or gene delivery, or as delivery carriers for protein molecules. Variations in chitosan molecular weight, chitosan concentration, cPP weight ratio and solution pH value were examined systematically for their effects on nanoparticle size, intensity of surface c
endency of particle aggregation so as to enable speedy fabrication of chitosan nanoparticles with predetermined properties. The canoparticles exhibited a high positive surface charge across a wide pH range, and the isoelectric point (IEP) of the nanoparticle
o be at pH 9.0. Detailed imaging analysis of the particle morphology revealed that the nanoparticles possess typical shapes of pe.g., pentagon and hexagon), indicating a similar crystallisation mechanism during the particle formation and growth process.emonstrates that systematic design and modulation of the surface charge and particle size of chitosan–TPP nanoparticles cachieved with the right control of critical processing parameters, especially the chitosan to TPP weight ratio.2005 Elsevier B.V. All rights reserved.
Chitosan is a non-toxic biodegradable polycationic poly-er with low immunogenicity. It has been extensively
nvestigated for formulating carrier and delivery systemsor therapeutic macrosolutes, particularly genes and proteinolecules primarily because positively charged chitosan cane easily complexed with negatively charged DNAs and pro-
eins[1,2]. Chitosan can effectively bind DNA and protectt from nuclease degradation[3,4]. It has advantages of notecessitating sonication and organic solvents for its prepara-
ion, therefore minimizing possible damage to DNA duringhe complexation process.
Furthermore, there is evidence demonstrating that catpolymers play an important role in both membrane adhe[5] and lysosomal escape[6] of the encapsulated DNA, prviding a potential explanation for the superiority of polymmediated gene transfer relative to naked DNA administrain many applications. These hybrid DNA–chitosan systcan be classified into two categories which differ in thmechanism of formation and morphology: complexesnanospheres.
Gentle mixing, followed by incubation, of chitosand DNA solutions generated ‘broad’ distributionschitosan–DNA particulate complexes with mean sbetween 100 and 600 nm, depending on the moleweight of the chitosan. Since particle formation was elicsolely by the tropism of the two oppositely charged mamolecules for one another, these particles were te
66 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
‘complexes’. The simplicity of chitosan–DNA complexes isboth an advantage and a drawback. Though such complexesare extremely easy to synthesize, the fact remains that theirtransfection efficacy is significantly below that of cationicliposomes in vitro and viral vectors in vivo. Aside fromN/P ratio and chitosan molecular weight, there remainfew parameters in the synthesis protocol which can bemodulated in an effort to augment transfection. To addressthis issue, investigators sought to develop more sophisticatedDNA-loaded chitosan nanoparticles.
The application of DNA–chitosan nanoparticles hasadvanced in vitro DNA transfection research, and data havebeen accumulating that shows their usefulness for genedelivery [7,8]. The therapeutic efficacy of the nanoparticlescould be due to their ability to protect the therapeutic agentfrom degradation due to lysosomal enzymes.
Due to their sub-cellular and sub-micron size,chitosan–TPP nanoparticles can penetrate deep into tissuesthrough fine capillaries, cross the fenestration present in theepithelial lining (e.g., liver)[9]. This allows efficient deliveryof therapeutic agents to target sites in the body. Also, bymodulating nanoparticles characteristics, such as enzymaticdegradation rate, size and surface charge density, one cancontrol the release of a therapeutic agent from nanoparticlesto achieve desired therapeutic level in target tissue forrequired duration for optimal therapeutic efficacy[6].
n asn fec-t laterh le-u , andi ctione rti-c rentp tingi en-s y toe
re ine ter-i ugs[ nts,e ofm ara-t s byi an-i om-p hast lentp ularc omep mostp llinga ttrac-t thed rma-
tion under mild conditions; homogeneous and adjustable sizeand a positive surface charge that can be easily modulatedand a great capacity for the association of peptides, proteins,oligonucleotides, and plasmids[11].
