The data are reported
as arbitrary units (AU) mgꢀ1 of cellular protein and represent
mean ꢁ standard deviation for triplicate samples.
A solution of nanoplex (500 ml) containing 2 mg of pDNA in
DMEM was added to each well. After incubation for 0.25, 0.5,
1, and 2 h, the cells were washed with 1 ꢂ PBS (3 ꢂ 500 ml) and
fixed with 4% paraformaldehyde solution for 10 min at 4 1C.
Then, the fixed cells were counter-stained with a blue nuclear
dye, DAPI, and the coverslips were mounted on glass slides
with a fluorescence-free Mounting Medium (UltraCruzt,
Santa-Cruz Biotechnology, USA). All confocal images were
captured using a Zeiss LSM 510 inverted laser-scanning
confocal microscope.
DNase I protection assay
Fluorescence activated cell sorting (FACS) analysis
The ability of the PNIm to protect the condensed pDNA from
nuclease attack was assessed by the DNase I assay. Native
pDNA and PNIm 10(6)/pDNA nanoplexes (at w/w ratio 1)
were incubated at 37 1C for 0.25, 0.5, 1 and 2 h with DNase I
(Sigma, USA) (1 ml, 1 unit mlꢀ1 in a buffer containing 100 mM
Tris, 25 mM MgCl2 and 5 mM CaCl2). Treatment with PBS
alone served as a control. After incubation, a solution of
EDTA (5 ml, 100 mM) was added and the mixture incubated
at 75 1C for 10 min to inactivate DNase I. The mixture was
further incubated for 2 h (room temperature) with heparin
(10 ml, 5 mg mlꢀ1) to release bound DNA from the cationic
polymer. Subsequently, samples were electrophoresed and
visualized, as described above (DNA release assay). The
amount of pDNA released from complexes after heparin
treatment was estimated by densitometry.
FACS analysis was performed to examine the GFP expression
at the individual cell level 36 h post-transfection. Briefly,
HEK293 cells were seeded in 24-well plates, and after 16 h,
the medium was aspirated and cells washed with phosphate
buffer saline (1 ꢂ PBS, twice). The transfection was performed
as described above. Similarly, pDNA complexes were
prepared with LipofectamineTM following manufacturers’
protocols, added onto the cells and incubated for 36 h.
Subsequently, transfected cells were washed with PBS (1 ꢂ 1 ml)
and harvested by trypsinization. Cells were recovered by
centrifugation at 5000 rpm for 5 min at 4 1C, the supernatant
was removed, the pellet was washed with 1 ꢂ PBS (2 ꢂ 500 ml)
and resuspended in 1 ꢂ PBS (1.0 ml). The percentage of
EGFP-expressing cells was determined to quantify trans-
fection efficiency by flow cytometry equipped with Cytosoft
Software (Guavas EasyCytet Plus Flow Cytometry System,
USA). The percentage of transfected cells was obtained by
determining the statistics of cells fluorescing above the control
level, where non-transfected cells were used as the control.
Approximately, 5000 cells were analyzed to generate data for
statistical purpose.
In vivo gene expression
For intravenous injection in Balb/c mice, 25 mg of the Luciferase
vector (pGL-3 control vector) and DNA complexes of PNIm
10(6) and PEI at a w/w ratio of 1 using normal saline as the
medium (final volume of 100 ml) were incubated for 30 min at
25 ꢁ 1 1C. These complexes were injected through tail vein
using an insulin syringe (40 U, needle size of 0.3 ꢂ 8 mm).
After 7 d on normal diet, the mice were sacrificed and liver,
spleen, kidney, lung, heart and brain were dissected out,
washed with chilled normal saline, weighed, chopped and
homogenate was made (25% w/v, 1 ꢂ lysis buffer). After three
cycles of freezing and thawing, the homogenates were centri-
fuged at 5000 rpm for 10 min at 41 C and 100 ml supernatant
was used to measure luciferase activity. Luciferase activity was
represented as RLU per mg of total protein. All the experi-
ments were carried out in duplicate.
DNA release assay
PEI was complexed with pDNA (1.5 mg) at a w/w ratio, at
which it exhibited the highest transfection efficiency, and was
incubated for 30 min, as described above. Subsequently, an
aqueous solution of heparin, an anionic polymer, was added in
different amounts varying from 0.5–20 U for the release of
plasmid DNA, which was bound to the cationic polymer. The
samples were then incubated for 20 min, electrophoresed
(100 V, 1 h) on 1% agarose gel, stained with ethidium bromide
and visualized on
a
UV transilluminator using a Gel
Statistical analysis
Documentation system. The amount of DNA released from
complexes after heparin treatment was estimated by densitometry.
Likewise, the assay was repeated with the PNIm 10 series.
