Evaluation of engineered PEI nanoparticles for gene delivery
1. INTRODUCTION
Positively charged lipids and polymers electrostatically interact with the
negatively charged phosphate-groups of the DNA, resulting in the formation of nano-
sized complexes, commonly referred to as lipoplexes and polyplexes, respectively.
Compared to the viral delivery systems, the non-viral systems are characterized by
low transfection efficiencies [1]. However, the non-viral delivery systems are
considered to be superior in terms of safety and scale-up issues. Moreover, the latter
systems are highly versatile, i.e. by varying the composition of the non-viral carrier
systems and by the chemical conjugation of additional functionalities, one can adjust
the properties of the non-viral systems and tailor them to the desired application [2-4]
such as the conjugation of poly (ethylene glycol) (PEG) to the surface of preformed
polyplexes has been demonstrated to prolong its pharmacokinetics [5]. It is expected
that the continuous optimization of the non-viral nucleic acid delivery systems
ultimately will enable them to rival with the virus-based systems.
A widely explored cationic polymer for transfection is polyethylenimine, with
its mechanism extensively studied and tested among various cell types. Although
unmodified bPEI (25 kDa) has high transfection efficiency, its applications in vivo has
not met with the desired results. The major reasons for such findings may be
attributed to the cytotoxicity of PEI and non-specific interactions with serum proteins
[6, 7]. Researchers have tried to improve the delivery profile of PEI with respect to
cytotoxicity and reactivity with serum proteins by employing hydrophilic coatings
such as polyethyleneglycol (PEG) or polyethyleneoxide (PEO) to form graft or
copolymers with polycations [8-13]. Over the past few years, PEG-coated
nanoparticles have shown great potential as long circulating systems after
intravenous administration [14].
In the present chapter, a nanoparticulate system has been designed based on
bPEI (25 kDa), phosphoethanolamine and PEG600. Polyethyleneglycol was converted
in to its bis (aldehyde) derivative and allowed to react with phosphoethanolamine to
form PEG600-bis (iminoethylphosphate) (PiP), which was subsequently allowed to
interact electrostatically with the amino groups of bPEI (25 kDa) resulting in the
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Evaluation of engineered PEI nanoparticles for gene delivery
formation of nanoparticles. A small series of PEI-PiP (PPiP) nanoparticles were
prepared by varying the concentration of PiP crosslinker and characterized for their
particle size and zeta potential. The ability to protect complexed DNA against
nucleases was investigated in vitro and the cellular trafficking of the projected
nanoparticles was studied in HeLa cells. PPiP nanoparticles were further evaluated
for their cytotoxicity and efficacy to deliver plasmid DNA (pDNA) in various
mammalian cells in vitro and compared with the standard transfection agents.
2. EXPERIMENTAL PROCEDURES
Preparation of PEI-PEG-bis(iminoethylphosphate (PPiP) nanoparticles
(i) Synthesis of ethanolamine-O-phosphate
In a round bottom flask, orthophosphoric acid (9.5g, 80.8 mmol) and ice (10g)
mixture was reacted with ethanolamine (5g, 81.9 mmol) with constant stirring. After
2h, the resulting reaction mixture was concentrated in vacuo and the oily residue, thus
obtained, was heated under vacuum on oil bath at 185oC for 6h. The flask was cooled
and water (1ml) was added. The resulting mixture was left overnight at 4oC for
crystallization. The solid was filtered and dried by suction to obtain the product,
ethanolamine-O-phosphate, in ~45% yield, as yellow crystalline solid, which was
characterized by FTIR.
IR (KBr), ν (cm-1): 1645 (NH2 deformation), 1460 (C-H deformation of CH2-O), 1086 (P-
OCH2)
(ii) Synthesis of PEG600 bis(aldehyde)
PEG600 (2g, 3.33 mmol) was dissolved in acetone (25ml) and added 2-
iodoxybenzoic acid (5.6g, 20 mmol). The reaction mixture was refluxed at 56oC for 1h
followed by cooling to room temperature and filtration to get rid of unreacted 2-
iodoxybenzoic acid. The filtered cake was washed with acetone (2 x 10 ml) and the
combined filtrates were concentrated in vacuo to obtain PEG600-bis (aldehyde) in ~95%
yield, which was characterized by FTIR.