Therefore, ability to control and modulate the propertiesof chitosan–TPP nanoparticles, most importantly particle sizeand density of surface charge, is central in determining genetransfection efficiency. It is important that these characteris-tic properties be predictably produced and easily modulatedin a flexible and reliable nano fabrication process with highyield and particle stability. It is therefore the focus of thispaper to report on how systematically manipulating process-ing parameters in the TPP initiated chitosan gelation to obtainpredictable and optimal nanoparticle properties for desiredapplications in relation to gene/protein delivery.
2. Materials and experimental methods
2.1. Materials
Three different molecular weight chitosan, derived fromcrab shell, in the form of fibrils flakes were obtainedfrom Sigma–Aldrich [Catalogue No. LMW 448869, MMW448877, HMW 419419]. The degree of deacetylation for thelow molecular weight chitosan (LMW Chitosan), mediumm lec-u %,a pur-c err
2
teri-a hasi mongt rs, iti d form pro-t rifyt therea eforea chi-t ditionso crossd l toe rticlef thisw vig-o olidc hi-t ash -t thec thor-o
The major drawback associated with using chitosaon-viral gene delivery system is the relatively low trans
ion rate in comparison to viral vectors, even though theas its own limitations in patient safety, difficulty in scap production, and possible toxicity, immune responses
nflammatory responses. It is understood that transfefficacy of cationic polymers depends primarily on: (i) pale size, which determines their intracellular uptake, diffeathways of their uptake, intracellular trafficking and sor
nto different intracellular compartments, and (ii) the intity of particle surface charge which influence their abilitfficiently condensate DNA and interact with cell.
Stable and reproducible chitosan nanoparticles wearly days formulated via chemical cross-linking in wa
n-oil emulsion system for entraping and delivering dr10]. However, the negative effects of cross-linking age.g., glutaraldehyde, on cell viability and the integrityacromolecular drugs led to the development of prep
ion method under mild conditions. Preparation methodonically cross-linking cationic chitosan with specific polyons were particularly successful as, aside from its clexation with negatively charged polymers, chitosan
he ability to gel spontaneously on contact with multivaolyanions due to the formation of inter- and intramolecross-linkage mediated by these polyanions. Among solyanions investigated, tripolyphosphate (TPP) is theopular because of its non-toxic property and quick gebility. The chitosan–TPP nano system exhibits some a
ive features which render them promising carriers forelivery of macromolecules. These features include fo
olecular weight chitosan (MWM chitosan) and high molar weight chitosan (HMW chitosan) is 86.6%, 84.7nd 82.5%, respectively. Sodium Tripolyphosphate washased from Sigma–Aldrich Chemical Co. Ltd. All otheagents used were of analytical grade.
.2. Purification of chitosan
Since medical applications of animal derived biomals entail an inherent risk of protein contamination which
n recent years aroused great awareness and anxiety ahe public, drug companies, and the industry regulatos of utmost importance to ensure that chitosan intende
edical applications is of the highest purity and free ofein contamination. It is therefore decided to further puhe purchased chitosan materials and examine whetherre changes in chemical as well as physical properties bnd after purification. The origin and purity of purchased
osan material depends on its source, season, and conf the chemical deacetylation process, which may vary aifferent suppliers. Further purification process is cruciansure that the starting chitosan material for nanopa
abrication possesses the highest purity and integrity. Inork, purchased chitosan materials were subjected to arous purification process which involved mixing the shitosan flakes in 1 M NaOH solution, allowing 1 g of cosan for 10 ml NaOH solution. This solid–liquid mixture weated and continuously stirred for 2 h at 70◦C, and then fil
ered using a Buchner funnel. Chitosan was insoluble inaustic solution, and the recovered flakes were washedughly 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 Macetic acid solution which was filtered using a filter paperto remove residues of insoluble particles. One molar NaOHsolution was used to adjust pH value of the filtrate to pH 8.0,resulting in purified chitosan in the form of white precipi-tates. The precipitated chitosan was washed thoroughly usingdeionized water, and the product was vacuum-dried at roomtemperature for 24 h. The dried samples were used for FT-IRanalysis and preparation of the chitosan–TPP nanoparticles.
