Gene delivery efficacy of polyethyleneimine-introduced chitosan shell/poly(methyl methacrylate) core nanoparticles for rat mesenchymal stem cells.

Abstract

This work investigated polyethyleneimine (PEI)-introduced chitosan (CS) (CS/PEI) nanoparticles as non-viral carrier of plasmid DNA for rat mesenchymal stem cells (MSCs). The CS/PEI nanoparticles were prepared by the emulsifier-free emulsion polymerization of methyl methacrylate monomer induced by a small amount of t-butyl hydroperxide in the presence of different concentrations of PEI mixed with CS. The resulting nanoparticles were characterized by their surface properties and buffering capacity. In vitro gene transfection was also evaluated. The introduction of PEI affected the surface charge, dispersing stability and buffering capacity of the nanoparticles. The CS/PEI nanoparticles formed a complex upon mixing with a plasmid DNA of luciferase. The complex enhanced the level of gene transfection and prolonged the time period of expression for MSCs, compared with those of plasmid DNA-original CS and PEI nanoparticles. Cytotoxicity of CS/PEI complexes with plasmid DNA was significantly low, depending on the amount of PEI introduced. It is concluded that the CS/PEI nanoparticle was a promising carrier for gene delivery of MSCs.

Full text

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Title
Gene Delivery Efficacy of Polyethyleneimine-Introduced
Chitosan Shell/Poly(methyl Methacrylate) Core Nanoparticles
for Rat Mesenchymal Stem Cells
Author(s)
Pimpha, Nuttaporn; Sunintaboon, Panya; Inphonlek, Supharat;
Tabata, Yasuhiko
Citation
JOURNAL OF BIOMATERIALS SCIENCE-POLYMER
EDITION (2010), 21(2): 205-223
Issue Date2010
URLhttp://hdl.handle.net/2433/148426
Right© Koninklijke Brill NV, Leiden, 2010
TypeJournal Article
Textversionpublisher
KURENAI : Kyoto University Research Information Repository
Kyoto University

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Journal of Biomaterials Science 21 (2010) 205–223
brill.nl/jbs
Gene Delivery Efficacy of Polyethyleneimine-Introduced
Chitosan Shell/Poly(methyl Methacrylate) Core
Nanoparticles for Rat Mesenchymal Stem Cells
Nuttaporn Pimphaa, Panya Sunintaboonb, Supharat Inphonlekband
Yasuhiko Tabatac,∗
aNational Nanotechnology Center, Thailand Science Park, Paholyothin Rd., Pathumthani, 12120,
Thailand
bDepartment of Chemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand
cDepartment of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences,
Kyoto University, 53 Kawara-cho Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
Received 15 October 2008; accepted 19 January 2009
Abstract
This work investigated polyethyleneimine (PEI)-introduced chitosan (CS) (CS/PEI) nanoparticles as non-
viral carrier of plasmid DNA for rat mesenchymal stem cells (MSCs). The CS/PEI nanoparticles were
prepared by the emulsifier-free emulsion polymerization of methyl methacrylate monomer induced by
a small amount of t-butyl hydroperxide in the presence of different concentrations of PEI mixed with CS.
The resulting nanoparticles were characterized by their surface properties and buffering capacity. In vitro
gene transfection was also evaluated. The introduction of PEI affected the surface charge, dispersing stabil-
ity and buffering capacity of the nanoparticles. The CS/PEI nanoparticles formed a complex upon mixing
with a plasmid DNA of luciferase. The complex enhanced the level of gene transfection and prolonged the
time period of expression for MSCs, compared with those of plasmid DNA–original CS and PEI nanoparti-
cles. Cytotoxicity of CS/PEI complexes with plasmid DNA was significantly low, depending on the amount
of PEI introduced. It is concluded that the CS/PEI nanoparticle was a promising carrier for gene delivery of
MSCs.
© Koninklijke Brill NV, Leiden, 2010
Keywords
Nanoparticles, chitosan, polyethyleneimine, mesenchymal stem cells, non-viral carrier
*To whom correspondence should be addressed. Tel.: (81-75) 751-4121; Fax: (81-75) 751-4646; e-mail:
yasuhiko@frontier.kyoto-u.ac.jp
© Koninklijke Brill NV, Leiden, 2010DOI:10.1163/156856209X415503