IR (KBr), ν (cm-1): 2876 (C-H stretching of CHO), 1729 (C=O stretching)
(iii) Synthesis of PEG600 bis(iminoethylphosphate) (PiP)
The titled compound was prepared by reacting PEG600 bis (aldehyde) with
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Evaluation of engineered PEI nanoparticles for gene delivery
ethanolamine-O-phosphate. To an aqueous solution of ethanolamine-O-phosphate
(1g, 7.5 mmol, 25ml) was added a solution of PEG600-bis (aldehyde) (1.5g, 2.5 mmol),
dissolved in water (10ml), dropwise over a period of 15 min with continuous stirring.
The reaction was allowed to stir for 2h followed by the addition of solid sodium
cyanoborohydride (0.6g, 10 mmol). Stirring was continued for 12h and then the
solution was concentrated in vacuo to reduce the total volume to 10ml. The resulting
reaction mixture was extracted with butanone (6x5ml), the organic phase collected
and concentrated to obtain PEG600 bis (iminoethylphosphate) (PiP) in ~79% yield,
which was characterized by FTIR.
IR (KBr), ν (cm-1): 1081 (P-OCH2), 1252 (C-N stretching)
(iv) Synthesis of PEI-PEG bis (iminoethylphosphate) (PPiP) nanoparticles
To an aqueous solution of PEI (50mg, 1mg/ml) was added an aqueous solution
of PiP (31.25 mg, 1mg/ml, for 10% crosslinking) dropwise over a period of 1h with
vigorous stirring. Stirring was continued for 2h and then the solution was subjected to
exhaustive dialysis (48h) against water with intermittent change of water. The
dialyzed solution was lyophilized to obtain PPiP nanoparticles (10% crosslinked).
Likewise, other PPiP nanoparticles (20, 30, 40 and 50% crosslinked) were prepared.
The percentage of crosslinking in PPiP nanoparticles was calculated by estimating
inorganic phosphorous, as described in chapter IV. These nanoparticles were further
characterized by FTIR.
IR (KBr), ν (cm-1): 3375 (NH2 stretching), 3126 (P-NH), 1377 (C-N stretching), 1098 (P-
OCH2)
Particle size and zeta potential measurements
The particle size and zeta potential of PPiP nanoparticles and their DNA
complexes were measured, as described in Chapter II.
DNA retardation assay
The DNA binding ability of PPiP nanoparticles was assessed in 0.8% agarose
gel, as outlined in Chapter II. pDNA was mixed with PPiP nanoparticles at different
w:w ratios, i.e. 0.83, 1.66, 2.5, 3.3, 4.16 and 5.0.
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Evaluation of engineered PEI nanoparticles for gene delivery
In vitro transfection
The ability of PPiP nanoparticles to deliver pDNA coding for GFP was assessed
in mammalian cells (HEK293, HepG2, HeLa) at different weight ratios, viz., 0.83, 1.66,
2.5, 3.33 and 5.0 and compared with those of bPEI (25 kDa), GenePORTER 2TM,
SuperfectTM and FugeneTM (v:w ratios , as mentioned in Chapter IV). DNA complexes
of PPiP, PEI, GenePORTER 2TM, SuperfectTM and FugeneTM were prepared and cells
were transfected, as described in Chapter II.
In vitro cytotoxicity
The PPiP/DNA, PEI/DNA, GenePORTER 2TM/DNA, SuperfectTM/DNA and
FugeneTM/DNA complexes were assessed for cytotoxicity in cell lines (HEK293,
HepG2 and HeLa) following the protocol described in Chapter II.
DNase protection assay
The capability of the projected nanoparticles to protect DNA against nucleases
was examined by DNase I assay. The PPiP (8.1%)/DNA complex (w:w ratio 2.5) was
treated with DNase I at different time intervals and compared with pDNA alone
(0.6µg/25μl). The experiment was carried out following the procedure outlined in
Chapter II.
Intracellular trafficking
PPiP (8.1%) nanoparticles were labeled with TRITC and its uptake in HeLa
cells at different time points monitored. The proposed study was undertaken by
following the steps already outlined in Chapter II.