2.3. Preparation of chitosan–TPP nanoparticles
Chitosan solutions of different concentration and molecu-lar weight were prepared by dissolving purified chitosan withsonication in 1% (w/v) acetic acid solution until the solutionwas transparent. Once dissolved, the chitosan solution wasdiluted with deionized water to produce chitosan solutionsof different concentrations at 0.05%, 0.10%, 0.15%, 0.20%,0.25%, and 0.30% (weight/volume). Tripolyphosphate wasdissolved in deionized water at the concentration 0.7 mg/ml.
The chitosan solution was flush mixed with an equal vol-ume of TPP solution and the formation of chitosan–TPPnanoparticles started spontaneously via the TPP initiatedionic gelation mechanism. The nanoparticles were formedat selected chitosan to TPP weight ratios of 3:1, 4:1, 5:1, 6:1and 7:1. The nanoparticle suspensions were gently stirred for6 rthera
2
am-p rkin-E itha uip-mA aClp Theis encys si
2c
ydis-p iclesw tru-m
2
clesw sionE o.,
Netherlands). One drop of dilute chitosan–TPP nanoparti-cles solution was syringe placed on a carbon film 300 meshcopper grid, allowing to sit until air-dried. The sample wasstained with 1 M uranyl acetate solution for 5 s at 7◦C beforeviewing on the TEM.
3. Results and discussion
3.1. Purification and characterisation of suppliedchitosan material
FT-IR was used to identify if there were variations inchemical functional groups present at the surface of chi-tosan samples of different molecular weight, and to deter-mine variations among purified and unpurified chitosan sam-ples. By comparing the characteristic transmittance spec-trum of different chitosan samples, it is possible to ascertainchanges in the constituent surface functional groups (e.g.,
NH2, CH2NH) during the purification process, an indi-cation of removal of impurities from the purchased chitosanmaterial.
Fig. 1 compares the transmittance spectrum of purifiedHMW chitosan with the original supplied materials. The con-trasting difference in the spectrum evidently demonstratest chi-t ovalo . Noa rtic-u eaksw thiss Wa thantp osans ealsv unc-t , butn upst
F rifiedH
0 min at room temperature before being subjected to funalysis and applications.
.4. FT-IR
In FT-IR analysis of both purified and raw chitosan sles, transmittance spectra were obtained using a Pelmer FT-IR spectrometer (SPECTRUM 1000) fitted wn attenuated total reflectance mode (ATR) cell. The eqent was positioned in a laboratory maintained at 25± 1◦C.small chitosan sample (7.0–9.0 mg) was placed on a N
late and subjected to light within the infrared spectrum.nstrument operated with a resolution of 4 cm−1 and 128cans were collected for each sample. The IR absorbcans were analysed between 700 and 4000 cm−1 for change
n the intensity of the sample peaks.
.5. Measurement of size and zeta potential ofhitosan–TPP nanoparticles
Measurement of physical size, zeta potential and polersity (size distribution) of the chitosan–TPP nanopartere performed using a 3000HSA Zetasizer (Malvern Insents, England).
.6. Morphology observation
The morphological characteristics of the nanopartiere examined using a high resolution TEM (Transmislectron Microscope) machine (Tecnai F-20, Phillips C
he changes in surface chemistry of the original suppliedosan material after purification, indicating possible remf impurities, such as protein molecules and pigmentsttempts were made in this paper to identify what palar chemical bonds are associated with the spectrum phich require additional verification beyond the scope oftudy. The variation in the transmittance spectrum for LMnd MMW chitosan samples was much less pronounced
he HMW chitosan before and after purification.Fig. 2com-ares the FT-IR transmittance spectrum of purified chitamples of different molecular weight. The figure revariations in corresponding peak values of the same fional groups among different molecular weight samplesot significant variations in presence of the functional gro
hemselves.
ig. 1. Representative FT-IR transmittance spectra of purified and unpuMW chitosan samples.
