# Cationic Polybutyl Cyanoacrylate Nanoparticles  for DNA Delivery

Authors: Jinghua Duan; Yangde Zhang; Wei Chen; Chengrong Shen; Mingmei Liao; Yifeng Pan; Jiwei Wang; Xingming Deng; Jinfeng Zhao
Journal: Journal of Biomedicine and Biotechnology
 (2009)

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## Abstract

To enhance the intracellular delivery potential of plasmid DNA using nonviral vectors, we used polybutyl cyanoacrylate (PBCA) and chitosan to prepare PBCA nanoparticles (NPs) by emulsion polymerization and prepared NP/DNA complexes through the complex coacervation of nanoparticles with the DNA. The object of our work is to evaluate the characterization and transfection efficiency of PBCA-NPs. The NPs have a zeta potential of 25.53 mV at pH 7.4 and size about 200 nm. Electrophoretic analysis suggested that the NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed that the NPs exhibit a low cytotoxicity to human hepatocellular carcinoma (HepG2) cells. Qualitative and quantitative analysis of transfection in HepG2 cells by the nanoparticles carrying plasmid DNA encoding for enhanced green fluorescent protein (EGFP-N1) was done by digital fluorescence imaging microscopy system and fluorescence-activated cell sorting (FACS). Qualitative results showed highly efficient expression of GFP that remained stable for up to 96 hours. Quantitative results from FACS showed that PBCA-NPs were significantly more effective in transfecting HepG2 cells after 72 hours postincubation. The results of this study suggested that PBCA-NPs have favorable properties for nonviral delivery.


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## Body

## 1. Introduction

Hepatocellular carcinoma (HCC) is a liver cancer
prevalent in Asia, especially in the Mainland China [1, 2]. As an
established human hepatocarcinoma cell line with epithelial morphology, HepG2 cells
are used routinely for a variety of biochemical and cell biological studies [3–5].The barrier to gene delivery, nuclease degradation in the lysosomal
compartment, has been the focus of many investigators. Important research has
been moving toward the development of polycation-based gene-delivery systems
(such as polylysine conjugates) designed to minimize nuclease degradation through
the design of vectors with the capacity to escape the endosomal-lysosomal pathway
[6–8]. Behr and others introduced the concept of the “proton sponge” and hypothesized
that polymers with buffering capacities between 7.2 and 5.0, such as polyethylenimine
(PEI) and imidazole-containing polymers, could buffer the endosome and
potentially induce its rupture. Protein-expression levels mediated by the
polycationic proton-sponge polymer, PEI, were at least 10-fold greater than polylysine
alone [9–12]. However, protein expression
after delivery of plasmid DNA to the cell nucleus depends on the processes of
transcription and translation. Cytotoxic gene-delivery systems may compromise
these processes and potentially limit protein expression. These situations are perhaps the most prevalent in
the nonviral polycationic gene-delivery class in which the polycationic nature
of the delivery system can lead to cytotoxicity. To drive gene therapy
ultimately into the clinic, improved delivery systems, or vectors, must deliver
DNA to the cell in a transcriptionally active form and must fulfill all
regulatory agency mandates to be considered safe for use in humans [13–16].PBCA particles are extensively investigated for gene and drug
delivery, but now most investigated stabilizers for PBCA lead to negatively
charged particles [17]. In an attempt to provide a positive charge to a
colloidal system, chitosan has been used in the preparation and stabilization
of polyester nanocapsules [18, 19], nanoparticles [20], submicron-sized emulsions
[18], microcapsules [21], and liposome [22]. Chitosan is a cationic
high-molecular-weight heteropolysaccharide composed mainly of b-(1,4)-2-deoxy-2-amino-D-glucopyranose
units and partially of b-(1,4)-2-deoxy-2-acetamido-D-glucopyranose. Because of favorable biological properties such as
biodegradability, biocompatibility, and nontoxicity, chitosan has attracted
great attention in pharmaceutical and biomedical fields [23–28]. Chitosan also increases the transcellular and paracellular transport across
epithelium [29].The aim of the study was to develop positively charged polybutyl cyanoacrylate (PBCA)-NPs in the presence of chitosan for the potential use as a targeting gene delivery system. Physicochemical
characteristics of the prepared nanoparticles were examined by dynamic light
scattering, transmission electron microscopy, and Fourier transform
infrared (FT-IR) spectroscopy. The transfection efficiency of the prepared
NPs into HepG2 cells was examined in vitro using digital
fluorescence imaging microscopy system and fluorescence-activated cell sorting
(FACS).


## 2. Materials and Methods

### 2.1. Materials

Chitosan (degree of deacetylation = 90%) was purchased
from (Shanghai Bio Life Science & Technology Co., Ltd. Shanghai, China).
Butyl cyanoacrylate (BCA) monomer was synthesized by (Guangzhou Baiyun Medical
Adhensive Co., Ltd. Guangzhou, China). HepG2
cell lines were provided by ourselves. All other chemicals used were of
analytical reagent grade and without further purification. Ultrapure water was
used for the preparation of all solutions.


### 2.2. PBCA Nanoparticles

PBCA-NPs were prepared by emulsion polymerization as described in detail elsewhere [30–35].
Briefly, the desired amount (100 μL) of BCA monomer was dropped into an acidic solution of
chitosan adjusted with 1 mol/L hydrochloric acid at room temperature, pH 1.5,
under constant magnetic stirring, stirring was maintained for at least 6 hours
until the polymerization was complete. The colloidal suspension obtained was
brought to pH value of 5.5 by adding 0.5 mol/L NaOH.