Statistical analysis wherever needed was carried out by one-
way analysis of variance followed by Student’s t-test after
ascertaining homogeneity of variance and normality of data.
A value of P o 0.05 was considered statistically significant.
JMP version 6.0.0, Statistical DiscoveryTM from SAS was used
for analysis.
Confocal laser scanning microscopy (CLSM)
The path of cellular entry was monitored using confocal
microscopy. The best working sample PNIm 10(6) was labeled
with TRITC to block nearly B1% of total amines in the
nanoparticles using a reported procedure from the lab.20
pDNA (0.3 mg) was labeled using YOYO-1 iodide (2 ml,
1 mM solution in DMSO) and stored at ꢀ20 1C. HeLa cells
were seeded (1.5 ꢂ 105 cells per well) on circular glass cover-
slips in a 6-well plate, grown overnight to B70% confluence.
Conclusions
In this study, we showed the effect of imidazole grafting
through an epoxy end-based linker on BDE crosslinked PEI
nanoparticles. The grafting was induced in such a way so as
not to reduce the overall charge on PEI but to convert one
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form to another. Additional delocalized charge was intro-
duced in the form of an imidazole ring in order to decrease
the cytotoxic effect and impart more buffering into the system.
The effect of increased buffering on the transfection efficiency
of the resulting nanoparticles was studied. It was found that
introduction of imidazole groups resulted in enhancement of
transfection efficiency of crosslinked nanoparticles. This
enhancement was attributed to the increased buffering in the
pH range 3–10. Among the series of nanoparticles prepared in
this study, PNIm 10(6) was found to be B2–17 folds superior
compared to PN-2, PEI and commercial transfection reagents
tested in this study, in terms of transfection efficiency in vitro.
siRNA studies involving PNIm 10(6) led to B90% suppres-
sion of gene expression, indicating the efficient delivery of
siRNA. The projected particles were also able to efficiently
carry the desired gene in vivo. These studies thus suggest that
PNIm 10(6) tested in the present study has a potential for
future applications in gene delivery.
PAMAM dendrimers in B16f10 melanoma cells, J. Controlled
Release, 2007, 117, 291–300.
11 S. Nimesh, A. Aggarwal, P. Kumar, Y. Singh, K. C. Gupta and
R. Chandra, Influence of acyl chain length on transfection
mediated by acylated PEI nanoparticles, Int. J. Pharm., 2007,
337, 265–274.
12 X. Gao, R. Kuruba, K. Damodaran, B. W. Day, D. Liu and S. Li,
Polyhydroxylalkyleneamines:
a class of hydrophilic cationic
polymer-based gene transfer agents, J. Controlled Release, 2009,
137, 38–45.
13 S. Nimesh, A. Goyal, V. Pawar, S. Jayaraman, P. Kumar,
R. Chandra, Y. Singh and K. C. Gupta, Polyethylenimine nano-
particles as efficient transfecting agents for mammalian cells,
J. Controlled Release, 2006, 110, 457–468.
14 A. Pathak, P. Kumar, K. Chuttani, S. Jain, A. K. Mishra,
S. P. Vyas and K. C Gupta, Gene expression, biodistribution
and pharmacoscintigraphic evaluation of chondroitin sulfate–PEI
nanoconstructs mediated tumor gene therapy, ACS Nano, 2009, 3,
1493–1505.
15 S. Patnaik, A. Aggarwal, S. Nimesh, A. Goel, M. Ganguli,
N. Saini, Y. Singh and K. C. Gupta, PEI-alginate nanocomposites
as efficient in vitro gene transfection agents, J. Controlled Release,
2006, 114, 398–409.
16 G. Borchard, Chitosans for gene delivery, Adv. Drug Delivery Rev.,
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17 P. Midoux, C. Pichon, J. J. Yaouanc and P. A. Jaffres, Chemical
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Acknowledgements
Authors are thankful to Dr. Naresh Singh for his help in
CLSM experiments. Financial support from the DBT
(No. BT/PR8344/NNT/28/05/06) and CSIR (NWP-35) to
KCG and UGC-SRF (10-2(05)2006(i)-E.U.II) to RG is grate-
fully acknowledged.
19 A. Swami, A. Aggarwal, A. Pathak, S. Patnaik, P. Kumar,
Y. Singh and K. C. Gupta, Imidazolyl-PEI modified nanoparticles
for enhanced gene delivery, Int. J. Pharm., 2007, 335, 180–192.
20 A. Swami, R. Goyal, S. K. Tripathi, N. Singh, N. Katiyar,
A. K. Mishra and K. C. Gupta, Effect of homobifunctional
crosslinkers on nucleic acids delivery ability of PEI nanoparticles,
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