68 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
The purification study demonstrates convincingly that fur-ther purification of supplied chitosan materials is essentialespecially with high molecular weight chitosans.
3.2. Modulation of particle size
Particle size is one of the most significant determinantin mucosal and epithelial tissue uptake of nanoparticles andin the intracellular trafficking of the particles[6]. Smallersize nanoparticles (∼100 nm) demonstrated more than 3-fold greater arterial uptake compared to larger nanoparticles(∼275 nm) in an ex vivo canine carotid artery model[12,13]as the smaller nanoparticles were able to penetrate throughoutthe sub-mucosal layers while the larger size micron-particleswere predominantly localized in the epithelial lining. Prabhaet al.[14] investigated the gene transfection levels of differ-ent size fractions of PLGA nanoparticles and found that thelower size nanoparticle fraction produced a 27-fold highertransfection in COS-7 cells and 4-fold higher transfection inHEK 293 cells for the same dose of nanoparticles. These stud-ies also suggested that uniform particle size distribution areimportant to enhance the nanoparticle-mediated gene expres-sion.
Chitosan’s ability of quick gelling on contact with polyan-ions relies on the formation of inter- and intramolecularc ticlesa sans TPPp distri-b ly onc ing,i
arti-c ratest stlya , andt rela-t
Fig. 3. Effect of chitosan concentration from 0.05% to 0.30% (w/v) on par-ticles size with three different chitosan molecular weight. Chitosan to TPPmass ratio = 5:1,T= 20± 1◦C, pH 5.0.
The effect of chitosan to TPP weight ratio on particle sizewas also very prominent (Fig. 4), showing a linear increase ofsize with increasing chitosan to TPP weight ratio within thetested chitosan to TPP ratio range. These linear relationshipsprovide a simple processing window for manipulating andoptimising the nano size for intended applications.
3.3. Modulation of particle surface charge
Chitosan has a rigid crystalline structure through inter- andintra- molecular hydrogen bonding. Chitosan molecules inaqueous solutions adopt extended conformation with a moreflexible chain because of the electrostatic charge repulsionbetween the chains. When chitosan and TPP were mixed witheach other in dilute acetic acid, they spontaneously formedcompact nano complexes with an overall positive surfacecharge, and the density of the surface charge is reflected bymeasured zeta potential values.
F arti-c ,T
ross-linkages mediated by these polyanions. Nanoparre formed immediately upon mixing of TPP and chitoolutions as molecular linkages were formed betweenhosphates and chitosan amino groups. Size and sizeution of the chitosan–TPP nanoparticles depend largeoncentration, molecular weight, and conditions of mix.e., stirring or sonication.
Fig. 3shows the effect of chitosan concentration on ples size at three different molecular weight. It demonsthat the size of HMW chitosan nanoparticles was moffected by the increased chitosan solution concentration
he increase in size with concentration showed a linearionship within the tested range.
ig. 4. Effect of chitosan to TPP mass ratio from 3:1 to 7:1 on ples size with three different chitosan molecular weight.c= 0.50% (w/v)= 20±1◦C, pH 5.0.
Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73 69
Fig. 5. Effect of chitosan to TPP mass ratio on particle zeta potential withthree different chitosan molecular weight.c= 0.50% (w/v),T= 20± 1◦C,pH 5.0.
It was reported that the ability of nanoparticles to escapethe endo-lysosomes was dependent on the surface charge ofthe nanoparticles[15,16]. Nanoparticles which show transi-tion in their surface charge from anionic at pH 7 to cationicin the acidic endosomal pH (pH 4–5) were found to escapethe endosomal compartment whereas the nanoparticles whichremain negatively charged at pH 4–5 were retained mostlyin the endosomal compartment. Thus, by varying the surfacecharge, one could potentially be able to direct the nanoparti-cles either to lysosomes or to cytoplasm[6]. The efficacy ofnanoparticles as drug carriers is also closely related to theirinteraction, predominantly influenced by surface charge, withproteins and enzymes in different body fluids. Calvo et al.[17]analysed the interaction phenomenon between lysozyme, apositively charged enzyme that is highly concentrated inmucosas, and poly-�-caprolactone coated nanoparticles, andfound that the interaction of lysozyme with the nanoparticlesand their consequent degradation was highly dependent ontheir surface charge.