### 2.3. Fourier Transform Infrared (FT-IR) Spectrometer

FT-IR spectra were recorded on a
spectrophotometer TENSOR 27 (Bruker, Billerica, Mass, USA). Test samples used for the FT-IR analysis
first were dried and ground into a powder. The powder then was mixed with KBr
and pressed into a disk. The samples were scanned from 400–4000 cm−1.


### 2.4. Particle Size and Zeta Potential

The size of PBCA-NPs was assessed using a dynamic light
scattering spectrophotometer Zetasizer 1000HSA (Malvern Instruments
Ltd., Worcestershire, UK). The colloidal suspension of the NPs was diluted with
deionized distilled water, and the particle size analysis was carried out at a
scattering angle of 90°C
and a temperature of 25°C.The zeta potential was measured on Zetasizer Nano system
(Malvern Instruments Ltd., Worcestershire, UK). The measurement was done in 10 mmol/L NaCl
using disposable zeta cells using the general purpose protocol at 25°C. The instrument
was calibrated routinely with a −50 mV latex standard. The mean zeta potential was
determined using phase analysis light scattering technique.


### 2.5. Transmission Electron Microscopy (TEM) Observation

Particle morphology of PBCA-NPs was observed by transmission electron microscopy (TEM) using a Hitachi H-600 at 50 kV. 1 mL dispersion was diluted with 1 mL demineralized
water and a drop of it was placed onto a collodion support on copper grids (200 mesh). About 2 minutes
of deposition, the grid was tapped with a filter paper to remove surface water
and negatively stained by using a sodium phosphotungstate for 5 seconds. The grid was allowed to
dry further for 10 minutes and was then examined with the electron microscope.


### 2.6. Preparation of NP/DNA Complexes

EGFP-N1
driven by immediate early promoter of cytomegalovirus (CMV) was purchased from (Clontech
Laboratories, Palo Alto, Calif, USA). The plasmids were propagated inEscherichia
coli, extracted by the alkali lysis technique [36], and purified by an EZ-10
Spin Column Plasmid Mini-Preps kit (Bio Basic Inc, Calif, USA) according to the manufacturer's instruction.
DNA purity was determined by measuring the optical
density (OD), and DNA with OD260/OD280 ≥ 1.8 was used.NP/DNA complexes were prepared through the complex coacervation
of the cationic polymer (PBCA-NPs) with the EGFP-N1
plasmid. Microliters of pDNA (100μg/mL) were added into PBCA-NPs solution, which had been
filtrated through a 0.45 μm filter and mixed with them in 10 mmol/L phosphate buffer (pH 7.4). The mixed liquor was standing
for 30 minutes at room temperature and the complexes were used for further
study. Complexes formation was confirmed by electrophoresis on a 1.0% agarose
gel with TBE buffer (89 mmol/L Tris, 89 mmol/L boric acid, and 2 mmol/L EDTA pH
8.0) at 75 V for 90 minutes. DNA was visualized by staining gels with ethidium
bromide (0.5 μg/μL). Images were acquired using an UV transilluminator (VILBER LOURMAT,
France).


### 2.7. Stability of NP/DNA Complexes

Nanoparticles suspension or naked DNA was incubated with 4μL DNase I (1 U/μL) in DNase I Buffer, which consisted of 100 mmol/L Tris-HCl, 25 mmol/L MgCl2, and 1 mmol/L CaCl2 at 37°C for 1hours. Samples of naked DNA, undigested and digested
complexes were loaded onto a 1.0% agarose gel in Tris-borate EDTA buffer at pH
8.0. The samples were run on the gel at 75 V for 90 minutes; the gel was stained
with ethidium bromide (0.5 μg/μL).


### 2.8. Cell Uptake Study

HepG2 cells were incubated in Dulbecco's modification of eagle's
medium (DMEM, Beijing Solarbio Science & Technology Co., Ltd, Beijing,
China) at 37°C, 5% CO2 with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin.A confluent layer of approximately 8000 HepG2 cells per
well was incubated with 20 microliters
fluorescent NP suspension, the final concentration of NPs in the serum-free medium was 210μg/mL, in the 6-well black plates at 37°C. After 6 hours of
incubation, the cells were washed three times with cold PBS (pH 7.4) to eliminate
excess particles which were not entrapped by the cells. The uptake of NPs by the HepG2 cells was examined with fluorescence microscopy Leica DM
LB2 (Leica Microsystems Wetzlar GmbH, Germany). In
the control experiment, there were no fluorescent NPs in the medium.To preparing for
flow cytometry, after digested with the lyse
solution containing 0.25% trypsin, the cocultivated cells were harvested and
rinsed with cooled D-Hank's solution and centrifuged (1000 rpm, 5 minutes) to discard cell
fragments. The cells were further washed three times with PBS and centrifuged (1000 rpm, 5 minutes) to
remove the uncombined nanoparticles. The obtained cells were diluted to single-cell suspension stabilized with 1% formaldehyde at 4°C. Processed single-cell suspensions
were analyzed on an FACS Calibur flow cytometer (BD, Franklin
Lakes, NJ, USA) using a 488 nm argon laser and FL1 bandpass emission (530
± 20 nm) for the green fluorescence. HepG2 cells
were gated by sideward scatter versus forward scatter (SSC/FSC) plots. Fluorescence
measurements were done in the FL1 channel.


