Professional Documents
Culture Documents
Received 14 March 2005; received in revised form 6 June 2005; accepted 8 June 2005
Abstract
This work investigates the polyanion initiated gelation process in fabricating chitosan–TPP (tripolyphosphate) nanoparticles in the size
range of 100–250 nm intended to be used as carriers for the delivery of gene or protein macromolecules. It demonstrates that ionic gelation
of cationic chitosan molecules offers a flexible and easily controllable process for systematically and predictably manipulating particle size
and surface charge which are important properties in determining gene transfection efficacy if the nanoparticles are used as non-viral vectors
for gene delivery, or as delivery carriers for protein molecules. Variations in chitosan molecular weight, chitosan concentration, chitosan to
TPP weight ratio and solution pH value were examined systematically for their effects on nanoparticle size, intensity of surface charge, and
tendency of particle aggregation so as to enable speedy fabrication of chitosan nanoparticles with predetermined properties. The chitosan–TPP
nanoparticles exhibited a high positive surface charge across a wide pH range, and the isoelectric point (IEP) of the nanoparticles was found
to be at pH 9.0. Detailed imaging analysis of the particle morphology revealed that the nanoparticles possess typical shapes of polyhedrons
(e.g., pentagon and hexagon), indicating a similar crystallisation mechanism during the particle formation and growth process. This study
demonstrates that systematic design and modulation of the surface charge and particle size of chitosan–TPP nanoparticles can be readily
achieved with the right control of critical processing parameters, especially the chitosan to TPP weight ratio.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Chitosan nanoparticles; Nanoparticle surface charge; Nanoparticle morphology; Ionic gelation; Gene delivery
0927-7765/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2005.06.001
66 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
‘complexes’. The simplicity of chitosan–DNA complexes is tion under mild conditions; homogeneous and adjustable size
both an advantage and a drawback. Though such complexes and a positive surface charge that can be easily modulated
are extremely easy to synthesize, the fact remains that their and a great capacity for the association of peptides, proteins,
transfection efficacy is significantly below that of cationic oligonucleotides, and plasmids [11].
liposomes in vitro and viral vectors in vivo. Aside from Therefore, ability to control and modulate the properties
N/P ratio and chitosan molecular weight, there remain of chitosan–TPP nanoparticles, most importantly particle size
few parameters in the synthesis protocol which can be and density of surface charge, is central in determining gene
modulated in an effort to augment transfection. To address transfection efficiency. It is important that these characteris-
this issue, investigators sought to develop more sophisticated tic properties be predictably produced and easily modulated
DNA-loaded chitosan nanoparticles. in a flexible and reliable nano fabrication process with high
The application of DNA–chitosan nanoparticles has yield and particle stability. It is therefore the focus of this
advanced in vitro DNA transfection research, and data have paper to report on how systematically manipulating process-
been accumulating that shows their usefulness for gene ing parameters in the TPP initiated chitosan gelation to obtain
delivery [7,8]. The therapeutic efficacy of the nanoparticles predictable and optimal nanoparticle properties for desired
could be due to their ability to protect the therapeutic agent applications in relation to gene/protein delivery.
from degradation due to lysosomal enzymes.
Due to their sub-cellular and sub-micron size,
chitosan–TPP nanoparticles can penetrate deep into tissues 2. Materials and experimental methods
through fine capillaries, cross the fenestration present in the
2.1. Materials
epithelial lining (e.g., liver) [9]. This allows efficient delivery
of therapeutic agents to target sites in the body. Also, by Three different molecular weight chitosan, derived from
modulating nanoparticles characteristics, such as enzymatic crab shell, in the form of fibrils flakes were obtained
degradation rate, size and surface charge density, one can from Sigma–Aldrich [Catalogue No. LMW 448869, MMW
control the release of a therapeutic agent from nanoparticles 448877, HMW 419419]. The degree of deacetylation for the
to achieve desired therapeutic level in target tissue for low molecular weight chitosan (LMW Chitosan), medium
required duration for optimal therapeutic efficacy [6]. molecular weight chitosan (MWM chitosan) and high molec-
The major drawback associated with using chitosan as ular weight chitosan (HMW chitosan) is 86.6%, 84.7%,
non-viral gene delivery system is the relatively low transfec- and 82.5%, respectively. Sodium Tripolyphosphate was pur-
tion rate in comparison to viral vectors, even though the later chased from Sigma–Aldrich Chemical Co. Ltd. All other
has its own limitations in patient safety, difficulty in scale- reagents used were of analytical grade.
