Characterization of a Protein - Chitosan Complex

Chitosan particles are of interest for use as drug carriers, and the size is an important characteristic for drug loading and efficacy. The characterisation shows a consistent relationship between increasing size and decreasing zeta potential


Chitosan (CS) is a chain-like biodegradable polysaccharide, which has recently been studied extensively for use as a vector for therapeutic protein or gene delivery. Chitosan will form spherical particles from 100 nm to 600 nm in diameter depending on the molecular weight of the CS, with addition of tripolyphosphate (TPP) as a cross linking agent (figure 1). The size and surface charge of the CS-TPP complex particle formed affects the capacity and efficiency of the drug carrier and is dependent on the concentration of the reagents and environmental conditions such as pH and temperature [1-3]. In this work, the size and zeta potential of the CS-TPP complex is studied using light scattering techniques.

Figure 1: The molecular structure of chitosan (upper), tripolyphosphate (lower left) and a TEM picture of a CS-TPP complex particle (lower right, the scale is 100 nm).
mrk1379 fig 1

The CS-TPP particles were measured using the Zetasizer Nano, which combines dynamic light scattering and electrophoretic light scattering techniques.

The hydrodynamic diameter D(h) of the complex particles was measured by dynamic light scattering at an angle of 173°. The average D(h) was obtained from the Stokes-Einstein equation  

mrk1379 equation1

where k is the Boltzmann constant, T the absolute temperature, ƞ the viscosity of the liquid and D the diffusion coefficient of the particles, which was calculated by cumulants analysis of the first order correlation function (g1) [4].

The zeta potential of the complex particles was determined by measuring the change in the frequency of the scattered light at a forward angle due to motion of the particles in an applied electric field. From the frequency information, the electrophoretic mobility of the particle, UE, can be obtained. This can be related to zeta potential by the Henry Equation:

mrk1379 equation2

Where Ɛ is the dielectric constant, ζ is the zeta potential, ƞ is the viscosity, f(κa) is the Henry function, a is the radius of the particle and κ is the reciprocal of the Debye length. In the case of an aqueous solution, where ka>>1, the Smoluchowski approximation (where f(ka) = 1.5) is used [5].


CS was purchased from Sigma-Aldrich and used without further purification. The molecular weight of the CS was not specified due to its wide distribution. However, the viscosity was indicated as 200 mPa.s. TPP and Harpin protein [6] were purified and provided by Nanjing Agriculture University. The isoelectric point of the Harpin protein occurs at pH 4.7. When pH < 4.7, the protein has a positive charge on the surface, while when pH > 4.7, the protein is negatively charged.

To prepare the sample, CS was dissolved in acetic acid aqueous solution with pH around 3. The concentration of the CS was fixed at 1mg/ml. The solution was stirred for 1 hour and filtered to remove any sediment before measurement.

The complex without protein was prepared by adding various amounts of the cross linking agent, TPP, into the CS solution to create nanoparticles of various sizes. The ratio of the CS to TPP by weight was recorded. A complex with protein was prepared by firstly adding Harpin protein to the CS solution, adjusting the pH above 5.5 and then adding TPP solution. In this way, the Harpin protein was incorporated into the CS shell. Adjustment of the pH was made by the addition of NaOH solution.

Results and Discussion

CS-TPP Complex

The size and zeta potential of the CS-TPP particles without addition of protein at different CD/TPP ratios were measured as a function of pH. With increasing pH, the size of the particles (CS/TPP = 6:1) increased from 180 nm to around 350 nm (figure 2). The zeta potential of the particle is positive in the measured pH range from 4.8 to 6.3 indicating that protonation of the -NH2 group determines the surface charge. The measured zeta potential decreases with increasing pH, which appears independent of the CS/TPP ratio. The decreasing zeta potential with increasing pH is due to the reduction in the degree of protonation of the amino group on the CS.