Organ distribution studies
The in vivo fate of PPiP (8.1%)/DNA complex was studied in Balb/C mice, as
described in Chapter II
3. RESULTS AND DISCUSSION
The unique chemical properties of PEI underscore its potential as a vector for
gene delivery. The high charge density in PEI is responsible for condensing DNA into
particles small enough to be endocytosed efficiently. Also, the amines in PEI exhibit
proton sponge mechanism that helps in the release of the DNA complexes from
endosomes. However, the presence of excessive charge on the backbone of PEI
126
(iii)
H2NOH H3PO4
(- H2O)H3N
OP
OH
OO
(i)
A
HO-CH2-PEG600-CH2OHIBX
OHC-PEG600-CHO(ii)
B
H3NO
P
OH
OO
+ OHC-PEG600-CHO
CH2-PEG 600-CH2NH
O
NH
O
PP
O H
OO
O
OHO
C
NaBH 3(CN)
Evaluation of engineered PEI nanoparticles for gene delivery
imparts the toxicity to the polymer, which limits its potential in vivo applications. The
present study was undertaken to develop an efficient and versatile PEI nanoparticle-
based transfection reagent that could be used for in vitro and in vivo gene delivery.
For this purpose, a novel crosslinker, based on polyethyleneglycol, was designed and
synthesized by taking into account of the beneficial properties of PEG in biological
applications. The crosslinker, PEG600 bis (iminoethylphosphate) (PiP), was prepared by
reacting PEG600 bis (aldehyde) with ethanolamine-O-phosphate to generate bis
(Schiff’s base), which was subjected to reduction with sodium cyanoborohydride to
obtain PEG600 bis (iminoethylphosphate) (PiP). It was subsequently used to prepare a
small series of PEI nanoparticles by varying its amount (Scheme 1), which were
characterized by FTIR. The bands at 3126 (P-NH) and 1098 (P-OCH2) confirmed the
incorporation of the projected crosslinker in PEI nanoparticles (PPiP).
Percent ionic linking in PPiP nanocomposites
The percent crosslinking in PPiP (Table 1) nanoparticles was determined by
colorimetric estimation of inorganic phosphorous content in nanoparticles by the
procedure, as described in chapter IV, section 4.2.2.
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Evaluation of engineered PEI nanoparticles for gene delivery
(iv)
Scheme 1: Preparation of linkers and PEI nanoparticles
Table 1: Percent amines ionically linked to PP in PPIP nanoparticles
S.No Attempted ionic interaction (%) Observed proportion ionic interaction (%)
1. 8 6.4
2. 10 7.24
3. 12 7.7
4. 14 8.1
5. 16 9.08
6. 18 10.06
H2NNH
HN
NH
HN
N
HN
H2N
NH
NH2
NH2
mn
NH
H2NNH
H2N N
N
HNHN
NH
NH3
NH2
mn
NH2
H2NNH
HN
HN
N
HNHN
NH
NH2
NH2
mn
O
O
P
P
OHO
O
O
OH
O
O
O
P
P
OHO
O
OOH
O-
PEG-linker
PEG-linker
NH2NH
HN
NH
NH2N
NH2
m nO
OPP
OH
OHOO
OHHOPEG-linker+
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Evaluation of engineered PEI nanoparticles for gene delivery
Size and zeta potential of PPIP nanoparticles
PPiP nanoparticles and their DNA complexes were analyzed for particle size
by dynamic light scattering (DLS) studies (Table 2) and atomic force microscopy
(AFM). The particle size of PPiP nanoparticles was found to be in the range of 56-116
nm, as shown by DLS measurements. AFM measurements reveal spherical PPiP
nanoparticles (Fig. 1). As expected, PPiP nanoparticles showed a smaller particle size
in AFM compared to DLS measurements due to hydrodynamic diameter of the
swollen nanoparticles in DLS studies. All PPiP nanoparticles and PPiP/DNA
complexes carried a positive zeta potential value (Table 2). The positive zeta potential
of nanoparticle/DNA complex helps in the interaction with cell membrane resulting in
internalization of the complex. However, the zeta potential of PPiP was lower than the
native PEI, which might be accredited to the masking of positive charge in PPiP
nanoparticles. With increase in the content of PEG600 bis (ethanolamine-O-phosphate)
crosslinker in PPiP nanoparticles, there was a decrease in zeta potential (Table 2).