up production, and possible toxicity, immune responses, and
inflammatory responses. It is understood that transfection 2.2. Purification of chitosan
efficacy of cationic polymers depends primarily on: (i) parti-
cle size, which determines their intracellular uptake, different Since medical applications of animal derived biomateri-
pathways of their uptake, intracellular trafficking and sorting als entail an inherent risk of protein contamination which has
into different intracellular compartments, and (ii) the inten- in recent years aroused great awareness and anxiety among
sity of particle surface charge which influence their ability to the public, drug companies, and the industry regulators, it
efficiently condensate DNA and interact with cell. is of utmost importance to ensure that chitosan intended for
Stable and reproducible chitosan nanoparticles were in medical applications is of the highest purity and free of pro-
early days formulated via chemical cross-linking in water- tein contamination. It is therefore decided to further purify
in-oil emulsion system for entraping and delivering drugs the purchased chitosan materials and examine whether there
[10]. However, the negative effects of cross-linking agents, are changes in chemical as well as physical properties before
e.g., glutaraldehyde, on cell viability and the integrity of and after purification. The origin and purity of purchased chi-
macromolecular drugs led to the development of prepara- tosan material depends on its source, season, and conditions
tion method under mild conditions. Preparation methods by of the chemical deacetylation process, which may vary across
ionically cross-linking cationic chitosan with specific polyan- different suppliers. Further purification process is crucial to
ions were particularly successful as, aside from its com- ensure that the starting chitosan material for nanoparticle
plexation with negatively charged polymers, chitosan has fabrication possesses the highest purity and integrity. In this
the ability to gel spontaneously on contact with multivalent work, purchased chitosan materials were subjected to a vig-
polyanions due to the formation of inter- and intramolecular orous purification process which involved mixing the solid
cross-linkage mediated by these polyanions. Among some chitosan flakes in 1 M NaOH solution, allowing 1 g of chi-
polyanions investigated, tripolyphosphate (TPP) is the most tosan for 10 ml NaOH solution. This solid–liquid mixture was
popular because of its non-toxic property and quick gelling heated and continuously stirred for 2 h at 70 ◦ C, and then fil-
ability. The chitosan–TPP nano system exhibits some attrac- tered using a Buchner funnel. Chitosan was insoluble in the
tive features which render them promising carriers for the caustic solution, and the recovered flakes were washed thor-
delivery of macromolecules. These features include forma- oughly and dried at 40 ◦ C for 12 h.
Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73 67
The NaOH treated chitosan flakes were dissolved in 0.1 M Netherlands). One drop of dilute chitosan–TPP nanoparti-
acetic acid solution which was filtered using a filter paper cles solution was syringe placed on a carbon film 300 mesh
to remove residues of insoluble particles. One molar NaOH copper grid, allowing to sit until air-dried. The sample was
solution was used to adjust pH value of the filtrate to pH 8.0, stained with 1 M uranyl acetate solution for 5 s at 7 ◦ C before
resulting in purified chitosan in the form of white precipi- viewing on the TEM.
tates. The precipitated chitosan was washed thoroughly using
deionized water, and the product was vacuum-dried at room
temperature for 24 h. The dried samples were used for FT-IR 3. Results and discussion
analysis and preparation of the chitosan–TPP nanoparticles.