### 2.9. Cytotoxicity Evaluation by MTT Assay

Cytotoxicity of the
NPs was determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) MTT assay in HepG2 cells. This assay is
based on the ability of living cells to reduce a water-soluble yellow dye, MTT,
to a purple colored water-insoluble formazan product by mitochondrial enzyme succinate
dehydrogenase. The cells were maintained in DMEM supplemented with 10% FBS in
5% CO2 incubator at 37°C. Eight thousand cells were seeded per
well in 96-well microtiter plates followed by incubation for 24 hours. The
growth medium was replaced with a fresh one containing 0.1–8.0 μg/μL NPs. After 24-hour
incubation, 20 μL of MTT solution (5 mg/mL in PBS, pH 7.4) was added to cells followed by further
incubation for 4 hours. Thereafter, the media was removed and cells were rinsed
with PBS. The formazan crystals formed were dissolved using dimethyl sulfoxide
(DMSO) (150 μL/well) and absorbance was read at 570 nm on a multiskan spectrum (Electro Thermo, Milford, Mass, USA). Cell viability
was determined as a percentage of the negative control (untreated cells).


### 2.10. in Vitro Transfection

For the qualitative analysis of transfection,
HepG2 cells were grown in 6-well culture
plates containing a Corning circular glass cover-slip with seeded1×105 cells seeded per well and
incubated until the cover rate reached 60–80%. The NP/DNA
complexes were dispersed in serum-free medium and untreated cells were used as
a negative control. After filtration, the NP/DNA complexes were added at a
concentration equivalent to 2 μg of EGFP-N1
plasmid DNA per well, and incubated with the cells at 37°C and 5% CO2 atmosphere for 6 hours. The culture was
removed from each well and replaced with regular growth medium including 10%
FBS. The cover slips were washed three times with PBS at 96 hours
posttransfection and mounted on to microscopic slides containing a drop of antifade mounting
medium (Beyotime Institute of Biotechnology, Shanghai, China). The expression
of GFP in the cells was observed by a fluorescence microscope.For the quantitative analysis of transfection, HepG2 cells were
grown to semiconfluence in a 6-well cell culture plate and transfected with
EGFP-N1 plasmid DNA encapsulated PBCA-NPs and compared with cells treated with
serum-free media alone. The NP/DNA complexes were dispersed in serum-free media
and added to each of the six wells at a concentration of 2μg of plasmid DNA per well. The cells are allowed to incubate for a period
of 6 hours at 37°C in a 5% CO2 incubator and then replaced by fresh growth medium. The adherent cells are
trypsinized at time intervals of 24, 48, 72, and 96 hours postincubation, centrifuged,
and fixed with 100 μL of 4% formalin buffer solution.
The fluorescence of the GFP produced in the transfected cell was detected by a flow cytometer equipped
with an argon 488 laser. The FL1 channel was used to detect the cells
expressing GFP fluorescence.


## 2.1. Materials

Chitosan (degree of deacetylation = 90%) was purchased
from (Shanghai Bio Life Science & Technology Co., Ltd. Shanghai, China).
Butyl cyanoacrylate (BCA) monomer was synthesized by (Guangzhou Baiyun Medical
Adhensive Co., Ltd. Guangzhou, China). HepG2
cell lines were provided by ourselves. All other chemicals used were of
analytical reagent grade and without further purification. Ultrapure water was
used for the preparation of all solutions.


## 2.2. PBCA Nanoparticles

PBCA-NPs were prepared by emulsion polymerization as described in detail elsewhere [30–35].
Briefly, the desired amount (100 μL) of BCA monomer was dropped into an acidic solution of
chitosan adjusted with 1 mol/L hydrochloric acid at room temperature, pH 1.5,
under constant magnetic stirring, stirring was maintained for at least 6 hours
until the polymerization was complete. The colloidal suspension obtained was
brought to pH value of 5.5 by adding 0.5 mol/L NaOH.


## 2.3. Fourier Transform Infrared (FT-IR) Spectrometer

FT-IR spectra were recorded on a
spectrophotometer TENSOR 27 (Bruker, Billerica, Mass, USA). Test samples used for the FT-IR analysis
first were dried and ground into a powder. The powder then was mixed with KBr
and pressed into a disk. The samples were scanned from 400–4000 cm−1.


## 2.4. Particle Size and Zeta Potential

The size of PBCA-NPs was assessed using a dynamic light
scattering spectrophotometer Zetasizer 1000HSA (Malvern Instruments
Ltd., Worcestershire, UK). The colloidal suspension of the NPs was diluted with
deionized distilled water, and the particle size analysis was carried out at a
scattering angle of 90°C
and a temperature of 25°C.The zeta potential was measured on Zetasizer Nano system
(Malvern Instruments Ltd., Worcestershire, UK). The measurement was done in 10 mmol/L NaCl
using disposable zeta cells using the general purpose protocol at 25°C. The instrument
was calibrated routinely with a −50 mV latex standard. The mean zeta potential was
determined using phase analysis light scattering technique.