The zeta potential of chitosan–TPP nanoparticlesincreased linearly with increasing chitosan to TPP weightratio from 3:1 to 7:1 (Fig. 5). Again, this simple linear rela-tionship could be easily explored for modulating the particlesurface charge density to facilitate the adhesion propertiesand transport properties of the nanoparticles.
The effect of chitosan concentration on zeta potential wasa io of5 les zetap tion.
3
aver-aa cidic
Fig. 6. Effect of chitosan concentration on particle zeta potential at threedifferent chitosan molecular weight. Chitosan to TPP mass ratio = 5:1,T= 20± 1◦C, pH 5.0.
medium, the amine groups will be positively charged, con-ferring to the polysaccharide a high charge density[19].Therefore, the surface charge density of chitosan moleculesis strongly dependent on solution pH[20,21], and the ioniccross-linking process for the formation of chitosan–TPPnanoparticles is pH-responsive, providing opportunities tomodulate the formulation and properties of the chitosan–TPPnanoparticles.
To study the effects of changing environmental pH val-ues on nanoparticle size and zeta potential, chitosan–TPPnanoparticles formulated with MMW chitosan at fixed con-centration of 0.15% (w/v) and different chitosan to TPP massratio between 4:1 and 7:1 were examined at varying chitosansolution pH values. The variations in particle size and zetapotential with chitosan solution pH are shown separately inFig. 7A and B. The nanoparticles formed at solution pH 5.5had a smaller size but a higher particle zeta potential.Fig. 7Balso demonstrated an interesting trough in zeta potential val-ues at pH 5.0.
The surface charge reversal of nanoparticles in the acidicsolution of endo-lysosomes is proposed as the mechanismresponsible for the endo-lysosomal escape of the nanoparti-cles into cytoplasmic compartment for effective release andgene expression[22]. Surface charge reversal occurs whenprotons or hydronium ions from bulk solution are transferredto nanoparticle surface under acidic conditions, resulting ani hichw then abili-s s intoc
pro-d zetap idings re-d MH ar-
lso investigated at a fixed chitosan to TPP weight rat:1. The results inFig. 6show that, unlike the trend of particize increase with increasing chitosan concentration, theotential decreased with increasing chitosan concentra
.4. The effect of solution pH
Chitosan is a weak base polysaccharide, having ange amino group density of 0.837 per disaccharide unit[18],nd insoluble at neutral and alkaline pH values. In an a
ncreased surface charge density and zeta potential, would allow stronger electrostatic interactions betweenanoparticles and tissue cells, leading to localized destation of the cell membrane and escape of nanoparticleytoplasmic compartment.
Low molecular weight chitosan–TPP nanoparticlesuced at solution pH 5.5 were tested for their size andotential response to changing pH values of the resolution medium by simply adjusting the solution pH to a petermined value by titration with either 1 M NaOH or 1Cl solutions.Fig. 8A and B showed that both measured p
70 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
Fig. 7. (A) Effect of solution pH on MMW chitosan–TPP particle size at dif-ferent chitosan to TPP mass ratio.c= 0.15% (w/v),T= 20± 1◦C. (B) Effectof solution pH value on MMW chitosan–TPP nanoparticles zeta potential.c= 0.15% (w/v),T= 20± 1◦C.