3.1. Purification and characterisation of supplied
2.3. Preparation of chitosan–TPP nanoparticles chitosan material
Chitosan solutions of different concentration and molecu- FT-IR was used to identify if there were variations in
lar weight were prepared by dissolving purified chitosan with chemical functional groups present at the surface of chi-
sonication in 1% (w/v) acetic acid solution until the solution tosan samples of different molecular weight, and to deter-
was transparent. Once dissolved, the chitosan solution was mine variations among purified and unpurified chitosan sam-
diluted with deionized water to produce chitosan solutions ples. By comparing the characteristic transmittance spec-
of different concentrations at 0.05%, 0.10%, 0.15%, 0.20%, trum of different chitosan samples, it is possible to ascertain
0.25%, and 0.30% (weight/volume). Tripolyphosphate was changes in the constituent surface functional groups (e.g.,
dissolved in deionized water at the concentration 0.7 mg/ml. NH2 , CH2 NH) during the purification process, an indi-
The chitosan solution was flush mixed with an equal vol- cation of removal of impurities from the purchased chitosan
ume of TPP solution and the formation of chitosan–TPP material.
nanoparticles started spontaneously via the TPP initiated Fig. 1 compares the transmittance spectrum of purified
ionic gelation mechanism. The nanoparticles were formed HMW chitosan with the original supplied materials. The con-
at selected chitosan to TPP weight ratios of 3:1, 4:1, 5:1, 6:1 trasting difference in the spectrum evidently demonstrates
and 7:1. The nanoparticle suspensions were gently stirred for the changes in surface chemistry of the original supplied chi-
60 min at room temperature before being subjected to further tosan material after purification, indicating possible removal
analysis and applications. of impurities, such as protein molecules and pigments. No
attempts were made in this paper to identify what partic-
2.4. FT-IR ular chemical bonds are associated with the spectrum peaks
which require additional verification beyond the scope of this
In FT-IR analysis of both purified and raw chitosan sam- study. The variation in the transmittance spectrum for LMW
ples, transmittance spectra were obtained using a Perkin- and MMW chitosan samples was much less pronounced than
Elmer FT-IR spectrometer (SPECTRUM 1000) fitted with the HMW chitosan before and after purification. Fig. 2 com-
an attenuated total reflectance mode (ATR) cell. The equip- pares the FT-IR transmittance spectrum of purified chitosan
ment was positioned in a laboratory maintained at 25 ± 1 ◦ C. samples of different molecular weight. The figure reveals
A small chitosan sample (7.0–9.0 mg) was placed on a NaCl variations in corresponding peak values of the same func-
plate and subjected to light within the infrared spectrum. The tional groups among different molecular weight samples, but
instrument operated with a resolution of 4 cm−1 and 128 not significant variations in presence of the functional groups
scans were collected for each sample. The IR absorbency themselves.
scans were analysed between 700 and 4000 cm−1 for changes
in the intensity of the sample peaks.
Table 2
Measured particles size and zeta potential with different chitosan molecular weight and concentration
LMW chitosan Size (nm) Zeta (mv) MMW chitosan Size (nm) Zeta (mv) HMW chitosan Size (nm) Zeta (mv)
(%) (w/v) (%) (w/v) (%) (w/v)
0.05 143.2 49.2 0.05 156.1 46.8 0.05 162.7 41.3
0.10 152.1 46.8 0.10 163.7 44.4 0.10 176.1 37.4
0.15 159.2 45.6 0.15 170.7 40.3 0.15 208.9 36.9
0.20 172.8 44.3 0.20 181.5 39.8 0.20 216.8 35.1
0.25 181.9 42.7 0.25 192.2 37.8 0.25 234.2 34.1
0.30 189.6 40.7 0.30 209.8 35.3 0.30 257.0 33.2
0.35 273.2 39.4 0.35 310.2 33.8 0.35 362.3 32.6
0.40 387.2 37.1 0.40 427.5 32.7 0.40 478.0 31.8
0.45 519.6 36.8 0.45 546.3 32.1 0.45 566.6 31.0
0.50 604.3 34.2 0.50 654.3 30.4 0.50 697.3 29.5
0.55 692.1 33.3 0.55 721.5 29.0 0.55 748.5 27.8
0.60 726.6 32.7 0.60 783.2 28.6 0.60 828.0 26.3
0.65 740.6 32.1 0.65 848.3 27.5 0.65 898.5 25.9
0.70 846.7 30.8 0.70 881.0 25.3 0.70 976.8 23.2
0.75 858.9 30.0 0.75 936.8 25.1 0.75 1654.9 15.9
0.80 893.6 28.5 0.80 988.6 24.8 0.80 2400.5 10.6
0.85 908.6 26.7 0.85 2371.0 15.2 0.85 5116.8 8.0
0.90 938.7 26.2 0.90 2742.7 12.2 0.90 – –
0.95 998.6 24.3 0.95 4276.9 7.6 0.95 – –
1.00 1819.3 13.5 1.00 – – 1.00 – –
1.10 2059.0 11.2 1.10 – – 1.10 – –
1.20 2851.6 7.6 1.20 – – 1.20 – –
Chitosan to TPP mass ratio = 4:1, T = 20 ± 1 ◦ C, pH = 5.0.