## 2.5. Transmission Electron Microscopy (TEM) Observation

Particle morphology of PBCA-NPs was observed by transmission electron microscopy (TEM) using a Hitachi H-600 at 50 kV. 1 mL dispersion was diluted with 1 mL demineralized
water and a drop of it was placed onto a collodion support on copper grids (200 mesh). About 2 minutes
of deposition, the grid was tapped with a filter paper to remove surface water
and negatively stained by using a sodium phosphotungstate for 5 seconds. The grid was allowed to
dry further for 10 minutes and was then examined with the electron microscope.


## 2.6. Preparation of NP/DNA Complexes

EGFP-N1
driven by immediate early promoter of cytomegalovirus (CMV) was purchased from (Clontech
Laboratories, Palo Alto, Calif, USA). The plasmids were propagated inEscherichia
coli, extracted by the alkali lysis technique [36], and purified by an EZ-10
Spin Column Plasmid Mini-Preps kit (Bio Basic Inc, Calif, USA) according to the manufacturer's instruction.
DNA purity was determined by measuring the optical
density (OD), and DNA with OD260/OD280 ≥ 1.8 was used.NP/DNA complexes were prepared through the complex coacervation
of the cationic polymer (PBCA-NPs) with the EGFP-N1
plasmid. Microliters of pDNA (100μg/mL) were added into PBCA-NPs solution, which had been
filtrated through a 0.45 μm filter and mixed with them in 10 mmol/L phosphate buffer (pH 7.4). The mixed liquor was standing
for 30 minutes at room temperature and the complexes were used for further
study. Complexes formation was confirmed by electrophoresis on a 1.0% agarose
gel with TBE buffer (89 mmol/L Tris, 89 mmol/L boric acid, and 2 mmol/L EDTA pH
8.0) at 75 V for 90 minutes. DNA was visualized by staining gels with ethidium
bromide (0.5 μg/μL). Images were acquired using an UV transilluminator (VILBER LOURMAT,
France).


## 2.7. Stability of NP/DNA Complexes

Nanoparticles suspension or naked DNA was incubated with 4μL DNase I (1 U/μL) in DNase I Buffer, which consisted of 100 mmol/L Tris-HCl, 25 mmol/L MgCl2, and 1 mmol/L CaCl2 at 37°C for 1hours. Samples of naked DNA, undigested and digested
complexes were loaded onto a 1.0% agarose gel in Tris-borate EDTA buffer at pH
8.0. The samples were run on the gel at 75 V for 90 minutes; the gel was stained
with ethidium bromide (0.5 μg/μL).


## 2.8. Cell Uptake Study

HepG2 cells were incubated in Dulbecco's modification of eagle's
medium (DMEM, Beijing Solarbio Science & Technology Co., Ltd, Beijing,
China) at 37°C, 5% CO2 with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin.A confluent layer of approximately 8000 HepG2 cells per
well was incubated with 20 microliters
fluorescent NP suspension, the final concentration of NPs in the serum-free medium was 210μg/mL, in the 6-well black plates at 37°C. After 6 hours of
incubation, the cells were washed three times with cold PBS (pH 7.4) to eliminate
excess particles which were not entrapped by the cells. The uptake of NPs by the HepG2 cells was examined with fluorescence microscopy Leica DM
LB2 (Leica Microsystems Wetzlar GmbH, Germany). In
the control experiment, there were no fluorescent NPs in the medium.To preparing for
flow cytometry, after digested with the lyse
solution containing 0.25% trypsin, the cocultivated cells were harvested and
rinsed with cooled D-Hank's solution and centrifuged (1000 rpm, 5 minutes) to discard cell
fragments. The cells were further washed three times with PBS and centrifuged (1000 rpm, 5 minutes) to
remove the uncombined nanoparticles. The obtained cells were diluted to single-cell suspension stabilized with 1% formaldehyde at 4°C. Processed single-cell suspensions
were analyzed on an FACS Calibur flow cytometer (BD, Franklin
Lakes, NJ, USA) using a 488 nm argon laser and FL1 bandpass emission (530
± 20 nm) for the green fluorescence. HepG2 cells
were gated by sideward scatter versus forward scatter (SSC/FSC) plots. Fluorescence
measurements were done in the FL1 channel.


## 2.9. Cytotoxicity Evaluation by MTT Assay

Cytotoxicity of the
NPs was determined by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) MTT assay in HepG2 cells. This assay is
based on the ability of living cells to reduce a water-soluble yellow dye, MTT,
to a purple colored water-insoluble formazan product by mitochondrial enzyme succinate
dehydrogenase. The cells were maintained in DMEM supplemented with 10% FBS in
5% CO2 incubator at 37°C. Eight thousand cells were seeded per
well in 96-well microtiter plates followed by incubation for 24 hours. The
growth medium was replaced with a fresh one containing 0.1–8.0 μg/μL NPs. After 24-hour
incubation, 20 μL of MTT solution (5 mg/mL in PBS, pH 7.4) was added to cells followed by further
incubation for 4 hours. Thereafter, the media was removed and cells were rinsed
with PBS. The formazan crystals formed were dissolved using dimethyl sulfoxide
(DMSO) (150 μL/well) and absorbance was read at 570 nm on a multiskan spectrum (Electro Thermo, Milford, Mass, USA). Cell viability
was determined as a percentage of the negative control (untreated cells).