Further, on complexing with DNA, the zeta potential of nanoparticle/DNA complexes
showed a decline (Table 2).
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Evaluation of engineered PEI nanoparticles for gene delivery
Table 2: Size and Zeta potential of PPIP nanoparticles
Figure 1: Characterization of PPiP nanoparticles and PPiP/DNA complex by AFM. 2-3µl of nanoparticle solution or nanoparticle/DNA complex was deposited on a freshly split untreated mica strip and images were recorded in acoustic mode.
DNA retardation assay
The DNA binding efficacy of PPiP nanoparticles was analyzed on 0.8% agarose
gel. DNA complexes of PPiP nanoparticles and bPEI (25 kDa), prepared at different
weight ratios, were electrophoresed. At a weight ratio of 0.5, PEI completely retarded
the electrophoretic mobility of DNA, whereas, as expected, the PPiP nanoparticles
required a higher weight ratio to retard the same amount of DNA (Fig. 2). Presence of
S. No.
Samples
Average particle size in nm (PDI)
Zeta potential (mV) Ratio ofNano-
composites : DNA (w:w)
Nano-composites
(in H2O)
DNA loaded Nano-
composites (in H2O)
Nano-composites
(in H2O)(+)
DNA loaded Nano-
composites(in H2O)
(+)1. PPiP (6.4%) 106 ± 2.15
(0.574)125 ± 3.05
(0.577)22.0 ± 1.07 19.8 ± 1.54 5:3
2. PPiP (7.24%) 97.0 ± 2.01 (0.505)
115 ± 3.41 (0.467)
20.6 ± 1.32 18.2 ± 0.947 5:3
3. PPiP (7.7%) 92.7 ± 2.24 (0.442)
106 ± 2.37 (0.384)
18.3 ± 0.478 16.9 ± 0.914 5:3
4. PPiP (8.1%) 87.23 ± 1.36 (0.614)
99.4 ± 1.978 (0.448)
16.4 ± 0.43 15.1 ± 0.27 5:3
5. PPiP (9.08%) 65.0 ± 0.98 (0.532)
78.4 ± 2.07 (0.577)
14.5 ± 0.61 12.09 ± 0.17 5:3
6. PPiP (10.06%) 59 ± 1.91 (0.486)
69.0 ± 0.42 (0. 443)
12.2 ± 0.91 9.9 ± 0.65 5:3
PPiP (8.1%) Av size 60 nm
PPiP (8.1%)/DNA complex Av size78 nm
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Evaluation of engineered PEI nanoparticles for gene delivery
PEG600 bis (ethanolamine-O-phosphate) in the nanoparticles partially masked the
positive charge on PEI resulting in the requirement of higher amounts of PPiP
nanoparticles to retard electrophoretic mobility of DNA. Moreover, it was observed
that on increasing the percentage of crosslinking in the series, the amount of
nanoparticles required to retard a fixed amount of DNA also increased indicating that
the degree of crosslinking slightly affected the pDNA complexing efficacy of PPiP
nanoparticles.
In vitro transfection
To assess the gene transfer ability of the projected PPiP nanoparticles,
transfection experiments were carried out on HEK293, HepG2 and HeLa cells using
plasmid containing reporter gene encoding green fluorescence protein (GFP).
Transfection studies were carried out both in the absence and presence of serum. In
the absence of serum, PPiP (8.1%) nanoparticles scored a 2.4-folds higher GFP
Figure 2: Gel retardation assay of PPiPA/DNA and PEI/DNA complexes. pDNA (300ng) was incubated with increasing amounts of nanoparticles in 5% dextrose and incubated for 20 min. Samples were electrophoresed through 0.8% agarose gel at 100V for 45min. The values mentioned correspond to the w:w ratio of nanoparticles/DNA in a 20 μl reaction.