ticle size and zeta potential are very sensitive to the changingpH values of the residing aqueous environment, indicating thesurface density of protonised amino groups and the degreeof protonisation are reversibly responsive to changing solu-tion pH values. The increase in measured average particle sizecould be caused mainly by particle aggregation when solutionpH value increased, rather than by further growth of the indi-vidual particle size after initial formation. The sharp increasein size at pH > 6.0 suggests that the degree of protonisationat surface of the particles were reduced, decreasing elec-trostatic repulsion between the particles thereby increasingthe probability of particle aggregation. The idea of depro-tonisation of the particle surface was supported by resultspresented inFig. 8B which shows a continual decrease inthe positive zeta potential well before the pH value reachedpH 6.0. Fig. 8B also shows that an isoelectric point forthe chitosan–TPP nanoparticles is located at around pH 9.0.Gelling of the nanoparticle colloidal system could easilyoccur when the overall particle surface charge is neutral atthe isoelectric point. The gelling mechanism in relation toswinging pH values has been investigated for producing smartresponsive nanoparticle systems for targeted drug delivery[20,21].
Fig. 8. (A) Responsive particle size change in relation to changing resid-ing solution pH values from 3.2 to 12.2. LMW chitosan–TPP nanoparticlesproduced at conditionsc= 0.50% (w/v), chitosan to TPP mass ratio = 5:1,T= 20± 1◦C, pH 5.5. (B) Responsive particle zeta potential change inrelation to changing residing solution pH values from 3.2 to 12.2. LMWchitosan–TPP nanoparticles produced at conditionsc= 0.50% (w/v), chi-tosan to TPP mass ratio = 5:1,T= 20± 1◦C, pH 5.5.
3.5. Stability of the chitosan–TPP nanoparticle system
The chitosan–TPP nanoparticle colloidal system is ther-modynamically unstable, especially at unfavourable solu-tion pH conditions and at high particle concentrations,because of high surface energy associated with the nanoscale dimensions.Fig. 9shows size growth kinetics of MMWchitosan–TPP particles at a dilute chitosan concentration0.15% (w/v). Ionic gelation and growth of the chitosan–TPPnanoparticles were completed within the first 60 min withsubsequently slight increase in particle size over the next24 h. No apparent aggregation of particles was observedduring this period at constant temperature and solutionpH.
However when the initial chitosan concentration wasincreased over and above a critical concentration, large aggre-gates formed instantaneously. The large aggregates wereobserved using the TEM imaging technique (Section3.6),
Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73 71
Fig. 9. Kinetics of chitosan–TPP nanoparticle size growth.c= 0.15% (w/v),chitosan to TPP mass ratio = 5:1,T= 20± 1◦C, pH 5.0.
and the critical aggregation concentration was studied usingZetasizer measurement which showed a drastic size increaseaccompanied by a sudden large reduction in zeta potential atthe critical concentration. The critical chitosan concentrationfor the spontaneous formation of aggregates depends on solu-tion pH and chitosan molecular weight.Tables 1 and 2showthat the critical chitosan concentration for LMW, MMW andHMW chitosan is 0.65%, 0.25%, 0.15% (w/v) at pH 4.0, and1.00%, 0.85%, 0.75% (w/v) at pH 5.0, respectively.
3.6. Morphological characteristics of chitosan–TPPnanoparticles
The morphological characteristics of the LMW chitosan–TPP nanoparticles were examined using the TEM technique.
Table 1Measured particles size and zeta potential at different chitosan molecular weight and concentration
72 Q. Gan et al. / Colloids and Surfaces B: Biointerfaces 44 (2005) 65–73
Fig. 10. TEM image of a single LMW chitosan–TPP nanoparticle.
TEM image of single chitosan–TPP nanoparticles (Fig. 10)reveals that the nanoparticles have a size range between 140and 250 nm which conforms with the size measurement byphoton correlation spectroscopy using Zeasizer 3000HAS.Fig. 11 shows the image of an aggregate of four distinc-tive single particles with clear joining boundaries formedalongside the regular geometry of the proximate polyhedron(pentagon and hexagon) shaped particles. Different to pastreported works[9,23], the evidence of the formation of poly-hydrons, instead of spheres, at nano-metric scale suggests anucleation through ionic gelation followed by semi-crystalformation and growth.