## 2.10. in Vitro Transfection

For the qualitative analysis of transfection,
HepG2 cells were grown in 6-well culture
plates containing a Corning circular glass cover-slip with seeded1×105 cells seeded per well and
incubated until the cover rate reached 60–80%. The NP/DNA
complexes were dispersed in serum-free medium and untreated cells were used as
a negative control. After filtration, the NP/DNA complexes were added at a
concentration equivalent to 2 μg of EGFP-N1
plasmid DNA per well, and incubated with the cells at 37°C and 5% CO2 atmosphere for 6 hours. The culture was
removed from each well and replaced with regular growth medium including 10%
FBS. The cover slips were washed three times with PBS at 96 hours
posttransfection and mounted on to microscopic slides containing a drop of antifade mounting
medium (Beyotime Institute of Biotechnology, Shanghai, China). The expression
of GFP in the cells was observed by a fluorescence microscope.For the quantitative analysis of transfection, HepG2 cells were
grown to semiconfluence in a 6-well cell culture plate and transfected with
EGFP-N1 plasmid DNA encapsulated PBCA-NPs and compared with cells treated with
serum-free media alone. The NP/DNA complexes were dispersed in serum-free media
and added to each of the six wells at a concentration of 2μg of plasmid DNA per well. The cells are allowed to incubate for a period
of 6 hours at 37°C in a 5% CO2 incubator and then replaced by fresh growth medium. The adherent cells are
trypsinized at time intervals of 24, 48, 72, and 96 hours postincubation, centrifuged,
and fixed with 100 μL of 4% formalin buffer solution.
The fluorescence of the GFP produced in the transfected cell was detected by a flow cytometer equipped
with an argon 488 laser. The FL1 channel was used to detect the cells
expressing GFP fluorescence.


## 3. Results and Discussion

### 3.1. Preparation and Characterization of PBCA-NPs

We synthesized the nanoparticles by dropping monomer into an acidic solution of chitosan. We obtained
uniform particles by adding BCA monomer, pH 1.5, at room temperature, to 0.5%
(w/v) chitosan during high-speed vortexing.FT-IR analysis was performed to confirm the
chitosan-graft-PBCA. Figure1 shows a comparison between the FT-IR spectra of
chitosan(A), chitosan-coated PBCA(B), and PBCA(C). After chitosan-stabilized NPs
had been extracted, the residue still possessed a -CN stretch (2253.84 cm−1) and a
carbonyl stretch (1752.93 cm−1) corresponding to
those of PBCA and a hydrogen-bonded OH stretch (3461.64 cm−1)
corresponding to that of chitosan. The amide band at 1640.57 cm−1
that was observed in chitosan shifts to 1635.89 cm−1. These
observations suggest that chitosan, especially the NH2 group, may initiate the
BCA monomer in the acidic polymerization medium, leading to chitosan chemically
coupled to PBCA to form chitosan-stabilized NPs.Figure 1
(A) Fourier transform IR spectra of chitosan; (B) chitosan-coated PBCA-NPs;  (C) PBCA-NPs.The size
distribution and zeta potential of the NPs in NaCl solution were investigated by dynamic light scattering. Dynamic size distribution and zeta potential may play important roles in
determining the fate of NPs after administration [37].
Figure 2(a) shows a unimodal
particle size distribution (number average) between 100 and 200 nm. It was reported that large
particles hardly reach the cells [38], while smaller particles tended to
accumulate in the tumor sites due to the enhanced permeability and retention (EPR)
effect and a greater internalization was also observed [6]. Additionally, gene carriers
with a diameter larger than 200 nm are readily scavenged nonspecifically by
monocytes and the reticuloendothelial system [39].(a) Size distribution and (b) zeta potential of the prepared chitosan-coated PBCA-NPs.
(a)(b)The results of Figure2(b) indicated that PBCA-NPs carry
positive charge with a zeta potential of about 25 mV. PBCA-NPs have a very
negative zeta potential because of the adsorption of anions from the aqueous
polymerization medium, but chitosan is a polycationic biopolymer, positive
charge PBCA-NPs may have been produced by coating them with chitosan [40, 41].In theory, more pronounced zeta potential values, being
positive or negative, tend to stabilize particle suspension due to the
electrostatic repulsion forces between particles with the same electric charge
prevents the aggregation of the spheres [42]. Consequently, the presence of the
positively charged groups on the particles could lead to electrical charge
interactions between the negatively charged surface cells and the particles [43].TEM images shown in Figure3 reveal the surface morphology
and spherical shape of the prepared particles. The actual diameter of the
nanoparticles observed by TEM was approximately 200 nm and was found to be
similar to the values obtained by measurements based on light scattering. The
plasmid is protected from DNase degradation in this formulation [44].Figure 3
Transmission electron micrograph of the prepared chitosan-coated PBCA nanoparticles.


### 3.2. Complex Formation of PBCA-Nps with Pegfp-N1 Plasmid DNA

pDNA complex with
previously prepared nanoparticles was achieved by the formation of ion-pairs between
the positively charged amino groups located on the particle surface and the
negatively charged phosphodiester backbone of the plasmid DNA [45]. The electrophoretic mobility of DNA, complex with PBCA-NPs, decreases
gradually on the agarose gel with increasing charge ratio (Figure 4). However,
in the control experiment, there was no DNA in agarose gel. This method also allowed showing the dependency
of this interaction on pH, reflecting the deprotonation of the modified silica
particles at alkaline pH (data not shown). The particle-DNA binding was also
inhibited by the presence of high salt concentrations (data not shown). This suggests
that this interaction is based on electrostatic forces, resulting in
interpolyelectrolyte complexes.Figure 4
Agarose gel electrophoresis of NP/DNA complexes. Lane 1: DNA
molecular weight marker; lane 2: naked plasmid DNA; lane 3: chitosan-coated
PBCA-NPs; lanes 4–7 correspond to DNA with progressively increasing
proportions of NPs at the charge ratio of 5:1, 10:1, 15:1, and 30:1 (w/w),
respectively.