expression in HepG2 cells compared to bPEI (25 kDa), whereas the expression was
~1.3, 3.34 and 5 folds higher than GenePORTER 2TM, SuperfectTM and FugeneTM,
respectively (Fig. 3). In HEK cells, the GFP expression of PPiP (8.1%) formulation was
~1.7, 2.8 and 4 folds higher than GenePORTER 2TM (GP2), FugeneTM (F) and
SuperfectTM (SF), respectively (Fig. 3 and 4). Likewise, the GFP expression in HeLa
cells was found to be higher in PPiP (8.1%) formulation compared to unmodified PEI,
SuperfectTM, FugeneTM and GenePORTER 2TM (Fig. 3). More importantly, in the
PPiP(7.24%)1.66 2.5 3.3 4.16
PPiP(7.7%)1.66 2.5 3.33 4.16
PPiP(8.1%) 1.66 2.5 3.3 4.16
PPiP(9.08%) 2.5 3.3 4.16
PPiP(10.06%)2.5 3.33 4.16 5.0
PPiP(6.4%)0.83 1.66 2.5
PEI 0.33 0.45 0.5 1.0
pDNA300ng
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Evaluation of engineered PEI nanoparticles for gene delivery
presence of serum, PPiP (8.1%) formulation exhibited a 1.5-5 folds higher GFP
expression compared to GenePORTER 2TM, FugeneTM and SuperfectTM in HEK cells
(Fig. 5).
0.83 1.66 2.5 3.33 5.0
w:w ratio of nanoparticle:DNA
Fl. intensi
tyA.U. x 10
3/mg
of protei
n
0
200
400
600
PPiP 6.4%PPiP 7.24%
PPiP 7.7%PPiP 8.1%PPiP 9.08%
PPiP 10.06PEIGP2F
SF
0
200
400
600
PPiP 6.4%PPiP 7.24%PPiP 7.7%PPiP 8.1%PPiP 9.08%PPiP 10.06PEIGP2FSF
HepG2
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Evaluation of engineered PEI nanoparticles for gene delivery
Figure 3: GFP fluorescence intensity of HepG2, HeLa and HEK293 cells transfected with PPiP/DNA, PEI/DNA, GP2/DNA, SF/DNA and F/DNA complexes in the absence of serum. The results represent the mean of three independent experiments performed in triplicates. The abscissa represents the w:w or v:w ratio of nanoparticles/DNA or commercial reagents/DNA, respectively. The w:w ratios of (i) unmodified PEI/DNA used were 0.66, 1 and 1.66, and v/w ratio of (ii) GP2/DNA was 5:3 , (iii) SF/DNA was 2:1 and (iv) F/DNA was 4:1.
Fl. intensit
yA.U. x
103/mg of
protein
0
200
400
600
PPiP 6.4%
PPiP 7.24%
PPiP 7.7%
PPiP 8.1%
PPiP 9.08%
PPiP 10.06
PEI
GP2
SF
F
0
200
400
600
PPiP 6.4%
PPiP 7.24%
PPiP 7.7%
PPiP 8.1%
PPiP 9.08%
PPiP 10.06
PEI
GP2
SF
F
HeLa
0.83 1.66 2.5 3.33 5.0w:w ratio of nanoparticle:DNA
0.83 1.66 2.5 3.33 5.0w:w ratio of nanoparticle:DNA
0
200
400
600
800
PPiP 6.4%
PPiP 7.24%
PPiP 7.7%
PPiP 8.1
PPiP9.08
PPiP 10.06
PEI
GP2
SF
F
0
200
400
600
800
PPiP 6.4%
PPiP 7.24%
PPiP 7.7%
PPiP 8.1
PPiP9.08
PPiP 10.06
PEI
GP2
SF
F
HEK293
Fl. intensi
tyA.U. x 10
3/mg
of protein
133
Evaluation of engineered PEI nanoparticles for gene delivery
Figure 4: Fluorescent microscopy of HEK293 cells transfected with PPiP/DNA, PEI/DNA, GenePORTER2/DNA, Superfect/DNA and Fugene/DNA complexes at the w:w ratio of nanoparticles/DNA required for the maximum transfection efficiency. Images were recorded at 10× magnification as observed under UV, C-F1 epifluorescence filter of fluorescent microscope.