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N. Pimpha et al. / Journal of Biomaterials Science 21 (2010) 205–223
1. Introduction
Mesenchymal stem cells (MSCs) have the potential to induce the regeneration
of mesenchymal tissues, such as bone, cartilage, muscle, ligament, tendon, adi-
pose and marrow stroma tissues. The cells can be differentiated effectively into
the adipocytic, chondrocytic, or osteocytic lineages [1–3]. Based on the biologi-
cal aspects, MSC-based therapy is one of the promising approaches to achieve the
regeneration and repairing of tissues [4–11]. It is expected for more effective cell
therapy to use MSC genetically modified for the functional activation. For example,
Meinel et al. studied the cell therapy of human MSC genetically engineered with
adenovirus, containing a human BMP-2 gene in the combination with silk scaffold
[12]. Ahn et al. examined PEI as a carrier of gene transfection for human adipose
tissue-derived stem cells [13]. Uludag and co-workers reported the effectiveness of
an amphiphilic poly(L-lysine)-palmitic acid in the plasmid DNA carrier for bone
marrow stromal cells [14]. Moreover, Hosseinkami et al. have prepared cationized
polysaccharides foranon-viralgenecarriertoenhancetheDNAexpressionofMSC
[15] and demonstrated the therapeutic efficacy in acute cardiac infarction [16].
To effectively transfect cells, various viral and non-viral carriers have been ex-
tensively investigated. Compared with the viral carrier, although a non-viral one is
less effective in term of gene transfection, it has superior safety profiles and low im-
munogenicity. Therefore, it a great challenge to develop effective non-viral carriers
for successful gene therapy.
Among the non-viral carriers, cationic polymers have attracted great attention,
since they can complex with plasmid DNA through the electrostatic interaction.
Moreover, their properties can easily be modified by changing the constituents
of monomer, controlling the polymerization conditions, or chemically introduc-
ing functional groups to the polymers. In addition, the composition can be easily
manipulated by the conjugation with a targeting ligand or intracellular trafficking
enhancer [17]. Polyethyleneimine (PEI) is one of the most promising non-viral
carriers because it shows a proton sponge effect [18]. However, for the clinical
application, PEI has some problems to be resolved, such as its biodegradability and
toxicity. Consider on biodegradability and toxicity, much attention has been given
to chitosan (CS), since it possesses interesting properties such as biocompatibil-
ity, biodegradability, film forming ability, gelation characteristics, and bioadhesion.
Moreover, because of its polycationic nature, CS has been extensively investigated
for carrier and delivery systems of negatively charged agents. CS is also a good
candidate for a plasmid DNA carrier that has been extensively investigated, but the
transfection efficiency is still low. It has been reported that the low ability of gene
transfection is due to the instability of CS–DNA complexes, which results in the
aggregation at the outside of cell membrane, and, consequently, poor cellular inter-
nalization [19]. Moreover, no buffering effect necessary for the endosomal escape
of complex is another disadvantage [20–22]. It is practically necessary to develop
a new non-viral carrier of gene transfection by combining the material advantage
of PEI and CS.