### 3.3. Enzyme Protection

Behr introduced the concept of the “proton
sponge” and hypothesized that polymers with buffering capacities between 7.2
and 5.0 could buffer the endosome and potentially induce its rupture [46]. As the chitosan molecule is a weak base with a pKa value of the D-glucosamine residue
about 6.2–7.0 [47], we
hypothesize NP/DNA complexes formation can resist enzymatic degradation. The results were analyzed by using agarose gel
electrophoresis. Figure 5 shows that free-plasmid DNA was degraded completely
into small fragments and could not be visualized in the agarose gel, however, NP/DNA
complexes digested by DNase I were still visualized around the sample pore.Figure 5
Electrophoretic mobility analysis NP/DNA complexes following
DNase I   digestion. Lane 1: EGFP-N1 plasmid DNA; lane 2: lane 1 + digestion;
lane 3: NP/DNA complex at the charge of 5:1; lane 4: lane 3 + digestion;
lane 5: NP/DNA complex at the charge of 15:1; lane 6: lane 5 + digestion.


### 3.4. Cytotoxicity Evaluation of NP/DNA  Complexes

In vitro
toxicity of NP/DNA complexes was evaluated by MTT assay in HepG2 cells using
increasing doses of NPs. MTT assay shows that the cytotoxicity of the NPs
depended on their concentration ranging from 0.1–8.0μg/μL
(Figure 6). The statistic analysis demonstrates that
there are significant differences between concentrations (one-way ANOVA, P<.001). These data can indicate that the NPs below 2 μg/μL had little adverse effect on the HepG2 cells viability,
suggesting that the doses of in vitro HepG2 cells uptake study less
than 0.5 μg/μL presented no toxicity. This is
important because most of the cationic polymers and lipids, which are commonly used
for gene transfection, have toxic effects on cells at a higher concentration
due to electrostatic interaction with negatively charged cellular membrane [48, 49].
Thus, the PBCA-NPs is a promising carrier for gene delivery with low cytotoxicity.
However, its long-term in vivo toxicity and immunogenicity should be further
investigated.Figure 6
Cytotoxicity of cationic chitosan coated PBCA-NPs analyzed by MTT
assay, 24 hours posttreatment (n=8). Columns, mean; bars,
SD, P<.001 as evaluated by one-way ANOVA using SPSS 17.0 version.


### 3.5. Uptake of the Functionalized PBCA-NPs

In order to study cellular uptake of NPs in vitro, the
use of fluorescently or radioactively labeled NPs is the most common
experimental approach found in the literature. Fluorescent labeling, which we
used the prepared fluorescent positive PBCA-NPs
in this study, was chosen for the present study to avoid exposure of the
samples to radioactive materials. Fluorescent labeling makes cellular uptake of
NPs readily detectable by fluorescence microscopy. The extent of particle
uptake can then be determined by flow cytometry. After incubation of HepG2
cells with the fluorescent PBCA-NPs
for 6 hours, the FACS measurements showed significantly higher mean
fluorescence intensity of 75.06% for cells incubated with the positively
charged fluorescent PBCA-NPs with respect to
the control particles (see Figure7).(a) Quantitative analysis intracellular fluorescence of HepG2 cells;
(b) endocytosised with fluorescent chitosan-coated PBCA-NPs by flow cytometry.
(a)(b)Figure8 shows fluorescent
microscopic images of HepG2 cell monolayers after the NPs uptake experiments, which strongly support the previous quantitative
measurements of the cellular uptake of the NPs
by showing strong fluorescence in the cell. However, no fluorescence can be
detected from the image of the control cells.Biocompatibility of functionalized chitosan-coated PBCA-NPs (×200). (a) HepG2 cell endocytosised fluorescent chitosan-coated PBCA-NPs;
(b) HepG2 cell without fluorescent chitosan-coated PBCA-NPs.
(a)(b)


### 3.6. in Vitro Gene Transfection and Expression in HepG2 Cells

In order to
evaluate the effect of PBCA-NPs on transferring DNA, we investigated the
transfection efficiency of complexes containing pDNA in HepG2 cells to find
gene expression level. The concentration of PBCA-NPs used in this experiment was
much less than 0.5μg/μL, which would not affect the cell viability obviously according to the
result mentioned in cell viability assay.The pictures of fluorescence microscope to detect the green fluorescence
sent out by transfected cells indicated relatively higher transfection
efficiencies of NP/DNA complexes (Figure9(a)). To determine the gene transfer capability
of NPs in vitro exactly, flow cytometry experiments were conducted to determine
the EGFP transfection levels in HepG2 cells. It was found that the transgene
expression levels increased with the time, and reached a climax at 72 hours,
then decreased. The results of the complexes are fused with cell membrane easily
through electrostatical interaction can be explained by the fact that complexes
possess the positive potential in neutral conditions.(a) Qualitative evaluation of EGFP-N1 transgene expression in HepG2
cells with chitosan-coated PBCA-NPs, fluorescent image (×200) of the cells was
obtained after 96 hours upon incubation. (b) Quantitative evaluation  of
EGFP-N1 transgene expression in HepG2 cells as a function of time measured
by flow cytometric analysis with chitosan-coated PBCA-NPs.
HepG2 cells without any treatment were used as control.
(a)(b)


