1. introductionshodhganga.inflibnet.ac.in/bitstream/10603/2457/15/15... · 2012. 6. 20. ·...

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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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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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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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

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(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)


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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(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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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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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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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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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

132


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

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


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


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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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


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


HepG2

135


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


HEK293

136


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

137


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

138


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