72 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
Fig. 11. TEM image of an aggregate of four single LMW chitosan–TPP Fig. 13. TEM image of a LMW chitosan–TPP nanoparticles incorporating
nanoparticles with distinctive polyhedron shapes. BSA molecules.
Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73 73
doubling the size of chitosan–TPP particles. The BSA incor- [3] Z. Cui, R.J. Mumper, Chitosan-based nanoparticles for topical
porated particles possessed a typical spherical shape and genetic immunization, J. Control. Release 75 (2001) 409–419.
smooth surface, which confirms the findings by Xu and Du [4] L. Illum, I. Jabbal-Gill, M. Hinchcliffe, A.N. Fisher, S.S. Davis,
Chitosan as a novel nasal delivery system for vaccines, Adv. Drug
[23], and Janes et al. [1]. Future work will investigate the Deliv. Rev. 51 (2001) 81–96.
encapsulation mechanism and efficiency of protein molecules [5] K.A. Mislick, J.D. Baldeschwieler, Evidence for the role of proteo-
in the ionic initiated chitosan–TPP nanoparticle formation glycans in cation-mediated gene transfer, Proc. Natl. Acad. Sci. USA
process, and the release kinetics of protein molecules from 93 (1996) 12349–12354.
the particle. [6] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug and
gene delivery to cells and tissue, Adv. Drug Deliv. Rev. 55 (2003)
329–347.
[7] K. Corsi, F. Chellat, L. Yahia, J.C. Fernandes, Mesenchymal stem
4. Conclusion cells, MG63 and HEK293 transfection using chitosan–DNA nanopar-
ticles, Biomaterials 24 (2003) 1255–1264.
The formation of high yield chitosan–TPP nanoparticles [8] K. Romoren, B.J. Thu, O. Evensen, Immersion delivery of plasmid
with predetermined nano-metric size and surface charge den- DNA. II. A study of the potentials of a chitosan based delivery sys-
tem in rainbow trout (Oncorhynchus mykiss) fry, J. Control. Release
sity can be simply manipulated and controlled by varying the 85 (2002) 215–225.
key processing conditions of chitosan concentration, chitosan [9] S.V. Vinagradov, T.K. Bronich, A.V. Kabanov, Nanosized cationic
to TPP weight ratio, and solution pH value. Within the tested hydrogels for drug delivery: preparation, properties and interactions
range of conditions, the increase in particle size and parti- with cells, Adv. Drug Deliv. Rev. 54 (2002) 223–233.
cle zeta potential showed a simple linear relationship with [10] Y. Ohya, M. Shiratani, H. Kobayashi, T. Ouchi, Release behav-
ior of 5-fluorouracil from chitosan-gel nanospheres immobilizing
increasing chitosan to TPP weight ratio, but the zeta poten- 5-fluorouracil coated with polysaccharides and their cell specific
tial at fixed chitosan to TPP ratio showed a linear decrease cytotoxicity, Pure Appl. Chem. A 31 (1994) 629–642.
with increasing chitosan concentration. Solution pH value [11] X.Z. Shu, K.J. Zhu, A new approach to prepare tripolyphos-
and chitosan concentration also had profound influence on the phate/chitosan complex beads for controlled drug delivery, Int. J.
stability of the nanoparticle system, and the critical chitosan Pharm. 201 (2000) 51–58.