Figure 5: GFP fluorescence intensity of HEK293 cells transfected with PPiP/DNA, PEI/DNA, GP2/DNA, SF/DNA and F/DNA complexes, at optimal w:w ratio of nanocomposites/DNA, in the presence of serum.
Cytotoxicity
For efficient transfection, the delivery vector should be non-toxic to cells.
Cationic polymers are well known to destabilize and ultimately rupture the cell
membrane due to strong electrostatic interactions between amine groups of the
0
100
200
300
400
500
PPiP 6.4%PPiP 7.24%PPiP 7.7%
PPiP 8.1%PPiP 9.08%PPiP 10.06%PEI
GP2SFF
0.83 1.66 2.5 3.33 5.0w:w ratio of nanoparticle:DNA
Fl. intensi
tyA.U. x
103/mg of
protein
HEK293
GenePORTER 2 FugenebPEI (25 kDa) Superfect
PPiP 8.1% PPiP 9.08%PPiP 7.7%PPiP 7.2%PPiP 6.4%
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Evaluation of engineered PEI nanoparticles for gene delivery
polymer and cellular compartments or accumulation of non-degraded polymer in cell.
Therefore, to investigate the in vitro cytotoxicity of PPiP nanoparticles, mammalian
cells were transfected with PPiP/DNA complexes, as described above. Cells treated
with PPiP/DNA complexes were found to be <80% viable (Fig. 6). In HeLa cells, PPiP
(8.1%)/DNA complexes showed <80% viability at w:w ratio 0.25:0.3 and decreased
with increasing dose of DNA complex. The cell viability of PEI/DNA complex treated
cells was found to be comparatively low. This observation might be explained on the
basis of reduction in positive charge of PEI in PPiP nanoparticles. The cell viability of
PPiP nanoparticles increased with increase in the degree of crosslinking until 8.1%,
beyond which, the cell viability started decreasing. A similar trend in cell viability
profiles was observed in HEK293 and HepG2 cells.
0
30
60
90
120
PPiP 6.4%
PPiP 7.24%
PPiP 7.7%
PPiP 8.1%
PPiP 9.08%
PPiP 9.08%
PEI
GP2
SF
F
0
30
60
90
120
PPiP 6.4%
PPiP 7.24%
PPiP 7.7%
PPiP 8.1%
PPiP 9.08%
PPiP 9.08%
PEI
GP2
SF
F
HeLa
0.83 1.66 2.5 3.33
w:w ratio of nanoparticle:DNA
Cel
l Via
bilit
y (%
)
0
30
60
90
120
PPiP 6.4%PPiP 7.24%PPiP 7.7%PPiP 8.1%PPiP 9.08%PPiP 10.06%PEIGP2SFF
Cel
l Via
bilit
y (%
)
0.83 1.66 2.5 3.33
w:w ratio of nanoparticle:DNA
HepG2
135
Evaluation of engineered PEI nanoparticles for gene delivery
Figure 6: Cytotoxicity of PPiP/DNA, PEI/DNA, GP2/DNA, SF/DNA and F/DNA complexes in HEK293, HepG2 and HeLa cells. Each point represents the mean of three independent experiments performed in triplicates. The abscissa represents the w:w or v/w ratio of nanoparticles/DNA or commercial reagents/DNA, respectively.
DNase protection assay
The susceptibility of bound pDNA towards nucleases was assessed by DNase I
assay. In order to substantiate the fact, the assay was carried out and analyzed by
0.8% agarose gel electrophoresis. The efficacy of PPiP (8.1%) nanoparticles to protect
complexed DNA against nucleases at different time points was assessed in vitro.
pDNA and PPiP (8.1%)/DNA complex were treated with DNase I and the complex
was subsequently treated with heparin to release the complexed DNA and the quality
of DNA checked on agarose gel. The gel analysis showed that the released DNA
remained appreciably intact (78.9%) even after 2h treatment with DNase I (Fig. 7)
implying that PPiP (8.1%) nanoparticles efficiently protected complexed DNA against
nucleases, which is considered to be an important requisite for efficient gene delivery
in vivo.
Intracellular trafficking
The PPiP (8.1%) nanoparticles were labeled with TRITC and their intracellular
trafficking studied in HeLa cells. The cells were treated with labeled PPiP
nanoparticles and incubated at different time intervals. After 15min of treatment, very
few particles were seen inside the cell, which increased after 30min of uptake (Fig. 8).