## 3.1. Preparation and Characterization of PBCA-NPs

We synthesized the nanoparticles by dropping monomer into an acidic solution of chitosan. We obtained
uniform particles by adding BCA monomer, pH 1.5, at room temperature, to 0.5%
(w/v) chitosan during high-speed vortexing.FT-IR analysis was performed to confirm the
chitosan-graft-PBCA. Figure1 shows a comparison between the FT-IR spectra of
chitosan(A), chitosan-coated PBCA(B), and PBCA(C). After chitosan-stabilized NPs
had been extracted, the residue still possessed a -CN stretch (2253.84 cm−1) and a
carbonyl stretch (1752.93 cm−1) corresponding to
those of PBCA and a hydrogen-bonded OH stretch (3461.64 cm−1)
corresponding to that of chitosan. The amide band at 1640.57 cm−1
that was observed in chitosan shifts to 1635.89 cm−1. These
observations suggest that chitosan, especially the NH2 group, may initiate the
BCA monomer in the acidic polymerization medium, leading to chitosan chemically
coupled to PBCA to form chitosan-stabilized NPs.Figure 1
(A) Fourier transform IR spectra of chitosan; (B) chitosan-coated PBCA-NPs;  (C) PBCA-NPs.The size
distribution and zeta potential of the NPs in NaCl solution were investigated by dynamic light scattering. Dynamic size distribution and zeta potential may play important roles in
determining the fate of NPs after administration [37].
Figure 2(a) shows a unimodal
particle size distribution (number average) between 100 and 200 nm. It was reported that large
particles hardly reach the cells [38], while smaller particles tended to
accumulate in the tumor sites due to the enhanced permeability and retention (EPR)
effect and a greater internalization was also observed [6]. Additionally, gene carriers
with a diameter larger than 200 nm are readily scavenged nonspecifically by
monocytes and the reticuloendothelial system [39].(a) Size distribution and (b) zeta potential of the prepared chitosan-coated PBCA-NPs.
(a)(b)The results of Figure2(b) indicated that PBCA-NPs carry
positive charge with a zeta potential of about 25 mV. PBCA-NPs have a very
negative zeta potential because of the adsorption of anions from the aqueous
polymerization medium, but chitosan is a polycationic biopolymer, positive
charge PBCA-NPs may have been produced by coating them with chitosan [40, 41].In theory, more pronounced zeta potential values, being
positive or negative, tend to stabilize particle suspension due to the
electrostatic repulsion forces between particles with the same electric charge
prevents the aggregation of the spheres [42]. Consequently, the presence of the
positively charged groups on the particles could lead to electrical charge
interactions between the negatively charged surface cells and the particles [43].TEM images shown in Figure3 reveal the surface morphology
and spherical shape of the prepared particles. The actual diameter of the
nanoparticles observed by TEM was approximately 200 nm and was found to be
similar to the values obtained by measurements based on light scattering. The
plasmid is protected from DNase degradation in this formulation [44].Figure 3
Transmission electron micrograph of the prepared chitosan-coated PBCA nanoparticles.


## 3.2. Complex Formation of PBCA-Nps with Pegfp-N1 Plasmid DNA

pDNA complex with
previously prepared nanoparticles was achieved by the formation of ion-pairs between
the positively charged amino groups located on the particle surface and the
negatively charged phosphodiester backbone of the plasmid DNA [45]. The electrophoretic mobility of DNA, complex with PBCA-NPs, decreases
gradually on the agarose gel with increasing charge ratio (Figure 4). However,
in the control experiment, there was no DNA in agarose gel. This method also allowed showing the dependency
of this interaction on pH, reflecting the deprotonation of the modified silica
particles at alkaline pH (data not shown). The particle-DNA binding was also
inhibited by the presence of high salt concentrations (data not shown). This suggests
that this interaction is based on electrostatic forces, resulting in
interpolyelectrolyte complexes.Figure 4
Agarose gel electrophoresis of NP/DNA complexes. Lane 1: DNA
molecular weight marker; lane 2: naked plasmid DNA; lane 3: chitosan-coated
PBCA-NPs; lanes 4–7 correspond to DNA with progressively increasing
proportions of NPs at the charge ratio of 5:1, 10:1, 15:1, and 30:1 (w/w),
respectively.


## 3.3. Enzyme Protection

Behr introduced the concept of the “proton
sponge” and hypothesized that polymers with buffering capacities between 7.2
and 5.0 could buffer the endosome and potentially induce its rupture [46]. As the chitosan molecule is a weak base with a pKa value of the D-glucosamine residue
about 6.2–7.0 [47], we
hypothesize NP/DNA complexes formation can resist enzymatic degradation. The results were analyzed by using agarose gel
electrophoresis. Figure 5 shows that free-plasmid DNA was degraded completely
into small fragments and could not be visualized in the agarose gel, however, NP/DNA
complexes digested by DNase I were still visualized around the sample pore.Figure 5
Electrophoretic mobility analysis NP/DNA complexes following
DNase I   digestion. Lane 1: EGFP-N1 plasmid DNA; lane 2: lane 1 + digestion;
lane 3: NP/DNA complex at the charge of 5:1; lane 4: lane 3 + digestion;
lane 5: NP/DNA complex at the charge of 15:1; lane 6: lane 5 + digestion.