As previously described in Section3.5, the formation oflarge nanoparticle aggregates depends on chitosan concen-tration and solution pH.Fig. 12shows the TEM image of a
F TPPn
Fig. 12. TEM image of a large aggregate of LMW chitosan–TPP nanopar-ticles.
large aggregate formed with many distinctive single nanopar-ticles, each still possessing a similar nano-metric dimensionas being shown inFig. 10.
Fig. 13shows a TEM image of a chitosan–TPP particleincorporating protein molecules of bovine serum albumin(BSA). 0.03 mg/ml BSA molecules were added to an equalvolume of LMW chitosan solution (1.5%, w/v) in acidic acidand gently mixed for 1 h before TPP solution (0.5%, w/v) wasadded to make up chitosan to TPP mass ratio at 5:1. The BSAincorporated nanoparticles have a size range of 300–350 nm,
F tingB
ig. 11. TEM image of an aggregate of four single LMW chitosan–anoparticles with distinctive polyhedron shapes.
ig. 13. TEM image of a LMW chitosan–TPP nanoparticles incorporaSA 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-porated particles possessed a typical spherical shape andsmooth surface, which confirms the findings by Xu and Du[23], and Janes et al.[1]. Future work will investigate theencapsulation mechanism and efficiency of protein moleculesin the ionic initiated chitosan–TPP nanoparticle formationprocess, and the release kinetics of protein molecules fromthe particle.
4. Conclusion
The formation of high yield chitosan–TPP nanoparticleswith predetermined nano-metric size and surface charge den-sity can be simply manipulated and controlled by varying thekey processing conditions of chitosan concentration, chitosanto TPP weight ratio, and solution pH value. Within the testedrange of conditions, the increase in particle size and parti-cle zeta potential showed a simple linear relationship withincreasing chitosan to TPP weight ratio, but the zeta poten-tial at fixed chitosan to TPP ratio showed a linear decreasewith increasing chitosan concentration. Solution pH valueand chitosan concentration also had profound influence on thestability of the nanoparticle system, and the critical chitosanconcentrations for spontaneous formation of large particleaggregates at pH 5 were found to be 0.65%, 0.25%, 0.15%( 5.0f o-e unda
derd gu-l mi-c andg cturew ichb ationa
A
inga
R
icles. 47
lec-odyrm.
[3] Z. Cui, R.J. Mumper, Chitosan-based nanoparticles for topicalgenetic immunization, J. Control. Release 75 (2001) 409–419.
[4] L. Illum, I. Jabbal-Gill, M. Hinchcliffe, A.N. Fisher, S.S. Davis,Chitosan as a novel nasal delivery system for vaccines, Adv. DrugDeliv. Rev. 51 (2001) 81–96.
[5] K.A. Mislick, J.D. Baldeschwieler, Evidence for the role of proteo-glycans in cation-mediated gene transfer, Proc. Natl. Acad. Sci. USA93 (1996) 12349–12354.
[6] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug andgene delivery to cells and tissue, Adv. Drug Deliv. Rev. 55 (2003)329–347.
[7] K. Corsi, F. Chellat, L. Yahia, J.C. Fernandes, Mesenchymal stemcells, MG63 and HEK293 transfection using chitosan–DNA nanopar-ticles, Biomaterials 24 (2003) 1255–1264.
[8] K. Romoren, B.J. Thu, O. Evensen, Immersion delivery of plasmidDNA. II. A study of the potentials of a chitosan based delivery sys-tem in rainbow trout (Oncorhynchus mykiss) fry, J. Control. Release85 (2002) 215–225.
[9] S.V. Vinagradov, T.K. Bronich, A.V. Kabanov, Nanosized cationichydrogels for drug delivery: preparation, properties and interactionswith cells, Adv. Drug Deliv. Rev. 54 (2002) 223–233.