[12] C. Song, V. Labhasetwar, X. Cui, T. Underwood, R.J. Levy, Arterial
concentrations for spontaneous formation of large particle uptake of biodegradable nanoparticles for intravascular local drug
aggregates at pH 5 were found to be 0.65%, 0.25%, 0.15% delivery: results with an acute dog model, J. Control. Release 54
(w/v) at pH 4.0, and 1.00%, 0.85%, 0.75% (w/v) at pH 5.0 (1998) 201–211.
for LMW, MMW and HMW chitosan, respectively. The iso- [13] M.P. Desai, V. Labhasetwar, G.L. Amidon, R.J. Levy, Gastrointesti-
electric point of the chitosan–TPP nanoparticles was found nal uptake of biodegradable microparticles: effect of particle size,
Pharm. Res. 13 (1996) 1838–1845.
at around pH 9.0. [14] S. Prabha, W.Z. Zhou, J. Panyam, V. Labhasetwar, Size depen-
Morphological study of the nanoparticles formed under dency of nanoparticles-mediated gene transfection: Studies with
different conditions revealed the formation of regu- fractionnated nanoparticles, Int. J. Pharm. 244 (2002) 105–
larly shaped polyhydron particles, an indication of semi- 115.
crystallisation mechanism during the particle formation and [15] W. Paul, C. Sharma, Chitosan a drug carrier for the 21st century
S.T.P, Pharm. Sci. 10 (2000) 5–22.
growth, suggesting the particles were of compact structure [16] V. Dodane, D. Vilivalam, Pharmaceutical applications of chitosan,
with orderly molecular arrangement, the discovery of which Pharm. Sci. Technol. Today 16 (1998) 246–253.
bears important implications on gene/protein encapsulation [17] P. Calvo, J.L. Vila-Jato, M.J. Alonso, Effect of lysozyme on the
and release mechanisms. stability of polyester nanocapsules and nanoparticles: stabilization
approaches, Biomaterials 18 (1997) 1305–1310.
[18] F.-L. Mi, H.-W. Sung, S.-S. Shyu, Synthesis and characterization of
biodegradable TPP/genipin co-crosslinked chitosan gel beads, Poly-
Acknowledgement mer 44 (2003) 6521–6530.
[19] K.W. Leong, H.Q. Mao, V.L. Truong-Le, DNA-polycation
To the European Social Funding programme for providing nanospheres as non-viral gene delivery vehicles, J. Control. Release
a scholarship to Colette Cochrane for this project. 53 (1998) 183–193.
[20] J.A. Ko, H.J. Park, S.J. Hwang, Preparation and characterization
of chitosan microparticles intended for controlled drug delivery, J.
Pharm. 249 (2002) 165–174.
References [21] X.Z. Shu, K.J. Zhu, Novel PH–sensitive citrate cross-linked chi-
tosan film for controlled drug release, Int. J. Pharm. 212 (2001)
[1] K.A. Janes, P. Calvo, M.J. Alonso, Polysaccharide colloidal particles 19–28.
as delivery systems for macromolecules, Adv. Drug Deliv. Rev. 47 [22] K. Makino, H. Ohshima, T. Kondo, Transfer of protons from bulk
(2001) 83–97. solution to the surface of poly(l-lactide) microcapsules, J. Microen-
[2] S.C.W. Richardson, H.V.J. Kolbe, R. Duncan, Potential of low molec- capsul. 3 (1986) 195–202.
ular mass chitosan as a DNA delivery system: biocompatibility, body [23] Y. Xu, Y. Du, Effect of molecular structure of chitosan on pro-
distribution and ability to complex and protect DNA, Int. J. Pharm. tein delivery properties of chitosan nanoparticles, Int. J. Pharm. 250
178 (1999) 231–243. (2003) 215–226.