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207
In this study, CS/PEI nanoparticles were prepared by an emulsifier-free emul-
sion polymerization in the presence of PEI and chitosan. The PEI/CS weight ratio
in preparation was changed systematically to evaluate the effect on particle size,
surface charge and buffering capacity. Following the incubation with MSCs, the
level of gene expression and the expression period were evaluated and compared
with those of CS and PEI nanoparticles.
2. Materials and Methods
2.1. Materials
CS was purchased from Seafresh Chitosan Lab. (Thailand). The degree of deacety-
lation was ca. 85%, and the weight-average molecular weight of CS was ca.
45 × 103, determined by a viscometric method. Branch polyethyleneimine (PEI;
molecular weight 750 × 103) was purchased from Aldrich. Methyl methacrylate
(MMA) and t-butyl hydroperoxide (TBHP) were purchased from Fluka. TBHP was
used as obtained. MMA was purified to remove inhibitors using a column packed
with alumina adsorbent. PEI was diluted with deionized water to 10 wt% and care-
fully adjusted with HCl until the pH 7. Deionized (DI) and autoclaved water were
sterilized and used throughout the study. All other chemicals were commercially
available and of analytical grade.
2.2. Methods
2.2.1. Preparation of PEI-Introduced Chitosan Nanoparticles
The emulsifier-free emulsion polymerization was carried out by the aid of the
amine/TBHP initiating system as illustrated in Fig. 1. Both PEI and chitosan have
plenty of amine groups on their backbones capable of forming initiating system
with TBHP. The resulting radicals on the nitrogen atoms of amines were then
propagated with vinyl monomers. By using this polymerization method, core–shell
nanoparticles can be formed with PEI and chitosan as a shell and vinyl polymer as
a core [23]. The synthetic procedure was briefly described as follows: The poly-
merization was conducted in a 100-ml water-jacketed glass reactor equipped with a
reflux condenser, nitrogen inlet, thermostat water bath, and magnetic stirrer. A to-
tal reaction volume of 50 ml was generally used. CS (0.5 g), predissolved in 1%
acetic acid, and deionized water were mixed with PEI (10 wt%, pH 7) solution. The
weight ratio of CS to PEI was varied (0.5:0, 0.5:0.1, 0.5:0.3, 0.5:0.5 and 0:0.5). The
mixture was purged with nitrogen gas for 30 min. Water was pumped through the
jacketed reactor from a thermostat water bath, which was controlled at 80 ± 1◦C.
The MMA monomer (1 g) was added, followed by an addition of TBHP aqueous
solution (1 g, 5 × 10−3M) to initiate polymerization. Then, the polymerization
was continued for 2 h under nitrogen atmosphere. The homopolymer and unreacted
monomer were removed by Soxhlet extraction.
2.2.2. Determination of Monomer Conversion and Solid Content
The monomer conversion and solid content were determined gravimetrically. About

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N. Pimpha et al. / Journal of Biomaterials Science 21 (2010) 205–223
Figure 1. Scheme of emulsifier-free emulsion polymerization in the presence of different concentra-
tionsofPEImixedwithCSunderN2atmosphereat80◦Cfor2hforCS/PEInanoparticlespreparation.
2 g nanoparticle latex prepared was taken into a pre-weighed aluminum pan. Af-
ter complete evaporation in a fume-hood, the air-dried latex was further dried in
a vacuum oven at 70◦C. The monomer conversion was calculated from the weight
of monomers initially added and dried polymers obtained:
Conversion (%)
?Weight of dried latex−Weight of PEI and/or CS used
?Weight of dried latex
2.2.3. Nanoparticle Characterization
The hydrodynamic diameter and polydispersity index (PDI) of nanoparticles were
measured using a dynamic light scattering (DLS) method with a laser particle size
analyzer (Malvern Instruments, UK) at 25◦C. Then, the latex samples were dropped
into the sample chamber with continuous stirring until the suitable concentration
was attained, which was in the range of 1–5 wt%. The measurements were repeated
three times. The surface charge of nanoparticles was measured by electrophoretic
light scattering (ELS), using a zetasizer (NanoZS 4700, Malvern Instruments) in
1 mM NaCl solution at room temperature. The measurements were done at a wave-
length of 633 nm at 25◦C with a scattering angle of 90◦. The results reported were
the mean of three determinations. The morphology of nanoparticles was observed
with scanning electron microscope (SEM, S-2500, Hitachi, Japan). To prepare the
sample, 50 µl nanoparticle (10 mg/ml) dispersion was dropped on a cover glass and
dried in a dust-free environment. The dried specimens were stuck on the sample
=
Weight of monomer feed
?
×100,
Solid content (%) =
Weight of latex
?
×100.