[10] Y. Ohya, M. Shiratani, H. Kobayashi, T. Ouchi, Release behav-ior of 5-fluorouracil from chitosan-gel nanospheres immobilizing5-fluorouracil coated with polysaccharides and their cell specificcytotoxicity, Pure Appl. Chem. A 31 (1994) 629–642.
[11] X.Z. Shu, K.J. Zhu, A new approach to prepare tripolyphos-phate/chitosan complex beads for controlled drug delivery, Int. J.Pharm. 201 (2000) 51–58.
[12] C. Song, V. Labhasetwar, X. Cui, T. Underwood, R.J. Levy, Arterialuptake of biodegradable nanoparticles for intravascular local drugdelivery: results with an acute dog model, J. Control. Release 54
[ esti-size,
[ epen-with
105–
[ ntury
[ san,
[ theation
[ n ofPoly-
[ nelease
[ ationy, J.
[ chi-001)
[ ulkn-
[ ro-. 250
w/v) at pH 4.0, and 1.00%, 0.85%, 0.75% (w/v) at pHor LMW, MMW and HMW chitosan, respectively. The islectric point of the chitosan–TPP nanoparticles was fot around pH 9.0.
Morphological study of the nanoparticles formed unifferent conditions revealed the formation of re
arly shaped polyhydron particles, an indication of serystallisation mechanism during the particle formationrowth, suggesting the particles were of compact struith orderly molecular arrangement, the discovery of whears important implications on gene/protein encapsulnd release mechanisms.
cknowledgement
To the European Social Funding programme for providscholarship to Colette Cochrane for this project.
eferences
[1] K.A. Janes, P. Calvo, M.J. Alonso, Polysaccharide colloidal partas delivery systems for macromolecules, Adv. Drug Deliv. Rev(2001) 83–97.
[2] S.C.W. Richardson, H.V.J. Kolbe, R. Duncan, Potential of low moular mass chitosan as a DNA delivery system: biocompatibility, bdistribution and ability to complex and protect DNA, Int. J. Pha178 (1999) 231–243.
nal uptake of biodegradable microparticles: effect of particlePharm. Res. 13 (1996) 1838–1845.
14] S. Prabha, W.Z. Zhou, J. Panyam, V. Labhasetwar, Size ddency of nanoparticles-mediated gene transfection: Studiesfractionnated nanoparticles, Int. J. Pharm. 244 (2002)115.
15] W. Paul, C. Sharma, Chitosan a drug carrier for the 21st ceS.T.P, Pharm. Sci. 10 (2000) 5–22.
16] V. Dodane, D. Vilivalam, Pharmaceutical applications of chitoPharm. Sci. Technol. Today 16 (1998) 246–253.
17] P. Calvo, J.L. Vila-Jato, M.J. Alonso, Effect of lysozyme onstability of polyester nanocapsules and nanoparticles: stabilizapproaches, Biomaterials 18 (1997) 1305–1310.
18] F.-L. Mi, H.-W. Sung, S.-S. Shyu, Synthesis and characterizatiobiodegradable TPP/genipin co-crosslinked chitosan gel beads,mer 44 (2003) 6521–6530.
19] K.W. Leong, H.Q. Mao, V.L. Truong-Le, DNA-polycationanospheres as non-viral gene delivery vehicles, J. Control. R53 (1998) 183–193.
20] J.A. Ko, H.J. Park, S.J. Hwang, Preparation and characterizof chitosan microparticles intended for controlled drug deliverPharm. 249 (2002) 165–174.
21] X.Z. Shu, K.J. Zhu, Novel PH–sensitive citrate cross-linkedtosan film for controlled drug release, Int. J. Pharm. 212 (219–28.
22] K. Makino, H. Ohshima, T. Kondo, Transfer of protons from bsolution to the surface of poly(l-lactide) microcapsules, J. Microecapsul. 3 (1986) 195–202.
23] Y. Xu, Y. Du, Effect of molecular structure of chitosan on ptein delivery properties of chitosan nanoparticles, Int. J. Pharm(2003) 215–226.