Figure 2: The dependence of the size and zeta potential of CS-TPP complex ratios as a function of pH
mrk1379 fig 2

The increase in the particle size with increasing pH could be due to particle aggregation and not further growth of the individual particle after initial formation [3]. This hypothesis is supported by the tendency of the zeta potential to decrease with increasing pH, thereby reducing the electrostatic repulsion between the particles and rendering the dispersion less stable to aggregation.

CS-TPP-Harpin complex

The zeta potential and size of the CS-TPP-Harpin complex was measured with various amounts of the Harpin protein incorporated. The pH of the medium was 6.3, where the Harpin protein is negatively charged. In figure 3, it can be seen that the zeta potential decreases with increasing protein content, while the size of the complex increases. The decrease in the zeta potential can be explained by the neutralization of the positive charge on the CS by the negatively charged Harpin protein. The more Harpin protein added, the less positive charge on the complex, and the lower the zeta potential. The increasing size could be a result of particle aggregation due to the reduced zeta potential.

Figure 3: The dependence of the size and zeta potential of the CS-TPP-protein complex on the concentration of the protein. The CS/TPP ratio is 10:1
mrk1379 fig 3

Changing the CS/TPP ratio has an impact on both the particle size and zeta potential, which is illustrated in figure 4. With increasing CS/TPP ratio, the size of the particles decreases, and the zeta potential of the particles increases. TPP is negatively charged and acts as a crosslinking agent, which binds the CS chain to form a particle. In addition, the negative charge on the TPP will neutralize some of the positive charge on the CS. Therefore the increase in the zeta potential with the CS/TPP ratio can be explained as due to an increase in the amount of positive charge on the surface of the particles. The decrease in the size with increasing CS/TPP ratio could be due to two factors. One factor is that at low CS/TPP ratio, where there is more TPP, the TPP not only acts as an intra-particle crosslink agent, but also plays a role as an inter-particle crosslinking agent. This may form large aggregates. Another reason could be that at low CS/TPP ratio, the zeta potential is relatively low, which decreases the stability of the dispersion to aggregation.

Figure 4: The dependence of the size and zeta potential of the CS-TPP complex on the mass ratio of CS/TPP. The pH is 6.3 and the concentration of Harpin protein is 0.1mg/ml
mrk1379 fig 4


The size and zeta potential of the CS-TPP complex particles with and without incorporation of Harpin protein were measured by dynamic light scattering and laser Doppler electrophoresis, respectively.

The measured complexes have a positive surface charge under the measurement conditions investigated. The surface charge and resultant zeta potential is influenced by the pH of the medium, the amount of protein added and the CS/TPP ratio. The pH influences the charge and zeta potential by changing the chemical equilibrium governing the charge of the amino and hydroxyl group on CS. Both TPP and Harpin protein are negatively charged in the range of pH studied. Addition of the protein and increasing the relative amount of TPP can decrease the zeta potential due to charge neutralization.

The measured system always shows an increase in size with decreasing zeta potential. The larger particle size could originate from the formation of particle-particle aggregates, due to a decrease in electrostatic repulsion between the particles.

In this study, we have shown that the size and surface charge of the CS- TPP complex can be manipulated by changing the amount of the additive and the environmental parameters. This should be of interest for future studies on controlled drug release.


 [1] K.L. Douglas, C.A. Piccirillo, M. Tabrizian (2006) J. Controlled Rel. 115, 354-361.

[2] M. George, T.E. Abraham (2006) J. Controlled Rel. 114, 1-14.

[3] G. Quan, W. Tao, C. Colette and M. Paul (2005) Colloids and surfaces B: Biointerfaces 44, 65-73.

[4] International Organisation for Standardisation,Standard ISO22412:2008 Particle size analysis, Dynamic Light Scattering (DLS)

[5] Measurement and Interpretation of Electrokinetic Phenomena (2005) Pure Appl. Chem. 77, 1753-1805.

[6] O. Jonghee et al (2007) J. Biol. Chem. 282, 18, 13601-13609.


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