## 3.4. Cytotoxicity Evaluation of NP/DNA  Complexes

In vitro
toxicity of NP/DNA complexes was evaluated by MTT assay in HepG2 cells using
increasing doses of NPs. MTT assay shows that the cytotoxicity of the NPs
depended on their concentration ranging from 0.1–8.0μg/μL
(Figure 6). The statistic analysis demonstrates that
there are significant differences between concentrations (one-way ANOVA, P<.001). These data can indicate that the NPs below 2 μg/μL had little adverse effect on the HepG2 cells viability,
suggesting that the doses of in vitro HepG2 cells uptake study less
than 0.5 μg/μL presented no toxicity. This is
important because most of the cationic polymers and lipids, which are commonly used
for gene transfection, have toxic effects on cells at a higher concentration
due to electrostatic interaction with negatively charged cellular membrane [48, 49].
Thus, the PBCA-NPs is a promising carrier for gene delivery with low cytotoxicity.
However, its long-term in vivo toxicity and immunogenicity should be further
investigated.Figure 6
Cytotoxicity of cationic chitosan coated PBCA-NPs analyzed by MTT
assay, 24 hours posttreatment (n=8). Columns, mean; bars,
SD, P<.001 as evaluated by one-way ANOVA using SPSS 17.0 version.


## 3.5. Uptake of the Functionalized PBCA-NPs

In order to study cellular uptake of NPs in vitro, the
use of fluorescently or radioactively labeled NPs is the most common
experimental approach found in the literature. Fluorescent labeling, which we
used the prepared fluorescent positive PBCA-NPs
in this study, was chosen for the present study to avoid exposure of the
samples to radioactive materials. Fluorescent labeling makes cellular uptake of
NPs readily detectable by fluorescence microscopy. The extent of particle
uptake can then be determined by flow cytometry. After incubation of HepG2
cells with the fluorescent PBCA-NPs
for 6 hours, the FACS measurements showed significantly higher mean
fluorescence intensity of 75.06% for cells incubated with the positively
charged fluorescent PBCA-NPs with respect to
the control particles (see Figure7).(a) Quantitative analysis intracellular fluorescence of HepG2 cells;
(b) endocytosised with fluorescent chitosan-coated PBCA-NPs by flow cytometry.
(a)(b)Figure8 shows fluorescent
microscopic images of HepG2 cell monolayers after the NPs uptake experiments, which strongly support the previous quantitative
measurements of the cellular uptake of the NPs
by showing strong fluorescence in the cell. However, no fluorescence can be
detected from the image of the control cells.Biocompatibility of functionalized chitosan-coated PBCA-NPs (×200). (a) HepG2 cell endocytosised fluorescent chitosan-coated PBCA-NPs;
(b) HepG2 cell without fluorescent chitosan-coated PBCA-NPs.
(a)(b)


## 3.6. in Vitro Gene Transfection and Expression in HepG2 Cells

In order to
evaluate the effect of PBCA-NPs on transferring DNA, we investigated the
transfection efficiency of complexes containing pDNA in HepG2 cells to find
gene expression level. The concentration of PBCA-NPs used in this experiment was
much less than 0.5μg/μL, which would not affect the cell viability obviously according to the
result mentioned in cell viability assay.The pictures of fluorescence microscope to detect the green fluorescence
sent out by transfected cells indicated relatively higher transfection
efficiencies of NP/DNA complexes (Figure9(a)). To determine the gene transfer capability
of NPs in vitro exactly, flow cytometry experiments were conducted to determine
the EGFP transfection levels in HepG2 cells. It was found that the transgene
expression levels increased with the time, and reached a climax at 72 hours,
then decreased. The results of the complexes are fused with cell membrane easily
through electrostatical interaction can be explained by the fact that complexes
possess the positive potential in neutral conditions.(a) Qualitative evaluation of EGFP-N1 transgene expression in HepG2
cells with chitosan-coated PBCA-NPs, fluorescent image (×200) of the cells was
obtained after 96 hours upon incubation. (b) Quantitative evaluation  of
EGFP-N1 transgene expression in HepG2 cells as a function of time measured
by flow cytometric analysis with chitosan-coated PBCA-NPs.
HepG2 cells without any treatment were used as control.
(a)(b)


## 4. Conclusions

From these investigations, it is evident that this method
forms uniform cationic PBCA nanospheres which
can bind DNA readily by electrostatic interaction. Chitosan cannot only
chemically couple to PBCA giving the net positive surface charge, but also
produced particles with uniform size and spherical shape, as observed by TEM. By
applying emulsion polymerization technique, it is possible to prepare
fluorescent dye labeled PBCA-NPs. In the present study, we identify that
chitosan-coated PBCA-NPs are positive charge and can be used as nonviral
carrier for gene transfection.Compared with negatively charged NPs, positively charged NP/DNA complexes might prevent destabilization by cation adsorption, give a better
controlled release of plasmid, favorably interact with negatively charged
tissues and provide site-specific target in vivo. Therefore, positively charged
targeting gene delivery vehicles may be considered as very promising gene
carriers for various administration routes.



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*Source: 149254-2009-03-11.xml*