Download pH-Responsive Calcium Phosphate- Polymer Nanoparticles as a

pH-Responsive Calcium PhosphatePolymer Nanoparticles as a Drug
Delivery System in Gene Therapy
Doerdelmann, Gregor (University of Duisburg-Essen)
Kozlova, Diana (University of Duisburg-Essen)
Matthias, Epple (University of Duisburg-Essen)
Introduction Calcium phosphate (CaP) as the
inorganic part of hard tissue is known for its
biocompatibility.1 In addition, CaP nanoparticles
have a high affinity to nucleic acids and are
efficiently taken up by cells and subsequently
dissolved in lysosomes at a pH below five.2
Therefore, CaP nanoparticles are a promising
delivery system for nucleic acids (e.g. siRNA or
DNA) in gene therapy. To additionally protect
nucleic acids that are adsorbed to the surface of
CaP nanoparticles from nucleases (RNase and
DNase), theses nanoparticles can be encapsulated
into a polymeric matrix. The pharmaceutical
polymer Eudragit® E100 is a copolymer based on
dimethylaminoethyl methacrylate (DMAM), butyl
methacrylate, and methyl methacrylate. Its cationic
character and solubility under acidic conditions
enhance the cellular uptake and the endosomal
escape by the proton sponge effect, respectively.3
The synthesis and characterization of calcium
phosphate nanoparticles loaded with siRNA and
encapsulated into a nanoparticulate matrix of
Eudragit® E100 is presented. In addition, the
efficiency of this nanoparticulate carrier system is
shown by in in vitro gene silencing experiments.
Materials and Methods
Calcium phosphate nanoparticles loaded with
nucleic acids were synthesized by a rapid
precipitation method. For the encapsulation of
these nucleic acid loaded calcium phosphate
nanoparticles into a matrix of Eudragit®-E100, a
water-in-oil-in-water (W1/O/W2) double emulsion
solvent evaporation method was used. The size and
surface charge of the resulting nanoparticles were
analyzed by scanning electron microscopy and
dynamic light scattering. The cellular uptake by
HeLa cells as well as the gene silencing efficiency in
an enhanced green fluorescent protein (eGFP)
expressing HeLa cell line were determined by
fluorescence microscopy. The cytotoxicity of the
nanoparticulate carrier system was analyzed by the
MTT-test.
Results
The applied W1/O/W2 double emulsion solvent
evaporation method yields spherical calcium
phosphate-E100 nanoparticles loaded with siRNA
with a diameter of 200 nm and a positive zeta
potential as shown by scanning electron microscopy
(SEM) and dynamic light scattering (DLS). Cellular
uptake studies showed that the nanoparticles are
efficiently taken up by HeLa cells with no cytotoxic
effects. Gene silencing experiments on enhanced
green fluorescent protein (eGFP) expressing HeLaCells showed an effective knock down of eGFP.
Discussion and Conclusion
The W1/O/W2 double emulsion solvent evaporation
technique proved to be suitable to synthesize
spherical cationic calcium phosphate Eudragit®
E100-nanoparticles loaded with siRNA. Cellular
uptake studies showed that these particles are
capable of inducing the proton sponge effect while
being not cytotoxic as shown by the MTT-test.
Furthermore, gene silencing experiments on HeLaCells expressing enhanced green fluorescent protein
(eGFP) showed an effective gene silencing. Thus,
this new nanoparticulate carrier system is a
promising delivery agent for nucleic acids in the
transfection of cells or gene therapy without the
cytotoxic effects of commonly used cationic
polymers (e.g. polyethyleneimine).
Figure 1: Synthesis of nucleic acid-loaded calcium phosphate nanoparticles (A) and their
encapsulation into a Eudragit®-E100 matrix by the W1/O/W2-emulsion technique (B). Structure and
composition
of
a
Eudragit®-E100
particle
which
carries
drug-loaded
calcium
phosphate
nanoparticles (C).
Figure 2: Gene silencing experiments on HeLa-Cells (left) expressing the enhanced green
fluorescent protein (eGFP). After transfection with calcium phosphate/E100 nanoparticles, which
carry anti-eGFP siRNA, the eGFP encoding gene is down regulated effectively (right).
References
1. Dorozhkin, S. V.; Epple, M. Biological and Medical Significance of Calcium Phosphates,
Angew. Chem. Int. Ed. 2002, 41, 3130-3146. 2. Neumann, S.; Kovtun, A.; Dietzel, I. D.;
Epple, M.; Heumann, R. The use of size-defined DNA-functionalized calcium phosphate
nanoparticles to minimise intracellular calcium disturbance during transfection,
Biomaterials. 2009, 30, 6794-6802. 3. Nel, A. E.; MÁ¤dler, L.; Velegol, D.; Xia, T.; Hoek, E.
M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Understanding
biophysicochemical interactions at the nano-bio interface, Nat. Mater. 2009, 8, 543-557.