0
30
60
90
120
PPiP 6.4%PPiP 7.24%PPiP 7.7%PPiP 8.1%PPiP 9.08%PPiP 10.06PEIGP2SFF
Cel
l Via
bilit
y (%
)
0.83 1.66 2.5 3.33 5.0
w:w ratio of nanoparticle:DNA
HEK293
136
Evaluation of engineered PEI nanoparticles for gene delivery
Figure 7: DNase I protection assay. PPiP/DNA complex was treated with DNase I at a w:w 1.66 for different time intervals. The complexed DNA was released by treating the samples with heparin. The release of DNA was monitored in 0.8% agarose.
After 45min, the particles were observed entering the nucleus. The concentration of
TRITC labeled particles entering nucleus increased with time and the particles were
detected inside the nucleus even after 3h of treatment. The results imply the uptake of
nanoparticles alone, by cell and eventually by the nucleus.
Figure 8: Intracellular trafficking of labeled PPiP (8.1%) nanoparticles in HeLa cells by confocal microscopy. (A) 30min (B) 60min (C) 90min (D) 2h (E) 3h. The first quadrant (I) shows the cells observed under TRITC filter (laser Helium-Neon 1mW), the second quadrant (II) shows images captured under DAPI filter (laser Diode 25mW) and the third quadrant (III) represents the overlaid images.
AI II III
B I II III
C IIIIII D IIIIII
E IIIIII
PPiP (8.1)with Dnase I
0.5h 1h 2h
pDNAwith Dnase I
0.5h 1h 2h
PPiP (8.1)without DNase I 0.5h 1h 2h
pDNA without Dnase I0.5h 1h 2h
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Evaluation of engineered PEI nanoparticles for gene delivery
Body distribution in mice
The body distribution of PPiP (8.1%)/ DNA complex delivered intravenously
was studied over a period of 24h in Balb/C mice. PPiP (8.1%)/DNA complex was
radiolabeled, as described in Chapter II, and injected intravenously through a tail-
vein. Retention of radioactivity in the liver was highest with 35.9 ± 2.6% ID after 1h,
which declined to 20.3 ± 1.9% ID after 24h (Table 3). In the kidney, the radioactivity
was low with 1.52 ± 0.62 % ID at 1h and 0.7 ± 0.09 after 24h (Table 3). In other tissues,
viz., lung, spleen and heart, the level of radioactivity was found to be much lower
than liver and declined between 1 and 24h (Table 3). The results on passage and
retention of radiolabeled PPiP (8.1%)/DNA complex in mice (Table 3) confirmed that
the complex was retained inside the body for at least 24h and that the PPiP
(8.1%)/DNA complex exhibited differential distribution that might provide clues for
tissue-specific targeting.
Table 3: Biodistribution pattern of radiolabeled PPiP/DNA complex
Organ or tissue PPiP (8.1%)/DNA complex %ID
1h 3h 6h 24hBlood 3.12 ± 0.3 1.95 ± 0.29 1.39 ± 0.18 0.5 ± 0.1
Heart 0.66 ± 0.04 0.6 ± 0.05 0.47 ± 0.1 0.27 ± 0.07
Lungs 18.4 ± 1.7 15.8 ± 1.9 13.1 ± 2.1 10.6 ± 2.3
Liver 35.9 ± 2.6 30.7 ± 1.92 26.4 ± 2.0 20.3 ± 1.9
Spleen 7.7 ± 0.98 5.5 ± 1.1 4.8 ± 1.3 3.8 ± 0.73
Kidney 1.52 ± 0.62 1.05 ± 0.56 0.88 ± 0.24 0.7 ± 0.09
Stomach 0.26 0.1 0.22 ± 0.12 0.2 ± 0.03 0.18 ± 0.08
Intestine 0.94 ± 0.15 0.7 ± 0.15 0.63 ± 0.12 0.5 ± 0.1
Brain 0.06 ± 0.002 0.04 ± 0.002 0.03 ± 0.01 0.03 ± 0.001
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Evaluation of engineered PEI nanoparticles for gene delivery
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